Animals

Female C57BL/6 mice (12 weeks old, weighing 21–25 g) were purchased from the Laboratory Animal Center of China Medical University. Mice were housed with free access to water and food. The indoor temperature was controlled at 21 ± 1 °C, the relative humidity was 50% ± 10%, and the light cycle was 12 hours (8,00–20:00). All animal procedures were approved by the Animal Ethics Committee of Shengjing Hospital of China Medical University. All experimental procedures were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978) and the ARRIVE guidelines on the Care and Use of Experimental Animals. Fifteen pregnant female mice were individually housed and observed daily for parturition, deemed as postnatal day (PD) 0. The entire cage was treated as one, and the 15 cages were randomly divided into a control (Con) group, a maternal separation (MS) group, a maternal separation + fluoxetine (MS + Flu) group, an LPS-treated (LPS) group and an LPS-treated + fluoxetine (LPS + Flu) group. Eight male pups from each group were randomly selected as experimental subjects on PD17. The body weight of mice was measured on PD31 before the behavioral tests. The experimental design and drug treatment schedule are shown in Fig. 1A.

Maternal separation (MS)

Pups in MS group and MS + Flu group received maternal separation between PD2 and PD17. The method is improved according to the previous description [22]. Pups were separated from their mothers twice for 3 hours every day, during which time the mice should be observed and kept warm. Each cage of mice was removed in two batches in rotation to reduce the enhancement of maternal care. When the mice were transferred, some of the bedding with the mother’s scent was also transferred.

LPS and fluoxetine administration

Mice in LPS group and LPS + Flu group were treated with LPS (2 mg/kg, L2880, Sigma–Aldrich, St. Louis, MO, USA) by i.p. injection for 5 consecutive days. Mice in MS + Flu group and LPS + Flu group were treated with fluoxetine hydrochloride (10 mg/kg, 343,290, Sigma–Aldrich) by i.p. injection for 14 consecutive days. Both LPS and fluoxetine were dissolved in normal saline. Mice in the Con group and MS group were treated with saline by i.p. injection during PD17 to PD31, and that in the LPS group were treated with saline by i.p. injection during PD22 to PD31.

Behavior tests

Behavior tests were performed after drug administration and two tests were conducted daily in the following order: open field test, elevated plus-maze, forced swimming test and tail suspension test. Mice were acclimated to the testing room for 2 h before testing. Behavior test data were recorded by the SMART™ tracking software program (San Diego Instruments, San Diego, CA, USA).

Open field test (OFT)

The OFT was used to evaluate the depressive-like behaviors of mice performed as previously reported [23]. The OFT consisted of an empty square arena (40 × 40 × 30 cm) constructed of plastic with a white base. The central region is 20 × 20 cm. Mice were placed individually in the corner of the OFT apparatus, and spontaneous activities were recorded for 10 min using the SMART™ tracking program. After each test, the arena was cleaned with 75% ethanol to eliminate odor cues.

Elevated plus-maze (EPM)

The EPM test was carried out as previously reported to evaluate the anxiety-like behaviors of mice [24]. Mice were tested in a cross-shaped maze consisting of two open arms (50 × 10 cm), two closed arms (50 × 10 cm) and a central region (10 × 10 cm). Each mouse was placed in the central region of the maze and allowed to explore for 5 min. The time spent in each arm was recorded using the SMART™ tracking program. After each test, the arena was cleaned with 75% ethanol to eliminate odor cues.

Forced swimming test (FST)

The FST was performed as previously reported [25]. The test device consisted of a transparent cylindrical glass container (10 cm in diameter, depth of 22 cm) filled with water (23 °C to 25 °C) and a video camera in front of the container. The mice could not touch the bottom of the container with their hind legs. The test was conducted for 6 min: the first 2 min was an adaptation phase, after which the immobility of the mouse in the water was recorded for 4 min (immobility refers to the mouse’s body floating with the absence of any movement except for those necessary for keeping the nose above water). FST data were recorded by the SMART™ tracking software program.

Tail suspension test (TST)

The TST test was performed as previously described to assess depressive-like behavior [26]. Mice were suspended by their tail (50 cm distance from the floor) using adhesive tape at 1 cm from the tip of the tail. The TST test was conducted for 6 min, and the duration of immobility in the last 4 min was recorded. TST data were recorded by the SMART™ tracking software program.

Animal tissue extraction

After completing the behavior tests, mice were anesthetized with isoflurane, and blood from portal vein and vena cava was centrifuged as previously reported [27] and serum samples were stored in a − 80 °C freezer. Mice were then decapitated after cervical dislocation. The right hippocampus was separated and stored in a − 80 °C freezer.

Protein extraction and quantification

As previously reported [28], the extracted mouse tissue were detergent-extracted on ice using radioimmunoprecipitation assay (RIPA) lysis buffer (P0013B, Beyotime, Shanghai, P R China) with 1 mM phenylmethanesulfonyl fluoride (ST506, PMSF, Beyotime), disrupted on ice for 30 min, and then fragmented with ultrasonication. The lysates were collected and centrifuged at 21000 g for 15 min. Total proteins were quantified using a BCA protein assay kit (P0012, Beyotime).

Elisa

ELISA was performed as previously reported [28]. The levels of IL-1β, IL-6, TNF-α, 5-HT, ACTH and CORT were investigated using assay kits (Tab S2) and following the manufacturer. Each sample (5× dilution) was used 50 μl for detection, and the absorbance at 450 nm was measured. The concentration was calculated according to the standard curve (Fig. S1-S6).

Western blotting

Western blotting was performed as previously reported [25]. Equal amounts of protein (up to 30 μg) from the treated mice were separated by 10% SDS–PAGE and transferred to PVDF membranes. Transferred blots were blocked with nonfat milk for 2 hours and then incubated overnight at 4 °C with primary antibody (1:1000). Blots were subsequently washed and incubated with goat anti-rabbit (ZB-2301, Zsgb-Bio, 1:5000) and goat anti-mouse (ZB-2305, Zsgb-Bio, 1:5000) secondary antibodies for 2 hours. The antibodies were listed in Table S1. Protein bands were detected with ECL reagent (WBKLS0500, Merck Millipore). Chemiluminescent signals were detected and analyzed using a Tanon-5500 chemiluminescent imaging system (Tanon Science and Technology Co., Ltd., Shanghai, P R China). The intensity of the bands was analyzed using ImageJ 1.49 software (National Institutes of Health, Bethesda, MD, USA). Full-length blots/gels are presented in Supplementary Fig. S7.

Immunofluorescence

Thirty μm thick brain tissue sections were washed with PBS. Then the sections were blocked by 8% BSA for 2 h and then treated overnight at 4 °C with primary antibody (Iba1, 1:200, Wako, Osaka, Japan). Brain sections were subsequently washed and incubated with Alexa Flour 488 (1:500, Thermo Fisher, Waltham, MA, USA) for 2 h. Sections were washed and incubated with DAPI (Absin, Shanghai, P R China) for 5 min. After washing, the sections were transferred to slides, and glass coverslips were mounted using mounting medium. Images were captured using Leica TCS SP8 laser scanning confocal microscope. The number of microglia was determined by partition counting, and the cell size was determined by Sholl analysis.

Sholl analysis

Sholl analysis was performed as previously reported [29]. Projected z-stack image with orthogonal views were obtained by Leica TCS SP8 laser scanning confocal microscope in 1 μm steps. The z-stack images were split into single channels using Fiji and stored as 8-bit images. The estimated geometric centre was marked using the point tool in Fiji and the image was analysed with the Fiji plugins Bitmap Sholl Analysis (http://fiji.sc/Sholl_Analysis, version 3.6.8). The manual method was carried out by digitally tracing rings centred at the soma centre and intersections counted. All Sholl analyses were carried out at 2 μm intervals to a maximum radius of 24 μm.

Statistical analysis

All data are expressed as the mean ± standard deviation (SD). Statistical analysis of data was performed using one-way analysis of variance (ANOVA) and Tukey′s multiple comparisons test. A P value of < 0.05 was considered significant. GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA) was used for statistical analysis. All detailed statistical data are provided in Table S3.