Subjects

Six to eight week-old male C57BL/6J mice were purchased from the Animal Center of Jilin University. Drd2-cre mice were kind gifts from Prof. Dongmin Yin (Key Laboratory of Brain Functional Genomics, Ministry of Education and Shanghai, School of Life Science, East China Normal University, Shanghai, China). Specific information on Drd2-cre mice is available from the Mutant Mouse Regional Resource Center https://www.mmrrc.org/ (MMRRC # 017263-UCD). Drd2-cre mice and wild-type mice used in our studies were 6–8 week-old male mice obtained by crossing Drd2-cre transgenic mice with wild-type (C57BL/6J) mice. The mice were housed in 25.5 × 15 × 14 cm cages for 3 days to ensure they were acclimated to the laboratory environment before the study, with a temperature of 23 ± 1 °C, humidity of 40–50%, 12-hour light and dark cycles (7:00–19:00 light cycle), and ad libitum food and water. The mice were randomly assigned to each group. All procedures were conducted according to the standards of the Laboratory Animal Guideline for Ethical Review of Animal Welfare (GB/T 35892-2018) and under protocols approved by the Institutional Animal Care and Use Committee of Jilin University.

Experimental design

To investigate the effects of 5-d UMS on depression-like behavior and its related mechanisms, mice were divided into two groups: control group and 5-d UMS group. To investigate the effects of Rasd2 overexpression (or knock-down) on depression-like behavior and its related mechanisms, mice were divided into ov-control group and ov-Rasd2 group (or sh-control group and sh-Rasd2 group), and 5-d UMS were performed 15 days after virus administration. To explore the effects of PrL-NAc core (NAcc) circuit on depression-like behavior and its related mechanism, mice were first divided into control group, hM3Dq group and hM4Di group according to different viral administrations. After 15 days of viral administration, each mouse was exposed to 5-d UMS. Then saline or CNO was administered 30 min before the beginning of behavioral experiments and the above three groups were divided into saline + control, saline + hM3Dq, saline + hM4Di, CNO + control, CNO + hM3Dq and CNO + hM4Di groups. To investigate the effects of Rasd2 overexpression in DRD2PrL-NAcc neurons on depression-like behavior, mice were divided into two groups according to genotype: WT group and Drd2-cre group. Each group was injected with the same virus.

Surgery

Mice were anesthetized with pentobarbital sodium (65 mg/kg, i.p., Dingguo Changsheng Biotechnology, Beijing, China) and placed in a stereotaxic frame (RWD Life Science, Shenzhen, China). Based on the Paxinos and Franklin mouse brain atlas [21], the NAcc (AP: +1.60; ML: ±1.50; DV: −4.40; mm relative to bregma [22, 23]) and mPFC PrL (AP: +2.10; ML: ±0.35; DV: −2.30; mm relative to bregma [24, 25]) were targeted in separate groups of mice and Fluorogold (or virus) was bilaterally injected into each location at a rate of 0.2 μL/min. After each injection, the needle was left in place for an additional 5 min and then slowly withdrawn. Details of the procedure can be found in a published reference [26].

5-d UMS

Mice received 2–3 of the following stressors each day for 5 consecutive days on a semi-randomized schedule: foot shock for 1 h (0.3 mA, 18 seconds duration at 8-second intervals), restraint for 2 h, exposure to continuous noise (approximately 85 dB [27]) for 8 h, food deprivation for 16 h, water deprivation for 16 h, cage tilt (45 degrees) for 16 h, bedding replaced with damp bedding (1 cm of sawdust and 100 ml of water) for 16 h, bedding (sawdust) removed for 16 h, placed in the cage with 1 cm water for 6 h. The same stressor was not applied on consecutive days.

DREADDs

Mice were anesthetized with pentobarbital sodium (65 mg/kg, i.p., Dingguo Changsheng Biotechnology) and the virus (0.3 μL; CaMKIIa-Cre virus: pAAV2/Retro-CaMKIIa-EGFP-P2A-Cre-WPRE, 3.28E + 12 V.G./mL, Obio Technology) was bilaterally injected into the NAcc and then another virus was bilaterally injected into the PrL region of the mPFC (0.3 μL; control: pAAV2/8-hSyn-DIO-mCherry, 1.34E + 13 V.G./mL; hM4Di: pAAV2/8-hSyn-DIO-hM4D(Gi)-mCherry, 4.43E + 13 V.G./mL; hM3Dq: pAAV2/8-hSyn-DIO-hM3D(Gq)-mCherry, 5.13E + 13 V.G./mL, Obio Technology). 5-d UMS was performed 15 days after the virus injection. Behavioral experiments were performed 30 min after clozapine-N-oxide (CNO) administration (5.0 mg/kg, i.p. [28,29,30], c0832, Sigma-Aldrich, MI, USA, dissolved in saline at a concentration of 0.5 mg/mL [31]).

Behavioral experimentsOpen field test

The circular acrylic apparatus (height: 16 cm) was divided by three concentric circles of diameters 12 cm, 29.4 cm, and 48 cm respectively. Each circle was divided into essentially equal-sized areas. The number of areas in the inner, middle, and outer circles were 1, 5, and 10, respectively. At the beginning of the test, mice were placed individually in the center of a circular acrylic apparatus. The open field test lasted 6 min and was digitally recorded (DCR-SX83E camera, Sony, Shanghai, China). Line crosses (the number of grid lines crossed by the mouse’s four paws), rearing (the number of times the mouse stood with its front paws off the ground) and the number of entries in the center square of the apparatus were determined by an observer blinded to the treatment conditions. The total distance moved in the open field test was analyzed by video tracking software (Noldus, Wageningen, the Netherlands, EthoVision XT 13). Details of the procedure can be found in published references [16, 32].

Forced swimming test

Mice were individually placed in a cylindrical container (height: 25 cm; diameter: 11 cm) containing 12 cm of water (temperature: 25 ± 1 °C). The test lasted 6 min and was digitally recorded by a camera (Sony, DCR-SX83E). The first two minutes were considered as acclimatization time. Behavior was only assessed during the last 4 min of the test. The time spent immobile (defined by lack of any movement except that necessary to keep the head out of the water), swimming (defined by swimming movements parallel to the sides of the container), and climbing (defined by climbing movements with the mouse oriented perpendicularly toward the sides of the container) [33], as well as defecation (number of fecal boli), were analyzed by an observer blinded to the treatment conditions. Details of the procedure can be found in published references [16, 32].

Sucrose preference test

Mice were adapted to consuming 1% sucrose solution for 2 days before the formal test (mice were given two bottles of 1% sucrose solution per test cage). Mice were deprived of water for 12 h, then two weighed bottles (one for 1% sucrose solution and one for water) were placed in each cage. The bottles were weighed at several time points, 07:00, 08:00, 09:00, 10:00, 13:00, and 19:00. The starting positions of the two bottles were initially randomized, exchanged after each weighing, and the sucrose solution and water consumption were measured at the noted time-points. Sucrose preference was calculated as sucrose solution consumption / (sucrose solution consumption + water consumption) × 100%. Details of the procedure can be found in published references [16, 34].

Tail suspension test

Tape was placed 2 cm from the tip of the mouse’s tail and the mouse was affixed to a horizontal metal bar in an inverted state with the head approximately 20 cm above a benchtop. The behavior of mice over 5 min was recorded by a camera (Sony, DCR-SX83E). Immobility time (the time the mouse stopped all struggling movements, in a vertical position, and immobile) was analyzed in the last 4 min of the test by an observer blinded to treatment conditions. Details of the procedure can be found in published references [16, 34, 35].

Enzyme-linked immunosorbent assay

Blood was collected by retro-orbital bleeding and placed at room temperature for 1 h, followed by centrifugation at 3000 rpm for 10 min. Then the serum was collected and stored at −80 °C until analysis. The serum concentration of corticosterone was quantified according to the recommended protocol (FY2061-A corticosterone kit, Feiya Biotechnology, Jiangsu, China). The optical density of the samples was recorded at 450 nm using a microtiter plate reader (Thermo Scientific, MA, USA, Varioskan Flash) for 15 min, and the concentration was determined from the regression line for a standard curve run at the same time.

Western blotting

Mice were decapitated after being anesthetized with pentobarbital sodium (65 mg/kg, i.p., Dingguo Changsheng Biotechnology). The whole brain was removed and placed on dry ice in a stainless-steel brain matrix (RWD Life Science, 68707), and the brain was cut into 1 mm thick coronal slices using a razor blade [36]. Bilateral NAc and mPFC were dissected according to the mouse brain atlas [21] and stored at −80 °C until further analysis. The tissue was homogenized in RIPA buffer (R0020, Solarbio, Beijing, China) with 1% phenylmethylsulphonyl fluoride solution, and the supernatant was obtained after centrifugation at 12,000 rpm for 20 min at 4 °C. After mixing with the loading buffer, the sample vials were placed in boiling water for 5 min. The sample was loaded onto 5% sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) gels and proteins separated by electrophoresis at 110 constant voltage (Bio-Rad, CA, USA PowerPac, Universal). To analyze target proteins, the gels were transferred to polyvinylidene difluoride membranes (IPVH00010, Millipore, MA, USA) at 110 constant voltage for 1 h and then followed by blocking with 5% skim-milk (dissolved in Tris-buffered saline (TBS)) for 2 h. The membranes were incubated in TBS containing primary antibody at 4 °C overnight: RASD2 (1:800, rabbit polyclonal antibody, ab67277, Abcam, Cambridge, UK); DRD1 (1:800, rabbit polyclonal antibody, DF7097, Affinity, OH, USA); DRD2 (1:1000, rabbit polyclonal antibody, ab130295, Abcam); DARPP-32 (PPP1R1B; 1:1000, rabbit monoclonal antibody, #2306, Cell Signaling Technology, MA, USA); GluA1 (GRIA1; 1:2000, rabbit polyclonal antibody, ab109450, Abcam); β-actin (ACTB; 1:2000, mouse monoclonal antibody, HC201, TransGen Biotech, Beijing, China); cAMP (1:2000, rabbit polyclonal antibody, ab26322, Abcam); PKA (PRKACA; 1:3000, rabbit polyclonal antibody, ab75991, Abcam); and GAPDH (1:3000, mouse monoclonal antibody, HC301, TransGen Biotech). After washing in TBST (TBS containing 0.1% Tween-20), the membranes were incubated in TBS containing secondary antibody for 1 h at room temperature (anti-rabbit: 1:2000, ZB2301, ZSBG-Bio, Beijing, China; or anti-mouse: 1:5000, ZB2305, ZSBG-Bio; as appropriate). After washing with TBST, bands were detected with enhanced chemiluminescence supersensitive developer solution (WBKLS0500, Millipore) and analyzed by Image J software, version 1.52.

Co-immunoprecipitation

In the co-immunoprecipitation procedure, the steps for brain tissue acquisition and protein extraction were the same as for western blotting. Co-immunoprecipitation was quantified using the recommended protocol (abs995, Absin, Shanghai, China). Firstly, 5 μL of Protein A and 5 μL of Protein G were added to 500 μL of sample and incubated for 60 min at 4 °C, followed by centrifugation at 12,000 × g for 1 minute at 4 °C, and removal of the supernatant. 1 μg of primary antibody (RASD2, rabbit polyclonal antibody, RHES-101AP, FabGennix, TX, USA; or Rabbit IgG, abs20035, Absin; as appropriate) was added to the supernatant and incubated overnight at 4 °C with gentle mixing. After washing with 500 μL wash buffer (1×), the samples were centrifuged at 12,000 × g for 1 min and the precipitate was retained. Forty μL of SDS sample buffer (1×) was added to the precipitate and then the samples were placed in a water bath for 5 min at 95–100 °C, followed by centrifugation at 14,000 × g for 1 minute. Finally, the samples were analyzed by western blotting (primary antibody: RASD2, 1:500, RHES-101AP, rabbit polyclonal antibody, FabGennix; DRD2, 1:500, sc-5303, mouse monoclonal antibody, Santa Cruz Bio, TX, USA).

Immunohistochemistry

On the 3rd day after the last 5-d UMS, 90 min after the last behavioral experiment, the whole brains of mice were removed for immunostaining. Mice were anesthetized by intraperitoneal injection with pentobarbital sodium (65 mg/kg, Dingguo Changsheng Biotechnology), and then perfused transcardially with saline followed by 4% paraformaldehyde at 4 °C. After perfusion, mouse brains were removed, post-fixed in 4% paraformaldehyde for 24 h at 4 °C, and then transferred to 30% sucrose solution in 0.1 M PBS until sinking. Next, the brain tissue was placed in a tinfoil box containing Tissue-Tek O.C.T. Compound (#4583, SAKURA, CA, USA), and stored at −80 °C for further analysis. Coronal sections (30 μm thickness) of brain tissue were taken using a cryostat (Leica, Heidelberg, Germany, CM1860). The brain slices were washed in PBS, transferred to PBS containing 0.6% hydrogen peroxide and incubated at room temperature for 15 min. After washing with PBS, the brain slices were transferred to a solution containing the primary antibody (c-Fos, 1:1000, mouse monoclonal antibody, sc-166940, Santa Cruz Bio; RASD2, 1:500, rabbit polyclonal antibody, ab67277, Abcam), 2% normal goat serum (abs933, Absin), 0.05% sodium azide and 0.3% Triton X-100 PBS, and incubated at 4 °C for 72 h. After washing with PBS, the slices were transferred to a solution containing secondary antibody (goat anti-rabbit (HRP conjugated), 1:400, ZB2010, ZSBG-Bio; or goat anti-mouse (HRP conjugated), 1:400, CW0102S, CWBIO, Beijing, China; as appropriate) and 0.3% Triton X-100 PBS, incubated at room temperature for 75 min, and then washed in PBS. The brain slices were incubated using the VECTASTAIN ABC kit (PK4002, Vector Laboratories, CA, USA) for 75 min and then washed with PBS, followed by processing with the DAB Substrate Kit (BL732A, Biosharp Life Sciences, Anhui, China), and incubated for 10 min. After washing with PBS, slices were placed on microscope slides and stained in 0.2% neutral red solution for 3 min. The reaction was terminated with ddH2O, and the slides were soaked for 15 s each in 70%, 95%, and 100% ethanol solutions, followed by xylene for 1 min. Slides were sealed with neutral gum. The staining was observed and captured by microscope (Olympus, Japan, IX73/BX51WI). Counting of c-Fos nuclei was conducted by individuals blinded to experimental conditions. Each count is the average of triplicate measures. Details of the procedure can be found in a published reference [26].

Immunofluorescence

The methods of obtaining and fixing brain tissue followed procedures described for immunohistochemistry. Brain tissues were coronally sectioned on a cryostat (Leica, CM1860) at a thickness of 15 μm, attached to slides, dried at room temperature, and stored at −20 °C for later processing. After washing with PBS, the slices were placed in citrate buffer (1×) (CW0128S, CWBIO) and microwaved to retrieve antigens. Slices were transferred to a solution containing 10% normal goat serum in 0.3% Triton X-100 PBS and incubated at room temperature for 2 h. Brain slices were transferred to solutions containing a primary antibody in 0.15% Triton X-100 PBS and 1.5% normal goat serum, and incubated at 4 °C overnight. The primary antibodies used were: c-Fos (1:50, mouse monoclonal antibody, sc-166940, Santa Cruz Bio; 1:500, Rabbit monoclonal, ab214672, Abcam), Fluorescent Gold (1:1000, rabbit polyclonal antibody, AB153-I, Millipore Sigma, PA, USA), RASD2 (1:200, rabbit polyclonal antibody, ab67277, Abcam), DRD2 (1:50, mouse monoclonal antibody, sc-5303, Santa Cruz Bio). After incubation in primary antibodies, slices were washed with PBS and incubated with the secondary antibody in 0.15% Triton X-100 PBS for 1 h at room temperature. The secondary antibodies used were: FITC Goat Anti-Rabbit (1:1000, RS0004, ImmunoWay Biotechnology), Alexa Fluor® 594 Goat Anti-Mouse (1:200, ab150116, Abcam), Alexa Fluor® 488 Goat Anti-Mouse (1:2000, ab150113, Abcam), Cy3 Goat Anti-Rabbit (1:100, AS007, ABclonal Technology, Wuhan, China). Following DAPI staining for 5 min, the slices were washed with PBS and sealed with 30% glycerol. The staining was observed and captured by microscope (IX73/BX51WI, Olympus). The positive cells with fluorescence were counted by Image J software, version 1.52. Each count is the average of triplicate measures.

Statistical analysis

No statistical methods were used to predetermine sample sizes in mouse studies, but our sample sizes are similar to those reported in previous publications [30, 37, 38]. Data were represented as mean ± s.e.m. and were analyzed using GraphPad Prism software (version 8.0.1) and SPSS (version 23). For all two group experiments, two-tailed unpaired t-tests were used. For more than 2 group experiments, two-way and three-way ANOVA were used to compare the effects of factorial designs. When a significant difference was obtained in ANOVA, post hoc comparisons were performed between means using Tukey’s HSD test. p < 0.05 was considered statistically significant. The Shapiro–Wilk test was used to evaluate the normality of the data. All statistical tests were two-sided.