Stress dynamically modulates neuronal autophagy to gate depression onset

Animals and ethical approval

All mice (aged 7–15 weeks, if not specified) were bred in our colony (that is, Atg7^{flox flox} lines⁵⁰ and GFP-LC3 lines) or purchased from SLAC (that is, C57BL/6J and CD-1 mice). CD-1 mice (4–6 months old, retired breeders) were single-housed, and all other mice were housed 4–5 per cage if not specified. All animals were subjected to a 12-h light–dark cycle (light on from 7:00 to 19:00) with food and water ad libitum. All mice were housed in a stable environment (23–25 °C ambient temperature and around 50% humidity). Only male mice were used in this study. All animal research and experimental procedures were approved by the Animal Care and Use Committee of the animal facility at Zhejiang University.

Bulk RNA-seq

RNA-seq was performed with fresh LHb, vHippo, mPFC, VTA, lateral hypothalamus and NAc samples from CRS and naive mice tested using FST 2 days before euthanasia. We divided CRS and naive mice into two groups, respectively. Namely CRS mice with high FST scores (FST immobility for more than 110 s, n = 3), CRS mice with low FST scores (50 s less than FST immobility less than 110 s, n = 3), naive mice with high FST scores (50 s less than FST immobility less than 80 s, n = 3), naive mice with low FST scores (FST immobility less than 50 s, n = 3). On the third day, mice were euthanized after anaesthesia with 1% pentobarbital NEMBUTAL (100 mg kg⁻¹) and perfused with 20 ml of ice-cold phosphate-buffered saline (PBS, pH 7.4). The brains were collected quickly on an ice plate and moved to liquid nitrogen for 25 s. Frozen brains were fitted into stainless steel mouse brain matrices and sectioned with blades into roughly 1-mm coronal slices containing the LHb (−1.5 to −1.9 mm from bregma), vHippo (−2.9 to −3.3 mm from bregma), mPFC (+1.9 to +1.5 mm from bregma), VTA (−2.9 to −3.3 mm from bregma), lateral hypothalamus (−0.6 to −1.0 mm from bregma) and NAc (+1.3 to +0.9 mm from bregma). Slices were immersed in ice-cold PBS and relevant brain regions were carefully micro-dissected on an ice plate under microscope. The samples were put into a marked Eppendorf tube and were then quickly transported into liquid nitrogen for subsequent mRNA sequencing under customized service of LC-Bio Technology. After the final transcriptome was generated, StringTie and ballgown were used to estimate the expression levels of all transcripts and determine mRNA expression abundance by calculating the FPKM value. Genes differential expression analysis was performed by DESeq2 software. The genes with P < 0.05 and absolute fold change greater than or equal to 1.2 were considered differentially expressed genes. Differentially expressed genes were then subjected to enrichment analysis of KEGG pathways. z-score values stand for the up- or downregulation of all genes enriched to this pathway. Bioinformatic analysis was performed using the OmicStudio tools at https://www.omicstudio.cn/tool. KEGG analysis and statistics of mRNA expression level were compared only between CRS mice with high FST scores and naive mice with low FST scores. For correlation analysis, all mice were included.

snRNA-seq

The LHb from CRS and naive mice was transferred to tissue storage solution (130-100-008, Miltenyi), and the suspension after filtration, centrifugation and resuspension was loaded onto the Chromium single-cell controller (10X Genomics) to generate single-cell gel beads in the emulsion according to the manufacturer’s protocol by LC-Bio Technology. Postprocessing and quality control were carried out by 10X Cell Ranger package (v.1.2.0, 10X Genomics). The bioinformatics analysis of snRNA-seq was conducted by LC-Bio Technology. Clustering analysis was carried out with standard seurat package procedures at a resolution of 1.20. The clusters were then assigned to neuronal and glial cell types by their canonical gene markers. Syp marked neuron clusters, Mag marked oligodendrocyte clusters, Gja1 marked astrocyte clusters and C1qc marked microglia clusters. To further explain differentially expressed autophagy-related genes among these cell types, we used gene set variation analysis to analyse the ‘Autophagosome Formation’ pathway.

Western blotting

Samples were collected using the same protocol as described in ‘Bulk RNA-seq’. After micro-dissection, LHb (−1.5 to −1.9 mm from bregma), vHippo (−2.9 to −3.3 mm from bregma), mPFC (+1.9 to +1.5 mm from bregma), VTA (−2.9 to −3.3 mm from bregma), lateral hypothalamus (−0.6 to −1.0 mm from bregma) and NAc (+1.3 to +0.9 mm from bregma), lateral septum (+0.9 to +0.5 mm from bregma), dorsal raphe nucleus (−4.2 to −4.6 mm from bregma), median raphe nucleus (−4.2 to −4.6 mm from bregma), arc (−1.2 to −1.5 mm from bregma), BLA (−1.3 to −1.9 mm from bregma) and liver samples were transferred to ice-cold lysis buffer (0.32 M sucrose, 10 mM HEPES pH 7.4, 2 mM EDTA, 1 mM PMSF, 1× Protease inhibitors, 1× Phosphatase inhibitors) and homogenized using ultrasonic cell grinder. Part of the lysates were taken as a total protein sample and incubated for 30 min on ice with 10% SDS. Remaining lysates were centrifuged at 1,000g for 10 min to remove pelleted nuclear fraction. The supernatant was then taken and centrifuged again at 15,000g for 30 min. The new supernatant was discarded and the pellet was resuspended in RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 1× Protease inhibitors, 1× Phosphatase inhibitors) as crude synaptosome sample that would be subsequently incubated on ice for 30 min. Samples were diluted in 5× SDS–PAGE loading buffer and were boiled at 95 °C for 5 min. Next, the samples were loaded on an SDS (8–15%) polyacrylamide gradient gel and transferred onto a polyvinyl difluoride membrane. The blots were incubated at 4 °C overnight with one of the following primary antibodies: rabbit anti-p62 (1:2,000, PM045, MBL), rabbit anti-Beclin-1 (1:1,000, 3495, CST), mouse anti-Atg7 (1:1,000, 67341-1-Ig, Proteintech), mouse anti-ubiquitin (1:1,000, 3936, CST), rabbit anti-p-AMPKα (Thr172) (1:1,000, 2535, CST), rabbit anti-p-mTOR (1:1,000, ab137133, Abcam), rabbit anti-p-p70S6 Kinase 1-T421/S424 (1:1,000, AP0502, ABclonal), rabbit anti-p-Akt (1:1,000, 4058, CST), rabbit anti-Cleaved Caspase-3 (1:1,000, 9664, CST), rabbit anti-GluA1 (1:1,000, ab31232, Abcam), rabbit anti-GluA2 (1:1,000, 11994-1-AP, Proteintech), rabbit anti-GluN1 (1:1,000, ab109182, Abcam), rabbit Anti-GABA_A Receptor alpha 1 (1:1,000, ab252430, Abcam), mouse anti-β-actin (1:5,000, MA5-15739, Thermo Fisher Scientific) or rabbit anti-α-tubulin (1:2,000, 11224-1-AP, Proteintech). The next day, horseradish peroxidase (HRP)-conjugated secondary antibodies, including anti-mouse IgG, HRP-linked antibody (1:10,000, 31430, Thermo Fisher Scientific) and anti-rabbit IgG, HRP-linked antibody (1:5,000, 31460, Thermo Fisher Scientific) were revealed using an ECL kit (Immobilon Ultra Western HRP Substrate, MILLIPORE) for protein detection. Selected films were scanned and quantified using BioRad Image Lab software (v.6.0). Some blots then were incubated in stripping buffer (Beyotime) for 20 min at room temperature followed by three washes with tris-buffered saline with Tween. After being reblocked, membranes were re-incubated with another primary antibody overnight at 4 °C. The following steps were the same as described above. β-actin and α-tubulin bands were used for normalization.

For gel source data, see the Supplementary Information.

Immunohistochemistry

Mice (7–8 weeks) that experienced ARS or CRS were anaesthetized and perfused with 20 ml of PBS followed by 20 ml of 4% paraformaldehyde (PFA) in PBS. After decapitation, brains were dissected and transferred to 4% PFA for postfixation for 24 h at 4 °C. The fixed brains were then dehydrated in 30% sucrose in PBS at 4 °C for 24 h and sectioned into 20-μm sections with a microtome (Thermo Scientific, NX50). Free-floating slices were washed three times in PBS, then moved onto microscope slides and permeabilized with 0.5% Triton X-100 in PBS for 20 min at room temperature. Slices were rinsed three times and then preserved in blocking buffer (5% normal goat serum) for 1 h at room temperature. Some sections were incubated with primary antibodies (mouse anti-LC3, 1:200, A17424, ABclonal; or rabbit anti-NeuN, 1:500, A19086, ABclonal) for 24 h at 4 °C to stain LC3 and NeuN, and other sections were incubated with primary antibodies (rabbit anti-LC3, 1:1,000, ab192890, Abcam; mouse anti-PSD95, 1:500, ab13552, Abcam; or guinea pig anti-NeuN, 1:500, ABN90, Sigma-Aldrich) for 48 h at 4 °C to stain LC3, PSD95 and NeuN. These were then rinsed three times with PBS and incubated with secondary antibodies for 1 h at room temperature (goat anti-mouse, 1:100, AS008, ABclonal or 1:1,000, A-21235, Thermo Fisher Scientific; goat anti-rabbit, 1:1,000, AS060, ABclonal, or 1:1,000, A-11008, Thermo Fisher Scientific; goat anti-guinea pig, 1:1,000, A-21435, Thermo Fisher Scientific) and finally rinsed again three times with PBS. For GFP-LC3 mice, 4,6-diamidino-2-phenylindole (DAPI) staining was applied for visualization of cells. Fluorescent image acquisition was performed with Leica SP8 Laser confocal microscope. Three slices (anterior, middle and posterior) and two views of each hemisphere of LHb were selected for counting. Confocal z-stack images were collapsed using the Z Projection-Max intensity function in Image J. LC3 puncta positive neurons were counted manually.

RNA isolation and real-time qPCR

Immediately after dissection, RNA was extracted using RNeasy Kit (AC0202-B, Shandong Sparkjade Biotechnology) according to the instruction protocol. Following extraction, RNA quality was evaluated by measuring the absorption at 230, 260 and 280 nm by the Nano Drop 2000 Spectrophotometer (Thermo Scientific). A ratio of A260/A280 = 1.8/2.1 was considered high purity. Complementary DNA (cDNA) synthesis was done using cDNA Synthesis Kit (R323-01, Vazyme), which was then used as a template for real-time qPCR. Real-time qPCR was carried out in a LightCycler 96 (Roche) using SYBR Green Pro Taq HS (AG11701, ACCURATE BIOTECHNOLOGY) according to the standard protocols. Melting curve analysis was performed to ensure a single peak for each PCR product. The gene expression levels were normalized by the average levels of Actb. All measurements were performed in triplicate and analysed using the 2^−ΔΔCt method. Primers (5′–3′) used were as follows: Actb, GACGGCCAGGTCATCACTATTG and AGGAAGGCTGGAAAAGAGCC; Atg7, GCTGCTGAGATCTGGGACAT and GAGATGTGGAGATCAGGACCAG; Atg10, AGGTCAGGGCGAGCGA and CCATCGCCTATCTGCTGTGA; Atg5, TGTGCTTCGAGATGTGTGGTT and GTCAAATAGCTGACTCTTGGCAA; Atg4d, CCCCGGCATTCACTGTACTT and TGGCAAAGGCCATCTCCAG; Atg12, TGAATCAGTCCTTTGCCCCTand CATGCCTGGGATTTGCAGT; Becn1, ATACTGTTCTGGGGGTTTGCG and GTCTCTCCTTTTTCCACCTCTTC; LC3, TTATAGAGCGATACAAGGGGGAG and CGCCGTCTGATTATCTTGATGAG; Mtor, CAGACTGGCTCTTGCTCATAA and GCTGGAAGGCGTCAATC; AMPK, GTCAAAGCCGACCCAATGATA and CGTACACGCAAATAATAGGGGTT.

Electron microscopy

Mice were anaesthetized with 1% Pentobarbital NEMBUTAL (100 mg kg⁻¹, Sigma-Aldrich) and perfused with 4% PFA in PBS. The brains were collected quickly and fitted into stainless steel mouse brain matrices and sectioned with blades into roughly 1-mm coronal slices containing the LHb (−1.5 to −1.9 mm from bregma). The LHb samples were postfixed with 2.5% glutaraldehyde in 0.1 M PBS (pH 7.4) for 12 h at 4 °C. Samples were then washed three times (10 min each) in 0.1 M PBS, treated with 1% OsO₄ in 0.1 M PBS for 40 min and washed three times (10 min each) in ddH₂O. Slices were then treated with 2% uranyl acetate in ddH₂O for 30 min. Finally, sections were successively dehydrated in 50, 70 and 90% ethanol (15 min each), then 100% ethanol (20 min each), followed by 100% acetone twice (20 min each). Sections were embedded in EPON resin and sliced into sections at 85 nm thickness. We inspected 15 random neurons (Thermo Scientific Talos L120C Electron Microscopy) and manually counted the autophagosomes, which are distinguished from other organelles by their oval or round shapes and double-membrane structures^8,37.

Immunoelectron microscopy

Mice were anaesthetized with 1% Pentobarbital NEMBUTAL (100 mg kg⁻¹, Sigma-Aldrich) and perfused with 4% PFA and 0.1% glutaraldehyde in 0.1 M phosphate buffer. The brains were removed quickly and fixed in the same fixative overnight at 4 °C. Fixed brains were then cut into 50-μm sections using a Leica VT1200S vibratome and the slides were kept in the same fixative for 2 h at room temperature. Free-floating 50-μm brain sections were collected and washed three times (15 min each) in phosphate buffer. To eliminate unbound aldehydes, sections were incubated in 50 mM glycine for 30 min, then rinsed with phosphate buffer for 15 min. The brain slides were permeabilized by 0.05% Triton X-100 in phosphate buffer for 15 min, and washed for another 15 min in phosphate buffer. The slides were incubated in 0.1% BSA-cold water fish gelatin (Ctm) in PBS for 30 min. Subsequently, primary antibody was then added with incubation buffer and slides were left for 1 h at room temperature, followed by an overnight incubation with primary antibody (rabbit anti-GluA2 1:100, 11994-1-AP, Proteintech) at 4 °C. The following day, slides was washed six times (10 min) with 0.1% BSA-C^tm in PBS. The slides were then incubated with Nanogold-labelled Fab’ goat anti-rabbit (1:50, N-24916, Thermo Fisher Scientific) for 1 h at room temperature, adjusted to 4 °C overnight. On the fourth day, slides were washed six times (10 min) with 0.1% BSA-C^tm in PBS and washed twice (10 min) in phosphate buffer. The slides were fixed again with 2.5% glutaraldehyde in phosphate buffer for 2 h and washed three times (10 min) in phosphate buffer. The slides were washed six times (5 min) in ddH₂O thoroughly. For silver using nanogold, slides were washed three times (5 min) in 0.02 M sodium citrate buffer and then underwent silver enhancement (HQ silver enhancement kit, Nanoprobes). After silver enhancement, the slides were washed six times (10 min) thoroughly with ddH₂O and incubated in ddH₂O overnight at 4 °C. On the fifth day, slides were washed three times (10 min) in PBS and placed in 1% OsO₄ for 40 min. The slides were then washed three times (10 min) with ddH₂O and placed in 2% Uranyl acetate for 30 min. Finally, the slides were washed three times (10 min) again, and were dehydrated with ethanol, 50% ethanol for 15 min, 70% ethanol for 15 min, 90% ethanol for 15 min, 100% ethanol twice (20 min), followed by 100% acetone twice (20 min). After incubation in EPON 812 mixed with acetone (1:1) for 2 h, the slides were embedded in EPON, and regions of interest were sectioned at 90 nm using an ultramicrotome (LEICA EM UC7). The slices were observed and photographed using transmission electron microscopy (Tecnai Spirit Bio-Twin) with an accelerating voltage of 120 kV, charge-couple device camera (Gatan, 15081901W0832) at the Center of CyroElectron microscopy, Zhejiang University. Quantitative analyses were performed to establish degree of GluA2 immunoreactivity colocalized within autophagosomes (quantified from 15 randomly selected neurons) in LHb from mice treated with ARS or CRS.

STORM

Four weeks after adeno-associated virus (AAV)-mCherry-EGFP-LC3 injection surgery, mice with or without surgery were restrained in 50-ml conical tubes for 2 h. They were then anaesthetized immediately and perfused with PBS followed by 4% PFA in PBS. The brains were dissected, postfixed in 4% PFA at 4 °C for 24 h, and then dehydrated in 30% sucrose in PBS for 48 h. Brains were sectioned into 20-μm slices using a microtome (Thermo Scientific NX50) and preserved in PBS. Free-floating slices were preserved in blocking buffer (5% normal goat serum and 0.3% Triton X-100 in PBS) for 1 h at room temperature. Slices were then incubated with primary antibodies (rabbit anti-GluA2, 1:250, 11994-1-AP, Proteintech, with or without mouse anti-LC3, 1:100, 83506, CST) in antibody dilution (5% normal goat serum, 0.3% Triton X-100, 1% bovine serum albumin in PBS) overnight at 4 °C. Slices were then washed with PBS (three times for 10 min) and incubated with secondary antibodies (goat anti-rabbit, 1:1,000, AS060, ABclonal, with or without goat anti-mouse, 1:1,000, A11001, Invitrogen) in antibody dilution for 2 h at room temperature. Finally, slices were washed with PBS (3 × 10 min) and incubated for 30 min at room temperature. The slice was immersed into STORM imaging buffer and LHb, Arc and BLA were immediately imaged with a Nikon N-STORM super-resolution system (Nikon Instruments) equipped with a ×100 oil immersion objective (CFI Apo ×100 oil, numerical aperture 1.49, TIRF WD 0.12) and Andor camera (Ixon 897 back-illuminated EMCCD). The buffer was freshly prepared before imaging and contained 7 μl of oxygen-scavenging GLOX buffer (14 mg glucose oxidase, 50 μl of 17 mg ml⁻¹ catalase in 200 μl of 10 mM Tris and 50 mM NaCl, pH 8.0), 35 μl of 1 M MEA buffer, and 7 μl of β-mercaptoethanol. A conventional wide-field fluorescence image was acquired with low laser power to identify the imaging objects and focal plane. The 647, 561 and 488 nm laser was turned to maximum power to turn off the fluorophores and trigger photo-switching. Nikon NIS Element v.4.51 software was used to perform three-dimensional reconstruction.

TUNEL assay

Three weeks after AAV-hSyn-mCherry-2A-Cre or AAV2/9-hSyn-MCS-mCherry-3×Flag injection surgery, mice were anaesthetized and perfused with PBS followed by 4% PFA in PBS. The brains were dissected, postfixed in 4% PFA at 4 °C for 24 h, and then dehydrated in 30% sucrose in PBS for 48 h. Brains were subsequently sliced into 50-μm sections. Terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay was performed with the TUNEL Apoptosis Detection Kit (40307ES50, Yeasen) according to the manufacturer’s instructions. Brain slices were rinsed twice with PBS (15 min each), followed by treatment with 100 μl of Proteinase K solution (20 μg ml⁻¹) at room temperature for 40 min, and then washed three times (5 min each) with PBS. For positive controls, each slice was incubated in 100 μl of 1× DNase I Buffer and 100 μl of DNase I (10 U ml⁻¹) for 5 min and 10 min, respectively, then washed three times (5 min each) with dH₂O. All slices were further equilibrated in 100 μl of 1× equilibration buffer at room temperature for 15 min and treated with 50 μl of TdT incubation buffer (34 μl of ddH₂O, 10 μl of 5× equilibration buffer, 5 μl of Alexa Fluor 488-12-dUTP Labelling Mix, 1 μl of Recombinant TdT Enzyme) at 37 °C for 1 h. Subsequently, the slices were washed five times (5 min each), first with PBS, then with 0.1% Triton X-100 in PBS and finally with PBS three times. DAPI staining solution (Solarbio) was used to visualize nuclei. Fluorescent images were acquired with an Olympus Slideview VS200 microscope and Olympus Fluoview FV3000 confocal microscope. The TUNEL signals (TUNEL/AAV virus/DAPI colabelled) and virus-infected neurons (AAV virus/DAPI colabelled) were counted manually with Fiji (v.2.1.0/1.53 c) within the LHb.

Viral vectors

AAV2/9-hSyn-oChIEF-tdTomato (titre 1.52 × 10¹³ vector genomes (v.g.) ml⁻¹, Taitool Bioscience), AAV2/9-hSyn-mCherry-2A-Cre (titre 4.71 × 10¹³ v.g. ml⁻¹, OBiO Technology), AAV2/9-hSyn-shRNA-Atg5-ZsGreen (titre 1.7 × 10¹² v.g. ml⁻¹, Hanbio), AAV2/9-hEF1α-DIO-hM4Di-mCherry (titre 1.35 × 10¹³ v.g. ml⁻¹, Taitool Bioscience), AAV2/9-hEF1α-DIO-mCherry (titre 1.25 × 10¹³ v.g. ml⁻¹, Taitool Bioscience), AAV2/9-EF1α-DIO-GCaMP6s (titre 5.94 × 10¹³ v.g. ml⁻¹, OBiO Technology), AAV2/9-hSyn-GCaMP6s (titre 1.67 × 10¹³ v.g. ml⁻¹, Taitool Bioscience), AAV2/9-hSyn-MCS-mCherry (titre 5.00 × 10¹³ v.g. ml⁻¹, OBiO Technology) were diluted to 1.00 × 10¹² v.g. ml⁻¹. AAV2/5-hSyn-shRNA-Atg7-EGFP (titre 5.47 × 10¹³ v.g. ml⁻¹, Sunbio Medical Biotechnology), AAV2/5-hSyn-EGFP (titre 7.8 × 10¹³ v.g. ml⁻¹, Sunbio Medical Biotechnology), AAV2/5-hSyn-shRNA-BECN1-mCherry (titre 3.67 × 10¹³ v.g. ml⁻¹, Sunbio Medical Biotechnology), AAV2/9-CMV-mCherry-EGFP-LC3 (titre 1 × 10¹³ v.g. ml⁻¹, Hanbio) were diluted to 5.00 × 10¹² v.g. ml⁻¹. AAV2/9-hSyn-Cre-EGFP (titre 1.67 × 10¹³ v.g. ml⁻¹, Taitool Bioscience) were diluted to 5.00 × 10¹¹ v.g. ml⁻¹. The cholera toxin subunit B (CTB-647) (0.2%, Invitrogen) was dissolved in PBS.

Stereotaxic surgery, virus injection and optic fibre and/or cannula implantations

For stereotaxic surgery, C57BL/6J mice or Atg7^flox/flox mice (7–8 weeks) were anaesthetized. Viruses were bilaterally injected with a glass pipette mounted on a stereotactic frame into one of the following brain regions: LHb (±0.46 mm medial–lateral, −1.72 mm anterior–posterior from bregma, −2.70 mm dorsal–ventral from the dura), vHippo (±2.80 mm medial–lateral, −3.0 mm anterior–posterior from bregma, −3.80 mm dorsal–ventral from the dura), lateral hypothalamus (±1.00 mm medial–lateral, −0.75 mm anterior–posterior from bregma, −4.78 mm dorsal–ventral from the dura). Volumes of virus or CTB ranged between 100 and 200 nl per hemisphere, infused at a rate of 100–150 nl min⁻¹. Injection pipette was withdrawn from the brain 10 min after the infusion. After surgery, mice were placed on a heating pad to recover from anaesthesia.

For optic fibre implantation, optic fibres (200-μm diameter, 0.37 numerical aperture, Inper Ltd) were bilaterally implanted 250 μm above the viral injection site and cemented onto the skull using dental cement. Viral expression profiles were verified by histology after completion of all experiments as shown in Extended Data Fig. 6h–k.

For cannulae implantation, a double guide cannulae (side-by-side distance 1.0 mm, RWD) was implanted bilaterally over the LHb (±0.46 mm mediolateral; −1.72 mm anterior–posterior from bregma and −2.20 mm dorsoventral from the dura) of C57BL/6J mice.

Cannula infusion experiment

For cannula implantation, a double guide cannulae was implanted over the bilateral LHb. tBP, Dyngo-4a, tBP with Dyngo-4a or TAT–GluA2_3Y, or vehicle was microinjected into the LHb 30 min before each behavioural test. For sustained effect test, behavioural tests were conducted at 24 h, on the seventh, 18th and 19th days following tBP infusion into the LHb of CRS mice. For prophylactic effect test, local tBP infusions occurred before the first, fifth and ninth days of restraint during 14-day CRS. SBI or vehicle was microinjected into the LHb before ARS. The double injector cannulae were connected with the micro-syringe using a polyethylene pipe filled with mineral oil. tBP (Selleck, 20 μg μl⁻¹, ref. ¹²) was dissolved in saline, and SBI (MCE, 5 μM) was dissolved in 10% DMSO, 40% PEG300, 5% Tween-80 and 45% saline. For all cannula infusion experiments, 150 nl of each drug was infused into the LHb bilaterally through the double injector cannulae. The injector cannulae were left in the LHb for an extra 4–5 min to allow adequate local drug diffusion and minimize the spread of the drug along the cannula track. The drug infusion sites were verified after all behavioural tests with CTB-647 (150 nl per side) injected into both sides of the LHb. Fluorescent image acquisition was performed with an Olympus Slideview VS200 microscope slide scanning system. Data were used only from mice with accurate injection sites.

Optogenetic manipulation

A blue light laser of 465-nm intensity was delivered at 5 mW, 2-ms pulse width. Mice were placed in the home cage and received 40 Hz phasic photostimulation as previously described²⁹ (40 Hz, five pulses per second, 10 min at intervals of 5 min, a total of 2 h) or LFS protocol (1 Hz, 15 min).

Intraperitoneal injection

Paroxetine-HCl (APE×BIO, 5 mg kg⁻¹) was dissolved in saline. For acute experiment, CRS mice were injected with paroxetine-HCl or vehicle 1 day before euthanasia or behavioural tests. For the chronic experiment, CRS mice were injected with paroxetine-HCl or vehicle for seven consecutive days and euthanized the next day, whereas Atg7^LHb−/− or Atg7^LHb+/+ mice with CRS were injected with paroxetine-HCl or vehicle for seven consecutive days before behavioural tests. For the ketamine experiment, CRS mice were injected with ketamine (Drug reference materials laboratory of the Third Research Institute of Ministry of Public Security, 10 mg kg⁻¹) or vehicle 1 or 24 h before euthanasia, and Atg7^LHb−/− mice were injected with ketamine or vehicle 24 h before behavioural tests. Rapamycin (MCE, 10 mg kg⁻¹) was dissolved in 10% DMSO, 40% PEG300, 5% Tween-80 and 45% saline. For behavioural tests and western blot, rapamycin or vehicle were injected for three consecutive days, and an extra 1 h before euthanasia or behavioural test.

Behavioural assays

All behavioural tests were conducted in a sound attenuation room to minimize external auditory stimuli.

Restraint stress

Mice were placed in 50-ml conical tubes that were drilled with several small holes (2 mm in diameter) for ventilation and were subjected to ARS (2 h) or CRS (2 h of restraint stress for 14 consecutive days). During fibre photometry, each mouse was gently grasped by one of the experimenter’s hands that was wearing a cotton glove for 10 s, and the tail was immobilized with the other hand²⁹.

Social defeat stress

As previously described^29,36, CD-1 aggressors were screened for aggressive behaviours towards intruders. Mice were placed in the home cage of a CD-1 aggressor and attacked for 5–10 min and then housed with the aggressor for 24 h, separated by a perforated transparent partition in the middle of the cage. Chronic social defeat stress lasted for ten consecutive days and mice were exposed to a new aggressor each day. For fibre photometry recording, tested mice were recorded during the 5 min of social defeat stress.

Footshock stress

As previously described^29,30, mice were placed in a shock box and allowed to freely explore the box for 5 min. Electrical footshocks (0.8 mA, 1–3 s duration) were then delivered at random intervals (1–15 s). Chronic footshock stress consisted of 360 footshocks within 60 min and was given for two consecutive days. For acute footshock stress, mice were subjected to 15 random footshocks within 3 min. For fibre photometry, mice were recorded during the footshock stress.

FST

Mice were gently and individually placed in a cylinder (12 cm in diameter and 25 cm in height) filled with water (23–25 °C) and allowed to swim for 6 min under normal illuminating conditions. The depth of water was adjusted to prevent the mice from touching the bottom of the cylinder with their hind limbs or tails. The behaviour of the mice was videotaped from the side. The immobile duration within the last 4 min was analysed by an experimenter blinded to the animal treatments. Immobility was defined as a state in which the mice remained floating with only necessary movements to keep balance in the water.

SPT

Mice were single housed followed by 2 days of adaptation with two bottles of water, after which mice were water deprived for 24 h before the test. During the test, mice were given one bottle of water and one bottle of 1% sucrose solution. Acute SPT was measured for the first 3 h in the active phase of mice, with the positions of the two bottles switched every 0.5 h to eliminate any baseline place preference. The consumption of water and sucrose was recorded by an experimenter blinded to the animal treatments. Sucrose preference was defined by the average percentage of sucrose consumption relative to total fluid consumption in 3 h. For chronic SPT, the sucrose consumption percentage was continuously monitored for the next 2 days, while the bottle positions were switched at the light phase and consumption was measured every 12 h to ensure balanced measurements across different positions and phases.

OFT

OFT was conducted before all other behavioural tests. The mice were gently placed in the centre of a square arena (40 × 40 × 40.5 cm), with the illumination above the centre brighter than the peripheral area. A video camera positioned directly above the area was used to track the movement of each mouse by means of Any-maze software (Stoelting).

SIT

An OFT box containing a wire-mesh cage was used on one side. Video tracking software (Any-maze, Stoelting) was used to measure the amount of time the experimental mouse spent in the social interaction zone (15 × 15 cm) surrounding the wire-mesh cage within the arena. The open field arena and wire-mesh enclosures were thoroughly cleaned between each experimental mouse. Mice were individually placed in the centre of the OFT box and allowed to explore freely for 2.5 min (without social target present, known as the ‘no target’ phase). The experimental mouse was then removed, an unfamiliar c57 mouse was placed under the mesh cage (known as the ‘target’ phase). The experimental mouse was then returned to the OFT box and allowed to explore for another 2.5 min, during which time spent interacting with the social target was measured. The social interaction (SI) ratio was calculated as: SI ratio = ((time in SI zone in target phase)/(time in SI zone in no target phase)) × 100.

Fibre photometry

Following 3–5 weeks of viral expression, GCaMP6s fluorescence was detected using a fibre photometry system (Inper Ltd). The Ca²⁺-dependent signal was detected by a 470-nm LED (20 μW) whereas the Ca²⁺-independent isosbestic signal was detected by a 410-nm LED (20 μW). A video camera (PHILIPS, p506) was installed above the centre of the arena to record the behaviours of tested mice. We applied a screen recorder to synchronize behavioural events and fluorescence signals (EV Capture). All mice expressing GCaMP6s were allowed to accommodate to the new environment for at least 1 hr before the recording sessions. When mice were quiet but stayed alert, a 10-min baseline was recorded. After that, each mouse was exposed to acute stressors and their neuronal Ca²⁺ responses were recorded.

Fibre photometry data analysis

To calculate the change ratios of the fluorescence, raw data were analysed by using a processing software (Inper Ltd) and MATLAB code (Thinker Tech Nanjing Biotech). We segmented the photometry data based on specific behavioural events within individual trials. Movement artefacts were corrected by recording the fluorescent signal that is stimulated with a 410-nm LED. This signal elicited by the 410-nm LED was linearly scaled using least-squares regression to minimize the difference between signals elicited by the 410- and 470-nm LEDs. We then subtracted the scaled 410-nm signal from the 470-nm signal to exclude the movement artefacts and obtain bleaching-corrected signals. We defined the change ratios of fluorescence (ΔF/F₀) by calculating (F − F₀)/F₀, in which F₀ was the averaged value of baseline fluorescence signals recorded before acute stress. ΔF/F ratio was presented with heat maps or as average plots with a shaded area indicating the standard error of the mean. We also calculated the area under the curve for each trial to compare the baseline response (−2–0 s) with the response to stress (0–5 s).

Slice preparation

The animals were anaesthetized with 1% pentobarbital NEMBUTAL (100 mg kg⁻¹) and then perfused with 20 ml of ice-cold cutting solution (oxygenated with 95% O₂ and 5% CO₂), a modified ACSF containing 210 mM sucrose, 125 mM NaCl, 2.5 mM KCl, 25 mM NaHCO₃, 1.25 mM NaH₂PO₄, 1 mM MgCl₂,1 mM CaCl₂, 25 mM glucose and 1 mM sodium pyruvate. After decapitation, the brains were dissected rapidly and transferred to ice-cold oxygenated cutting solution. LHb-containing coronal slices (300-μm thickness) were cut in ice-cold oxygenated cutting solution with a Leica VT1200S vibratome and then incubated in a recovery chamber containing normal incubation ASCF (125 mM NaCl, 2.5 mM KCl, 25 mM NaHCO₃, 1.25 mM NaH₂PO₄, 1 mM MgCl₂, 1 mM CaCl₂, 25 mM glucose) at 32 °C. Incubation ACSF was continuously bubbled with a gas mixture of 95% O₂ and 5% CO₂. Slices were allowed to recover for at least 1 h and then incubated at room temperature before electrophysiology recordings.

In vitro electrophysiology

Whole-cell patch-clamp recordings were performed on LHb neurons at 30 °C, which was monitored with an automatic temperature controller (TC-324C, Warner Instruments). Slices were continuously perfused with ACSF (same as the incubation solution) at 2–3 ml min⁻¹. The patch pipettes (5–6 MΩ) were pulled with a pipette puller (PC-100, Narishige) from borosilicate glass (Sutter Instrument). For all whole-cell patch recordings, the pipettes were filled with an internal solution containing 127 mM K-gluconate, 13 mM KCl, 4 mM Mg₃-ATP, 0.3 mM Na₃-GTP, 0.3 mM EGTA, 10 mM HEPES and 10 mM Na-phosphocreatine (pH 7.25). Recordings were performed with a MultiClamp 700B amplifier and pCLAMP v.10.6 software (Axon Instruments). Signals were amplified, and then filtered at 2 kHz and sampled at 10 kHz using a Digidata 1550B. The series resistance and capacitance were compensated automatically after a stable giga-seal. Cells were discarded if their series resistances changed more than 20%.

To record sEPSCs and sIPSCs, neurons were held at −50 mV in a voltage-clamp mode. To record spontaneous neuronal activity, neurons were held under current clamp (I = 0 pA). The excitability was measured by action potentials elicited by injecting a series of current pulses (500-ms duration, −100 to 150-pA intensity with an increment of 10 or 20 pA) under current clamp.

For plasticity experiments, repetitive control photostimulation (465-nm light laser, 5 mW) was applied at 0.1 Hz to induce evoked excitatory postsynaptic currents (EPSCs). LFS protocol (1 Hz, 15 min) was used for LTD induction, whereas the excitatory postsynaptic potentials (EPSPs)-burst dependent plasticity (EBDP) protocol (4–5 pulses per second, 40 Hz, 100 stimuli for 100 s) was performed to trigger LTP as previously described²⁹. For pharmacological experiments, drugs (SBI, 0.5 μM, Selleck; tBP, 0.1, 1 and 5 μM, Selleck; TAT–GluA2_3Y peptide, 10 μM, MCE; a designed tBP-scrambled (YGRKKRRQRRRGGVGNDFFINHETTGFATEW), 1 μM, MCE; TAT–GluA2_3Y-scrambled, 10 μM, MCE; dyngo-4a, 5 μM, MCE) were bath applied after 10 min of stable baseline recordings.

To measure the dosage effect of tBP (0.5, 1 and 5 μM) in LHb neurons from CRS mice, we designed an incubation experiment, in which tBP was bath perfused into the recording chamber and incubated for at least 10 min before recordings. The before and after effects of tBP on the same neuron were also measured to test the onset time of the drug effect.

Offline analysis of electrophysiological data was performed with Clampfit v.10.6 (Molecular Devices) and Mini Analysis Program (Synaptosoft).

The percentage of burst- and tonic-type spikes were obtained by the ratio of normalized tonic/bursting spike number to the total normalized spike number:

Statistical analysis and reproducibility

The required sample sizes were determined on the basis of our previous experience conducting similar experiments. For all experiments, mice were randomly grouped and investigators were blinded to group allocation during experimentation and data analysis. The offline data analysis was performed blindly with GraphPad Prism software v.8. Data points were excluded from analyses if the viral injection site or drug delivery site was out of the interested region. For all data, we first checked the normality and homogeneity of variance. The following statistical tests were used when applicable: two-sided paired or unpaired t-test, or two-sided unpaired t-test with Welch’s correction, one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test, uncorrected Fisher’s LSD test or Tukey’s multiple comparisons test, Pearson correlation test and two-way ANOVA. In the case of non-Gaussian distributed data, we used the two-sided Wilcoxon test, two-sided Mann–Whitney test, and the Kruskal–Wallis test with an uncorrected Dunn’s test. To compare the firing patterns and percentage of spikes of LHb neurons, we used the two-sided chi-square test and Fisher’s exact test. Data details are shown in the Source data. Data are presented as mean ± s.e.m. All statistical tests were two-sided and statistical significance was set to P < 0.05.

For Fig. 5i and Extended Data Fig. 11c, immunofluorescence data are representative of three independent experiments. All experiments were conducted using at least two cohorts of animals. The results were reproducible across cohorts and combined for the final analysis.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.