All mice used in this study were housed at room temperature, in a 12 h dark/light cycle (8:00 a.m. - 8:00 p.m.). Mice had free access to standard rodent chow and water. In order to avoid differenced arising due to age and gender, only 2-4 month old male mice were used in this study. Mice in same group were housed together, with 3-4 mice in each cage. Cx3cr1-GFP mice were used as heterozygous mice. Cx3cr1creER and iDTR mice 31 were purchased from Jackson Laboratory. Cx3cr1-GFP mice were a kind gift from Dr. Junwei Hao of Tianjin Medical University.
All animal experiments were approved by the Institutional Animal Care and Use Committee at the Beijing Institute of Basic Medical Sciences (Beijing, China).
Sertraline (Cat. S6319, Sigma-Aldrich, Germany) was administered by intragastric gavage (i.g.) at a concentration of 15 mg/kg. Tamoxifen (TAM; Cat. S1238, Selleck, USA) was dissolved in sunflower beads oil containing 5% ethanol. 10 mg tamoxifen administered once a day for three consecutive days.. For microglia depletion, 1 ug diphtheria toxin (DT; Cat. D0564, Sigma-Aldrich, Germany) injected intraperitoneally 3 weeks after TAM administration, once a day for three consecutive days. PLX3397 (Cat. S7818, Selleck, USA) was added to the diet at a concentration of 290 mg/kg and fed to the mice 21 days before delivering foot-shocks. Minocycline (Cat. S4226, Selleck, USA) was administered intragastrically at 40 mg/kg/day, 3 days before delivering foot-shocks. Sertraline, PLX3397, Minocycline were continually administered until all the behavior tests were completed.
All behavior tests were started at 10:00 a.m. and finished before 5:00 p.m. Each group contained eight or more mice and their littermates were used as control groups.
Open field test
Open field test was conducted inside a clear box (50 cm x 50 cm x 20 cm). Activity was automatically monitored by ANY-maze software (Global Biotech, USA). The apparatus was washed with a 75% ethanol solution before each mouse was introduced. Each mouse was recorded for 5 mins and total distance, average speed, time speed and distance travelled in center area (25 cm x 25 cm) were the parameters that were analyzed.
Elevated plus maze (EPM) test
The maze consisted of two open arms (35 cm × 5 cm) and two enclosed arms (35 cm × 5 cm × 15 cm) connected to a common central platform (5 cm × 5 cm). The apparatus was raised to a height of 50 cm from the floor and was lit by a dim light placed above the central platform. The maze was washed with a 75 % ethanol solution before each mouse was introduced. Time spent and distance traveled in open arms versus close arms was measured for a period of 5 min.
Electric foot-shock procedures
The procedure for electric foot-shock was adapted from Zhang et al. 32 and Qiu et al. 33. Electric foot-shocks were carried out in a fear-conditioning chamber (35 cm × 20 cm × 20 cm) (Jiliang Tech, China). After a 5 min adaptation period, 15 intermittent, inescapable foot-shocks were delivered to the mice (intensity: 0.8 mA; interval: 10 s; duration: 10 s). The control group mice were placed in the same chambers without stimulation for a total of 10 min to adapt to the same circumstance. Repeat stimulation on the second day enhances fear memory and increases the chances of developing PTSD. A 75% ethanol solution was used to wipe the chamber before each mouse was introduced, to avoid any effects of feces and odor.
Contextual freezing measurement
In the fear contextual test, mice who have previously experienced foot-shocks will freeze intermittently when exposed to the chamber where the foot-shocks were delivered. This freezing behavior is associated with context-induced fear memory 34. All mice were tested three times at different time points, with each test lasting for 5 min. The total cumulative freezing time and percentage of time spent frozen were recorded and analyzed by DigBehv software (Jiliang Tech, China).
Immunohistochemistry And immunofluorescence
Immunohistochemistry was performed as previously described 8. In brief, mouse brains were fixed with 4% paraformaldehyde after perfusion with saline. Fixed brains were dehydrated in 30% sucrose (in PBS). 20 mm thick coronal sections were cut throughout the whole brain and the sections were washed with PBS and incubated with 3% hydrogen peroxide (H2O2) for 15 min to inhibit endogenous peroxidases. After three washes with PBS, the sections were blocked for 1.5 hours with blocking buffer (0.3% Triton X-100 + 10% goat serum in PBS) at room temperature, followed by incubating with rabbit monoclonal anti-IBA1 (1:600; Cat. 019-19741, WAKO, Japan) and then visualized with biotinylated goat anti-rabbit IgG (1:200, Vectastain ABC kit, Vector Laboratories, USA), followed by streptavidin-conjugated horseradish peroxidase (Vectastain ABC kit, Vector Laboratories, USA) staining. The primary and secondary antibodies were diluted in blocking buffer. For immunohistochemistry, positive immunostaining was visualized with 3,30-diaminobenzidine (DAB kit, Zhongshanjinqiao, China). Stained sections were mounted onto slides and imaged using Nanozoomer (Hamamatsu, Japan). For immunofluorescence, Alexa Fluor 488-conjugated secondary antibody (1:400, Invitrogen, USA) was used. Nuclear morphology was visualized using Hoechst 33258 (Sigma, USA). Cx3cr1-GFP mice were stained only with the Hoechst. Immunofluorescence was imaged using a Nikon A1 confocal microscope (Nikon, USA).
Images were captured using Nanozoomer (Hamamatsu, Japan) and Nikon A1 confocal microscope (Nikon, USA). The number of microglia and their soma area were automatically measured at 20x magnification for a defined area by Image Pro Plus (Media Cybernetics, Inc)(export image--count and measure objects—select color—count—view measurement data). For precision, we counted 2-3 fields of the targeted brain area for each slice, 5 consecutive slices from each mouse were analyzed and the average microglial density was calculated.
The skeleton analysis was done using ImageJ software (National Institutes of Health, USA). The images prepared for skeleton analysis were captured as a Z-series stack (20 mm) using Nikon A1 confocal microscope. Z-stack images were condensed into a maximum intensity projection image and converted to 8-bit using an ImageJ plugin and then skeletonized using the Skeletonize (2D/3D) plugin. Microglial process number and length were analyzed using the AnalyzeSkeleton plugin. The resulting parameters were used as measures of microglia morphology.
Quantitative real-time PCR
The experiment was performed as previously described 7. Briefly, total RNA was extracted from mouse brain tissue using TRIzol (Thermo Fisher, USA). Reverse transcription was performed using random primers. Quantitative PCR was performed using UltraSYBR supermix with ROX (CWBIO, China) and detected by ABI QuantStudio 3(Thermo Fisher, USA) apparatus. The housekeeping gene ACTB was used as an endogenous control. Gene expression levels were expressed as 2-DCt. Primer sequences for QPCR are listed in Supplementary Table 2.
Tissue harvesting and single cell dissociation
After the first contextual fear response test (day 3), mice were sacrificed, their brains perfused with saline and the brain parenchyma harvested. Scissors were used to cut the brain parenchyma into small pieces, which were then digested for 30 min at 37° C in digestion buffer (PBS containing 2% FBS and 2 mg/ml collagenase Ⅳ). The samples were homogenized with a syringe and filtered through a 70 mm cell strainer. After centrifugation at 600 g for 6 min, the acquired pellet was resuspended in 37% Percoll (GE Healthcare, USA) in PBS. This suspension was subjected to gradient centrifugation, with the gradients spanning 70% Percoll in PBS, 30% Percoll in PBS and only PBS (2000 g for 30 min at 4° C). The immunocytes were collected at the 37-70% interphase and washed once in PBS. The samples were then ready for staining with the mass cytometry antibody.
The metal isotope-labeled antibodies used in mass cytometry were made using antibody-labeling kits from Fluidigm (Fluidigm, USA) and all the experiments were performed according to the manufacturer protocols. We first performed tests to ensure that all antibodies were effective and that the parameters were informative. Five different anti-CD45 antibodies conjugated with Pd-104, Pd-105, Pd-106, Pd-108 and Pd-110 were used to label live cells 35. Then, composite samples were incubated with a cocktail of primary antibodies. Barcoded composite samples were loaded onto a Helios mass cytometer (Fluidigm, USA) and the data were by analyzed with MATLAB (MathWorks, China) and Cytobank software (Cytobank, USA). The results were present in viSNE map, heat map and Flow Self Organizing Map (FlowSOM).
Statistical analyses were performed using ANOVA followed by Tukey’s post hoc test or by a two-tailed Student’s t-test, depending on the dataset. All values are expressed as mean ± SEM. * p < 0.05, ** p < 0.01 and *** p < 0.001 denote the significance thresholds.