使用了以下小鼠菌株：对于标准CSD实验
，从杰克逊实验室购买了7周龄的C57BL/6 J（库存号000664）小鼠。对于骨髓移植实验，从杰克逊实验室获得了4周大的B6.SJL-PTPRCA PEPCB/BOYJ（库存002014 ，B6 CD45.1）小鼠 。B6.129（CG）-CCR2TM2.1IFC/J（库存NO
。017586
，CCR2RFP）和B6.129×1-MMP8TM1OTIN/J（库存号005514，MMP8 - / - ）是繁殖的。四到六个月大的男性退休CD-1育种者（Charles River Laboratories ，CRL：CD1 [ICR]）被用作男性CSD的侵略者 。对于女性CSD实验
，雄性B6N.129S6（CG）-ESR1TM1.1（CRE）和/J（股票号017911，ERA-CRE ，ERA-CRE（也称为ESR1））是从Jackson Laboratory购买的
，并与CD-1女性交叉，以获得F1 MALES ，以获取F1 MALES
，该男性被用作战斗机
。从外部供应商购买的小鼠被允许居住到动物设施至少一周。将小鼠保持在12小时的光线：黑暗周期（在07:00的灯光上，在19:00开灯） ，并随意进入食物和水 。对于所有行为测试，允许小鼠适应至少1小时的测试室
。所有程序均根据《美国国家卫生研究院指南实验室动物的护理和使用指南》以及西奈山（ISMMS）机构动物护理和使用委员会的伊坎医学院进行。 　　对于雄性CSD111
，已退休的男性CD-1育种者（年龄：4-6个月）用作侵略者 。在每次失败之前，根据先前描述的标准 ，侵略者连续三天进行侵略行为
。在失败开始前两天
，CD-1小鼠被安置在穿孔的有机玻璃分区的一侧。在CSD连续10天内，实验小鼠（7-8周龄）与CD-1进行直接相互作用 ，每天10分钟（骨髓嵌合体队列5分钟）
，一天的其余时间放置在Plexiglass Diverider的另一侧，允许感官接触 ，允许直接接触 。雌性CSDS61,62的雄性侵略者如下：杂合子ERA-CRE小鼠在双侧注射CRE依赖性AAV-DIO-HM3D（GQ）-Dreadd（Addgene
，44361-AAV2）中的杂合ERA-CRE小鼠，中心症状。为了激活ERα+细胞 ，在每次失败之前30分钟施用了1.0 mg kg-kg-1氯氮平-N-氧化物（Tocris
，4936）的腹膜内注射（Tocris，4936）
。在有机玻璃隔板上配对无重的对照小鼠。失败的最后一天后 ，压力和无压力的对照小鼠被单独容纳（男性）或成对（女性） 。在CSDS实验期间，仔细检查了所有应力小鼠的伤口
，并排除了过度受伤的小鼠
。 　　亚阈值应力是CSDS范式的一种变体，用于揭示促敏度因子32 ，而无需引起无操纵小鼠的行为改变。将实验小鼠暴露于3×5分钟的直接物理相互作用与侵略性CD-1小鼠之间的直接物理相互作用
，但两次失败之间的间隔为15分钟 。最后一次失败后24小时，如下所述进行了SI测试
。 　　在红灯条件下最后一次失败会议后24小时进行SI测试。在行为套件的1小时习惯后 ，将小鼠放入有机玻璃舞台（42 cm×42 cm×42 cm
，全国性塑料）中，一端有一个小的网状外壳 。在最初的2.5分钟内 ，实验鼠标自由探索了竞技场
。然后将小鼠从随后用70％乙醇清洗的竞技场中取出
，然后将新型的社交目标（男性的CD-1和女性CSD的ERA-CRE）放入围栏中，并将实验小鼠放回竞技场中 ，再加上2.5分钟。使用Noldus Ethovision System（Noldus Information Technology
，版本11.0）跟踪运动活动并记录 。将Si比计算为在存在目标小鼠与不存在的目标小鼠附近的实验小鼠在附近（SI区）所花费的时间之间的比率
。Si比为≥1的小鼠显示出类似于无重理对照小鼠的行为特征，称为弹性 ，而Si比的小鼠 <1 were termed susceptible. To test social avoidance behaviour towards a juvenile mouse, SI test was performed as described above with a four- to six-week-old male juvenile mouse as a social target. Additional parameters that were measured were total locomotion and time spent in corners, calculated as the sum between the two corners opposite the wire enclosure. 　　Chronic variable stress was conducted in female mice as previously described63. For 21 days, mice were exposed to daily 1 h long stressors, consisting of either 100 mild foot shocks (0.45 mA), restraint stress in a 50 ml Falcon tube, or tail suspension. For the duration of the stress, mice were group housed. 　　Before injection, rMMP8 (Bio-techne, 2904-MP-010) was activated ex vivo for 1 h at 37 °C with 1 mM 4-aminophenylmercuric acetate (APMA) in mercury-containing assay buffer (Anaspec, AS-71154) and then diluted in 0.9% sterile saline solution (VWR, 101448-952). For the dose–response experiment, we injected three different doses 50, 100 and 200 µg kg−1, and blood was drawn 20 min after the injection via submandibular bleeding and 18 h post-injection using trunk blood. MMP8 was measured as described below. For the behavioural experiments, mice were injected with 100 µg kg−1 rMMP8 or APMA 20 min before the defeat bout. 　　Surgeries were performed as described previously23. In brief, 6 week-old C57BL/6 J mice were injected intraperitoneally with a mixture of ketamine hydrochloride (100 mg kg−1 body weight) and xylazine (10 mg kg−1 body weigh). After anaesthesia was confirmed, mice were placed on a stereotaxic instrument (David Kopf Instruments). For the Cldn5 knockdown experiment, we bilaterally injected 0.5 μl of virus (1.0 × 1011 infectious units per ml) expressing either AAV2/9-shRNA or AAV2/9-shRNA-Cldn5 into the NAc (coordinates from bregma: AP + 1.5 mm; ML ± 0.5 mm; DV − 4.4 mm). After 2 weeks of recovery, mice received doxycycline (2 mg ml−1 in drinking water) for another 2 weeks. For the hyaluronidase infusion experiment, 27 G guide cannulae were inserted bilaterally into the NAc (from bregma: AP + 1.5 mm; ML ± 0.5 mm; DV − 4.4 mm) and fixed onto the skull using dental cement (Grip cement; Dentsply). After two weeks of recovery, daily infusions of 5 U of hyaluronidase (Sigma-Aldrich, H1136) or saline were completed once daily for 10 consecutive days. All compounds and viruses were infused at a rate of 0.1 μl min−1 and allowed to passively diffuse for 5 min before removing the needles. 　　Bone marrow chimeras were generated as described10,28. To ablate the peripheral immune system of the host mouse, 5-week-old male B6 CD45.1 mice were irradiated with a total of 11 Gy, delivered in two doses of 5.5 Gy, 3–4 h apart (X-rad 320 Irradiator (Precision X-Ray)). Haematopoietic progenitor cells were isolated from the femur/tibia of either Mmp8−/− or Mmp8+/+ male donor mice (12 weeks old). One hour after the second dose of irradiation, 1 × 106 cells were injected retro-orbitally in mice anaesthetized with isoflurane. Host mice were then allowed to recover for a total of six weeks. Mice received antibiotic treatment (0.2% in drinking water) (Neomycin trisulfate, N1876, Sigma) during the first three weeks of recovery. The level of chimerism was assessed using flow cytometry, comparing CD45.1 (host) (mouse anti-CD45.1-PE-Cyanine7, clone A20, Invitrogen, 25-0453-81) and CD45.2 (donor) (mouse anti-CD45.2-BV421, clone 104, BD Bioscience, 562895) leukocytes, and measuring MMP8 in plasma (Abcam, ab206982). 　　sCPP was performed as described64. The experiment was done under red-light conditions after mice were habituated to the CPP room for 1 h. The CPP chamber (Med Associates) consisted of 3 different compartments: a neutral middle part, and two adjacent chambers, each with distinct floors (grid pattern) and walls. On the pre-test day, mice were allowed to explore all three chambers for 20 min and the time spent in each chamber was recorded. Based on these durations, mice were balanced to account for pre-test preferences. During the four consecutive conditioning days, mice were conditioned twice per day: In the morning, mice were placed in one chamber for 15 min with a novel, same-sex juvenile (4 to 5 weeks old) C57BL/6 J mouse (paired chamber). In the afternoon, the experimental mouse was put in the empty opposite chamber for the same amount of time (unpaired chamber). On the testing day, mice were again allowed to freely explore all chambers for 20 min and the time spent in each chamber was automatically recorded (Med Associates). 　　Sucrose preference test was performed to assess hedonic behaviour towards a sweet gustatory stimulus11. Mice were given access to two water bottles (50 ml conical tubes with sipper tops) for 24 h for habituation. Then, one water bottle was exchanged with a bottle containing 1% sucrose (Sigma, S0389) in drinking water. After 24 h, the bottle positions were swapped to prevent position bias. After another 24 h, sucrose preference was assessed as follows (based on weight of bottles): (sucrose (g)/total fluid (g)) × 100%. 　　The splash test, a test performed to assess self-care behaviour, was conducted under red-light conditions as described previously11. In brief, after 1 h of habituation to the testing room, a 10% sucrose solution was gently sprayed onto the lower back of the mouse. Behaviour was recorded for 5 min, and time spent grooming was scored. 　　The EPM was conducted to assess anxiety-like behaviours11. After 1 h of habituation to the testing room, mice were placed on an elevated cross-shaped maze for 5 min under red-light conditions. The four arms (two arms without and two arms with walls, each arm of the maze measuring 12 × 50 cm) were elevated 1 m above the floor. Behaviour was tracked using a Noldus Ethovision System (Noldus Information Technology, version 11.0). Parameters assessed included time spent in closed arms, open arms and in the centre. 　　Blood was collected directly into fluorescence-activated cell sorting (FACS) buffer (DPBS (Thermo Fisher Scientific, 14190144) containing 0.5% bovine serum albumin (Sigma-Aldrich, A9647) and 2 mM EDTA (Invitrogen, AM9260G)). Cells were pelleted and red blood cells (RBCs) were lysed using RBC lysis buffers (BD, 555899). Immune cells of the brain were isolated as previously described25. In brief, mice were anaesthetized with 10% chloral hydrate and transcardially perfused with ice-cold PBS (0.1 M). Brains were extracted, leptomeninges carefully removed and the brains then cut into small pieces using scissors in a total of 3 ml digestion buffer (RPMI (Thermo Fisher Scientific, 11875093) with 2% fetal bovine serum (Thermo Fisher Scientific, A3840001), 2 mM HEPES (Corning, 25-060-CI) and 0.4 mg ml−1 collagenase D (Roche, 12352200)). The cell suspension was then incubated for 30 min at 37 °C. Digestion was stopped by adding EDTA (Invitrogen, AM9260G) to a final concentration of 5 mM. Using blunt 18 G needles (BD, 303129), the cell suspension was gently homogenized, and the homogenate was passed through a 70 μm strainer (pre-wet with PBS) (Miltenyi Biotec, 130-095-823). Cells were pelleted, resuspended in 30% Percoll (Millipore Sigma, GE17-0891-01) and centrifuged for 30 min at 23,500g without brakes at 4 °C. The myelin layer was aspirated and the middle layer containing leukocytes was transferred into a conical tube. Cells were then washed and stained for 30 min on ice with a mix of metal-conjugated antibodies (Supplementary Table 1). After antibody staining, cells were incubated with cisplatin for 5 min at room temperature as a viability dye to enable exclusion of dead cells. Cells were then fixed in PBS containing 1.6% formaldehyde and a 1:4,000 dilution of Ir nucleic acid intercalator to label all nucleated cells. Immediately prior to acquisition, cells were washed in PBS, then in distilled water, and finally resuspended in distilled water containing a 1/10 dilution of Equation 4 Element Calibration beads (Fluidigm, SKU 201078). After routine instrument tuning and optimization, the samples were acquired on a CyTOF2 Mass Cytometer equipped with a Super Sampler fluidics system (Victorian Airships). The acquisition rate was <500 events per second. The resulting FCS files were concatenated and normalized using a bead-based normalization algorithm in the CyTOF acquisition software and uploaded to Cytobank (https://mtsinai.cytobank.org/cytobank/; Cytobank, Menlo Park, CA, v7.0). FCS files were manually pre-gated for CD45+ events, excluding dead cells, doublets and DNA-negative debris (Extended Data Fig. 1a). Data analysis was performed with Clustergrammer, a web-based tool for visualizing and analysing high-dimensional data (https://github.com/ismms-himc/LegendScreen_CyTOF). 　　For the mouse leukocyte subpopulation sequencing experiment, trunk blood was collected directly into FACS buffer. Samples were centrifuged and RBC lysis was performed (BD, 555899). After washing the cell pellet with ice-cold DPBS, Fc receptor blocking (rat anti-CD16/CD32, clone 2.4G2, BD Biosciences, 553141) was performed on ice for 30 min. Cells were pelleted and washed once. Leukocytes were then stained with the following antibodies (all at 1:400): rat anti-CD11b-PE-Cyanine7 (clone M1/70, BioLegend, 101215), rat anti-Ly6C-PerCP–Cy5.5 (clone HK1.4, BioLegend, 128027), rat anti-Ly6G-PE (clone 1A8, BioLegend, 127607), rat anti-B220-FITC (clone RA3-6B2, BioLegend, 103205) and rat anti-CD90.2-APC (clone 53-2.1, BioLegend, 140312) for 30 min on ice protected from light. After an additional wash, cells were sorted directly into Trizol (Themo Fisher Scientific, 15596026) by a BD FACSAria II cell sorter. Raw flow cytometry data were analysed using FlowJo software (FlowJo LLC, version 10.6.2). Samples were flash frozen on dry ice and stored at −80 °C. RNA was extracted using the RNeasy Micro Kit according to the manufacturer’s instructions (Qiagen, 74004). RNA quality, RNA integrity number (RIN) and RNA concentrations were assessed using Nanodrop (Thermo Fischer Scientific) and Bionalyzer (Agilent, 5067-1513). 500 pg of purified RNA was used for library preparation, which was performed using the SMARTer Stranded Total RNASeq Kit –2 - Pico Input Mammalian (Takara, 634413). Libraries were barcoded for multiplexing. Before sequencing, library quality and concentration were measured using Qubit Fluorometric Quantitation (Thermo Fisher). Libraries were sequenced (2 × 150 bp, paired-end reads configuration, v4 chemistry) on an Illumina HiSeq machine at a minimum of 30 million reads per sample. Sequencing was performed at Genewiz. Raw sequencing reads from the samples were mapped to mm10 using HISAT2 v2.1.065. Counts of reads mapping to genes were obtained using htseq-count v0.12.4 against Ensembl v90 annotation66. Differential expression analysis was done using DESeq2 v1.26.0 package67. The fold change threshold was set at 2 (that is, log2(fold change) > |1|). GO terms were determined using DAVID, version 6.868. Only GO terms with an adjusted P value < 0.05 (FDR) and an overall of > 5% (involved genes/total genes) were considered. 　　Leukocyte subpopulation frequencies from brain border regions were isolated as previously described69. Mice were anaesthetized with 10% chloral hydrate and transcardially perfused with ice-cold PBS (0.1 M). Leptomeninges, dura and choroid plexus were carefully dissected on ice. Meninges were digested in RPMI (Thermo Fisher Scientific, 11875093) with 1.4 U ml−1 Collagenase VIII (Sigma-Aldrich, C2139) and 1 mg ml−1 DNAse 1 (Thermo Fisher Scientific, EN0521) for 15 min at 37 °C. Digested dura and leptomeninges and undigested choroid plexus were passed through a 70 μm cell strainer (pre-wet with PBS) (Miltenyi Biotec, 130-095-823) into a 15 ml conical tube. Cells were pelleted (300g for 10 min at 4 °C) and washed once with ice-cold PBS. Cells were then resuspended in FACS buffer, Fc receptor binding was blocked (rat anti-CD16/CD32, clone 2.4G2, BD Biosciences, 553141) and cells were stained with a viability dye (Thermo Fisher Scientific, 65-0865-14) for 30 min. Cells were washed and stained with the following fluorophore-conjugated primary antibodies for 30 min on ice (all dilutions: 1:400): rat anti-CD11b–FITC (clone: M1/70, Invitrogen 11-0112-81), Armenian hamster anti-TCRβ-PerCP–Cy5.5 (clone: H57-597, Invitrogen, 45-5961-80), rat anti-Ly6C–APC (clone: HK1.4, Invitrogen, 17-5932-82), rat anti-Ly6G–eFluor 450 (clone: 1A8-Ly6g, Invitrogen, 48-9668-82), rat anti-CD19–PE (clone: IDE, BD Pharmingen, 553786), rat anti-CD45–PE–Cy7 (clone: 30-F11, BD Pharmingen, 552848). After an additional wash, cells were resuspended in FACS buffer and sorted by a BD FACSAria II using the 70 μm nozzle to sort cells into 1.5 ml Eppendorf tubes containing TRIzol LS with a sort speed of approximately 10,000 events per second. Raw flow cytometry data were analysed using FlowJo software (FlowJo, version 10.8.1). 　　Brain-trafficking monocytes and brain-resident myeloid cells were isolated based on previous published protocols70. Twenty-four hours after the SI test, mice were euthanized by injecting 10% chloral hydrate and perfused transcardially with ice-cold 0.1 M PBS (pH 7.4). Brains were rapidly dissected, leptomeninges carefully removed, and brains put in ice-cold PBS (for brain-trafficking monocyte RNA-sequencing experiment) or bilateral NAc tissue punches were obtained from 1 mm thick coronal slices using 1.2 mm punches (for resident myeloid cell RNA-sequencing experiment) (GE Healthcare Life Sciences, 1205×41). All the following steps were performed strictly on ice. For whole brains, tissue was cut into small pieces, for punches no shredding was needed. Tissue was then transferred to DPBS and homogenized with pestles (Sigma, D8938-1) in ice-cold PBS (20 stokes with pestle A, 20 stokes with pestle B). The cell suspension was then passed through a 70 μm cell strainer (pre-wet with PBS) (Miltenyi Biotec, 130-095-823) into a 15 ml conical tube. Cells were pelleted (300g for 5 min at 4 °C), resuspended in 10 ml of ice-cold 40% isotonic Percoll (Millipore Sigma, GE17-0891-01) (diluted in PBS) and centrifuged for 30 min at 500g at 4 °C with full acceleration and braking. The myelin layer was aspirated, then the cell pellet was washed with 10 ml of ice-cold PBS by centrifuging at 300g for 5 min at 4 °C. Cells were then resuspended in FACS buffer, Fc receptor binding was blocked (rat anti-CD16/CD32, clone 2.4G2, BD Biosciences, 553141) and then cells were stained with a viability dye (Thermo Fisher Scientific, 65-0865-14) for 30 min. Cells were washed and stained with a combination of the following fluorophore-conjugated primary antibodies: rat anti-CD45–BV510 (clone 30-F11, BioLegend, 103137), rat anti-CD11b–PerCP-Cyanine5.5 (clone M1/70, BioLegend, 101227), rat anti-Ly6C–APC-Cyanine7 (clone HK1.4, BioLegend, 128025), and rat anti-Ly6G–eFluor 450 (clone 1A8-Ly6g, Thermo Fisher Scientific, 48-9668-82) at a 1:400 dilution for 30 min on ice. After an additional wash, cells were sorted by a BD FACSAria II using the 70 μm nozzle to sort single cells into 96-well plates containing master mix (see below) with a sort speed of approximately 10,000 s−1. Raw flow cytometry data were analysed using FlowJo software (FlowJo LLC, version 10.6.2). All single-cell RNA-sequencing experiments were performed at the Single Cell Core Facility of the Sulzberger Columbia Genome Center, New York. Library preparation and RNA sequencing was performed as described previously71. In brief, cells were directly sorted into master mix, containing 1× Maxima Reverse Transcriptase Buffer (Thermo Fisher Scientific, EP0742), 40 U Maxima H Minus Reverse Transcriptase (Thermo Fisher Scientific, EP0751), 4 U SuperaseIN (Thermo Fisher Scientific, AM2694), 15% PEG (VWR, 97061-102), 1 μM TSO (Integrated DNA Technologies), and nuclease-free water. Template-switching reverse transcription was performed with adapter-linked oligo primers containing both cell- and molecule-specific barcodes (Supplementary Table 6). Excess primers were removed by adding 2 μl of Exonuclease I (Thermo Fisher Scientific, EN0581) mix to each well and incubated at 37 °C for 30 min, 85 °C for 15 min, 75 °C for 30 s. All wells were then pooled into a single 15 ml conical tube and cDNA was purified and concentrated with Dynabeads MyOne Silane beads (Thermo Fisher Scientific, 37002D). The cDNA was split into duplicate reactions of 25 μl cDNA, 25 μl of 2× HIFI HotStart Ready Mix (Kapa Biosystems, 07958927001), and 0.2 M SMART PCR Primer (Supplementary Table 6). PCR was performed as described above. cDNA was purified with AMPure XP beads (Beckman Coulter, A63880), visualized on an Agilent TapeStation and quantified with a Qubit II fluorometer (Thermo Fisher Scientific). Library preparation was performed using a modified protocol of the Nextera XT kit (Illumina, FC-131-1024), purified twice with AMPure XP beads (Beckman Coulter, A63880), and visualized and quantified as described above. Pooled, 3’-end libraries were sequenced on an Illumina NextSeq 500/550 apparatus. Reads were aligned to the mouse genome reference GRCm38 using STAR (version 2.5)72. Reads were assigned to cells and unique molecular identifiers73. The expression matrix for single-cell data was processed using the package Seurat v3.1.5 in R74. Features for which fewer than 3 cells were detected were removed, effectively excluding unexpressed features. Cells having at least 1,000 and at most 4,000 features were retained. Cells with more than 5% of reads mapping to mitochondrial genes were discarded. The NormalizeData function was used to log-normalize the dataset with a scale factor of 10,000. The top 2,000 most variable features across cells were found using the function FindVariableFeatures. The ScaleData function was applied to scale the dataset. The variable features were used to carry out dimensional reduction using principal components analysis. The optimal number of principal components to be used for dimensional reduction using UMAP was determined using ElbowPlot. FindNeighbors and FindClusters functions were utilized to construct a nearest neighbour graph and cluster cells in the dataset. UMAP was generated using the function DimPlot. The FindAllMarkers function was applied to determine markers for clusters in the UMAP plot. The FindMarkers function was used to carry out differential expression analysis for the three experimental groups. 　　Twenty-four hours after the SI test, Ccr2rfp+/− mice were injected with 10% chloral hydrate and transcardially perfused with ice-cold 0.1 M PBS followed by 4% paraformaldehyde (PFA) (Electron Microscopy Sciences, 15713 S). Intact brains were dissected out of the skull and post-fixed in 4% PFA in PBS at 4 °C for 18 h. Brains were then cleared and stained according to the iDISCO+ staining protocol (http://www.idisco.info). The primary rabbit anti-RFP antibody (Rockland, 600-401-379, 1:1,000) and the corresponding secondary antibody (donkey anti-rabbit IgG, Alexa Fluor 647, Thermo Fisher Scientific, A-31573, 1:1,000) were incubated with the brains for 7 days each at 37 °C. A LaVision light-sheet microscope with zoom body was used for sagittal half brain scanning with dynamic focus and a step size of 4 µm. Brain images were processed as previously described using ClearMap (version 1)31. RFP+ cells were quantified using the cell detection module, with cell detection parameters optimized and validated based on the intensity and shape parameters of the signal. The autofluorescence channel was aligned to the Allen Institute’s Common Coordinate Framework using the Elastix toolbox. Brain areas were collapsed into their parent regions prior to analyses. 　　Bilateral NAc tissue punches were briefly thawed on ice and digested for 60 min at 37 °C in 20 U ml−1 chondroitinase ABC (Sigma-Aldrich, C3667) in 25 mM Tris-buffered saline (Thermo Fisher Scientific, BP2471) with protease inhibitors (Thermo Fisher Scientific, 1861281). Samples were immediately cooled on ice, centrifuged for 20 min at 21,000g at 4 °C and the supernatant was transferred to a new tube. Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, 23227). Samples were flash frozen and stored at −80 °C. Total protein (20 μg) was separated by electrophoresis with a SDS–PAGE polyacrylamide gel (Bio-Rad, 4561034) and transferred to a PVDF membrane (Bio-Rad, 1704157). The membrane was blocked with 5 % non-fat dry milk (Bio-Rad, 1706404) in 0.1% Tween-20 (Sigma-Aldrich, P7949) in Tris-buffered saline (Thermo Fisher Scientific, BP2471, TBS-T) and incubated overnight with primary antibodies against aggrecan (1:1,000, Sigma-Aldrich, AB1031) or horseradish peroxidase (HRP) conjugated β-actin (1:2,000, Cell Signaling, 12262) in 5% non-fat dried milk (Sigma-Aldrich, A9647) in TBS-T. Membranes were then washed for 1 h with TBS-T and then incubated with secondary antibodies (1:10,000, anti-rabbit IgG HRP-linked, Cell Signaling, 7074) for 3 h at room temperature. After washing the membrane, visualization was performed using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, 32209) with an iBright CL1500 Imaging System (Thermo Fisher Scientific, A44114). Quantification was done with ImageJ (NIH, v1.53f51)75. Uncropped blots are available in Supplementary File 1. 　　RNA from fluorescence-activated cell-sorted monocytes was isolated using the RNeasy Micro Kit according to the manufacturer’s instructions (Qiagen, 74004). RNA quality and RNA concentrations were assessed using Nanodrop (Thermo Fischer Scientific). RNA was reversed transcribed to cDNA using qScript (QuantaBio, 95048-100) and the PCR reaction was performed using the SYBR Fast Advanced Master Mix system (Thermo Fisher Scientific, A46012) with the following primers (Integrated DNA Technologies): Mmp8 (Mm.PT.58.6942600, primer 1: AGGATCAGTGGAGTGAGAGAG; primer 2: CAAGGTATTGGAGGAGATGCTC); Gapdh (Mm.PT.39a.1, primer 1: GTGGAGTCATACTGGAACATGTAG; primer 2: AATGGTGAAGGTCGGTGTG). Gene expression analysis was done using the 2(–ΔΔCT) method76 and samples were normalized to the housekeeping gene Gapdh. 　　Brains were rapidly extracted from isoflurane-anaesthetized mice, and coronal sections (250 µm) were sliced using a Compresstome (VF-210-0Z, Precisionary Instruments) in cold (0–4 °C) sucrose-based artificial cerebrospinal fluid (aCSF) containing: 87 mM NaCl (Sigma-Aldrich, S7653), 2.5 mM KCl (Sigma-Aldrich, P9333), 1.25 mM NaH2PO4 (Sigma-Aldrich, 71507), 4 mM MgCl2 (Sigma-Aldrich, M2670), 0.5 mM CaCl2 (Sigma-Aldrich, C8106), 23 mM NaHCO3 (Sigma-Aldrich, S6297), 75 mM sucrose (Sigma-Aldrich, S7903), 25 mM glucose (Sigma-Aldrich, G7021). After 60 min in aCSF at 32 °C for recovery, slices were kept in oxygenated (95% O2, 5% CO2) aCSF containing 130 mM NaCl (Sigma-Aldrich, S7653), 2.5 mM KCl (Sigma-Aldrich, P9333), 1.2 mM NaH2PO4 (Sigma-Aldrich, 71507), 2.4 mM CaCl2 (Sigma-Aldrich, C8106), 1.2 mM MgCl2 (Sigma-Aldrich, M2670), 23 mM NaHCO3 (Sigma-Aldrich, S6297), 11 mM glucose (Sigma-Aldrich, G7021) at room temperature for the rest of the day and individually transferred to a recording chamber continuously perfused at 2 to 3 ml min−1 with oxygenated aCSF. Patch pipettes (4–6 MΩ) were pulled from thin wall borosilicate glass using a micropipette puller (P-97, Sutter Instruments) and filled with a potassium gluconate-based intra-pipette solution containing: 116 mM KGlu (Sigma-Aldrich, P1847), 20 mM HEPES (Sigma-Aldrich, H3375), 0.5 mM EGTA (Sigma-Aldrich, E0396), 6 mM KCl (Sigma-Aldrich, P9333), 2 mM NaCl (Sigma-Aldrich, S7653), 4 mM ATP (Sigma-Aldrich, A9187), 0.3 mM GTP (Sigma-Aldrich, 51120) (pH adjusted to 7.2 and osmolarity to 290 mOsm). Cells were visualized using an upright microscope with an IR-DIC lens and illuminated with a white light source (Scientifica). Excitability was measured in current-clamp mode by injecting incremental steps of current (0–300 pA, +20 pA at each step). For recording of sEPSCs, NAc MSNs were recorded from in voltage-clamp mode at −70 mV. Whole-cell recordings were performed using a patch clamp amplifier (Axoclamp 200B, Molecular Devices) connected to a Digidata 1550 LowNoise acquisition system (Molecular Devices). Signals were low pass filtered (Bessel, 2 kHz) and collected at 10 kHz using the data acquisition software pClamp 11 (Molecular Devices). Electrophysiological recordings were extracted using Clampfit 11 (Molecular Devices) and analysed with R (version: 3.6.1, http://www.R-project.org). All groups were counterbalanced by days after CSDS. All recordings were performed while blinded to the experimental conditions. 　　Study participants with MDD and healthy controls, as assessed according to SCID-577, were recruited through the Depression and Anxiety Center for Discovery and Treatment at the ISMMS. The ISMMS review board approved the study, and written informed consent was obtained from all participants prior to any study procedure. Participants were compensated for their time and effort. They provided demographic information and underwent a psychiatric evaluation using the SCID-5 conducted by trained study staff. Participants completed the Quick Inventory of Depressive Symptomatology-SR (QIDS-SR) to measure depressive symptom severity78. The Perceived Stress Scale27, a 10-item self-rating scale, was used to determine perceived stress levels. All participants underwent biochemistry and haematological laboratory testing, urine toxicology and pregnancy (if applicable) testing. At the time of enrolment, all participants were free of medications known to affect the immune system for at least two weeks. Participants were free of active infections or systemic illness. Subjects with concomitant unstable medical illnesses were excluded. Participants were free of current substances of abuse. On the day of blood draw, patients were fasted for at least 6 h. Blood was drawn into Vacutainer Gold Top 5 ml Silica Gel tubes (BD, 365968) for serum isolation, EDTA tubes (BD, 365975) to assess complete blood count and differential count (Sysmex XN-9100 Automated Hematology System) and into BD Vacutainer CPT tubes (BD, 362761) for the isolation of peripheral blood mononuclear cells (PBMCs). For serum, blood was allowed to clot for >30分钟，然后在4°C下以1,300克离心15分钟
，然后等分并存储在-80°C下。根据制造商的说明分离PBMC，并在液氮中冷冻保存 。分析当天 ，将所有PBMC仔细地在37°C的水浴中融化。将细胞颗粒（在4°C下为300g 10分钟）
，并用冰冷的PBS洗一次
。然后将细胞重悬于FACS缓冲液中。使用抗CD16/32（克隆2.4G2，Bio X细胞 ，BE0307）阻断FC受体结合
，并用活力染料（Thermo Fisher Scientific，65-0865-14）染色30分钟 。Cells were washed and stained with the following fluorophore-conjugated primary antibodies for 30 min on ice (all dilutions: 1:400): mouse anti-CD45–V500 (clone HI30, Fisher Scientific, BDB560779), mouse anti-CD19–PE–Cy7 (clone SJ25C1, Fisher Scientific, BDB560911), mouseanti-CD24–PE (clone ML5, Fisher Scientific, BDB560991), mouse anti-CD27–APC (clone L128), mouse anti-CD38 PerCP–Cy5.5 (clone HIT2, Fisher Scientific, BDB551400) and mouse anti-IgD–V450 (clone IA6-2, Fisher Scientific, BDB561309).洗涤细胞 ，然后重悬于FACS缓冲液中
，然后再在BD LSRFortessa细胞分析仪（BD Biosciences）上获得
。使用FACS Diva软件（BD，V.9）获取流式细胞仪数据。使用FlowJo软件（Flowjo LLC ，版本10.8.1）分析数据 。如描述的79进行B细胞亚型的门控
。 　　根据制造商的说明（鼠标MMP8：ABCAM
，AB206982; Human MMP8：R＆D Systems，DMP800B）进行酶联免疫吸附测定。对于脑裂解物 ，用Pierce BCA蛋白测定试剂盒（Thermo Fisher Scientific，23225）测量了总蛋白 。使用SoftMax Pro 5软件（分子设备）
，通过串行稀释曲线（分子设备）计算了Spectramax 340PC384微型读取器（分子设备）和MMP8的板读取板
。 　　根据制造商的说明（Millipore Sigma，McyTomag-70K）和MMMP9和MMP12（MMMP1和MMMP12测量） ，用Milliplex MAP小鼠细胞因子/趋化因子细胞因子/趋化因子细胞因子/趋化因子/趋化因子/趋化因子磁珠组合测定确定小鼠血浆细胞因子和趋化因子的小鼠血浆细胞因子和趋化因子
，并确定小鼠血浆细胞因子的血浆细胞因子和趋化因子，MMMP9和MMMP12（MMMP1）根据制造商确定 ，用小鼠磁磁珠测定测定小鼠血浆细胞因子和趋化因子MMMP1MAG-79K
，MMMP2MAG-79K）。 　　将小鼠注入10％氯氢酸盐，并在4°C下以0.1 m cacododylate缓冲液进行心铁灌注 ，然后在4°C下用0.5％PFA固定 。按照80,81,82进行了冻结的纤维片
，并冻结样品的冻结替代和低温嵌入
。通过浸入较高浓度的甘油（从PBS中的10％到30％）中浸透切片（v/v）。将切片迅速浸入液态丙烷中，通过通用冷冻固定系统KF80（Reichert-Jung）中的液氮（-190°C）冷却 。将样品浸入溶解在无水甲醇（-90°C ，24小时）中的1.5％乙酸铀酰中。温度从-90°C升至-45°C
，步骤为4°C/h
。用无水甲醇洗涤后，将样品用-45°C的Lowicryl HM20树脂（电子显微镜科学）浸润。用紫外线（360 nm）在-45°C下进行48小时的聚合 ，然后在0°C下进行24小时 。用涂层的粘合剂（电子显微镜科学）在Leica UC7 Ulmicrotome上用钻石刀切割超薄切片（80 nm），并在300网地铜网上安装
。使用Hitachi 7700电子显微镜（Hitachi High-Technologies Corporation America）拍摄图像（N = 10）
，该图像配备了XR81-B-M1-BT-FX，8百万像素数码相机（高级显微镜技术）。然后将图像导入Adobe Photoshop（Adobe ，2022）
，ECS是使用计算机平板电脑手动评分的 。评分是由两名对实验条件蒙蔽的独立研究人员进行的
。然后将图像进口到ImageJ（V1.53F51）75中，并计算出标记面积/总面积的百分比。 　　将小鼠注射10％氯氢酸盐 ，并在冰冷的0.1 M PBS（pH 7.4）上灌注冰冷的小鼠
，然后是冰冷4％PFA（电子显微镜科学，15713 s） 。将完整的大脑从头骨中解剖 ，并在4％PFA下4°C固定18小时
。然后将大脑在30％的蔗糖（Sigma
，S0389）中冷冻保护，冷冻并切成35μm的低温恒温器。将切片用PBS洗涤3次 ，并在阻塞溶液中孵育（3％正常驴血清（Jackson Immuno Research
，017-000-121），0.3％Triton X-100（Sigma ，T9284）在PBS中孵育2小时 。然后将切片在原始抗体（大鼠抗CD31
、1：300，Biolegend
，102501; Rabbit Anti-RFP，1：300 ，Rockland
，600-401-379; Goat Anti-RFP，1：200 ，Rockland
，200-101-379; Rabbit; Rabbit Anti-afi-i-afimific; rabifific; rabific;PA5-85767）在4°C下过夜
。第二天，将部分在PBS中用0.3％Tween-20（PBST（Sigma ，P7949））洗涤3次
，持续15分钟，然后与抗Rabbit-Cy2和抗Rab-Cy2和抗Rat-Cy5二级抗体孵育2 h（1：400 ，Jackson Immuno Research
，711-225-152-152-152和7112-12-15-15-15-1555）将切片再次用PBST洗涤3次。然后将切片安装在幻灯片上，隔夜隔断 ，脱水，并用DPX覆盖（电子显微镜科学
，13510） 。使用Zeiss LSM 780共聚焦显微镜对所有切片进行成像。使用Imaris软件（Oxford Instruments，v9.9）进行3D重建
。 　　根据制造商的说明（Thermo Fisher Scientific ，A39256）
，使用EZ-Link Sulfo-NHS-Biotin套件进行了小鼠RMMP8（Bio-Techne，2904-MP-010）的生物素化。使用Pierce C18自旋柱 ，7 K MWCO（Thermo Fisher Scientific
，89882）将生物素化的RMMP8与未结合生物素分离，该柱将恢复大于7 kDa的蛋白质和大分子 。将生物素化的RMMP8骨注射到麻醉的小鼠中
。循环2小时后 ，将小鼠安乐死并用冰冷的PBS灌注
，然后用4％PFA灌注。如在免疫组织化学和共聚焦显微镜部分中所述进行的脑组织加工和成像，并使用以下抗体：使用俄勒冈州绿色488中性蛋白生物素结合蛋白（Thermo Fisher Scientific ，A6374）可视化生物素 。使用兔子反纽恩（1：500
，ABCAM，AB177487）和大鼠抗CD31（1：300 ，Biolegend，102501）进行反染色
。 　　每个实验的详细统计信息可以在补充表2中找到。除非另有说明
，否则使用GraphPad Prism软件（GraphPad Software，版本9）或SPSS版本24（IBM ，SPSS）进行统计分析 。使用Grubbs的测试确定了离群值
，并将其排除在统计分析之外
。统计显着性水平设置为p <0.05。 　　有关研究设计的更多信息可在与本文有关的自然投资组合报告摘要中获得 。