For all experiments, male C57BL/6J mice (7–8 weeks, from Shanghai Laboratory Animal Center, Chinese Academy of Sciences, China) were fed ad libitum, allowed a 1 week habituation period before experimental manipulation, and housed at 23 ± 2°C on a 12 h light/dark cycle (lights on at 07:00). CD-1 retired breeders (male, 8–9 months, from Vital River Laboratories, Beijing, China) were used as the aggressors and were screened every 3 months to ensure their antagonistic interactions. This study was carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. The protocol was approved by Animal Ethics Committee of Shanghai Medical College, Fudan University, Shanghai, China (20120302-107).

The social defeat stress was conducted as previously described (Han et al., 2014). Briefly, every C57BL/6J mouse was individually introduced to the home cage of an unfamiliar aggressive CD-1 resident mouse for 5–10 min and exposed to physical defeat, after which it was housed together with the CD-1, but separated by a perforated plastic divider to allow for visual, olfactory and auditory contact for the remainder of 24 h. On the next day, the exposed mouse was transferred into a new cage that resided another unfamiliar aggressive CD-1 mouse. During the whole period of social defeat, these mice were subject to social defeat for 10 consecutive days (exposure to 10 different CD-1 mice). Control mice were kept alone in transparent plastic cages during the 10 days. For each pair of mice, the two transparent plastic cages were placed next to each other. This allowed for visual, olfactory, and auditory contact, without any physical contact. The schematic diagram of the experimental setting is shown in Figure 1A.

Escitalopram oxalate was obtained from Stru Chem (Suzhou, Jiangsu, P.R. China). After social defeat exposure, all animals were kept alone in home cages during the next 4 weeks with no stress. During the 4 weeks, susceptible mice were injected daily with escitalopram (10 mg/kg) or its vehicle (0.9% NaCl) intraperitoneally. Resilient mice were injected daily with the vehicle and the control mice received no injections.

Social interaction test was performed on day 11 and day 39, in a clean open arena (42 × 42 cm). Each test consisted of two 2.5 min sessions, separated by an interval of 30–60 s. In the first (also called No-Target) session, the C57BL/6J mouse was introduced to the arena with an empty mesh cage (10 × 6 cm) at one of its sides. In the second (Target) session, a mesh cage with an unfamiliar CD-1 mouse replaced the empty cage. Mesh cages allowed for visual and olfactory interactions (but no physical contact) between the test and target mice. The area of 26 × 14 cm, meaning 8 cm around the mesh cage, was defined as the interaction zone (IZ, Figure 1B). The time spent in the IZ was measured. The social interaction rate (SIR) represents the Target session time spent in the IZ divided by the No-Target session time spent in the IZ. Susceptible and resilient mice were segregated based on the SIR: mice with scores < 1 were defined as “susceptible,” and those with scores ≥1 were defined as “resilient.”

After social interaction test, blood collection was performed. Mice were anesthetized with ether and blood was withdrawn from retro-orbital plexus using capillary tubes. Blood was collected between 15:00 and 16:00 to avoid possible effects of circadian variations on serum corticosterone levels. Samples were collected into a separator tube and after clot formation, centrifuged at 3,000 × g for 10 min. Serum was extracted and stored at −20°C until use. The concentration of corticosterone was measured with enzyme-linked immunosorbent assay (ELISA) kits (ABCAM, ab108821) following the manufacturer's protocol. According to the sensitivity and precision of the assay, the minimum detectable dose of corticosterone was typically around 0.3 ng/mL and the intra-assay and inter-assay coefficient of variation was 5 and 7.1%, respectively. Each sample was run together with its duplicate. The mean value for each standard and sample is therefore based on two results, and the concentrations of samples were calculated from the standard curve.

After decapitation, the hippocampus and hypothalamus were quickly dissected and stored at −80°C until use. The hippocampus and hypothalamus were ultrasonically disrupted in RIPA lysis buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfonate, sodium orthovanadate, sodium fluoride, ethylene diamine tetraacetic acid, leupeptin, PMSF] followed by centrifugation at 12,000 × g for 20 min. For cytosolic and nuclear extraction, NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, #78833) were used following the manufacturer's protocol. The protein level was measured using the Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). Samples were separated on 12% acrylamide gels and then transferred onto polyvinylidene fluoride membranes. After blocking with 5% skim milk in tris-buffered-saline with tween (TBST, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.05% Tween-20) at 4°C for 2 h, the membranes were incubated with the primary antibody, anti-Glucocorticoid Receptor antibody (1:200, ABCAM, ab2768), horseradish peroxidase (HRP)-rabbit-anti-β-Actin (1:1,000, Cell Signal Technology, #12620), or anti-Histone H3 Antibody (1:1,000, Cell Signal Technology, #9715) a 4°C overnight. Then, the blots were washed in TBST and incubated in the appropriate secondary antibody, HRP-goat-anti-mouse IgG (1:10,000, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) or HRP-goat-anti-rabbit IgG (1:10,000, Jackson) at 4°C for 2 h. Western blot images were captured on an ImageQuant LAS4000 Mini Image Analyzer (GE Healthcare, Buckinghamshire, UK) and the band levels were quantified using Quantity One, version 4.62.

The hypothalamus was homogenized using Trizol. The total RNA was then reverse-transcribed using iScript™ cDNA Synthesis Kit (Bio-Rad, CA, USA) and the resulting cDNA were analyzed in quantitative polymerase chain reactions (qPCR). SYBR green detection was used according to the manufacturer's protocol (Bio-Rad, CA, USA). The 2^−ΔΔCT method was then used to convert ΔCT values to mRNA fold changes relative to the control group. The mRNA level of Crf were normalized with glyceraldehyde-3-phosphatedehydrogenase (GAPDH) mRNA level to exclude effects of varying RNA amounts. Primer sequences are as follows: Mus Crf, forward: GGG AGT CAT CCA GTT GTT T, reverse: GAG CTT ACA CAT TTC GTC C; Mus Gapdh, forward: AAA TGG TGA AGG TCG GTG TG, reverse: AGG TCA ATG AAG GGG TCG TT.

We conducted the first experiment (Figure 1C) to investigate the individual differences in the HPA axis response and GR protein expression between susceptible and resilient mice after exposure to social defeat stress for 10 days. We conducted the second experiment (Figure 4A) to observe, if escitalopram could counteract behavioral abnormalities, HPA axis response and GR protein expression of susceptible mice. We conducted the third experiment (Figure 4A) to study the individual difference of GR protein translocation in the hippocampus between susceptibility and resilience as well as the effect of escitalopram on GR protein translocation in the hippocampus of susceptible mice.

The data are presented as the mean ± standard error (SEM). All statistical analyses were performed and all graphs plotted using the GraphPad Prism 6.01 (GraphPad Software Inc., San Diego, CA, USA). The statistical significance of differences between groups was analyzed with a one-way analysis of variance (ANOVA) followed by the Tukey HSD. The statistical significance level was set at p < 0.05.

After exposure to social defeat stress, we carried out social interaction test to differentiate defeated mice. Only ~53% of the defeated C57 mice (n = 38) were classified as susceptible ones (n = 20) that exhibit social avoidance and the rest (n = 18) were identified as resilient ones that fail to develop social avoidance (Figure 1D). Besides, control mice and resilient mice behaved similarly [F_{(2, 55)} = 19.36, p = 0.7982] and both exhibited significantly higher SIR than susceptible mice [F_{(2, 55)} = 19.36, control mice vs. susceptible mice, p < 0.0001; resilient mice vs. susceptible mice, p < 0.0001, Figure 1D]. In our study susceptible mice showed obvious social avoidance, while the behavior of resilient mice resembled this of normal control mice.

To monitor the HPA axis response, we detected the mRNA expression of Crf in the hypothalamus and serum CORT concentration of mice. After exposure to social defeat stress, susceptible mice showed significantly more Crf mRNA expression than control and resilient mice [F_{(2, 15)} = 19.63, control mice vs. susceptible mice, p < 0.0001; resilient mice vs. susceptible mice, p = 0.0014; control mice vs. resilient mice, p = 0.2539, Figure 2A], and significantly more serum CORT than control mice [F_{(2, 18)} = 3.855, control mice vs. susceptible mice, p = 0.0380; resilient mice vs. susceptible mice, p = 0.7411; control mice vs. resilient mice, p = 0.1551, Figure 2B], which indicated that social defeat stress induced hypercortisolemia in susceptible mice. Despite some gains in Crf mRNA expression and serum CORT concentration in resilient mice, the differences were not significant as compared to control mice. The data demonstrate that HPA axis of resilient mice was activated slightly less than this of susceptible mice.

After exposure to social defeat stress, resilient mice showed significantly more GR protein expression than control and susceptible mice in the hippocampus [F_{(2, 18)} = 19.18, control mice vs. susceptible mice, p = 0.2382; resilient mice vs. susceptible mice, p = 0.0011; control mice vs. resilient mice, p < 0.0001, Figure 3A]. This result indicates that in response to social defeat stress, there was sufficient GR protein expression in resilient mice. Moreover, susceptible mice showed significantly less GR protein expression than control and resilient mice in the hypothalamus [F_{(2, 18)} = 32.23, control mice vs. susceptible mice, p < 0.0001; resilient mice vs. susceptible mice, p < 0.0001; control mice vs. resilient mice, p = 0.9119, Figure 3B]. These results indicate that susceptible mice exhibited significantly less GR protein expression than resilient mice in the hippocampus and hypothalamus.

After 4 weeks of treatment, escitalopram alleviated the social avoidance behavior of susceptible mice. However, susceptible mice administered with vehicle still exhibited significantly lower SIR than other groups [F_{(3, 57)} = 5.431, susceptible+saline mice vs. control mice, p = 0.0036; susceptible+saline mice vs. resilient+saline mice, p = 0.0226; susceptible+saline mice vs. susceptible+escitalopram mice, p = 0.0059, Figure 4B]. The data indicate that social avoidance behavior can continue for at least 4 weeks and that escitalopram could counteract the abnormal behavior induced by chronic social defeat stress.

The Crf mRNA expression in the hypothalamus of susceptible mice subsided 4 weeks after the last social defeat stress [F_{(3, 19)} = 4.191, susceptible+saline mice vs. control mice, p = 0.3006, Figure 5A]. Besides, it is noteworthy that susceptible mice administered with escitalopram exhibited significantly lower Crf mRNA expression than control mice in the hypothalamus [F_{(3, 19)} = 4.191, susceptible+escitalopram mice vs. control mice, p = 0.0129, Figure 5A]. The data indicate that escitalopram can attenuate the Crf mRNA expression in the hypothalamus. Additionally, even though serum CORT of the model groups also decreased to some extent, the serum CORT of susceptible mice administered with vehicle still kept a high level, compared with other groups [F_{(3, 18)} = 10.15, susceptible+saline mice vs. control mice, p = 0.0002; susceptible+saline mice vs. resilient+saline mice, p = 0.0063; susceptible+saline mice vs. susceptible+escitalopram mice, p = 0.0062, Figure 5B].

Susceptible mice administered with escitalopram expressed significantly more GR protein in the hippocampus than those administered with vehicle [F_{(3, 20)} = 23.73, susceptible+escitalopram mice vs. susceptible+saline mice, p < 0.0001], and showed significantly more GR protein expression than control mice to face past distressing events, just like resilient mice [F_{(3, 20)} = 23.73, susceptible+escitalopram mice vs. control mice, p < 0.0001; resilient+saline mice vs. control mice, p = 0.0398, Figure 6A], which indicates that the antidepressant could enhance the GR protein expression in the hippocampus. Combined with the result in Figure 4, this indicates that in response to a bitter memory from the past, such as social defeat, there was sufficient GR protein expression in resilient mice and susceptible ones administered with antidepressant, all of which engaged in active social interaction. However, there were no significant differences of GR protein expression in the hypothalamus [F_{(3, 20)} = 1.002, Figure 6B]. Thus, we next examined the GR protein translocation in the hippocampus by detecting its levels in the cytosolic and nuclear compartments.

After exposure to social defeat stress, susceptible mice exhibited significantly more cytosolic GR protein expression than control and resilient mice [F_{(2, 9)} = 17.90, control mice vs. susceptible mice, p = 0.0029; resilient mice vs. susceptible mice, p = 0.0009; control mice vs. resilient mice, p = 0.6774, Figures 7A,B], while resilient mice showed significantly more nuclear GR protein expression than control and susceptible mice in the hippocampus [F_{(2, 9)} = 7.821, control mice vs. susceptible mice, p = 0.6000; resilient mice vs. susceptible mice, p = 0.0104; control mice vs. resilient mice, p = 0.0479, Figures 7A,C]. These results indicate that there was more nuclear translocation of GR in the hippocampus of resilient mice than in that of susceptible mice.

There were no significant differences of cytosolic GR protein expression in the hippocampus 4 weeks after the last social defeat stress [F_{(3, 12)} = 0.4462, Figures 8A,B]. However, susceptible mice administered with escitalopram expressed significantly more nuclear GR protein in the hippocampus than those administered with vehicle [F_{(3, 12)} = 8.432, susceptible+escitalopram mice vs. susceptible+saline mice, p = 0.0410] and, similar to resilient mice, showed significantly more nuclear GR protein expression than control mice, [F_{(3, 12)} = 8.432, susceptible+escitalopram mice vs. control mice, p = 0.0019; resilient+saline mice vs. control mice, p = 0.0419, Figures 8A,C]. The data imply that escitalopram could increase nuclear translocation of GR and ultimately reverse social withdrawal behaviors.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer TK and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.