Saida Oubraim,Roh-Yu Shen,Samir Haj-Dahmane
Oxytocin excites dorsal raphe serotonin neurons and bidirectionally gates their glutamate synapses
Molecular neuroscience,Sensory neuroscience
Summary Oxytocin (OXT) modulates wide spectrum of social and emotional behaviors via modulation of numerous neurotransmitter systems, including serotonin (5-HT). However, how OXT controls the function of dorsal raphe nucleus (DRN) 5-HT neurons remains unknown. Here, we reveal that OXT excites and alters the firing pattern of 5-HT neurons via activation of postsynaptic OXT receptors (OXTRs). In addition, OXT induces cell-type-specific depression and potentiation of DRN glutamate synapses by two retrograde lipid messengers, 2-arachidonoylglycerol (2-AG) and arachidonic acid (AA), respectively. Neuronal mapping demonstrates that OXT preferentially potentiates glutamate synapses of 5-HT neurons projecting to medial prefrontal cortex (mPFC) and depresses glutamatergic inputs to 5-HT neurons projecting to lateral habenula (LHb) and central amygdala (CeA). Thus, by engaging distinct retrograde lipid messengers, OXT exerts a target-specific gating of glutamate synapses on the DRN. As such, our data uncovers the neuronal mechanisms by which OXT modulates the function of DRN 5-HT neurons.
Oxytocin excites dorsal raphe serotonin neurons and bidirectionally gates their glutamate synapses

Oxytocin (OXT) modulates wide spectrum of social and emotional behaviors via modulation of numerous neurotransmitter systems, including serotonin (5-HT). However, how OXT controls the function of dorsal raphe nucleus (DRN) 5-HT neurons remains unknown. Here, we reveal that OXT excites and alters the firing pattern of 5-HT neurons via activation of postsynaptic OXT receptors (OXTRs). In addition, OXT induces cell-type-specific depression and potentiation of DRN glutamate synapses by two retrograde lipid messengers, 2-arachidonoylglycerol (2-AG) and arachidonic acid (AA), respectively. Neuronal mapping demonstrates that OXT preferentially potentiates glutamate synapses of 5-HT neurons projecting to medial prefrontal cortex (mPFC) and depresses glutamatergic inputs to 5-HT neurons projecting to lateral habenula (LHb) and central amygdala (CeA). Thus, by engaging distinct retrograde lipid messengers, OXT exerts a target-specific gating of glutamate synapses on the DRN. As such, our data uncovers the neuronal mechanisms by which OXT modulates the function of DRN 5-HT neurons.

The nonapeptide oxytocin (OXT) is an evolutionarily conserved hormone well known for its role in parturition and the onset of lactation., In mammalian brain, OXT is mainly synthesized in magnocellular neurons of the hypothalamus, located in the supraoptic (SON), paraventricular (PVN),, and accessory nuclei., In addition to neurohypophysis afferences, oxytocinergic neurons project extensively to the forebrain, including medial prefrontal cortex (mPFC), septum, central amygdala (CeA), parts of the brainstem area and dorsal raphe nucleus (DRN). The physiological effects of OXT are largely mediated by a single OXT receptor (OXTR) belonging to Gq/11 G-protein-coupled receptor (GPCR) family and highly expressed throughout the brain., Consequently, by controlling the activity of complex neuronal networks, OXTRs regulate wide spectrum of behaviors including social,, maternal behaviors, anxiety-related behaviors, food intake, stress responses,, and pain. OXT interacts with others neurotransmitters, including serotonin (5-HT) to modulate an array of socio-affective behaviors. Indeed, the DRN, a major source of 5-HT to the forebrain, receives oxytocinergic fibers and expresses OXTRs., In rodent, activation of these receptors increases 5-HT release, reduces anxiety-related behaviors, enhances social reward, and decreases aggression behaviors., Oxytocinergic signaling in the DRN also regulates the normal development and expression of maternal social and affective behaviors., For instance, postpartum genetic deletion of DRN OXTRs increases pups loss after parturition, impairs nursing, increases aggression, and behavioral despair. In addition, numerous studies have reported that 5-HT regulates the function of PVN oxytocinergic neurons., This functional cross-talk between OXT and 5-HT systems is involved in several neurodevelopmental disorders, including autism spectrum disorders (ASD).,, Thus, animal and human studies have revealed an altered function of OXT and 5-HT systems in ASD.,, Importantly, pharmacological manipulations of OXT signaling improves social and emotional deficits associated with ASD, at least in part, by recruiting the 5-HT system.,,, Although the role of OXT-5-HT interaction in controlling socio-affective behaviors is well established, the precise mechanisms by which OXT controls the function of 5-HT neurons and synaptic dynamic in DRN remain unknown. Here, we show that activation of OXTRs excites DRN 5-HT neurons, alters their firing pattern, and induces target-specific depression and potentiation of glutamate synapses. This bidirectional gating is signaled by two distinct retrograde lipid messengers, the endocannabinoid 2-arachidonoylglycerol (2-AG) and arachidonic acid (AA), respectively. As such, the present study unravels novel cellular mechanisms by which OXT modulates the function of DRN 5-HT neurons.

All electrophysiological recordings were performed from neurons located in dorsal-medial (dmDRN) and ventral-medial (vmDRN) regions of the DRN. Serotonin neurons were identified using three criteria: (1) they display a resting membrane potential around −60 mV, (2) low-frequency and regular firing action potentials (1–2 Hz) following injection of suprathreshold depolarizing current pulses, and (3) a pronounced aflterhyperpolarizing potential (AHP)., Using a post-hoc tryptophan hydroxylase type 2 (Tph2) immunohistochemical labeling, we and others have previously shown that all DRN neurons that exhibit these properties were Tph2 positive neurons.

To assess the effects of OXT on the excitability of DRN 5-HT neurons, we first used cell-attached recordings and examined the effects of bath application of OXT on their spontaneous firing activity. As reported previously,, most sampled DRN 5-HT neurons were either quiescent or exhibit very low regular spontaneous firing (Control: 0.26 ± 0.04 Hz, n = 10, Figure 1A1). Administration of OXT (1 μM) increased the spontaneous firing activity in 77% of sampled DRN neurons (OXT: 0.54 ± 0.08 Hz, n = 10, p = 0.0064 vs. control, Figures 1A and 1B). In the remaining 23% of neurons, OXT shifted their firing pattern from slow regular to high burst firing (n = 4, Figure 1C). Next, using whole-cell recordings, we assessed the effect of OXT on the resting membrane potential and found that OXT (1 μM) induced a strong membrane depolarization that triggered action potential firing (n = 8, Figure 1D). In DRN 5-HT neurons clamped at −65 mV, and in the presence of voltage-dependent sodium channel blocker tetrodotoxin (TTX, 1 μM), OXT caused a significant inward shift of the holding current (IOXT), which was blocked by OXTR antagonist (d(CH₂)₅1,Tyr(Me)2,Orn⁸)-Oxytocin trifluoroacetate salt (OTA,10 μM) (OXTCTRL: 24.23 ± 5.46 pA; OXTOTA: 3.22 ± 2.84 pA, n = 6; p = 1.20E-4, Figure 1E). The inward current was also induced by a selective OXTR agonist (Thr⁴,Gly⁷)-Oxytocin (TGO,1 μM) and was blocked with OTA (1 μM) (TGOCTRL: 27.63 ± 3.73 pA, TGOOTA: 3.36 ± 2.90, n = 6; p = 3.90E-6, Figure 1F). Importantly, The OXT-induced inward current (Ioxt) was abolished by inhibition of postsynaptic G-protein signaling with guanosine-5'-(β-thio)-diphosphate (GDPβS) (IOXT GTP: 24.85 ± 4.53 pA, Ioxt GDPβS: 3.96 ± 1.62 pA, n = 7; p = 0.0033; Figure 1G), indicating that it was signaled by activation of G-protein. Collectively, these results demonstrate that OXT depolarizes and increases the excitability of putative DRN 5-HT neurons via activation of postsynaptic OXTRs.

We also interrogated the effects of OXT on the strength of glutamatergic inputs to putative DRN 5-HT neurons, by examining the impact of OXT on the amplitude of evoked excitatory postsynaptic currents (eEPSCs). We found that in a group of neurons (n = 18), OXT (1 μM) significantly depressed the amplitude of eEPSCs. This effect was blocked by OXTR antagonist OTA (10 μM) (OXTCTRL: 70.82 ± 1.97% of baseline, n = 18; OXTOTA: 101.93 ± 3.39% of baseline, n = 12; p = 1.99E-15; Figure 2A). Similar results were obtained using the selective OXTR agonist TGO (1 μM) (TGOCTRL: 68.74 ± 8.01%, n = 8; TGOOTA: 97.57 ± 4.99%, n = 5; p = 0.007; Figure 2B), thereby indicating that OXTRs signal the inhibition of eEPSCs. Examination of the paired pulse ratio (PPR) and coefficient of variance (CV) of eEPSCs, two independent measures of the probability of glutamate release, showed that the depression of eEPSCs was consistently accompanied by a significant increase in both PPR (Control: 1.11 ± 0.09; OXT: 1.29 ± 0.13, n = 6, p = 0.013, Figure 2C) and CV (Control: 0.31 ± 0.04, OXT: 0.42 ± 0.05, n = 6, p = 0.0046 control vs. OXT, Figure 2D), indicating that OXTRs depress glutamate DRN synapses by inhibiting glutamate release. In another group of 5-HT neurons, OXT (1 μM) was found to potentiate the amplitude of eEPSCs. This effect was also readily blocked by OTA (10 μM), a selective OXTR antagonist (OXTCTRL: 152.71 ± 11.19% of baseline, n = 8, OXTOTA: 103.95 ± 3.63% of baseline, n = 15; p = 6.18E-8; Figure 2E) and mimicked by selective OXTR agonist TGO (1 μM) (TGOCTRL: 144.81 ± 8.29%, n = 11; TGOOTA: 105.92 ± 6.89%, n = 7; p = 3.36E-4; Figure 2F). The OXTR-induced potentiation of eEPSCs was accompanied by a decrease in PPR (Control: 1.47 ± 0.15; OXT: 1.21 ± 0.11, n = 6, p = 0.025, vs. control, Figure 2G) and CV (control: 0.45 ± 0.04; OXT: 0.32 ± 0.06, n = 6, p = 0.037 vs. control, Figure 2H), indicating an increase in glutamate release. Collectively, these results indicate that OXT bidirectionally regulates the strength of glutamate synapses in different subpopulations of putative DRN 5-HT neurons.

To determine the mechanisms of OXT-induced depression of glutamate release, we first examined whether this effect was signaled by pre or postsynaptic OXTRs. To that end, we assessed the impact of inhibiting postsynaptic G-protein signaling with intracellular application of GDPβS on OXTR-induced inhibition of eEPSC amplitude. We found that postsynaptic intracellular application of GDPβS, which inhibited OXTRs as indicated by the blockade of IOXT (Figure 1F), also abolished the inhibition of eEPSCs (OXTGTP: 68.10 ± 1.18% of baseline, n = 10; OXTGDPβS: 98.88 ± 5.84% of baseline, n = 13; p = 2.54E-8; Figure 3A). This finding indicates that the OXT-induced depression of glutamate synapses is mediated by postsynaptic OXTRs and involves retrograde messengers. Next, because OXTRs are coupled to Gq/11 type G-protein, activation of which induces endocannabinoid (eCB) synthesis and release,, we examined whether the depression of glutamate release is signaled by retrograde eCB signaling. Blocking CB1Rs with AM 251 (3 μM) abolished the OXT-induced depression of eEPSC amplitude (OXTCTRL: 68.91 ± 1.24% of baseline, n = 10, OXTAM 251: 95.48 ± 3.20% of baseline, n = 12; p = 7.67E-12; Figure 3B). Collectively, these results indicate that activation of postsynaptic OXTRs depresses DRN glutamate synapses via retrograde eCBs. OXT receptors activate phospholipase C (PLC) leading to the formation of 1, 2 diacylglycerol (1,2 DAG),, which in turn is cleaved by diacylglycerol lipase (DAGL) to produce the eCB 2-arachidonoylglycerol (2-AG)., To test whether retrograde 2-AG mediates the depression of glutamate synapses, we examined the effect of PLC inhibitor U73122 on the OXTR-induced depression of eEPSCs. Treatment of DRN slices with U73122 (10 μM) but not its inactive analog U73433 (10 μM), blocked the OXTR-induced depression of eEPSC amplitude (OXTU73122: 111 ± 8.08% of baseline, n = 10, p = 0.20 vs. baseline; OXTU73343: 56.55 ± 2.95% of baseline, n = 9, p = 3.56E-7 vs. baseline; Figure 3C). Similar to the effect of PLC inhibitor, treatment of DRN slices with the DAGL inhibitor RHC80267 (50 μM) prevented the OXTR-induced inhibition of eEPSC amplitude (OXTCTRL: 60.32 ± 2.03% of baseline, n = 7; OXTRHC80267: 106.29 ± 7.25% of baseline, n = 12; p = 1.38E-6; Figure 3D). Collectively, these results indicate that activation of PLC/DAGL signaling cascade and the release of retrograde 2-AG mediates the OXTRs-induced depression of glutamate synapses of DRN 5-HT neurons.

To determine the mechanisms of OXT-mediated potentiation of glutamate synapses of putative DRN 5-HT neurons, we first assessed the role of postsynaptic OXTRs, by examining the effects of postsynaptic G-protein inhibition on OXT-induced potentiation of eEPSCs. Consistent with an effect mediated by postsynaptic OXTRs, our results revealed that intracellular application of GDPβS, which blocked IOXT (see Figure 1F), also abolished the potentiation of DRN glutamate synapses (OXTGTP: 156.48 ± 12.17% of baseline, n = 7; OXTGDPβS: 98.84 ± 4.39%, n = 13; p = 8.24E-8; Figures 4A1-A2). Because OXTRs are coupled to PLC signaling pathways, we next tested the involvement of this signaling cascade and found that inhibition of PLC with U73122 (5 μM), had no effect on OXT-induced potentiation of eEPSC amplitude (OXTU73343: 146.81 ± 11.99% of baseline, n = 7; OXTU73122: 157.26 ± 12.5% of baseline, n = 12; p > 0.5; Figures 4B1-B2). Similarly, inhibition of DAGL with RHC80267 (50 μM), did not affect the magnitude of OXT-induced potentiation of eEPSCs (OXTCTRL: 146.85 ± 7.01% of baseline, n = 5; OXTRHC80267: 157.56 ± 13.26% of baseline, n = 6; p > 0.5; Figures 4C1-C2). Collectively, these results indicate that while mediated by postsynaptic OXTRs, the OXT-induced potentiation of eEPSCs in putative DRN 5-HT neurons is not signaled by PLC/DAGL downstream cascade. Stimulation of OXTRs also activates cytosolic phospholipase A2 (cPLA2) and enhances the release of arachidonic acid (AA),, raising the possibility that cPLA2/AA signaling cascade could mediate the OXTR-induced potentiation of glutamate synapses. This possibility was tested by assessing the impact of inhibiting cPLA2 with AACOCF3 on the OXT-induced potentiation of EPSC amplitude. Results of this experiments showed that the cPLA2 inhibitor AACOCF3 (20 μM) abolished OXTR-induced potentiation of eEPSC amplitude (OXTCTRL: 141.36 ± 6.80% of baseline, n = 6, OXTAACOCF3: 102.95 ± 4.03% of baseline, n = 12; p = 3.62E-5; Figure 5A). In contrast, direct administration of AA (10 μM) mimicked the effect of OXT and induced potentiation of eEPSC amplitude (AA: 159.96 ± 8.15% of baseline, n = 9, p = 3.23E-6 vs. baseline; Figure 5B) similar to that obtained with OXT (OXT: 144.15 ± 6.97% of baseline, n = 6, p = 0.0018 vs. baseline; Figure 5B). Taken together, these results indicate that activation of cPLA2 and the increase in AA signaling mediate the OXTR-induced potentiation of glutamate synapses to DRN 5-HT neurons. Arachidonic acid is readily metabolized onto prostaglandins (PGs) by cyclooxygenase type 2 (COX2), an enzyme highly expressed in DRN neurons. These AA metabolites modulate synaptic strength in other brain areas via PG receptors, which are expressed in the DRN. Thus, we sought to determine the involvement of AA/COX2 downstream products by assessing the impact of COX2 inhibitors on OXTR-induced potentiation of DRN glutamate synapses. The results showed that the COX2 inhibitor N-(2-cyclohexyloxy-4-nitrophenyl) methanesulfonamide (NS398, 40 μM) had no effect on OXT-induced potentiation of eEPSC amplitude (OXTCTRL: 141.03 ± 6.90% of baseline, n = 6; OXTNS398: 133.32 ± 6.25% of baseline, n = 6; p > 0.5; Figure 5C), thereby ruling out the involvement of COX2 downstream products. AA can also directly inhibit voltage-gated potassium channels (Kv),, including presynaptic A-type channels,,, inhibition of which increases neurotransmitter release. Therefore, to test whether AA signals OXTR-induced potentiation of glutamate synapses by inhibiting A-type potassium, we examined whether blockade of these channels can mimic and occlude the effect of OXTRs. We found that bath application of 4-AP (50 μM), a blocker of A-type potassium channels, induced a robust potentiation of eEPSCs (4-AP: 270 ± 8.33%, n = 5, p = 0.010 vs. baseline, Figure 5D) and decreased the PPR (PPR baseline: 0.98 ± 0.06, PPR 4-AP: 0.69 ± 0.03, n = 5, p = 0.01 vs. baseline). Importantly, pretreatment of DRN slice with 4-AP occluded and prevented the OXTR-induced potentiation of glutamate synapses (OXTCTRL: 145.14 ± 10.23% of baseline, n = 6; OXT4-AP: 113.39 ± 7.59% of baseline, n = 7; p = 0.036; Figure 5E), suggesting that inhibition of K+ channels mediates the effects of OXT. Collectively, these results suggest that the OXT-induced potentiation of glutamate synapses in a subpopulation of putative DRN 5-HT neurons is mediated by activation of cPLA2 and generation of AA, which inhibits K+ channels and enhances glutamate release.

Accumulating evidence indicate that DRN 5-HT neurons are heterogeneous neurons clustered in different subpopulations with specific physiological and molecular properties.,, Each subgroup of 5-HT neurons preferentially project to distinct brain targets and mediate specific physiological functions., This raises the possibility that glutamate synapses of various subsets of DRN 5-HT projecting neurons may be differentially modulated by OXT. To test this possibility, we examined the effects of OXT on glutamate synapses of 5-HT neurons projecting to the medial prefrontal cortex (DRNmPFC), the lateral habenula (DRNLHb) and the central amygdala (DRNCeA), which are known to regulate stress homeostasis.,, We first targeted DRNmPFC neurons by injecting retrograde adeno-associated virus (AAV) expressing tdTomato (rgAAV-CAG-tdTomato) in the prelimbic (PL) and infralimbic (IL) subregions of the mPFC and performed whole-cell recordings from retrogradely labeled DRN neurons. Consistent with earlier reports,, we found that tdTomato-labeled DRNmPFC neurons were mainly clustered in the vmDRN between the medial longitudinal fasciculus (mlf) (Figure 6A1). Co-immunostaining of DRN sections for tryptophan hydroxylase 2 (Tph2) revealed that the vast majority (85.5 ± 10.5%) of DRN tdTomato labeled neurons were also Tph2 positive (Figure 6A1). Whole-cell recordings from these neurons showed that OXT (1 μM) potentiated (158.74 ± 8.88% of baseline, n = 6, p = 0.0013 vs. baseline, Figure 6A2) and depressed (75.50 ± 4.83 of baseline, n = 6, p = 0.0032 vs. baseline, Figure 6A3) the amplitude of eEPSCs in 80% and 20% of these neurons, respectively, indicating that OXTRs preferentially potentiate glutamate synapses of DRNmPFC neurons. Next, using similar approach, we targeted DRNLHb and DRNCeA neurons. We found that dtTomato-labeled DRNLHb neurons were scattered throughout the dmDRn, vmDRn, and lateral wings (Figure 6B1). Co-staining for Tph2 indicated that most of tdTomato neurons (76.6 ± 8.4%) were also Tph2 positive neurons (Figure 6B1). In contrast, dtTomato-labeled DRNCeA neurons were clustered in the vmDRN between the mlf, and most of these neurons were also Tph2 positive (82.01 ± 6.07%) (Figure 6C1). Bath application of OXT (1 μM) depressed the amplitude of eEPSC in all DRNLHb (56.49 ± 9.81% of baseline, n = 5, p = 0.0114 vs. baseline, Figure 6B2) and DRNCeA neurons (68.04 ± 6.09% of baseline, n = 7, p = 0.0017 vs. baseline, Figure 6C2), thereby indicating that glutamate synapses of these neurons are mainly inhibited by OXTRs.

The hormone OXT controls wide spectrum of socially driven behaviors via modulation of central 5-HT system. Dysfunctions of both OXT and 5-HT systems contribute to social and emotional deficits associated with neurodevelopmental disorders, such as autism spectrum disorder (ASD).,, However, the precise cellular mechanisms by which OXT regulates the function of DRN 5-HT neurons are not known. Here, we show that OXT via activation of OXTRs excites and alters the firing pattern of DRN 5-HT neurons. Beside these postsynaptic effects, our study reveals that OXT exerts target-specific gating of excitatory synaptic inputs to DRN by preferentially potentiating glutamate synapses of DRNmPFC neurons while inhibiting those impinging onto DRNLHb and DRNCeA neurons. The depression and potentiation of DRN glutamate synapses are signaled by two distinct retrograde lipid messengers, 2-AG and AA, respectively. As such, the present study unravels the cellular mechanisms by which OXT controls the function of DRN 5-HT neurons. The distribution pattern of OXTRs and oxytocinergic fibers in the DRN supports a key role of OXT in modulating DRN 5-HT neurons, which are implicated in the regulation of social and emotional behaviors.,, Indeed, the present study reveals that OXT robustly enhances the spontaneous activity and depolarizes DRN 5-HT neurons. These excitatory effects are readily blocked by OXTR antagonist and by inhibiting postsynaptic G-proteins demonstrating that they are mediated by activation of postsynaptic OXTRs. Interestingly, activation of these receptors has also been shown to depolarize and increase neuronal excitability in various brain areas, such as, the CA2 region of the hippocampus, the subiculum, and the lateral amygdala, thereby indicating that OXT largely exerts postsynaptic excitatory effects in the brain. In addition to the increased intrinsic excitability, OXT transforms firing pattern of putative DRN 5-HT neurons from regular to bursting activity. A similar effect has also been reported in CA2 pyramidal neurons. Most of DRN 5-HT neurons are quiescent or fire at slow and regular frequency,, and the probability to encounter burst firing neurons in the DRN is very low. The OXT-induced switch from low regular to high burst firing mode combined with the increased excitability could mediate the reported in vivo increase of 5-HT release and contribute, at least in part, to anxiolytic-like effects induced by activation of OXTRs., Furthermore, because activation of DRN 5-HT neurons increases sociability,, it is possible that the OXT-induced excitation of these neurons could encode the prosocial effects of OXT, though additional studies are required to directly test this notion. Along with the increased intrinsic excitability, OXT bidirectionally controls the strength of glutamate synapses of putative DRN 5-HT neurons. Depending on the subpopulation of 5-HT neurons, OXT can either depress or potentiate glutamate synapses via a decrease and increase in glutamate release, respectively. The OXT-induced decrease in glutamate release is consistent with previous studies showing that OXT pre-synaptically inhibits glutamatergic transmission in others brain areas.,, Mechanistically, the current study reveals that the OXT-induced depression of DRN glutamate synapses is prevented by inhibiting postsynaptic G-proteins, by blocking CB1Rs and by inhibiting PLC/DAGL cascade. These findings indicate that OXT inhibits glutamatergic transmission onto putative DRN 5-HT neurons via retrograde 2-AG signaling, an eCB known to controls the strength and plasticity of DRN glutamate synapses. Such conclusion agrees with numerous studies showing that OXTR are canonically coupled to Gαq/11-G-protein-coupled receptors, and with the notion that activation of these receptors increases the synthesis and release of 2-AG in several brain areas,,, including DRN. The finding that OXTRs inhibit the strength of glutamate synapses by recruiting retrograde eCB signaling reveals a functional link between OXT and eCB systems that could play a key role in mediating the behavior and physiological effects of OXT. Consistent with this concept, results from a previous study have shown that OXT enhances social reward, at least in apart, via a recruitment of brain eCB signaling. In a subset of putative DRN 5-HT neurons, activation of postsynaptic OXTRs potentiates glutamatergic transmission by enhancing glutamate release. This effect is not signaled by PLC/DAGL, but rather by activation of cPLA2/AA signaling cascade. The released AA retrogradely increases glutamate release by inhibiting voltage-gated A potassium channels (Figure 8). Several lines of evidence support this model. First, the OXT-induced potentiation of excitatory transmission is blocked by inhibiting postsynaptic G-protein function and cPLA2, but not PLC/DAGL signaling cascade. Second, manipulation that increases AA levels enhances glutamatergic transmission and mimics the effect of OXT. Finally, pharmacological blockade of voltage-dependent potassium channels mimics the OXT-induced increase in glutamate release and prevents additional potentiation of DRN glutamate synapses. While prior 4-AP application might not occlude any further effect of OXT in facilitating glutamate release, these results suggest their common mechanism and the direct involvement of AA and A-type potassium channels. Interestingly, such a functional model is consistent with previous studies showing that exogenous AA directly inhibits A-type potassium channels,, and potentiates glutamatergic transmission., Consequently, the current finding further supports the synaptic role of AA and suggest that OXTR-driven AA release serves as a retrograde messenger that gates synaptic transmission in the DRN. Given that OXTRs and cPLA2 are expressed in neurons of many others brain regions, it is possible that OXTR-driven AA signaling may be a general mechanism by which OXT potentiates central synapses. Altogether, our findings indicate that by engaging different membrane lipids (i.e. 2-AG, AA), OXT can bidirectionally control excitatory inputs to DRN 5-HT neurons. This control is target-specific, as OXT preferentially potentiates glutamatergic inputs to DRNmPFC neurons, while inhibiting the inputs to DRNLHb and DRNCeA neurons. Although the precise physiological roles of OXT-mediated bidirectional and target-specific synaptic gating in the DRN remain unknown, it is possible that, OXT serves as a filter that engage a specific set of neuronal circuits to encode complex social behaviors. In addition, given the involvement of DRN 5-HT system in effecting regulation, and sociability, the new evidence of OXT-mediated control of the excitability of 5-HT neurons and synaptic dynamic in the DRN may represent a potential neuronal mechanism by which OXT modulates social and emotional behaviors. Consistent with this notion, results from both animal and human studies have shown that OXT ameliorates social deficits in ASD via activation of 5-HT system.,,,, Furthermore, growing evidence indicate that eCB system regulates social behaviors, and that enhancing eCB signaling rescues social deficits observed in various animal models of ASD., The present finding that OXT regulates synaptic dynamic in the DRN by recruiting eCB signaling may represents an additional mechanism by which OXT control plethora of behaviors and physiological functions. Consequently, OXT-driven eCB signaling in the DRN could represent a target for future treatment strategies of social and emotional deficits associated with neurodevelopment disorders.

In this study, we show that OXT plays a key role in controlling the excitability of putative DRN 5-HT neurons and gating their glutamate synapses. Although our results unravel a cell-type-specific bidirectional control of DRN glutamate synapses, future studies using molecularly identified subset of DRN 5-HT neurons, combined with channelrhodopsin assisted circuit analysis, are required to further decipher the effects of OXT on the various DRN neuronal circuitries.



Samir Haj-Dahmane: sh38@buffalo.edu.

Further information and request for resources are available from the lead contact upon request.

All experiments and animal procedures used in this study were approved by the University at Buffalo Animal Care and Use Committee (IACUC) in accordance with the National Institutes of Health (NIH) Guideline for the Care and Use of Laboratory Animals. Male Sprague Dawley rats (4-7 weeks old) were kept in 12/12 h dark/light cycles with food and water ad libido at room temperature and standard humidity.

For neuronal retrograde labeling, a retrograde tracer rgAAV-CAG-tdTomato (Addgene cat # 59462, Addgene, Watertown, MA, USA) was used. The viral particles were injected in the prelimbic and infralimbic areas of the mPFC, in the central amygdala (CeA) or lateral Habenula (LHb) of male Sprague Dawley rats (3 to 4 weeks) under ketamine (60 mg/kg) and xylazine (2.5 mg/kg) anesthesia. Briefly, rats were positioned in stereotaxic frame (KOPF instruments, Tujunga, CA, USA) and small burr holes were drilled above the brain regions of interest. The viral suspension was bilaterally injected either in the medial prefrontal cortex (mPFC) (coordinates to bregma: AP+3, ML ± 0.6, DV -4 to brain surface: 500 nl/side, 100 nl/min), the (coordinates to bregma: AP-2, ML ± 4, DV -8 to brain surface: 300 nl/side, 100 nl/min), or (coordinates to bregma: AP-3.6, ML ± 0.6, DV -4.6 to brain surface: 300 nl/side, 100 nl/min). The injections were performed using a 33-gauge needle attached to 5 μl syringe (Hamilton, Reno, NV, USA) and driven by a Stoelting pump (Stoelting Co, Wood Dale, IL, USA). After the injections were completed, animals were allowed to recover on the heat pad in their home cage. Animal welfare was monitored daily after the procedure. Two to three weeks after surgery, rats were used for in vitro electrophysiological and immunohistochemical studies.

To generate coronal brain slices containing the DRN, rats were deeply anesthetized by isoflurane and decapitated using guillotine, the brain was removed and brainstem region containing the DRN was isolated and sliced (350 μm) using a vibratome (Leica VT1200S; Leica Biosystem, St Louis, MO, USA) in ice-cold cutting solution of the following composition (in mM): 110 Choline-Cl; 2.5 KCl; 0.5 CaCl2; 7 MgSO4; 1.25 NaH2PO4; 26.2 NaHCO3; 11.6 sodium L-ascorbate; 3.1 sodium pyruvate, 25 glucose and saturated with 95% O2/5% CO2. Slices were incubated in the cutting solution for 15 min at 35°C and then in regular ACSF of the following compositing (in mM: 119 NaCl; 2.5 CaCl2; 1.3 MgSO4; 1 NaH2PO4; 26.2 NaHCO3; 11 glucose and continuously bubbled with a mixture of 95% O2/5% CO2) for 30 min at 35°C. After recovery, slices were stored in holding chamber containing regular ACSF saturated with 95% O2/5% CO2 and maintained at room temperature for at least one hour before electrophysiological recordings. For electrophysiological experiments, slices were transferred to a recording chamber (Warner Instruments, Hamden, CT, USA) mounted on a fixed upright microscope and continuously perfused (2 to 3 ml/min) with regular ACSF solution saturated with 95% O2/5% CO2 and heated to 30 ± 1°C using a solution heater (Warner Instruments, Hamden, CT, USA). DRN neurons were visualized using Olympus BX51 microscope equipped with a 40X water-immersion objective, differential interference contrast (DIC) and fluorescence systems. Somatic whole-cell recordings from putative DRN 5-HT neurons were performed using patch electrodes with tip resistance of 3 - 5 mΩ, when filled with an internal solution containing (in mM): 120 potassium gluconate; 10 KCl; 10 Na2-phosphocreatine; 10 HEPES; 1 MgCl2; 1 EGTA; 2 Na2-ATP; 0.25 Na-GTP (pH 7.3 adjusted with KOH, osmolality 280 - 290 mOsm). In some experiments, TdTomato retrogradely labeled putative DRN 5-HT neurons were visualized using Texas Red fluorescence filter (λ = 540-580 nm) and targeted for whole-cell patch clamp recordings. Membrane current and voltage were amplified with an Axoclamp 2B or Multiclamp 700B amplifier (Molecular Devices, San Jose Union City, CA, USA), filtered at 3 kHz, digitized at 20 kHz with Digidata 1440 and acquired using pClamp 10.7 software (Molecular Devices, San Jose, CA, USA). Evoked excitatory postsynaptic currents (eEPSCs) were induced by single square-pulses (duration = 100 to 200 μs) delivered at 0.1 Hz and recorded from neurons voltage clamped at -70 mV in the presence of GABAA receptor antagonists picrotoxin (100 μM). To access paired-pulse ratio (PPR), pair of eEPSCs were triggered with an inter-stimulus interval of 50 ms. The intensity of the stimulus was adjusted to evoke 75 % of the maximal amplitude of eEPSCs. The cell-input resistance and access resistance (10 - 20 mΩ) were monitored online throughout the experiments using 10 mV hyperpolarizing voltage steps (500 ms duration). Recordings were discarded when the input and series resistance changed by more than 20 %.

Rats were deeply anesthetized with pentobarbital and transcardially perfused with 100 ml phosphate buffer saline (PBS) (0.9 %), followed by 250 ml ice-cold 4 % paraformaldehyde (PF) in 1X phosphate buffer (pH 7.4). Brains were removed and post-fixed in 4 % PF for an hour, and immersed in 10 % sucrose solution at 4°C. Coronal sections (40 μm) containing the DRn were cut using a microtome (American Optical 860 model). To block unspecific binding, brain sections containing DRn, were first incubated overnight at 4°C in PB solution containing 10% normal goat serum (NGS) and 0.5 % Triton X-10. The sections were then incubated overnight at 4 °C with the primary antibodies (Mouse anti-Tph2, 1:200 dilution, rabbit anti-RFP, 1:200 dilution, abcam, Waltham, MA, USA) in a PBS solution containing 1 % NGS and 0.5 % Triton X-10. Section were washed in PBS (10 min, 3 times) and incubated with the species-specific secondary antibodies (Goat anti-mouse Alexa 488, Goat anti-rabbit Alexa 594; 1:500, abcam, Waltham, MA, USA) for 5 hours at room temperature. After incubation with secondary antibodies, the sections were washed three time with PBS and mounted on glass slides for fluorescence imaging using Leica DMi8 fluorescence inverted microscope (Leica Microsystems Inc., Buffalo Grove, IL, USA).

Glutamate-mediated eEPSCs were analyzed using Clampfit 10.7 software (Molecular Devices, San Jose Union City, CA, USA). The amplitude of eEPSCs was determined by measuring the average current during a 2 ms time window at the peak of each eEPSC and subtracted from baseline current measured during a 5 ms before the stimulus artifact. All eEPSC amplitudes were normalized to the mean baseline amplitude recorded for at least 5 min before administration of OXTR agonists. For paired pulse experiments the paired pulse ratio (PPR = eEPSC2/eEPSC1) were averaged for at least 60 consecutive trials before and during activation of OXTRs. To determine the coefficient of variation (CV), the standard deviation (SD) and the mean amplitude of eEPSCs were calculated for at least 60 consecutive trials before and during administration of OXTR agonists. The CV was then given by the following ratio (SD) / (eEPSC mean amplitude). Statistical analysis was performed using Origin 8.0 software (OriginLab Co, Northampton, MA, United States). The results in the text and figures are expressed as mean ± SEM. Parametric paired t-test was used for within group comparison. For comparison between groups, analysis of variance (ANOVA) using post-hoc Bonferroni test was used. Statistical significance was set at p < 0.05.