Animals
Male C57BL/6J (6 weeks old) mice were purchased from the Experimental Animal Center of Air Force Medical University (Xi`an, China). They were housed in plastic cages under controlled laboratory conditions (temperature: 21–24℃; 12-h light/dark cycle: lights on 9 a.m.-9 p.m.) with food and water supplied ad libitum. After one-week acclimatization, mice were randomly divided into four groups with nine mice each, namely Group A (control group; plain water for 3-week), group B (dysbacteriosis group; water with ASC antibiotics for 3-week), Group C (saline group; water with ASC antibiotics for 3-week and then plain water for 2-week), Group D (probiotics group; water with ASC antibiotics for 3-week and then probiotics for 2-week). The ASC antimicrobial cocktail is a mixture of Ampicillin (Macklin, Shanghai, China), Streptomycin (Macklin, Shanghai, China), Clindamycin (Macklin, Shanghai, China), and sterile drinking water at a final concentration of 1 g/L as previously described(4, 11). While in Group C and D, there was a reverse course with ASC antibiotic mixture changed to plain water or probiotics (Live Combined Bifidobacterium and Lactobacillus Tablets, Inner Mongolia Shuang Qi Pharmaceutical Co., Ltd. PRC, Inner Mongolia, China) for another 2 weeks. The mice in each group were then subjected to gut microbiota analysis, a series of behavioral tests including anxiety, depression, pain response test and recognitive memory test and immunostaining for Fos protein.
To minimize environment stress, all mice in each group (N = 6 mice) were permitted to adapt to the environment of behavioral test room (12 h light/dark cycle, 22℃, 50% relative air humidity) 3 days before testing. Mice were subjected to a series of behavioral tests during the following weeks as shown in Fig. 1.
Anxiety-like behavior
Elevated plus maze (EPM). The following day, the mice were subjected to EPM test system consisted of two open arms (30 cm × 5 cm) and two closed arms (30 cm × 5 cm × 15 cm) as described in previous studies (29). They were individually placed in the central square platform (5 cm × 5 cm) 70 cm above the floor with the nose toward a closed arm and allowed to explore for 5 min. The time spent and total distance traveled were recorded using a motion tracking system and calculated by the analyzing system (Shanghai Mobile Datum Information Technology, Shanghai, China).
Open field test (OFT). The mice were placed in the OFT system, which was comprised of 8 square chambers (50 cm × 50 cm × 45 cm) (6). Their horizontal movement was detected by a motion tracking system and the central distance and total traveling distance were analyzed by the analysis software (Shanghai Mobile Datum Information Technology, Shanghai, China).
Depression-like behavior
Tail suspension test (TST). Mice were suspended 50 cm above the floor using adhesive tape placed approximately 1 cm from the tip of the tail for 5 min. The duration of immobility was monitored and recorded in seconds by a time recorder. Immobility of the mice was defined as the absence of escape-oriented movement and completely motionless while suspended.
Splash Test. This test was based on grooming behavior, which was performed by vaporization of the 10% of sucrose solution on the dorsal coat of mice as described in previous study(14). The latency and frequency of the grooming behavior was scored during 5 min and then analyzed.
Recognitive memory
Morris water maze test (MWMT). Learning and memory performance was evaluated in a circular tank (diameter, 120 cm) filled with white opaque water at approximately 21℃. A fixed platform (diameter, 10 cm) remained constant and submerged 1 cm below the water surface in a target quadrant. Reference cues of different colors and shapes were placed along the walls surrounding the tank. During the hidden platform testing, mice were gently placed into the tank and the entry point of four quadrants were randomly changed. Mice were gently guided to the hidden platform if they failed to find it within 60 s, and they were kept on the platform for 15 s. During training trails, the mouse behavior was recorded using a motion tracking system and the escape latency was calculated by the analyzing system (Shanghai Mobile Datum Information Technology, Shanghai, China) per testing day. On day 6, the hidden platform was removed and a probe test was performed. The swimming velocity and the number of crossing the area where the hidden platform used to be was recorded.
Pain behavior
Paw withdrawal thresholds test. The mice were kept in Lucite cubicles over a wire mesh and acclimated for 30 min as described in previous studies(26, 30). A series of Von Frey filaments (0.008, 0.02, 0.04, 0.16, 0.4, 0.6, 1, 1.4, 2 g) with various bending forces (according to 0.078, 0.196, 0.392, 1.568, 3.92, 5.88, 9.8, 13.72, 19.6 mN) were applied to the plantar surface of the hindpaw until the mice withdrew from the stimulus. The lowest force at which a withdrawal response was obtained was considered as the pas withdrawal threshold.
Spontaneous pain
Visceral pain were defined by mouse postures including licking of the abdomen without other grooming behavior, flattening the abdomen against the floor, abdominal retractions, and whole-body stretching as described in the previous study(6).
16S rRNA qPCR
The time points of fecal samples collected of the four groups were shown in Fig. 1. DNA was extracted from stool samples that snap-frozened on dry ice using the TIANamp Stool DNA kit (Tiangen Biotechnology company, Beijing, China) according to the manufacturer`s instructions. The abundance of specific bacterial groups was measured by qPCR using genus-specific 16S rRNA gene primers (Table 1) and the SuperReal PreMix Plus SYBR Green kit (Tiangen Biotechnology company, Beijing, China). qPCR was performed in a real-time PCR detection system (Bio-Rad, Hercules, CA). Bacterial DNA was quantified using standard curves constructed with reference bacteria specific for each bacterial group analyzed.
Fos mapping
Histology. Mice of the three groups were deeply anesthetized and then transcardially perfused with 0.9% saline (50 ml), followed by 4% paraformaldehyde (PFA, 100 ml, PH 7.4). Brains were extracted immediately, post-fixed in 4% PFA for 4 h, and cryoprotected by 0.1 M PB containing 4% (w/v) sucrose at 4℃ until cutting. 30 µm serial coronal sections were cut using a freezing microtome (Kryostat 1720, Leitz, Mannheim, Germany). Subsequently, the sections were washed with 0.01 M phosphate buffer solution (PBS, PH 7.4) and rinsed three times for 10 min. The sections were pre-incubated in blocking solution (PBS containing 1% bovine serum albumin, 0.3% Triton X-100) for 1 h, and then incubated with primary anti-c-Fos antiserum (1:400, Abcam, Cambridge, MA, USA) overnight. Next, the sections were rinsed and then incubated in an Alexa Fluor (AF) 488-conjugated Rabbit anti-mouse secondary antibody solutions (1:400) for 4 h at room temperature. The slices were then washed extensively with PBS, incubated with DAPI (D9542, Sigma, USA) at 1:1000 in PBS, and then mounted on microscope slides and cover-slipped. Images were acquired by using a virtual slide microscope (VS120, Olympus, Japan). The images were analyzed by using software CellSens Dimension (Olympus, Japan).
Statistical Analysis
All data were collected by experimenters who were blind to the conditions of the experimental animals. Statistical analysis was performed using GraphPad Prism 8 (version 8.0.2). Statistical significance was assessed by one-way ANOVA (Tukey`s multiple comparisons test) and two-way ANOVA (Sidak`s multiple comparisons test). Analyzed numbers (n) for each experiment are illustrated in the main text sections of corresponding figure legends. Data were presented as the Mean ± S.E.M. A threshold for statistical significance of P < 0.05 was chosen in this study.