Materials and reagents

C57BL/6 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd). Mice have free access to water and food on a 12:12 h light/dark cycle. Male and female mice were bred to generate pup litters for the experiments. In general, experiments are run in cohorts of 20–40 mice at one time.
Male CD1 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd). Mice were single housed with free access to water and food on a 12:12 h light/dark cycle. CD1 mice should be single housed for at least a week before aggression screening.
Surgical scissors and suture (RWD, catalog number: SP0009-M)
Isoflurane (RWD, catalog number: R510-22-10)
Cleaning solution (Oxytech, catalog number: D-50/500)

Equipment

Arena (Wuhan Yihong Technology Co., Ltd, catalog number: YH-OF-M/R): 48 cm × 48 cm × 40 cm with an empty wire mesh enclosure (10 cm × 10 cm × 15 cm) alongside the middle of one of the arena walls. The 10 cm wide area surrounding the wire mesh cage is called the social interaction zone; the opposing corners in the arena are called corner zones (Figure 1A).
Arena (Wuhan Yihong Technology Co., Ltd, catalog number: YH-OF-M/R): 48 cm × 48 cm × 40 cm with a center zone (20 cm × 20 cm) and four corner zones (10 cm × 10 cm) (Figure 1B)
Video recording equipment (EthoVision XT) (Figure 1C)
Stopwatch for timing

Figure 1. Schematic and physical objects of the social interaction arena (A), open field arena (B), and video-tracking equipment (C)

Software

EthoVision XT software (Noldus, https://www.noldus.com/ethovision-xt) or other video-tracking packages.
GraphPad Prism Software (version 7.0) or other statistical software.

Procedure

Aggression screening
1. Introduce a novel C57BL/6 mouse (8–16 weeks old) into the home cage of single-housed CD1 mouse (4–6 months old).
2. Record attack latencies of CD1 mice during a 3 min testing session.
3. Repeat the procedure for three consecutive days, using different C57BL/6 mouse for each CD1 mouse each day.
4. Select CD1 mice that attacked within 60 s for at least two consecutive days as aggressors.
5. Resect the gonads of selected CD1 aggressors (refer to Sophocleous and Idris, 2019) using surgical scissors and sutures and isoflurane, to avoid harmful behaviors against experimental mice in subsequent experiments.
6. Reuse CD1 aggressors for different pup litters for no longer than three months.

Early-life social defeat paradigm
1. Randomly assign mouse pup litters to control and experimental groups at postnatal day 15 (PD 15).
2. Introduce each pup of the experimental group into the home cage of an unfamiliar CD1 aggressor for a session of 30 min.
3. Place the pup litters back in their home cages with dams.
4. Repeat the aggression with different resident home cages every day from PD 15 to PD 24.
5. Keep pups in the control group undisturbed from PD 15 to PD 24.
6. Raise all experimental mice individually in their cage after the 10 day social defeat for a month.

Social interaction test (SIT)
1. Conduct the SIT on PD 55 between 10:00 and 14:00.
2. No target trial: introduce experimental and control mice into the arena while the wire mesh enclosure is empty for 300 s.
3. Analyze the time that experimental mice spend in the social interaction and corner zones with EthoVision XT.
4. Remove the experimental mice from the arena and place them into their home cage for 1 min.
5. Target trial: place an unfamiliar CD1 mouse into the wire mesh enclosure of the arena.
6. Introduce experimental mice into the arena for another 300 s.
7. Analyze the time that experimental mice spend in social interaction zone and corner zones with EthoVision XT.
8. Clean the arena and wire mech enclosure with odorless cleaning solution and wipe dry.

Open field test (OFT)
1. Conduct the OFT on PD 56 between 10:00 and 14:00.
2. Introduce each experimental mice into the arena for 10 min.
3. Analyze the time that experimental mice spend in the central and corner zones of the open field arena with EthoVision XT.
4. Analyze the total distance traveled by experimental mice with EthoVision XT.
5. Clean the arena with odorless cleaning solution and wipe dry.

Data analysis

Analyze all data with GraphPad Prism Software (version 7.0) or any other type of statistical software.
Assess the normality of the distribution with Kolmogorov–Smirnov test.
Present data of different groups as mean ± standard error of mean.
Use an unpaired 2-tailed Student’s t-test/two-way analysis of variance (ANOVA) for comparisons among groups.
Define statistical significance as p < 0.05.

Acknowledgments

W.D.N. designed the experiments. W.D.N. and Y.Z. performed the experiments. Y.Z. wrote the article and W.D.N. revised the manuscripts. We thank L.H.C. for his helpful review of this manuscript. We thank S.J.R. for their previous work on animal models of social defeat.

This research was supported by the National Natural Science Foundation of China (82102301 to W.D.N.), National Post-doctoral Program for Innovative Talents of China (BX20190226 to W.D.N.).

Competing interests

The authors declare that they have no conflicts of interest.

Ethics

All procedures involving animals were approved by the Association for Assessment and Accreditation of Laboratory Animal Care and the Institutional Animal Care and Use Committee of Sichuan University. All efforts were made to minimize the suffering of mice.

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