Esketamine alleviates depressive-like behavior in neuropathic pain mice through the METTL3-GluA1 pathway
In recent years, the incidence of neuropathic pain and depression, among other neurological and psychiatric disorders, has been gradually increasing, significantly impacting the well-being and societal engagement of patients (Fitzcharles et al. ; Silva et al. ; Hardy ; Dunne et al. ). The urgent demand for effective treatments for these conditions underscores the importance of in-depth research into the underlying pathological mechanisms and the search for viable therapeutic approaches. Recent studies have indicated that the utilization of esketamine, an antagonist of the N-methyl-D-aspartate (NMDA) receptor that operates in a non-competitive manner, demonstrates promising advantages for addressing neuropathic pain and manifestations similar to depressive tendencies (Yavi et al. ; Henter et al. ). However, there remains a lack of understanding regarding esketamine's molecular mechanisms in these disorders. Esketamine, the S-enantiomer of Ketamine, functions as a non-competitive NMDA receptor antagonist. Its pharmacological effects are primarily achieved by blocking glutamate receptors, resulting in analgesic, antidepressant, and anesthetic effects. Compared to Ketamine, Esketamine has stronger analgesic and antidepressant effects and fewer adverse reactions, leading to its growing application in managing treatment-resistant depression and specific pain conditions (Medeiros et al. ; Kheirkhah et al. ). Studies have shown that the Homer1a-mGluR5 signaling pathway may be targeted by Esketamine to ameliorate symptoms of depression provoked by neuropathic pain in rat models (Wang et al. , ). Moreover, Esketamine combined with electroconvulsive therapy (ECT) can activate the PI3K/Akt/GLT-1 pathway, significantly improving depressive symptoms (Zang et al. ). Within the scope of current research, m6A methylation, as an emerging RNA epigenetic modification, has grabbed notable focus for its involvement in governing biological and irregular mechanisms in the neural network (Weng et al. ; Zhang et al. ; Wang et al. , ). m6A methylation is primarily regulated by m6A methyltransferases (such as METTL3) and demethylases (Wen et al. ; Han et al. ). Variations in the expression levels of different m6A modification enzymes within the central nervous system have been linked to the onset and progression of neurological disorders (Zhang et al. ). Hence, the proposition is that esketamine could potentially affect the adjustment of m6A methylation modification, influencing neuropathic pain and depressive tendencies in mice. The amygdala, a critical brain region for emotional regulation and processing, exerts a considerable influence on the emergence of neuropathic pain and depression. AMPAR (AMPA receptor) serves as a primary mediator of neuronal synaptic transmission in the amygdala, with GluA1 being a crucial subunit of the AMPAR, directly influencing synaptic transmission efficiency (Herring et al. ; Kim et al. ; Gallina et al. ). Therefore, it is postulated that the synaptic expression of the GluA1 subunit of AMPAR in the amygdala may be influenced by esketamine, potentially altering neuropathic pain and depressive symptoms in mice. The objective of this research was to clarify the molecular pathways governing the regulation of AMPAR GluA1 subunit synaptic expression in the amygdala by esketamine-mediated m6A modification in mice exhibiting neuropathic pain-induced depressive-like behavior. Transcriptome sequencing and bioinformatics analysis were employed for this purpose, leveraging findings from relevant studies (Zeng et al. ; Wang et al. , ; Liu et al. ). The neuropathic pain mouse model was induced through spinal nerve ligation, followed by behavioral tests to assess esketamine's therapeutic effects on depressive-like behavior. Moreover, neuronal dendritic spine morphology and density in the amygdala region, along with the presence of m6A modification and the m6A modification enzyme METTL3 in neuropathic pain mice, were investigated, with a specific focus on the m6A modification levels of GluA1 mRNA and the association between METTL3 and GluA1 mRNA. This research endeavors to provide new molecular targets and therapeutic strategies for neuropathic pain and depression while improving the understanding of the functional roles and regulatory mechanisms of m6A methylation in the nervous system. These findings could pave the way for novel approaches to treating neurobehavioral disorders, offering hope for patients in need of effective interventions. 

Construction of neuropathic pain models in mice Beijing Vital River Laboratories in China supplied seventy-five 6-week-old male SPF C57BL/6N mice for the experiment. The mice resided in separate enclosures within an SPF animal establishment where the humidity ranged from 60 to 65% and the temperature maintained between 22 to 25 °C. Following a one-week adaptation period, the mice's wellbeing was evaluated prior to initiating the study. The Institutional Animal Ethics Committee granted approval for the experimental methods and animal handling guidelines. In compliance with the experimental demands, a total of five mice were chosen at random to form the Sham cohort, with the rest of the mouse subjects assigned for the generation of the SNI model. Generating the SNI model required inducing rapid anesthesia in the mice with a mixture of 5% isoflurane and 50% oxygen, followed by reducing the isoflurane concentration to 2% to maintain a stable anesthetic state. During the surgical procedure, close monitoring of the mice's respiratory rate and foot pad color was conducted to ensure proper anesthesia. Subsequently, the mice underwent ligation of the spinal nerves, with a small incision made at the L4-S1 level on the left side, removal of the paraspinal muscles from L5 and L6 transverse processes, and dissection of the left L5 and L6 spinal nerves to uncover the parallel L5 and L6 spinal nerves while lying on their back. Precise ligation of nerves was performed at a 2–5 mm interval from the distal aspect of the dorsal root ganglia utilizing 6–0 silk sutures, subsequently stitching the incision and surrounding skin with 5–0 silk sutures (Lin et al. ). The procedures for the Sham group mice were identical to those for the SNI group, except for the absence of ligation of the dorsal root ganglia. Following model establishment, Sucrose Preference Test (Days 1–2): Typically performed on the 7th day after model establishment and lasting two days. The experimental environment was kept quiet with moderate lighting to ensure the mice could freely consume water and sucrose solution. A significant reduction in sucrose preference (e.g. preference below 65%) was considered indicative of depressive-like behavior.Tail Suspension Test (Day 3): Conducted 24 h after the sucrose preference test. Mice were suspended by their tails, and their struggling and immobility times were recorded during a 6-min test. Reduced struggling time and increased immobility were considered signs of depressive-like behavior.Forced Swim Test (Day 4): Conducted 24 h after the tail suspension test. Mice were placed in a cylindrical container with sufficient water depth to prevent escape, and their swimming and immobility times were recorded over a 6-min test. Prolonged immobility during the later stages was indicative of depressive-like behavior.mice exhibiting depressive behavior were selected through behavioral assessments for subsequent experiments. Placement of intrathecal catheter and lentivirus infection After anesthetizing the mice, the occipital ridge was exposed. Using an 18-gauge needle, a midline incision was made through the atlanto-occipital membrane. Subsequently, a polyethylene-10 catheter was implanted into the gap and passed through the subarachnoid space to the dorsal aspect at the L4-L5 level. Following the placement of the intrathecal catheter, gene overexpression and silencing techniques mediated by lentivirus vectors were implemented according to the experimental groups. Specifically, for oe-METTL3, sh-METTL3, sh-GluA1, and their respective negative controls, lentivirus interference vectors pSIH1-H1-copGFP (sh-interference vector, SI501A-1, System Biosciences, USA) and overexpression vector pCDH-CMV-MCS-EF1α-copGFP (oe-overexpression vector, CD511B-1, System Biosciences, USA) were constructed for METTL3 and GluA1 based on lentivirus technology. The viral titers for sh-METTL3 and sh-GluA1 were 9 × 10 7 TU/mL and 8 × 10 7 TU/mL, respectively, while the viral titer for the oe-METTL3 overexpression vector was 1.2 × 10 8 TU/mL. Full-length mus-METTL3 (NM_019721.2) sequence was cloned into the lentivirus overexpression vector for oe-METTL3. The silencing sequences for METTL3 and GluA1 were as follows: sh-METTL3: CCTCAGTGGATCTGTTGTGAT; sh-GluA1: GCGACAGCTTTGAGATGACTT; oe-Scrambled: ATCGTACGATACGATACGTA; sh-Scrambled: GTGCGTTGCTAGTACCAAC (Jiang et al. , ; Lin et al. ). A 0.1 ml lentivirus solution was intrathecally injected into the mice for target gene silencing and overexpression. Animal grouping This research sought to analyze the consequences of esketamine and distinct gene regulations on the actions and cellular levels of mice. The mice underwent careful categorization into the subsequent groups: Sham + Saline, SNL + Saline, SNL + esketamine, Sham + oe-NC, SNL + oe-NC, SNL + oe-METTL3, Sham + Saline + sh-NC, SNL + Saline + sh-NC, SNL + esketamine + sh-NC, SNL + esketamine + sh-METTL3, SNL + esketamine + sh-GluA1, Sham + oe-NC + sh-NC, SNL + oe-NC + sh-NC, SNL + oe-METTL3 + sh-NC, and SNL + oe-METTL3 + sh-GluA1. In this experimental setup, esketamine and Saline underwent intrathecal injection with a 10 mg/kg dosage of esketamine in the treatment group (Liu et al. ; Kawazoe et al. ) and an equivalent volume of Saline as the control. The administering of the injection recurred at 3-day intervals for a total of 14 days (Rahman et al., ; Wu et al. ; Wang et al. , ). Mechanical allodynia behavioral testing Behavioral and sensitivity assessments were conducted on days 21, 23, 27, and 34 after model establishment. The "up and down" method was employed using a Von Frey filament to trigger responses in the plantar area of the mice's left hind paw, measuring their pain threshold. During the experiment, a 0.16 g Von Frey filament was applied to the paw for 5 stimulations, each lasting 2 s. The Von Frey filament was bent at a 45-degree angle to ensure adequate stimulus intensity, and the mouse's withdrawal response was observed. If the mouse exhibited behaviors like paw flicking, paw withdrawal, or paw licking during each stimulation, it was recorded as painful with the symbol "X"; if there were no avoidance behaviors, it was recorded as non-painful with the symbol "O". If the response was recorded as "O", a higher-grade Von Frey filament was used for stimulation, whereas if the response was "X", a lower-grade filament was used. The strength of the Von Frey filament was adjusted based on the mouse's response, ultimately resulting in the paw withdrawal threshold (measured in grams). A lower paw withdrawal threshold indicates higher sensitivity, whereas a higher threshold suggests lower sensitivity (Jiang et al. ). Behavioral testing for thermal hyperalgesia in mice Behavioral and sensitivity assessments were conducted on days 21, 23, 27, and 34 after model establishment. Mice were habituated on the thermal hyperalgesia testing apparatus. While the mice were in a quiet state, their left hind paw was irradiated with a certain intensity of heat radiation, but the maximum exposure time should not exceed 20 s to prevent skin burns. When mice experienced thermal pain, they would retract their paw to evade the heat. At this point, the heat source should be discontinued, and the paw withdrawal latency recorded. Each mouse underwent three measurements with intervals of 5–10 min between each trial. The average of the three measurements was taken as the final paw withdrawal latency for that particular mouse. By adjusting the irradiation intensity, the baseline paw withdrawal latency was maintained between 12 ± 2 s. A shorter paw withdrawal time indicates heightened sensitivity to thermal hyperalgesia; conversely, a longer time suggests reduced sensitivity to thermal hyperalgesia (Jiang et al. ). Experiment on forced swimming behavior Behavioral and sensitivity assessments were conducted on days 14 after model establishment. The experiment began by filling a 2 L beaker with 1.5 L of water and placing it in a water bath (40 cm × 40 cm × 50 cm). The water temperature in the beaker was adjusted to 25–26 degrees Celsius using a thermometer. The experiment comprised two stages: pre-adaptation and the actual experiment. In the pre-adaptation phase, mice were placed in the beaker for 15 min, then immediately removed, dried, and placed in cages for normal feeding and adaptation for 1–2 days (once a day). After the last pre-adaptation forced swimming trial, the mice were returned to their cages for 24 h of acclimatization. Subsequently, the experiment phase began by placing the mice in the beaker at the predetermined temperature for testing. A video recording system was positioned at the forefront of the experimental setup to record the time the mice remained still in the beaker. The total recording time was 6 min, with the initial 2 min designated for acclimation without recording any mouse activity. During the remaining 4 min, if the mice ceased active struggling, floated motionless, did not move their limbs, or if the hind legs were still for more than 1 s, they were considered still. Throughout the experiment, precise water temperature control was crucial, and mice were promptly dried after being removed from the water to prevent them from getting cold (Ebrahimi-Ghiri et al. ). The experimental environment was kept quiet to avoid any additional stress stimuli. Tail suspension test Behavioral and sensitivity assessments were conducted on days 14 after model establishment. Mice were suspended upside down on a hanging device 50 cm above the ground, with their tails secured with tape. The total time the mice remained completely immobile (without attempting to struggle) within a 6-min period was measured. The experiments were conducted in a soundproof room with soft lighting to minimize external disturbances. After each trial, fresh tape was applied to ensure consistent experimental conditions for each mouse. Open-field test (OFT) Behavioral and sensitivity assessments were conducted on days 14 after model establishment. Mice were placed in a white experimental box with each side measuring 40 cm, and their movements were automatically tracked by a camera positioned above the box. The total distance traveled by the mice served as a metric for assessing their motor abilities. Throughout the experiment, the frequency of mouse entries into the central area of the enclosure and the length of time spent in that zone were monitored to gauge their anxiety levels. Following the completion of the test, the experimental box was cleaned using 75% alcohol after each trial to prevent residual odors from affecting the test results (Tang et al. ). Sucrose preference (SP) test Behavioral and sensitivity assessments were conducted on days 14 after model establishment. Baseline measurements were conducted during the acclimation period. During this phase, individual mice were housed in distinct enclosures and sequentially provided access to a pair of containers filled with sugared liquid (1% weight/volume) and regular water for a duration of 12 h each. Prior to the preference test, an absence of food and water was enforced on the mice for a period lasting 12 h. In the preference trial, individual mice were placed in separate cages and offered unrestricted consumption of two bottles, one with sucrose water (1% concentration) and the other with water, each containing 200 mL, over a 12-h span. Evaluation of the intake of sugary liquid and regular water was accomplished through the measurement of bottle weights. The determination of the SP value involved the application of a specific formula: Sucrose intake (g) × 100% / [sucrose intake (g) + water intake (g)] (Li et al. ). Golgi staining method Implemented the Golgi staining method employing a Golgi staining kit (HTKNS1125NH, from Beijing Boleide Technology Development Co., Ltd.). The entire brain underwent immersion in the impregnating solution for a duration of 4 to 5 days, followed by a 24-h transfer in a solution at 4℃. After rinsing the brain tissue, coronal slices (100 μm) were obtained using a cryostat. Subsequently, the brain slices were dehydrated, cleared with xylene, and air-dried at room temperature (RT) in darkness. The optical microscope (BX50; Olympus Corp, located in Tokyo, Japan) was employed for capturing neural visuals of the amygdala nucleus. For the representative dendritic secondary branches of the amygdaloid nucleus neurons, 10 μm segments of dendrites were selected for quantitative analysis of spine number and density using Image-J software (Tian et al. ). RNA extraction and sequencing The synaptosome preparation procedure was as follows: Mice were rapidly sacrificed, and their brains were transferred into ice-chilled artificial cerebrospinal fluid (aCSF). Precise localization of the BLA was achieved by slicing brain samples into sections measuring 300–400 μm in thickness using a vibratome placed in an ice-filled container. Under a dissecting microscope and guided by a brain atlas, the BLA region was identified, and the tissue was transferred to a tube containing homogenization buffer. Homogenization of the tissue was achieved through the operation of a glass homogenizer, and the homogenate underwent sequential low- and medium-speed centrifugation. Ultracentrifugation in a Percoll or sucrose density gradient was then performed to isolate synaptosome-enriched fractions. Isolation of total RNA was conducted on synaptic samples obtained from the basolateral amygdala of mice, with the assistance of Trizol reagent (Invitrogen, Carlsbad, USA). The Nanodrop 2000 spectrophotometer (manufactured by Nanodrop in the USA) was used to evaluate both the concentration and purity levels of the RNA samples. Meeting certain conditions was mandatory for the samples to be deemed appropriate for future studies; these included having an RNA Integrity Number (RIN) above 7.0 and a 28S:18S ratio exceeding 1.5. A sum of 5 µg of RNA was applied in every sample. Initially, depleting ribosomal RNA (rRNA) from the total RNA was achieved by implementing the Ribo-Zero™ Magnetic Kit (Epicentre Technologies, Madison, Wisconsin, USA). Subsequently, preparation of Illumina libraries involved the use of the NEB Next Ultra RNA Library Prep Kit (#E7775, NEB, USA), leading to RNA fragmentation into approximately 300 bp fragments. Reverse transcription, initiated with random primers, allowed for the generation of the first-strand cDNA, subsequently advancing to the fabrication of second-strand cDNA within a reaction buffer incorporating dUTP Mix (10 ×). Repaired were the extremities of cDNA portions, involving the integration of poly(A) appendages as well as sequencing adaptors. Post ligation of Illumina sequencing adapters, the second cDNA strand was cleaved using the USER Enzyme from NEB based in the USA, leading to the development of strand-specific libraries. PCR amplification, as well as subsequent purification and enrichment processes, were applied to the library's DNA. Identification of the library was executed via the Agilent 2100 system, while quantification was accomplished using the KAPA Library Quantification Kit offered by KAPA Biosystems. Finally, Hunan Sanzhong Biotech Co., Ltd. oversaw the paired-end sequencing process utilizing the Illumina NextSeq CN500 sequencer. Improving quality control and aligning reference genomes for sequencing data analysis Assessment of the quality of the paired-end reads in the raw sequencing data was accomplished by employing FastQC software version 0.11.8. Processing of the raw data involved the use of Cutadapt software version 1.18, which facilitated the removal of Illumina sequencing adapters and poly(A) tail sequences. Subsequently, implementation of a Perl script facilitated the removal of reads containing more than 5% N content. Later, extracting reads with a base quality of 20 or greater, which represented 70% of the dataset, was accomplished through the utilization of the FASTX Toolkit software version 0.0.13. Rectifying paired-end sequences is achievable by utilizing BBMap software. Ultimately, the premium filtered reads underwent alignment with the mouse reference genome through hisat2 software version 0.7.12. Bioinformatics analysis The "edgeR" algorithm in R was applied to analyze differences in gene expression by manipulating the mRNA read count dataset. Defining the parameters to isolate differentially expressed genes (DEGs), the thresholds were stipulated as |log2FC|> 1 and P.value < 0.05. The analysis of gene enrichment involved the application of the "clusterProfiler" package in R for GO and KEGG pathway investigations. The m6A sites of the genes were predicted using the RMBase v2.0 website. Please refer to Figure for further details. Utilization occurred with the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database ( https://cn.string-db.org/ ). GluA1 was entered as the query gene, with the interaction score threshold set to 0.4, and the species limited to Mus musculus. Genes significantly interacting with GluA1 were filtered to construct the interaction network. Western blot Extraction of total proteins from tissues was performed with the application of RIPA lysis buffer supplemented with PMSF (P0013C, Biyuntian, Shanghai, China). Incubation of the samples on ice for a period of 30 min was followed by centrifugation at a force of 8000 g for a duration of 10 min at a temperature of 4℃, with the subsequent collection of the supernatant. Measurement of the overall protein concentration was conducted with the aid of a BCA assay kit (23,227, ThermoFisher, USA). Subsequently, treatment involved dissolving 50 µg of protein in 2 × SDS loading buffer followed by boiling at 100℃ for 5 min. The samples were progressed beyond SDS-PAGE gel electrophoresis, proceeded by their transfer onto a PVDF membrane and blockage employing 5% non-fat milk at RT for 1 h. The PVDF membrane underwent an overnight incubation at 4℃ with diluted primary antibodies against METTL3 (1:1000, ab195352, Abcam, Cambridge, UK), GluA1 (1:1000, #13,185, CST, USA), and GAPDH (1:2500, ab9485, Abcam, Cambridge, UK) as a loading control. Upon completion of three successive washes utilizing TBST over 10-min intervals, the membrane was exposed to an HRP-conjugated secondary antibody (goat anti-rabbit IgG H&L (HRP), 1:2000, ab97051, Abcam, Cambridge, UK) for 1 h. Upon cleansing, the membrane was positioned onto a fresh glass platform, with a fitting concoction of liquids A and B solutions from an ECL chemiluminescence detection kit (abs920, Aibico Biotechnology Co., Ltd., Shanghai, China) carefully spread on the membrane within a low-lit room. Image acquisition was performed applying the Bio-Rad imaging system sourced from the USA, followed by analysis utilizing Quantity One v4.6.2 software. The aim was to ascertain the relative protein content represented by the grayscale value of the respective protein bands normalized to GAPDH protein bands. Conducted thrice, the experiment yielded an average value through computation (Liu et al. ). Dot blot assays The extraction of total RNA from synaptosomes derived from the amygdala tissue of mice was accomplished through the use of TRIzol (15,596,026, ThermoFisher, USA). After the purification of total RNA, the subsequent step involved isolating mRNA by employing the mRNA purification kit (80,817, Beijing Boao Pike Biotechnology Co., Ltd., Beijing, China). The process involved in attaching the isolated mRNA onto Amersham Hybond-N + membrane, which is optimal for nucleic acid transfer, included denaturation under ultraviolet irradiation for a period of 7 min followed by immediate freezing on ice. Upon ultraviolet cross-linking completion, staining of the membrane was carried out with 0.02% methylene blue (M8030, Beijing Solibao Technology Co., Ltd., Beijing, China) and followed by an overnight incubation period with anti-m6A antibody (1:5000, ab284130, Abcam, Cambridge, UK). Ultimately, the membrane underwent visualization with Immobilon Western Chemilum HRP Substrate (Merck Millipore, Germany) (Cai et al. ). Me-RIP analysis The amygdala synaptic bodies from mice were subjected to RNA isolation through the application of TRIzol (ThermoFisher, USA, 15596026), followed by mRNA extraction from the isolated total RNA using an mRNA purification kit (Beijing BioPark Biotechnology Co., Ltd., Beijing, China, 80817). The organization of the experiment involved the addition of the anti-m6A antibody (Abcam, UK, Cambridge, 1:500, ab151230) or the anti-IgG antibody (Abcam, UK, Cambridge, 1:100, ab109489) into the IP buffer (20 millimolars Tris pH 7.5, 140 millimolars NaCl, 1% NP-40, 2 millimolars EDTA), with subsequent binding to protein A/G magnetic beads for a duration of one hour. Subsequently, the mRNA that was isolated and purified, together with the bead-antibody complex, underwent an overnight incubation at 4 °C in the IP buffer containing ribonuclease and protease inhibitors. The eluted RNA was cleansed through the phenol–chloroform method and subsequently examined for GluA1 mRNA levels utilizing RT-qPCR. The primers used were as follows: forward: ATGTGGAAGCAAGGACTCCG; reverse: GCTGACCACTCTGCCATTCT (Yang et al. ; Vu et al. ). PAR-CLIP Small nuclear ribonucleoprotein particles (snRNPs) are indispensable for pre-mRNA splicing by recognizing splice sites. To investigate the protein-RNA interactions in mouse amygdala tissue, 4-thiouridine (4SU) at a concentration of 200 mM was incubated with the tissue extract for 14 h. The samples were then crosslinked at 0.4 J/cm2 under 365 nm UV light. Subsequent immunoprecipitation was carried out using METTL3 antibody (1:50 dilution, ab195352, Abcam, Cambridge, UK) followed by radiolabeled precipitation of RNA with [γ−32-P]-ATP for visualization by autoradiography. PAR fragments were subjected to RT-qPCR analysis after proteinase K treatment for protein removal and RNA extraction, to assess GluA1 mRNA expression levels (Du et al. ; Yue et al. ). Immunohistochemistry (IHC) Following deparaffinization, the amygdala tissue sections of mice underwent a rehydration process using a series of alcohol gradients. Subsequently, they were immersed in distilled water for a duration of 2 min, exposed to a 3% hydrogen peroxide solution in methanol for 20 min, rinsed in distilled water for another 2 min, and then placed in 0.1 M PBS for 3 min. Retrieving antigens involved soaking the tissue sections in an antigen retrieval solution in a water bath and subsequently cooling them in tap water. Afterward, the sections received incubation with a blocking solution of normal goat serum (C-0005, Shanghai Haoran Biotechnology Co., Ltd., Shanghai, China) at RT for 20 min, followed by removal of excess liquid. Subsequently, the primary antibodies used for the sections were mouse METTL3 (ab195352, 1:500, Abcam, USA) and GluA1 (ab31232, 1:100, Abcam, USA), with an overnight incubation at 4 °C. After being rinsed three times in 0.1 M PBS for 5 min each, a secondary antibody, goat anti-rabbit IgG (ab6785, 1:1000, Abcam, UK), was applied and allowed to incubate at 37 °C for 20 min. Then, the sample was exposed to the active solution of horseradish peroxidase-conjugated avidin–biotin complex (0343-10000U, Beijing Yimo Biotechnology Co., Ltd, China) at 37 °C for 20 min, followed by visualization utilizing DAB (ST033, Guangzhou Weijia Technology Co., Ltd., China). Post-color development, the slides underwent a rinsing process and were subsequently counterstained employing hematoxylin (PT001, Shanghai Bogu Biotechnology Co., Ltd., China) for a duration of 1 min. Following this, they were treated with 1% ammonia water to attain a blue hue, dehydrated using a series of graded alcohol solutions, cleared with xylene, and finally mounted with neutral resin for microscopic analysis and imaging. The examination comprised counting 100 cells in each of the five high-power fields selected randomly on the slide. The slides were independently reviewed by two experienced pathologists. Positive IHC staining criteria: METTL3-positive cells are characterized by brown nuclear staining, while GluA1-positive cells exhibit brown membrane staining. Three repetitions were executed for every single experiment (Toyota et al. ; Cheng et al. ). Statistical analysis Utilizing the SPSS software (version 21.0, IBM, USA) facilitated the statistical analysis of the research findings. Continuous variables' descriptive statistics were depicted by mean and standard deviation measures. Preceding the initiation of correlation evaluations, assessments for the normality and homoscedasticity were performed. In cases where the data fulfilled the criteria for normal distribution and homogeneity of variances, we applied independent t-tests for comparisons between groups, one-way ANOVA or repeated measures ANOVA for assessing differences among several groups, and Tukey's post hoc examination for pairwise group comparisons. Highlighting the crucial aspect of statistical relevance, a p-value below 0.05 was deemed significant in the analysis. Utilization of the Graphpad Prism 8.4.0 software facilitated the graphical depiction of the statistical outcomes. 

Beijing Vital River Laboratories in China supplied seventy-five 6-week-old male SPF C57BL/6N mice for the experiment. The mice resided in separate enclosures within an SPF animal establishment where the humidity ranged from 60 to 65% and the temperature maintained between 22 to 25 °C. Following a one-week adaptation period, the mice's wellbeing was evaluated prior to initiating the study. The Institutional Animal Ethics Committee granted approval for the experimental methods and animal handling guidelines. In compliance with the experimental demands, a total of five mice were chosen at random to form the Sham cohort, with the rest of the mouse subjects assigned for the generation of the SNI model. Generating the SNI model required inducing rapid anesthesia in the mice with a mixture of 5% isoflurane and 50% oxygen, followed by reducing the isoflurane concentration to 2% to maintain a stable anesthetic state. During the surgical procedure, close monitoring of the mice's respiratory rate and foot pad color was conducted to ensure proper anesthesia. Subsequently, the mice underwent ligation of the spinal nerves, with a small incision made at the L4-S1 level on the left side, removal of the paraspinal muscles from L5 and L6 transverse processes, and dissection of the left L5 and L6 spinal nerves to uncover the parallel L5 and L6 spinal nerves while lying on their back. Precise ligation of nerves was performed at a 2–5 mm interval from the distal aspect of the dorsal root ganglia utilizing 6–0 silk sutures, subsequently stitching the incision and surrounding skin with 5–0 silk sutures (Lin et al. ). The procedures for the Sham group mice were identical to those for the SNI group, except for the absence of ligation of the dorsal root ganglia. Following model establishment, Sucrose Preference Test (Days 1–2): Typically performed on the 7th day after model establishment and lasting two days. The experimental environment was kept quiet with moderate lighting to ensure the mice could freely consume water and sucrose solution. A significant reduction in sucrose preference (e.g. preference below 65%) was considered indicative of depressive-like behavior.Tail Suspension Test (Day 3): Conducted 24 h after the sucrose preference test. Mice were suspended by their tails, and their struggling and immobility times were recorded during a 6-min test. Reduced struggling time and increased immobility were considered signs of depressive-like behavior.Forced Swim Test (Day 4): Conducted 24 h after the tail suspension test. Mice were placed in a cylindrical container with sufficient water depth to prevent escape, and their swimming and immobility times were recorded over a 6-min test. Prolonged immobility during the later stages was indicative of depressive-like behavior.mice exhibiting depressive behavior were selected through behavioral assessments for subsequent experiments. 

After anesthetizing the mice, the occipital ridge was exposed. Using an 18-gauge needle, a midline incision was made through the atlanto-occipital membrane. Subsequently, a polyethylene-10 catheter was implanted into the gap and passed through the subarachnoid space to the dorsal aspect at the L4-L5 level. Following the placement of the intrathecal catheter, gene overexpression and silencing techniques mediated by lentivirus vectors were implemented according to the experimental groups. Specifically, for oe-METTL3, sh-METTL3, sh-GluA1, and their respective negative controls, lentivirus interference vectors pSIH1-H1-copGFP (sh-interference vector, SI501A-1, System Biosciences, USA) and overexpression vector pCDH-CMV-MCS-EF1α-copGFP (oe-overexpression vector, CD511B-1, System Biosciences, USA) were constructed for METTL3 and GluA1 based on lentivirus technology. The viral titers for sh-METTL3 and sh-GluA1 were 9 × 10 7 TU/mL and 8 × 10 7 TU/mL, respectively, while the viral titer for the oe-METTL3 overexpression vector was 1.2 × 10 8 TU/mL. Full-length mus-METTL3 (NM_019721.2) sequence was cloned into the lentivirus overexpression vector for oe-METTL3. The silencing sequences for METTL3 and GluA1 were as follows: sh-METTL3: CCTCAGTGGATCTGTTGTGAT; sh-GluA1: GCGACAGCTTTGAGATGACTT; oe-Scrambled: ATCGTACGATACGATACGTA; sh-Scrambled: GTGCGTTGCTAGTACCAAC (Jiang et al. , ; Lin et al. ). A 0.1 ml lentivirus solution was intrathecally injected into the mice for target gene silencing and overexpression. 

This research sought to analyze the consequences of esketamine and distinct gene regulations on the actions and cellular levels of mice. The mice underwent careful categorization into the subsequent groups: Sham + Saline, SNL + Saline, SNL + esketamine, Sham + oe-NC, SNL + oe-NC, SNL + oe-METTL3, Sham + Saline + sh-NC, SNL + Saline + sh-NC, SNL + esketamine + sh-NC, SNL + esketamine + sh-METTL3, SNL + esketamine + sh-GluA1, Sham + oe-NC + sh-NC, SNL + oe-NC + sh-NC, SNL + oe-METTL3 + sh-NC, and SNL + oe-METTL3 + sh-GluA1. In this experimental setup, esketamine and Saline underwent intrathecal injection with a 10 mg/kg dosage of esketamine in the treatment group (Liu et al. ; Kawazoe et al. ) and an equivalent volume of Saline as the control. The administering of the injection recurred at 3-day intervals for a total of 14 days (Rahman et al., ; Wu et al. ; Wang et al. , ). 

Behavioral and sensitivity assessments were conducted on days 21, 23, 27, and 34 after model establishment. The "up and down" method was employed using a Von Frey filament to trigger responses in the plantar area of the mice's left hind paw, measuring their pain threshold. During the experiment, a 0.16 g Von Frey filament was applied to the paw for 5 stimulations, each lasting 2 s. The Von Frey filament was bent at a 45-degree angle to ensure adequate stimulus intensity, and the mouse's withdrawal response was observed. If the mouse exhibited behaviors like paw flicking, paw withdrawal, or paw licking during each stimulation, it was recorded as painful with the symbol "X"; if there were no avoidance behaviors, it was recorded as non-painful with the symbol "O". If the response was recorded as "O", a higher-grade Von Frey filament was used for stimulation, whereas if the response was "X", a lower-grade filament was used. The strength of the Von Frey filament was adjusted based on the mouse's response, ultimately resulting in the paw withdrawal threshold (measured in grams). A lower paw withdrawal threshold indicates higher sensitivity, whereas a higher threshold suggests lower sensitivity (Jiang et al. ). 

Behavioral and sensitivity assessments were conducted on days 21, 23, 27, and 34 after model establishment. Mice were habituated on the thermal hyperalgesia testing apparatus. While the mice were in a quiet state, their left hind paw was irradiated with a certain intensity of heat radiation, but the maximum exposure time should not exceed 20 s to prevent skin burns. When mice experienced thermal pain, they would retract their paw to evade the heat. At this point, the heat source should be discontinued, and the paw withdrawal latency recorded. Each mouse underwent three measurements with intervals of 5–10 min between each trial. The average of the three measurements was taken as the final paw withdrawal latency for that particular mouse. By adjusting the irradiation intensity, the baseline paw withdrawal latency was maintained between 12 ± 2 s. A shorter paw withdrawal time indicates heightened sensitivity to thermal hyperalgesia; conversely, a longer time suggests reduced sensitivity to thermal hyperalgesia (Jiang et al. ). 

Behavioral and sensitivity assessments were conducted on days 14 after model establishment. The experiment began by filling a 2 L beaker with 1.5 L of water and placing it in a water bath (40 cm × 40 cm × 50 cm). The water temperature in the beaker was adjusted to 25–26 degrees Celsius using a thermometer. The experiment comprised two stages: pre-adaptation and the actual experiment. In the pre-adaptation phase, mice were placed in the beaker for 15 min, then immediately removed, dried, and placed in cages for normal feeding and adaptation for 1–2 days (once a day). After the last pre-adaptation forced swimming trial, the mice were returned to their cages for 24 h of acclimatization. Subsequently, the experiment phase began by placing the mice in the beaker at the predetermined temperature for testing. A video recording system was positioned at the forefront of the experimental setup to record the time the mice remained still in the beaker. The total recording time was 6 min, with the initial 2 min designated for acclimation without recording any mouse activity. During the remaining 4 min, if the mice ceased active struggling, floated motionless, did not move their limbs, or if the hind legs were still for more than 1 s, they were considered still. Throughout the experiment, precise water temperature control was crucial, and mice were promptly dried after being removed from the water to prevent them from getting cold (Ebrahimi-Ghiri et al. ). The experimental environment was kept quiet to avoid any additional stress stimuli. 

Behavioral and sensitivity assessments were conducted on days 14 after model establishment. Mice were suspended upside down on a hanging device 50 cm above the ground, with their tails secured with tape. The total time the mice remained completely immobile (without attempting to struggle) within a 6-min period was measured. The experiments were conducted in a soundproof room with soft lighting to minimize external disturbances. After each trial, fresh tape was applied to ensure consistent experimental conditions for each mouse. 

Behavioral and sensitivity assessments were conducted on days 14 after model establishment. Mice were placed in a white experimental box with each side measuring 40 cm, and their movements were automatically tracked by a camera positioned above the box. The total distance traveled by the mice served as a metric for assessing their motor abilities. Throughout the experiment, the frequency of mouse entries into the central area of the enclosure and the length of time spent in that zone were monitored to gauge their anxiety levels. Following the completion of the test, the experimental box was cleaned using 75% alcohol after each trial to prevent residual odors from affecting the test results (Tang et al. ). 

Behavioral and sensitivity assessments were conducted on days 14 after model establishment. Baseline measurements were conducted during the acclimation period. During this phase, individual mice were housed in distinct enclosures and sequentially provided access to a pair of containers filled with sugared liquid (1% weight/volume) and regular water for a duration of 12 h each. Prior to the preference test, an absence of food and water was enforced on the mice for a period lasting 12 h. In the preference trial, individual mice were placed in separate cages and offered unrestricted consumption of two bottles, one with sucrose water (1% concentration) and the other with water, each containing 200 mL, over a 12-h span. Evaluation of the intake of sugary liquid and regular water was accomplished through the measurement of bottle weights. The determination of the SP value involved the application of a specific formula: Sucrose intake (g) × 100% / [sucrose intake (g) + water intake (g)] (Li et al. ). 

Implemented the Golgi staining method employing a Golgi staining kit (HTKNS1125NH, from Beijing Boleide Technology Development Co., Ltd.). The entire brain underwent immersion in the impregnating solution for a duration of 4 to 5 days, followed by a 24-h transfer in a solution at 4℃. After rinsing the brain tissue, coronal slices (100 μm) were obtained using a cryostat. Subsequently, the brain slices were dehydrated, cleared with xylene, and air-dried at room temperature (RT) in darkness. The optical microscope (BX50; Olympus Corp, located in Tokyo, Japan) was employed for capturing neural visuals of the amygdala nucleus. For the representative dendritic secondary branches of the amygdaloid nucleus neurons, 10 μm segments of dendrites were selected for quantitative analysis of spine number and density using Image-J software (Tian et al. ). 

The synaptosome preparation procedure was as follows: Mice were rapidly sacrificed, and their brains were transferred into ice-chilled artificial cerebrospinal fluid (aCSF). Precise localization of the BLA was achieved by slicing brain samples into sections measuring 300–400 μm in thickness using a vibratome placed in an ice-filled container. Under a dissecting microscope and guided by a brain atlas, the BLA region was identified, and the tissue was transferred to a tube containing homogenization buffer. Homogenization of the tissue was achieved through the operation of a glass homogenizer, and the homogenate underwent sequential low- and medium-speed centrifugation. Ultracentrifugation in a Percoll or sucrose density gradient was then performed to isolate synaptosome-enriched fractions. Isolation of total RNA was conducted on synaptic samples obtained from the basolateral amygdala of mice, with the assistance of Trizol reagent (Invitrogen, Carlsbad, USA). The Nanodrop 2000 spectrophotometer (manufactured by Nanodrop in the USA) was used to evaluate both the concentration and purity levels of the RNA samples. Meeting certain conditions was mandatory for the samples to be deemed appropriate for future studies; these included having an RNA Integrity Number (RIN) above 7.0 and a 28S:18S ratio exceeding 1.5. A sum of 5 µg of RNA was applied in every sample. Initially, depleting ribosomal RNA (rRNA) from the total RNA was achieved by implementing the Ribo-Zero™ Magnetic Kit (Epicentre Technologies, Madison, Wisconsin, USA). Subsequently, preparation of Illumina libraries involved the use of the NEB Next Ultra RNA Library Prep Kit (#E7775, NEB, USA), leading to RNA fragmentation into approximately 300 bp fragments. Reverse transcription, initiated with random primers, allowed for the generation of the first-strand cDNA, subsequently advancing to the fabrication of second-strand cDNA within a reaction buffer incorporating dUTP Mix (10 ×). Repaired were the extremities of cDNA portions, involving the integration of poly(A) appendages as well as sequencing adaptors. Post ligation of Illumina sequencing adapters, the second cDNA strand was cleaved using the USER Enzyme from NEB based in the USA, leading to the development of strand-specific libraries. PCR amplification, as well as subsequent purification and enrichment processes, were applied to the library's DNA. Identification of the library was executed via the Agilent 2100 system, while quantification was accomplished using the KAPA Library Quantification Kit offered by KAPA Biosystems. Finally, Hunan Sanzhong Biotech Co., Ltd. oversaw the paired-end sequencing process utilizing the Illumina NextSeq CN500 sequencer. 

Assessment of the quality of the paired-end reads in the raw sequencing data was accomplished by employing FastQC software version 0.11.8. Processing of the raw data involved the use of Cutadapt software version 1.18, which facilitated the removal of Illumina sequencing adapters and poly(A) tail sequences. Subsequently, implementation of a Perl script facilitated the removal of reads containing more than 5% N content. Later, extracting reads with a base quality of 20 or greater, which represented 70% of the dataset, was accomplished through the utilization of the FASTX Toolkit software version 0.0.13. Rectifying paired-end sequences is achievable by utilizing BBMap software. Ultimately, the premium filtered reads underwent alignment with the mouse reference genome through hisat2 software version 0.7.12. 

The "edgeR" algorithm in R was applied to analyze differences in gene expression by manipulating the mRNA read count dataset. Defining the parameters to isolate differentially expressed genes (DEGs), the thresholds were stipulated as |log2FC|> 1 and P.value < 0.05. The analysis of gene enrichment involved the application of the "clusterProfiler" package in R for GO and KEGG pathway investigations. The m6A sites of the genes were predicted using the RMBase v2.0 website. Please refer to Figure for further details. Utilization occurred with the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database ( https://cn.string-db.org/ ). GluA1 was entered as the query gene, with the interaction score threshold set to 0.4, and the species limited to Mus musculus. Genes significantly interacting with GluA1 were filtered to construct the interaction network. 

Extraction of total proteins from tissues was performed with the application of RIPA lysis buffer supplemented with PMSF (P0013C, Biyuntian, Shanghai, China). Incubation of the samples on ice for a period of 30 min was followed by centrifugation at a force of 8000 g for a duration of 10 min at a temperature of 4℃, with the subsequent collection of the supernatant. Measurement of the overall protein concentration was conducted with the aid of a BCA assay kit (23,227, ThermoFisher, USA). Subsequently, treatment involved dissolving 50 µg of protein in 2 × SDS loading buffer followed by boiling at 100℃ for 5 min. The samples were progressed beyond SDS-PAGE gel electrophoresis, proceeded by their transfer onto a PVDF membrane and blockage employing 5% non-fat milk at RT for 1 h. The PVDF membrane underwent an overnight incubation at 4℃ with diluted primary antibodies against METTL3 (1:1000, ab195352, Abcam, Cambridge, UK), GluA1 (1:1000, #13,185, CST, USA), and GAPDH (1:2500, ab9485, Abcam, Cambridge, UK) as a loading control. Upon completion of three successive washes utilizing TBST over 10-min intervals, the membrane was exposed to an HRP-conjugated secondary antibody (goat anti-rabbit IgG H&L (HRP), 1:2000, ab97051, Abcam, Cambridge, UK) for 1 h. Upon cleansing, the membrane was positioned onto a fresh glass platform, with a fitting concoction of liquids A and B solutions from an ECL chemiluminescence detection kit (abs920, Aibico Biotechnology Co., Ltd., Shanghai, China) carefully spread on the membrane within a low-lit room. Image acquisition was performed applying the Bio-Rad imaging system sourced from the USA, followed by analysis utilizing Quantity One v4.6.2 software. The aim was to ascertain the relative protein content represented by the grayscale value of the respective protein bands normalized to GAPDH protein bands. Conducted thrice, the experiment yielded an average value through computation (Liu et al. ). 

The extraction of total RNA from synaptosomes derived from the amygdala tissue of mice was accomplished through the use of TRIzol (15,596,026, ThermoFisher, USA). After the purification of total RNA, the subsequent step involved isolating mRNA by employing the mRNA purification kit (80,817, Beijing Boao Pike Biotechnology Co., Ltd., Beijing, China). The process involved in attaching the isolated mRNA onto Amersham Hybond-N + membrane, which is optimal for nucleic acid transfer, included denaturation under ultraviolet irradiation for a period of 7 min followed by immediate freezing on ice. Upon ultraviolet cross-linking completion, staining of the membrane was carried out with 0.02% methylene blue (M8030, Beijing Solibao Technology Co., Ltd., Beijing, China) and followed by an overnight incubation period with anti-m6A antibody (1:5000, ab284130, Abcam, Cambridge, UK). Ultimately, the membrane underwent visualization with Immobilon Western Chemilum HRP Substrate (Merck Millipore, Germany) (Cai et al. ). 

The amygdala synaptic bodies from mice were subjected to RNA isolation through the application of TRIzol (ThermoFisher, USA, 15596026), followed by mRNA extraction from the isolated total RNA using an mRNA purification kit (Beijing BioPark Biotechnology Co., Ltd., Beijing, China, 80817). The organization of the experiment involved the addition of the anti-m6A antibody (Abcam, UK, Cambridge, 1:500, ab151230) or the anti-IgG antibody (Abcam, UK, Cambridge, 1:100, ab109489) into the IP buffer (20 millimolars Tris pH 7.5, 140 millimolars NaCl, 1% NP-40, 2 millimolars EDTA), with subsequent binding to protein A/G magnetic beads for a duration of one hour. Subsequently, the mRNA that was isolated and purified, together with the bead-antibody complex, underwent an overnight incubation at 4 °C in the IP buffer containing ribonuclease and protease inhibitors. The eluted RNA was cleansed through the phenol–chloroform method and subsequently examined for GluA1 mRNA levels utilizing RT-qPCR. The primers used were as follows: forward: ATGTGGAAGCAAGGACTCCG; reverse: GCTGACCACTCTGCCATTCT (Yang et al. ; Vu et al. ). 

Small nuclear ribonucleoprotein particles (snRNPs) are indispensable for pre-mRNA splicing by recognizing splice sites. To investigate the protein-RNA interactions in mouse amygdala tissue, 4-thiouridine (4SU) at a concentration of 200 mM was incubated with the tissue extract for 14 h. The samples were then crosslinked at 0.4 J/cm2 under 365 nm UV light. Subsequent immunoprecipitation was carried out using METTL3 antibody (1:50 dilution, ab195352, Abcam, Cambridge, UK) followed by radiolabeled precipitation of RNA with [γ−32-P]-ATP for visualization by autoradiography. PAR fragments were subjected to RT-qPCR analysis after proteinase K treatment for protein removal and RNA extraction, to assess GluA1 mRNA expression levels (Du et al. ; Yue et al. ). 

Following deparaffinization, the amygdala tissue sections of mice underwent a rehydration process using a series of alcohol gradients. Subsequently, they were immersed in distilled water for a duration of 2 min, exposed to a 3% hydrogen peroxide solution in methanol for 20 min, rinsed in distilled water for another 2 min, and then placed in 0.1 M PBS for 3 min. Retrieving antigens involved soaking the tissue sections in an antigen retrieval solution in a water bath and subsequently cooling them in tap water. Afterward, the sections received incubation with a blocking solution of normal goat serum (C-0005, Shanghai Haoran Biotechnology Co., Ltd., Shanghai, China) at RT for 20 min, followed by removal of excess liquid. Subsequently, the primary antibodies used for the sections were mouse METTL3 (ab195352, 1:500, Abcam, USA) and GluA1 (ab31232, 1:100, Abcam, USA), with an overnight incubation at 4 °C. After being rinsed three times in 0.1 M PBS for 5 min each, a secondary antibody, goat anti-rabbit IgG (ab6785, 1:1000, Abcam, UK), was applied and allowed to incubate at 37 °C for 20 min. Then, the sample was exposed to the active solution of horseradish peroxidase-conjugated avidin–biotin complex (0343-10000U, Beijing Yimo Biotechnology Co., Ltd, China) at 37 °C for 20 min, followed by visualization utilizing DAB (ST033, Guangzhou Weijia Technology Co., Ltd., China). Post-color development, the slides underwent a rinsing process and were subsequently counterstained employing hematoxylin (PT001, Shanghai Bogu Biotechnology Co., Ltd., China) for a duration of 1 min. Following this, they were treated with 1% ammonia water to attain a blue hue, dehydrated using a series of graded alcohol solutions, cleared with xylene, and finally mounted with neutral resin for microscopic analysis and imaging. The examination comprised counting 100 cells in each of the five high-power fields selected randomly on the slide. The slides were independently reviewed by two experienced pathologists. Positive IHC staining criteria: METTL3-positive cells are characterized by brown nuclear staining, while GluA1-positive cells exhibit brown membrane staining. Three repetitions were executed for every single experiment (Toyota et al. ; Cheng et al. ). 

Utilizing the SPSS software (version 21.0, IBM, USA) facilitated the statistical analysis of the research findings. Continuous variables' descriptive statistics were depicted by mean and standard deviation measures. Preceding the initiation of correlation evaluations, assessments for the normality and homoscedasticity were performed. In cases where the data fulfilled the criteria for normal distribution and homogeneity of variances, we applied independent t-tests for comparisons between groups, one-way ANOVA or repeated measures ANOVA for assessing differences among several groups, and Tukey's post hoc examination for pairwise group comparisons. Highlighting the crucial aspect of statistical relevance, a p-value below 0.05 was deemed significant in the analysis. Utilization of the Graphpad Prism 8.4.0 software facilitated the graphical depiction of the statistical outcomes. 

Esketamine alleviates depressive characteristics resulting from neuropathic pain in mice Previous literature has reported that esketamine possesses antidepressant properties (Rotroff et al. ). In this study, referenced from Fig. A, delving into the antidepressant effects of esketamine, we induced depressive-like behavior in mice by establishing a neuropathic pain model (SNL model) to unravel the specific molecular mechanism responsibile (Guida et al. ; Lee et al. ). Through behavioral assessments, mice exhibiting depressive behaviors were selected for further experiments and treated with esketamine. On days 21, 23, 27, and 34 after modeling, mechanical allodynia and thermal hyperalgesia tests were performed to assess the mechanical and thermal pain thresholds in mice. On day 14 after modeling, the assessment of depressive behaviors involved the OFT, forced swim test, and tail suspension test. The results (Fig. B-G, Figure ) illustrated that, in relation to the sham group, mice in the SNL group displayed significant pain hypersensitivity, as indicated by a marked decrease in mechanical and thermal pain thresholds. Additionally, depressive-like behaviors were observed, encompassing a substantial rise in the duration of immobility in both forced swim and tail suspension assessments, a decrease in both the frequency of entries into and the duration spent within the central zone in the OFT, and a notable decrease in SP. Following treatment with esketamine, the above behavioral abnormalities in SNL model mice were significantly reversed. Specifically, mechanical and thermal pain thresholds were markedly enhanced, notable reduction in immobile time recorded during the forced swim and tail suspension tests, considerable growth was evident in both the frequency of entries towards the center and the duration stationed in the center during the OFT, and SP was markedly improved (Fig. D). Existing research suggests that ketamine can exert antidepressant effects by improving synaptic dysfunction (Tang et al. ). The investigation into the dendritic spine number and morphology of neurons in the brain's amygdala area was accomplished utilizing Golgi staining. The results (Fig. H) revealed that contrasted against the sham group, dendritic spine number and density in the amygdala region of the brain were noticeably lessened in mice of the SNL group. Following esketamine treatment in SNL model mice, there was a significant increase in dendritic spine number and density in the amygdala brain region. The above results indicate that esketamine can alleviate depressive-like behaviors induced by the SNL model in mice and correct the abnormalities in the quantity and morphology of neuronal dendritic spines in the amygdala brain region of the SNL group. The m6A methyltransferase METTL3 alleviates depressive-like behavior in mice with neuropathic pain by upregulating m6a modification levels Existing literature has reported the significant role of m6A modification in mammalian brain development (Wang et al. ), as well as the close association between m6A modification at synapses and synaptic function (Merkurjev et al. ). Through Dot blot assays for detecting m6A modification levels in synaptic extracts from the amygdala, the results depicted in Fig. A indicate that contrasted with the sham group, mice in the SNL group indicated a notable drop in m6A modification levels in synaptic extracts from the amygdala. Post esketamine therapy, there was a noticeable escalation in m6A modification levels. Therefore, it is speculated that esketamine may influence m6A modification levels by modulating proteins associated with m6A modification. To identify specific m6A modification-related proteins regulated by esketamine, we conducted transcriptome sequencing of synaptic regions in the amygdala of various groups of mice. Analyzing the transcriptome data highlighted 873 DEGs associated with synapses in both the Sham and SNL groups, with 562 genes upregulated and 311 genes downregulated (Fig. B). Similarly, in the SNL + Saline and SNL + esketamine groups, there were 1411 DEGs in the synapses, comprising 813 upregulated genes and 598 downregulated genes (Fig. C). By intersecting the downregulated genes in the SNL vs. Sham group with the upregulated genes in the SNL + esketamine vs. SNL + Saline group, we identified 17 candidate genes (Fig. D). Gene Ontology enrichment of these candidates revealed the involvement of METTL3, PICK1, and MYRF in glial cell differentiation (GO:0010001) (Fig. E). Particularly, the m6A methyltransferase METTL3 showed significantly decreased expression in SNL mice, which was markedly increased after esketamine treatment (Fig. F). Therefore, it is hypothesized that esketamine may impact m6A modification levels by regulating the m6A methyltransferase METTL3. Western blot and IHC results (Fig. G) demonstrated that when juxtaposed with the Sham group, the level of METTL3 protein expression in synaptic samples taken from the amygdala was markedly lower in the SNL group. However, esketamine treatment led to a substantial rise in METTL3 protein expression levels. The discovery suggests that esketamine has the potential to trigger the production of the METTL3 protein responsible for m6A methylation. Next, we conducted group treatments on mice through intrathecal injection. The results from the Western blot and IHC analysis (Fig. I) illustrated a noteworthy surge in the level of METTL3 protein in the amygdala synaptic extracts following METTL3 overexpression. Dot blot assays revealed a substantial elevation in the m6A modification levels within the amygdala synaptic extracts of mice after METTL3 overexpression (Fig. K). Behavioral assessments (Fig. L) demonstrated that mice exhibited a notable increase in mechanical and thermal pain thresholds upon METTL3 overexpression, along with a substantial downturn in immobility time during forced swimming and tail suspension tests. Moreover, the center witnessed increased entries and extended durations from mice, accompanied by a significant upsurge in sucrose consumption. Additionally, Golgi staining results (Fig. R) further showed that METTL3 overexpression led to a pronounced escalation in the count and concentration of neuronal dendritic spines in the amygdala brain region of mice. The collective discoveries indicate that the m6A methyltransferase METTL3 may mitigate neuropathic pain and symptoms of depression in mice through upregulation of m6A modification levels. Esketamine Alleviates Depressive Symptoms Associated With Neuropathic Pain in Mice by Activating METTL3 to Upregulate m6A Modification Levels. Investigating the influence of esketamine on depressive behaviors in neuropathic pain-afflicted mice by regulating METTL3, SNL model mice were grouped and treated with esketamine or esketamine combined with METTL3 silencing. Western blot and IHC analyses (Fig. A-B) showed that METTL3 protein expression in synaptosome extracts from the amygdala was significantly increased after esketamine treatment in SNL model mice. Silencing METTL3 reversed this increase in METTL3 protein levels. Dot blot assay results (Fig. C) demonstrated that m6A modification levels in synaptosome extracts from the amygdala were significantly elevated following esketamine treatment in SNL model mice, while METTL3 silencing reversed the esketamine-induced upregulation of m6A modification levels. These findings indicate that esketamine can upregulate m6A modification levels in synaptosome extracts from the amygdala by activating METTL3 expression. Behavioral assessments (Fig. D) revealed that esketamine therapy exhibited remarkable improvements in mechanical and thermal pain thresholds, decreased immobility period in forced swim and tail suspension trials, heightened central area entries and dwell time during the OFT, and significantly enhanced sucrose ingestion in SNL model mice. However, METTL3 silencing reversed these behavioral effects: notable reductions in both mechanical and thermal pain thresholds, substantial elevations in immobility durations observed in the forced swim and tail suspension evaluations, decreased frequencies of entries into the central area and reduced durations spent in the central region, as well as a marked decline in sucrose consumption. Golgi staining results (Fig. J) showed that esketamine treatment in SNL model mice substantially elevated the quantity and concentration of neuronal dendritic spines in the amygdala brain region, whereas METTL3 silencing reversed these effects. These findings demonstrate that esketamine alleviates depressive-like behaviors in neuropathic pain mice by activating the m6A methyltransferase METTL3. METTL3 facilitates synaptic expression of GluA1 by promoting m6A modification of AMPAR GluA1 subunit mRNA Candidate genes from Fig. D were further analyzed using NetworkAnalyst for KEGG enrichment, revealing the involvement of GluA1 and CHRNA5 in the Neuroactive ligand-receptor interaction pathway (Fig. A). Existing literature has linked m6A modification of GluA1 mRNA to synaptic function (Merkurjev et al. ), and transcriptomic analysis results show (Fig. B) significant downregulation of GluA1 (alias: Gria1) in SNL mice, with a notable increase in GluA1 expression following esketamine treatment. Through STRING database interrogation, related interacting genes of GluA1 were identified (Fig. C), and GO enrichment analysis indicated that GluA1 and its interacting genes primarily participate in the AMPA glutamate receptor complex (GO: 0032281) (Fig. D). Western blot and IHC analyses demonstrated (Fig. E-F) a notable drop in GluA1 protein expression in amygdala synaptic extracts of SNL group when juxtaposed with the sham group; conversely, the application of esketamine showed a marked increase in GluA1 protein expression. Me-RIP analysis of m6A modification levels of GluA1 mRNA showed (Fig. G) a considerable decrease in m6A modification levels in the amygdala synaptic extracts of GluA1 mRNA in the SNL group as opposed to the sham group, which significantly enhanced after esketamine treatment. These findings suggest that esketamine upregulates the m6A modification level of GluA1 mRNA. The noted outcomes demonstrate that esketamine has the ability to elevate the m6A modification levels of GluA1 mRNA by activating the expression of METTL3 in the amygdala synaptic extracts. Thus, it is postulated that the upregulation of m6A modification levels of GluA1 mRNA is associated with METTL3. Grouped treatment of SNL model mice was performed, and Western blot and IHC analyses showed (Fig. H) a significant increase in GluA1 protein expression in amygdala synaptic extracts following METTL3 overexpression. Me-RIP results indicated (Fig. J) a marked increase in m6A modification levels of GluA1 mRNA after METTL3 overexpression. The binding analysis using PAR-CLIP illustrated a substantial boost in METTL3's affinity for GluA1 mRNA following METTL3 overexpression, as depicted in Fig. K. The outcomes imply that METTL3 may bind to the mRNA of the AMPAR GluA1 subunit. These results suggest that METTL3 may facilitate m6A modification of GluA1 by binding to AMPAR GluA1 subunit mRNA, thereby promoting the synaptic expression of GluA1. Impact of METTL3 on depressive behavior in mice due to neuropathic pain via regulation of GluA1 By examining how METTL3 impacts depressive behaviors in mice suffering from neuropathic pain via controlling GluA1, SNL model mice were grouped and treated with METTL3 overexpression alone or METTL3 overexpression combined with GluA1 silencing. Western blot and IHC analyses (Fig. A-B) showed that METTL3 overexpression significantly increased METTL3 and GluA1 protein expression in synaptosome extracts from the amygdala. GluA1 silencing reversed the upregulation of GluA1 protein expression induced by METTL3 overexpression, with no effect on METTL3 expression. Behavioral assessments (Fig. C) revealed that METTL3 overexpression significantly increased mechanical and thermal pain thresholds, reduced duration of immobility in the forced swim and tail suspension tests, raised central entry count and time allocation within the center in the OFT, and markedly improved sucrose intake. However, GluA1 silencing reversed these behavioral effects: mechanical and thermal pain thresholds were drastically decreased, immobility time during the forced swim and tail suspension tests considerably escalated, center entries and time spent in the center significantly reduced, and sucrose intake markedly decreased. Golgi staining results (Fig. I) demonstrated that METTL3 overexpression notably amplified the amount and density of neuronal dendritic spines in the amygdala brain region. This effect was reversed by GluA1 silencing. These findings indicate that METTL3 mitigates symptoms of depression in mice with neuropathic pain by enhancing the expression of GluA1. Esketamine eases depressive behavior triggered by neuropathic pain in mice through METTL3/GluA1 stimulation For a more thorough examination of how esketamine impacts depressive symptoms related to neuropathic pain by altering METTL3/GluA1, we executed group interventions on mice experiencing the SNL model. Results obtained through Western blot indicated (Fig. A-B) that the expression of METTL3 and GluA1 proteins in the amygdala synaptosome extracts of mice significantly increased after esketamine treatment. Silencing GluA1 could reverse the upregulation of GluA1 protein expression by esketamine without affecting METTL3 expression. Behavioral results (Fig. C) demonstrated that after esketamine treatment, the mechanical and thermal pain thresholds of mice profoundly boosted, the immobility durations in forced swimming and tail suspension assessments markedly decreased, the quantity of center entries and the duration of center exploration significantly increased. Additionally, a marked elevation in sucrose ingestion was observed in the mice. Silencing GluA1 could reverse the aforementioned behavioral performance. Golgi staining results (Fig. I) showed that following esketamine treatment, the quantity and concentration of dendritic spines of neurons in the amygdala brain region of mice significantly increased. Silencing GluA1 could reverse the effects of esketamine on dendritic spine number and density. These findings indicate that activation of the METTL3/GluA1 pathways by esketamine shows effectiveness in alleviating depressive manifestations linked to neuropathic pain in mice. 

Previous literature has reported that esketamine possesses antidepressant properties (Rotroff et al. ). In this study, referenced from Fig. A, delving into the antidepressant effects of esketamine, we induced depressive-like behavior in mice by establishing a neuropathic pain model (SNL model) to unravel the specific molecular mechanism responsibile (Guida et al. ; Lee et al. ). Through behavioral assessments, mice exhibiting depressive behaviors were selected for further experiments and treated with esketamine. On days 21, 23, 27, and 34 after modeling, mechanical allodynia and thermal hyperalgesia tests were performed to assess the mechanical and thermal pain thresholds in mice. On day 14 after modeling, the assessment of depressive behaviors involved the OFT, forced swim test, and tail suspension test. The results (Fig. B-G, Figure ) illustrated that, in relation to the sham group, mice in the SNL group displayed significant pain hypersensitivity, as indicated by a marked decrease in mechanical and thermal pain thresholds. Additionally, depressive-like behaviors were observed, encompassing a substantial rise in the duration of immobility in both forced swim and tail suspension assessments, a decrease in both the frequency of entries into and the duration spent within the central zone in the OFT, and a notable decrease in SP. Following treatment with esketamine, the above behavioral abnormalities in SNL model mice were significantly reversed. Specifically, mechanical and thermal pain thresholds were markedly enhanced, notable reduction in immobile time recorded during the forced swim and tail suspension tests, considerable growth was evident in both the frequency of entries towards the center and the duration stationed in the center during the OFT, and SP was markedly improved (Fig. D). Existing research suggests that ketamine can exert antidepressant effects by improving synaptic dysfunction (Tang et al. ). The investigation into the dendritic spine number and morphology of neurons in the brain's amygdala area was accomplished utilizing Golgi staining. The results (Fig. H) revealed that contrasted against the sham group, dendritic spine number and density in the amygdala region of the brain were noticeably lessened in mice of the SNL group. Following esketamine treatment in SNL model mice, there was a significant increase in dendritic spine number and density in the amygdala brain region. The above results indicate that esketamine can alleviate depressive-like behaviors induced by the SNL model in mice and correct the abnormalities in the quantity and morphology of neuronal dendritic spines in the amygdala brain region of the SNL group. 

Existing literature has reported the significant role of m6A modification in mammalian brain development (Wang et al. ), as well as the close association between m6A modification at synapses and synaptic function (Merkurjev et al. ). Through Dot blot assays for detecting m6A modification levels in synaptic extracts from the amygdala, the results depicted in Fig. A indicate that contrasted with the sham group, mice in the SNL group indicated a notable drop in m6A modification levels in synaptic extracts from the amygdala. Post esketamine therapy, there was a noticeable escalation in m6A modification levels. Therefore, it is speculated that esketamine may influence m6A modification levels by modulating proteins associated with m6A modification. To identify specific m6A modification-related proteins regulated by esketamine, we conducted transcriptome sequencing of synaptic regions in the amygdala of various groups of mice. Analyzing the transcriptome data highlighted 873 DEGs associated with synapses in both the Sham and SNL groups, with 562 genes upregulated and 311 genes downregulated (Fig. B). Similarly, in the SNL + Saline and SNL + esketamine groups, there were 1411 DEGs in the synapses, comprising 813 upregulated genes and 598 downregulated genes (Fig. C). By intersecting the downregulated genes in the SNL vs. Sham group with the upregulated genes in the SNL + esketamine vs. SNL + Saline group, we identified 17 candidate genes (Fig. D). Gene Ontology enrichment of these candidates revealed the involvement of METTL3, PICK1, and MYRF in glial cell differentiation (GO:0010001) (Fig. E). Particularly, the m6A methyltransferase METTL3 showed significantly decreased expression in SNL mice, which was markedly increased after esketamine treatment (Fig. F). Therefore, it is hypothesized that esketamine may impact m6A modification levels by regulating the m6A methyltransferase METTL3. Western blot and IHC results (Fig. G) demonstrated that when juxtaposed with the Sham group, the level of METTL3 protein expression in synaptic samples taken from the amygdala was markedly lower in the SNL group. However, esketamine treatment led to a substantial rise in METTL3 protein expression levels. The discovery suggests that esketamine has the potential to trigger the production of the METTL3 protein responsible for m6A methylation. Next, we conducted group treatments on mice through intrathecal injection. The results from the Western blot and IHC analysis (Fig. I) illustrated a noteworthy surge in the level of METTL3 protein in the amygdala synaptic extracts following METTL3 overexpression. Dot blot assays revealed a substantial elevation in the m6A modification levels within the amygdala synaptic extracts of mice after METTL3 overexpression (Fig. K). Behavioral assessments (Fig. L) demonstrated that mice exhibited a notable increase in mechanical and thermal pain thresholds upon METTL3 overexpression, along with a substantial downturn in immobility time during forced swimming and tail suspension tests. Moreover, the center witnessed increased entries and extended durations from mice, accompanied by a significant upsurge in sucrose consumption. Additionally, Golgi staining results (Fig. R) further showed that METTL3 overexpression led to a pronounced escalation in the count and concentration of neuronal dendritic spines in the amygdala brain region of mice. The collective discoveries indicate that the m6A methyltransferase METTL3 may mitigate neuropathic pain and symptoms of depression in mice through upregulation of m6A modification levels. Esketamine Alleviates Depressive Symptoms Associated With Neuropathic Pain in Mice by Activating METTL3 to Upregulate m6A Modification Levels. Investigating the influence of esketamine on depressive behaviors in neuropathic pain-afflicted mice by regulating METTL3, SNL model mice were grouped and treated with esketamine or esketamine combined with METTL3 silencing. Western blot and IHC analyses (Fig. A-B) showed that METTL3 protein expression in synaptosome extracts from the amygdala was significantly increased after esketamine treatment in SNL model mice. Silencing METTL3 reversed this increase in METTL3 protein levels. Dot blot assay results (Fig. C) demonstrated that m6A modification levels in synaptosome extracts from the amygdala were significantly elevated following esketamine treatment in SNL model mice, while METTL3 silencing reversed the esketamine-induced upregulation of m6A modification levels. These findings indicate that esketamine can upregulate m6A modification levels in synaptosome extracts from the amygdala by activating METTL3 expression. Behavioral assessments (Fig. D) revealed that esketamine therapy exhibited remarkable improvements in mechanical and thermal pain thresholds, decreased immobility period in forced swim and tail suspension trials, heightened central area entries and dwell time during the OFT, and significantly enhanced sucrose ingestion in SNL model mice. However, METTL3 silencing reversed these behavioral effects: notable reductions in both mechanical and thermal pain thresholds, substantial elevations in immobility durations observed in the forced swim and tail suspension evaluations, decreased frequencies of entries into the central area and reduced durations spent in the central region, as well as a marked decline in sucrose consumption. Golgi staining results (Fig. J) showed that esketamine treatment in SNL model mice substantially elevated the quantity and concentration of neuronal dendritic spines in the amygdala brain region, whereas METTL3 silencing reversed these effects. These findings demonstrate that esketamine alleviates depressive-like behaviors in neuropathic pain mice by activating the m6A methyltransferase METTL3. 

Candidate genes from Fig. D were further analyzed using NetworkAnalyst for KEGG enrichment, revealing the involvement of GluA1 and CHRNA5 in the Neuroactive ligand-receptor interaction pathway (Fig. A). Existing literature has linked m6A modification of GluA1 mRNA to synaptic function (Merkurjev et al. ), and transcriptomic analysis results show (Fig. B) significant downregulation of GluA1 (alias: Gria1) in SNL mice, with a notable increase in GluA1 expression following esketamine treatment. Through STRING database interrogation, related interacting genes of GluA1 were identified (Fig. C), and GO enrichment analysis indicated that GluA1 and its interacting genes primarily participate in the AMPA glutamate receptor complex (GO: 0032281) (Fig. D). Western blot and IHC analyses demonstrated (Fig. E-F) a notable drop in GluA1 protein expression in amygdala synaptic extracts of SNL group when juxtaposed with the sham group; conversely, the application of esketamine showed a marked increase in GluA1 protein expression. Me-RIP analysis of m6A modification levels of GluA1 mRNA showed (Fig. G) a considerable decrease in m6A modification levels in the amygdala synaptic extracts of GluA1 mRNA in the SNL group as opposed to the sham group, which significantly enhanced after esketamine treatment. These findings suggest that esketamine upregulates the m6A modification level of GluA1 mRNA. The noted outcomes demonstrate that esketamine has the ability to elevate the m6A modification levels of GluA1 mRNA by activating the expression of METTL3 in the amygdala synaptic extracts. Thus, it is postulated that the upregulation of m6A modification levels of GluA1 mRNA is associated with METTL3. Grouped treatment of SNL model mice was performed, and Western blot and IHC analyses showed (Fig. H) a significant increase in GluA1 protein expression in amygdala synaptic extracts following METTL3 overexpression. Me-RIP results indicated (Fig. J) a marked increase in m6A modification levels of GluA1 mRNA after METTL3 overexpression. The binding analysis using PAR-CLIP illustrated a substantial boost in METTL3's affinity for GluA1 mRNA following METTL3 overexpression, as depicted in Fig. K. The outcomes imply that METTL3 may bind to the mRNA of the AMPAR GluA1 subunit. These results suggest that METTL3 may facilitate m6A modification of GluA1 by binding to AMPAR GluA1 subunit mRNA, thereby promoting the synaptic expression of GluA1. 

By examining how METTL3 impacts depressive behaviors in mice suffering from neuropathic pain via controlling GluA1, SNL model mice were grouped and treated with METTL3 overexpression alone or METTL3 overexpression combined with GluA1 silencing. Western blot and IHC analyses (Fig. A-B) showed that METTL3 overexpression significantly increased METTL3 and GluA1 protein expression in synaptosome extracts from the amygdala. GluA1 silencing reversed the upregulation of GluA1 protein expression induced by METTL3 overexpression, with no effect on METTL3 expression. Behavioral assessments (Fig. C) revealed that METTL3 overexpression significantly increased mechanical and thermal pain thresholds, reduced duration of immobility in the forced swim and tail suspension tests, raised central entry count and time allocation within the center in the OFT, and markedly improved sucrose intake. However, GluA1 silencing reversed these behavioral effects: mechanical and thermal pain thresholds were drastically decreased, immobility time during the forced swim and tail suspension tests considerably escalated, center entries and time spent in the center significantly reduced, and sucrose intake markedly decreased. Golgi staining results (Fig. I) demonstrated that METTL3 overexpression notably amplified the amount and density of neuronal dendritic spines in the amygdala brain region. This effect was reversed by GluA1 silencing. These findings indicate that METTL3 mitigates symptoms of depression in mice with neuropathic pain by enhancing the expression of GluA1. 

For a more thorough examination of how esketamine impacts depressive symptoms related to neuropathic pain by altering METTL3/GluA1, we executed group interventions on mice experiencing the SNL model. Results obtained through Western blot indicated (Fig. A-B) that the expression of METTL3 and GluA1 proteins in the amygdala synaptosome extracts of mice significantly increased after esketamine treatment. Silencing GluA1 could reverse the upregulation of GluA1 protein expression by esketamine without affecting METTL3 expression. Behavioral results (Fig. C) demonstrated that after esketamine treatment, the mechanical and thermal pain thresholds of mice profoundly boosted, the immobility durations in forced swimming and tail suspension assessments markedly decreased, the quantity of center entries and the duration of center exploration significantly increased. Additionally, a marked elevation in sucrose ingestion was observed in the mice. Silencing GluA1 could reverse the aforementioned behavioral performance. Golgi staining results (Fig. I) showed that following esketamine treatment, the quantity and concentration of dendritic spines of neurons in the amygdala brain region of mice significantly increased. Silencing GluA1 could reverse the effects of esketamine on dendritic spine number and density. These findings indicate that activation of the METTL3/GluA1 pathways by esketamine shows effectiveness in alleviating depressive manifestations linked to neuropathic pain in mice. 

In conclusion, preliminary conclusions can be inferred from the results discussed previously: Esketamine may potentially boost the synaptic expression of GluA1 by upregulating the m6A modification level of GluA1 subunit mRNA through the activation of the m6A methyltransferase METTL3, thereby inhibiting depressive tendencies in mice afflicted by neuropathic pain (Fig. ). This study sheds light on the therapeutic efficacy of esketamine in treating depressive symptoms in mice experiencing neuropathic pain. Neuropathic pain is a debilitating condition that profoundly affects the well-being of individuals, and the presence of depressive-like behavior further exacerbates the suffering of individuals with chronic pain (Finnerup et al. ; Rouwette et al. ; Fitzcharles et al. ). By delving deeper into the mechanisms of esketamine action, identification of fresh routes and goals aiming at mitigating neuropathic discomfort and correlated feelings of melancholy is viable (Yavi et al. ). This study, through transcriptome sequencing and bioinformatics analysis, revealed that esketamine may relieve symptoms of depression in neuropathic pain mice through the activation of the m6A methyltransferase METTL3, promoting m6A modification of the AMPAR GluA1 subunit, and increasing its synaptic expression. This discovery offers fresh perspectives on the exact mechanisms of esketamine's antidepressant effects. The discovery also suggests that modulating m6A modifications could influence neurotransmitter receptor expression, potentially controlling neuropathic pain and associated depressive-like behaviors. As a key ionotropic receptor, AMPAR facilitates swift stimulatory communication within the CNS. Increased synaptic expression of the GluA1 subunit enhances synaptic transmission and could ameliorate symptoms of neuropathic pain and depression (Ge and Wang ; Dore et al. ). Our study validated this hypothesis, showing that esketamine increases GluA1 synaptic expression through m6A modification. Regarding the mechanism by which esketamine influences METTL3, although evidence suggests that esketamine exerts its antidepressant effects through various signaling pathways, whether it directly regulates METTL3 expression and activity via NMDA receptors requires further investigation. Existing literature indicates that esketamine, as a non-competitive NMDA receptor antagonist, may indirectly affect related signaling pathways by reducing presynaptic inhibition and enhancing AMPAR activity (Gass et al. ; K V et al. ; Sial et al. ). However, our study did not explore the direct relationship between NMDA receptor pathways and METTL3. Since no NMDA receptor antagonists or upstream pathway inhibitors were used in this experiment, we cannot entirely rule out the potential influence of other pathways on METTL3. This limitation highlights an area for future research, where NMDA receptor antagonists or downstream molecular inhibitors could be utilized to further investigate whether esketamine activates METTL3 and subsequent m6A modification through NMDA receptors. Our research demonstrated that esketamine significantly reduced depressive-like behaviors in neuropathic pain mice, consistent with previous findings, further confirming its inhibitory effects on depressive-like behaviors in this context (Zanos and Gould ; Smith-Apeldoorn et al. ; Grabski et al. ). The primary emphasis of this research was on the impact of m6A modification on neuropathic pain and symptoms resembling depression. m6A modification, a prevalent RNA modification, has garnered increasing attention in recent years for its role in neural functions (Tao et al. ; Lei and Wang ; Tian et al. ). Esketamine, as a specific antidepressant, may positively impact the psychological state of pain patients, offering new avenues for integrated treatment. Additionally, our study emphasized the role of RNA methylation modifications in neurological disorders, paving the way for developing drugs targeting RNA-modifying enzymes to regulate neural functions (Barbieri and Kouzarides ). As our research was conducted exclusively on animal models, expanded clinical studies are essential to validate the generalizability of these findings to the human species. While animal models are invaluable for mechanism studies, differences between animal and human diseases require cautious interpretation and generalization of the results (Xiang et al. ). The research adopted the SNL model, acknowledged for its contribution to neuropathic pain research. Other models, encompassing the chronic constriction injury (CCI) and partial sciatic nerve ligation (PNL) models, offer unique features in representing behavioral and molecular mechanisms of neuropathic pain. Differences among models in neural inflammation and conduction changes may affect the generalizability of esketamine's effects on METTL3 and GluA1 regulation. While our study focused on a specific neuropathic pain model to investigate the mechanisms of esketamine, validating its effects in other models would enhance the general applicability of the findings. Due to time and resource constraints, we were unable to perform additional experiments using other models. In future studies, we plan to explore models such as the CCI model to further validate esketamine's effects. Neuropathic pain is frequently accompanied by depressive-like behaviors, imposing a substantial burden on patients' quality of life. However, current clinical research lacks comprehensive understanding and effective treatments for the emotional complications associated with neuropathic pain. Existing therapies primarily focus on pain management while neglecting emotional disorders, which are a significant reason why many patients continue to suffer from distress and depressive symptoms post-treatment (Vieira et al. ; Coelho et al. ). At the molecular level, research on specific biomarkers and potential targets for neuropathic pain-associated depression remains limited, particularly regarding the role of m6A methylation in this process. This study addresses these gaps by demonstrating for the first time that esketamine can upregulate m6A modification of the AMPAR GluA1 subunit mRNA in the amygdala, thereby regulating synaptic expression and effectively alleviating depressive-like behaviors in neuropathic pain mice. The unveiling of this insight provides fresh understandings regarding the regulatory processes of m6A methylation in depressive symptoms associated with neuropathic pain and implies that METTL3 could be a promising molecular target for the management of depression. Compared to traditional therapies, the molecular mechanism revealed in this study forms a basis for developing novel antidepressants, potentially improving overall therapeutic outcomes for patients with neuropathic pain. Through the authentication of the function of m6A modifications in the regulation of depression and pain, our study opens new possibilities for more precise treatment strategies for depression. Future clinical trials confirming esketamine's role in regulating METTL3 activity and GluA1 expression could enhance the efficacy of existing drugs and aid in the development of novel medications targeting the emotional symptoms of neuropathic pain. Additionally, further investigation into alternative drug targets may enhance the management of neuropathic pain and symptoms resembling depression. In conclusion, the investigation unveils a distinct approach towards understanding and potentially easing neuropathic pain and the correlated symptoms of depression. However, as research progresses, addressing various challenges will be crucial to ensure these findings translate into clinical applications, ultimately benefiting patients in need. Figure S2. Behavioral Assessment of Mice Before and After SNL Surgery. Note: (A) Forced swim test showing increased immobility time in SNL mice on days 21, 23, 27, and 34 after modeling; (B) Tail suspension test showing increased immobility time (no struggling attempts) in SNL mice on days 21, 23, 27, and 34 after modeling; (C) OFT showing reduced center entries and time spent in the center in SNL mice on days 21, 23, 27, and 34 after modeling; (D) Sucrose preference test showing reduced sucrose intake in SNL mice. * indicates significant difference between the two groups ( P < 0.05), with 5 mice in each group 

Below is the link to the electronic supplementary material. ESM 1 The Flowchart of Bioinformatics and Molecular Biology Experiments (JPG 2.75 MB) ESM 2 Behavioral Assessment of Mice Before and After SNL Surgery (JPG 393 KB) 

