Male C57BL/6J mice (∼24 g) and CD-1 male mice 7–8 months of age (∼45 g) were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (China). All mice were kept at a temperature under 22 ± 2°C and on a 12 h light and 12 h dark cycle with food and water available ad libitum. All animal handling and experimental procedures followed the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of the Fourth Military Medical University.
Chronic social defeat stress (CSDS) model
CSDS was performed as previously described . First, CD-1 mice were screened for aggressive behavior during social interactions for three consecutive days before the start of the social defeat sessions. They were then housed in the social defeat cage 24 h before the start of defeats on one side of a clear perforated Plexiglas divider (26.7 cm width × 48.3 cm depth × 15.2 cm height, pore size 0.6 cm × 45.7 cm × 15.2 cm). Experimental C57BL/6J mice were subjected to physical interactions with a novel CD-1 mouse for 10 min once per day over 10 consecutive days. After the interactions, the experimental C57BL/6J mice were transferred to the opposite side of the social defeat cage and allowed sensory contact over the subsequent 24 h period. Unstressed control C57BL/6J and CD-1 mice were individually placed into the same cages and rotated daily in a similar manner without exposure to the CD-1 mice. After the last interaction, all experimental C57BL/6J and CD-1 mice were singly housed for 24 h before the behavioral testing.
Social interaction test (SIT)
First, C57BL/6J mice were habituated to the testing suite for 1 h before testing. Second, the mice were placed in a square open-field arena (50 cm × 50 cm × 50 cm) with a small plastic cage placed at the middle of one side of the square for 2.5 min, and the movements of the mice were monitored and recorded automatically by a Sony camera and SMART V3.0-Panlab Harvard Apparatus; these movements were used as baseline exploratory behavior and locomotion in the absence of a social target. At the end of 2.5 min, the mouse was removed and returned to its home cage until the next stage, and the arena was wiped with 75% alcohol to remove any smells. Third, the movements of the mice in the presence of a novel social target inside the small cage were monitored and recorded for 2.5 min. Fourth, the time spent in the interaction and overall locomotion were compared between the two recordings. The SI ratio was calculated by dividing the time spent in the interaction zone with the target CD-1 mouse present by the time spent in the interaction zone without the presence of the target CD-1 mouse.
Sucrose preference test (SPT)
The SPT reflects anhedonia, a core symptom of depression. On the first day, the water bottle on the home cage was replaced by two 50 mL tubes with sipper tops filled with water, and mice were allowed 24 h of acclimation to the tubes before the start of testing. On the second day, water in one of the two tubes was replaced with a 1% sucrose solution. The placement of the two tubes was changed every 8 h in case of position preference. On the third day, the food and the two tubes were removed for 24 h to induce thirst in the mice. On the fourth day, the food and both tubes filled with water and 1% sucrose solution were replaced. Both tubes were weighed, and mice were allowed to drink ad libitum for 12 h. During the tests, the placement of the two tubes was switched 2 times. At the end of the testing, sucrose preference was calculated by dividing the total amount of sucrose consumed by the total amount of fluid consumed over the 12 h of sucrose availability.
Tail suspension test (TST)
The depressive behavior of the mice was analyzed with the TST. Mice were suspended individually by adhesive tape from a tail suspension experimental shelf. The mice were isolated from each other. The tape was placed 1 cm from the tip of the tail. The mice were observed for a period of 3.5 min, and the activity of the mice was monitored and recorded automatically by a Sony camera and SMART V3.0-Panlab Harvard Apparatus. Their immobility time, which was defined as the time spent completely motionless, was recorded.
A microdialysis probe (4-mm guide cannula length, 0.22-mm membrane outer diameter, 1-mm membrane length, MW cutoff 50 kD; Eicom Corp) was stereotaxically inserted into the mPFC (15° angle, 1.75 mm anterior and 0.75 mm lateral from bregma, and 1.5 mm ventral to the dura) through the cannula guide. Artificial cerebrospinal fluid (ACSF) (124 mM NaCl; 4.4 mM KCl; 2 mM CaCl2; 2 mM MgSO4; 25 mM NaHCO3; 1 mM KH2PO4; and 10 mM glucose; pH 7.4) was perfused at a flow rate of 1 µl/min using a microinjection pump. After equilibrium for 1 h, the mouse brain interstitial fluid was continuously collected into microvials for 4 h, and these interstitial fluid samples were subsequently lyophilized and redissolved in 20 µl of ACSF.
One sample (100 mg) was homogenized in 300 µl of water. Cold steel balls were placed into the mixture, which was then incubated on ice for 10 min. The steel ball was removed, 500 µl of pure methanol was added, and the mixture was vortexed at 2500 rpm for 5 min. Next, the mixture was centrifuged at 12,000 rpm at 4℃ for 10 min, and 600 µl of the supernatant was transferred to another centrifuge tube. Then, 100 µl of 5% methanol (95% water) was added to the dried product, mixed and centrifuged at 12000 rpm at 4°C for 10 min. The supernatant was collected for LC-MS/MS analysis. The analytical conditions were as follows: UPLC: column, Waters ACQUITY UPLC HSS T3 C18 (1.8 µm, 2.1 mm*100 mm); column temperature, 35℃; flow rate, 0.3 mL/min; injection volume, 1 µL; solvent system, water (0.01% methanolic acid): acetonitrile; gradient program for positive mode, 95:5 V/V at 0 min, 79:21 V/V at 3.0 min, 50:50 V/V at 5.0 min, 30:70 V/V at 9.0 min, 5:95 V/V at 10.0 min, and 95:5 V/V at 14.0 min; gradient program for negative mode, 95:5 V/V at 0 min, 79:21 V/V at 3.0 min, 50:50 V/V at 5.0 min, 30:70 V/V at 9.0 min, 5:95 V/V at 10.0 min, and 95:5 V/V at 14.0 min. The original data file obtained by LC-MS analysis was converted into mzML format by ProteoWizard software. Peak extraction, alignment and retention time correction were performed by the XCMS program. The “SVR” method was used to correct the peak area. The peaks were filtered with a deletion rate > 50% in each group of samples. Next, metabolic identification information was obtained by searching the laboratory’s custom-built database and integrating the public database and metDNA. Finally, statistical analysis was carried out with the R program.
The mPFC samples were collected and lysed in RIPA buffer with protease and phosphatase inhibitors (Roche). Protein levels were assessed with a Bradford assay with BSA as the standard. Approximately 10 µg of denatured proteins was separated by 10% SDS-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene difluoride (PVDF) membranes (Roche). Nonspecific binding was blocked with TBST (TBS-1% Tween20) with 5% (w/v) nonfat milk for 2 h at room temperature. PVDF membranes were then incubated overnight at 4°C in TBST with the following primary antibodies: rabbit anti-NR2A (1/500, ab106590, Abcam), rabbit anti-NR2B (1/500, ab28373, Abcam), rabbit anti-CSAD (1/1000, ab91016, Abcam), rabbit anti-CDO1 (1/1000, 12589-1-AP, Proteintech), rabbit anti-Syntaxin 1A (1:1000, #18572, Cell Signaling Technology), anti-PSD95 (1:1000, ab18258, Abcam), and mouse anti-β-actin (1:5000, 60008-1-ig, Proteintech). Membranes were then incubated at room temperature for 2 h in TBST with secondary antibodies (1/5000, Invitrogen). Protein bands were detected by chemiluminescence (Tanon, Shanghai, China) and quantified by densitometry with ImageJ (ImageJ 7.0 software). Protein levels were normalized to the level of β-actin as a control.
Mice were anesthetized with 5% pentobarbital and transcardially perfused with 20 ml of ice-cold PBS followed by 40 ml of 4% paraformaldehyde (PFA). Brain samples were postfixed in 4% PFA for 2 h, followed by an additional 48 h of dehydration in 30% sucrose at 4°C. Then, the brain samples were sectioned at 18 µm using a freezing microtome (CM-1950, Leica) at -20°C. Sections were washed with 0.01 mM PBS (pH 7.4) and blocked with 3% bovine serum albumin (3% BSA and 0.3% Triton-X in PBS) for 1 h at room temperature, followed by an overnight 4°C incubation with the following primary antibodies: rabbit anti-taurine (1/50, AB5022, Sigma-Aldrich) and rabbit anti-NR2A (1/500, ab106590, Abcam). After rinsing with PBS, the sections were incubated with the corresponding secondary antibodies conjugated with fluorochromes for 2 h in PBS with Alexa 594-AffiniPure donkey anti-rabbit IgG antibody (1/1000, Jackson). Next, the sections were incubated with Hoechst (1:1000) for another 10 min and then washed with 0.01 mM PBS. Finally, the sections were mounted and cover slipped with Fluoromount-G and stored at -20°C. A confocal microscope (Olympus, Japan) and FLUOVIEW software (ver. 1.7a, Olympus, Japan) were used for image acquisition.
The mouse brains were rinsed in bidistilled water and immersed in impregnation solution made by mixing equal volumes of commercial solutions (potassium dichromate, mercuric chloride, and potassium chromate) and stored for 1 week in darkness at room temperature. The blocks were then transferred into PBS. Subsequently, coronal sections were cut at a thickness of 150 µm, still in darkness, using a vibratome, and mounted on gelatinized slides in PBS. The slides were rinsed in bidistilled water, stained in the staining solution, dehydrated in successive baths of ethanol, cleared in xylene, and cover slipped with Permount TM Mounting Medium. A confocal microscope (Olympus, Japan) and FLUOVIEW software (ver. 1.7a, Olympus, Japan) were used for image acquisition. Images were acquired at a resolution of 2048 pixels in the X–Y–Z dimension. Z dimensions were variable. For the analysis of dendritic branches and spines, neurons were imaged using a 20× objective lens (numerical aperture = 0.75). The Z-dimensional increment was 2 µm. Neurons selected for analysis were randomly picked from at least 10 brain slices of three control, three CSDS, and three taurine-treated CSDS mice. The stack images were analyzed using Imaris software (version 7.7.1, serial number: 32mr-rfhf-7hbu-jb58, Bitplane, Switzerland). The total length of the dendrites and the volume of the cell body were automatically calculated. For Sholl analysis, spheres were constructed continuously from the center of the cell body with an increase in the radius of 50 mm. The number of intersections between each sphere and the dendrites was calculated for comparison. Compared with the use of 2D images, the use of 3D images for Sholl analysis can provide statistical results that are closer to the actual structure of the neurons, especially when analyzing dendrites with different angles. Spine shape was defined by the length of the spine and the widths of the spine neck and spine head, which allowed us to classify the spines into four types: stubby, mushroom, long thin, and filopodia. The stubby type had a length < 1 mm; the mushroom type had a length > 3 mm, and the maximum width of the head/the mean of the neck was > 2; the long thin type had a ratio of the mean width of the head/mean width of the neck of ≥ 1; and the rest of the spines were filopodia. Spine measurements were performed using a MATLAB-X Tension Spines Classifier in Imaris. All imaging data were analyzed by an investigator who was blinded to the experimental groups.
Mice were randomly assigned to the control, CSDS, and taurine-treated CSDS mouse groups. Analyses were performed in a manner blinded to treatment assignments in all experiments. Statistical analyses were performed using GraphPad Prism software v 7.0, and all data are presented as the mean ± SEM. Statistical significance was evaluated using Student’s t-test analysis or one-way ANOVA followed by the Tukey-Kramer post hoc test. P < 0.05 was used to determine significance where indicated.