2.1 Animals and housing

Male and female (8-week-old) C57BL/6 black mice (Harlan) were maintained in a pathogen-free, temperature-controlled (22°C ± 1°C) mouse facility on a reverse 12-/12-h light/dark cycle, with lights switched on at 8 pm. Male white ICR (CD-1) outbred mice (Harlan) were used as residents for the social defeat paradigm. Female white ICR (CD-1) outbred mice (in-house breeding) were used for the social crowding stress paradigm. All experimental animals were pair-housed with same-sex partners for the duration of the experiment. Home cage control animals were not housed with stressed animals but with other control animals. We did not monitor females' estrous cycle and used freely cycling females, as several studies suggest behavioural effects of exposure to stress may overshadow effects of different estrus cycle stages on behaviour (Dalla et al., 2008; Horovitz et al., 2014; Prendergast et al., 2014). All experimental animals were weighed weekly to monitor general health. The study contains data from several batches that served as internal control. The Institutional Animal Care and Use Committee (IACUC) of The Weizmann Institute of Science approved all procedures. Animals were given ad libitum access to food and water.

2.2 Female CSS paradigm

Combining elements from several social stress protocols, including resident–intruder, crowding stress and social instability, we generated a novel female mouse model of CSS (Figure 1b). Our model employed 8-week-old female C57BL/6, socially housed in groups and then in pairs in order to create highly familiar low aggression pairs.

During the 15 days of the social stress protocol, each experimental C57BL/6 female mouse was daily transported to a testing room where it was exposed to crowding by a variable number (5–9) of unfamiliar ICR female mice in a novel cage (see below description of maintaining the ICR females) for 1 h. For each stress session, the experimental mouse was allowed to interact directly with the unfamiliar ICR female mice for 30 min; mice behaviour included agonistic behaviour such as sniffing and grooming as well as antagonistic behaviour such as chasing, physical attacks and related vocalizations. For the remaining 30 min, the experimental mouse was placed inside a metal mesh within the same cage, enabling visual, olfactory and auditory interaction but no physical contact. In order to increase instability and unpredictability, stress sessions were conducted without habituation, each day at different times during the active dark phase and in different testing rooms. After each 1-h stress session, the experimental mice were returned to the home cage containing the same highly familiar partner. In this manuscript, we describe behavioural and physiological response of the experimental mice to social stress, although the behaviour of the partner mice is reported in a separate manuscript.

Control female C57BL/6 mice were housed in pairs and handled daily for 15 days.

The ICR female mice were housed in conditions of social instability for the duration of the social stress protocol: At the end of each stress session, ICR mice were randomly divided into groups of four until the following day. At the beginning of each stress session, ICR mice were dispersed in novel cages containing five to nine animals per cage. This procedure created high levels of arousal and exploration of conspecifics among ICR female mice and rendered female ICR mice more aggressive towards the C57BL/6 female experimental mice.

2.3 Male CSS paradigm

On the basis of chronic social defeat stress (CSDS) protocols in male mice (Berton, et al., 2006), we developed a protocol of CSDS that is comparable with the female protocol described above (Figure 1b). Briefly, our model employed 8-week-old male C57BL/6, housed in groups and then in pairs until 12 weeks old in order to create low aggression levels, as is known to occur in groups of males cohabiting for weeks (Haller et al., 1999). During the 15-day social stress protocol, each experimental C57BL/6 male mouse was transported daily to a testing room and spent 1 h in the home cage of an aggressive and unfamiliar ICR outbred mouse (Harlan); see below the description of maintaining the ICR males. During the first 5 min of that 1-h exposure, the mice were allowed to physically interact. During this time, the ICR mouse attacked the intruder mouse, and the intruder displayed subordinate posturing. For the remaining 55 min, the C57BL/6 experimental mouse was placed in a cage with a metal mesh, which enabled visual, olfactory and auditory interaction but no direct physical contact. After each 1-h stress exposure, the experimental mice were returned to their homecage, containing the same highly familiar mouse partner. In this manuscript, we describe behavioural and physiological response of the experimental mice to social stress, although the behaviour of the partner mice is reported in a separate manuscript.

Control male C57BL/6 mice were housed in pairs and handled daily for 15 days.

ICR male mice were housed alone in cages for 1 week prior to CSDS in order to establish territoriality.

3.1 Social interaction test

A SIT was conducted based on prior protocols (Berton et al., 2006; Krishnan et al., 2007). Briefly, 24 h following the last social stress session, each experimental mouse (either male or female) was placed in a dimly illuminated open field arena (white plastic box, 50 × 50 × 22 cm, 3–5 lux), and its trajectory was tracked for two consecutive sessions of 3 min each. Video recordings were performed as previously described (Regev et al., 2011). During the first session (‘no target’), the FC contained an empty wire mesh cage (circular, 10 cm diameter) located at one end of the field. During the second session (‘target’), the conditions were identical except that a social target animal (an unfamiliar same-sex ICR mouse) was introduced. Between the two sessions, the experimental mouse was removed from the arena and was placed in a temporary novel cage for approximately 1 min. An automated video-tracking software (Ethovision 9, Noldus) was used to determine the time spent by experimental mice in a defined ‘interaction zone’ (a semi-circle surrounding the mesh) during the ‘no target’ and ‘target’ conditions. An interaction ratio was calculated as follows:

3.2 DLT test

The DLT test takes advantage of the natural conflict of a rodent between the exploration of a novel environment and the aversive properties of a large, brightly lit open-field (Regev et al., 2011). The DLT test apparatus consists of a polyvinyl chloride box divided into a black dark compartment (14 × 27 × 26 cm) connected to a larger white 1,200 lux illuminated light compartment (30 × 27 × 26 cm). Mice were placed in the dark chamber, and behaviour was quantified during the 5-min test starting when the door connecting the dark and light chambers was opened. A video tracking system (VideoMot2; TSE Systems) was used to quantify time spent in the light compartment, the distance travelled in light area and number of dark–light transitions.

3.3 Forced swim test

When forced to swim in a narrow space from which there is no escape, mice adopt an immobile posture after an initial period of vigorous activity, which is considered a behavioural despair reaction. The FST is considered an index of ‘depressive-like symptoms’ and was performed as previously described (Issler et al., 2014). Briefly, mice were placed in plexiglas cylinders (13 cm diameter × 24 cm high) containing water (22°C ± 2°C) to a depth of 15 cm, their activity during 6 min was recorded, and immobility duration was quantified off-line with an automated video-tracking system (Ethovision 9, Noldus).

3.4 Fear conditioning

FC is a behavioural learning paradigm that allows organisms to learn to predict aversive events, by pairing an aversive event to a neutral stimulus (a foot shock paired with a neutral sounds). Previous studies reported effects of chronic stress on memory formation and retrieval using different conditioning protocols (e.g., Rodrigues et al., 2009; Roozendaal et al., 2009), as well as sex differences in fear memory learning and extinction that were related to anxiety disorders (McDermott et al., 2015). We therefore employed a long-term protocol of FC (Karpova et al., 2011) that allowed us to examine male and female behaviour during conditioning, extinction training and long-term fear memory retrieval. A computer-controlled fear-conditioning system (TSE Systems, Bad Homburg, Germany) monitored the procedure while measuring freezing behaviour (defined as lack of movement except for respiration for at least 3 s); the measure was expressed as a percentage of time spent freezing. FC and extinction took place in two different contexts: FC context (A) was a transparent plexiglas chamber cage (21 cm × 20 cm × 36 cm) with metal grids on floor and constant illumination (250 lux), whereas extinction context (B) was a black nontransparent plexiglas chamber (same size) with planar floor and no illumination except dim red light. Both Context A and Context B were cleaned before each session with 70% ethanol and 1% acetic acid, respectively. On Day 1, mice were conditioned using five pairings of the conditioned stimulus, CS (CS duration 30 s, 1 Hz, white noise, 80 dB) with the unconditioned stimulus, US (1-s foot-shock 0.6 mA, co-terminated with the CS); intertrial interval (ITI): randomly varying 20–120 s. On Day 2, conditioned mice were submitted to extinction training in Context B during which they received 12 presentations of the CS alone (ITI: randomly varying 30–60 s). On Day 10, mice were tested first for spontaneous recovery of fear (cue test) in Context B, and 2 h later were tested for context dependent fear renewal (context test) in Context A. Both tests included four presentations of the CS (ITI: randomly varying: 20–60 s).

3.5 ASR test

The ASR test reflects a transient motor response to a sudden unexpected stimulus, such as a loud noise. Animals suffering from anxiety and hyperarousal are found to express an enhanced startle response; the startle reaction is potentiated by previous stressful experiences such as foot shocks and is attenuated by prepulse inhibition (PPI: Koch, 1999). Startle response and PPI protocol were assessed in a Startle Response System (TSE Systems) using a protocol adapted from Neufeld-Cohen et al. (2010). Briefly, mice were placed in a small plexiglas and wire mesh cage on top of a vibration-sensitive platform in a sound-attenuated, ventilated chamber. A high-precision sensor, integrated into the measuring platform, detected movement. Two high-frequency loudspeakers inside the chamber produced all the audio stimuli. The session began with 5-min acclimation to white background noise (65 db) maintained through the whole session. Thirty-two startle stimuli (120 db, 40 ms in duration with a randomly varying ITI of 12–30 ms) were presented interspersed with an additional 40-ms startle stimuli randomly preceded by 40 ms prepulses of either 74, 78 or 82 dB. Latency to peak startle response and response amplitude was measured both in response to startle stimuli and in response to startle stimuli preceded by prepulses.

3.6 Corticosterone measurement

Plasma was extracted from blood samples that were collected by tail bleed under basal conditions at two time points: 1 h before onset of the active phase (7 am) and 1 h after offset of the active phase (9 pm). Blood samples were centrifuged immediately (3,500 rpm for 25 min at 4°C) and extracted plasma was stored at −80°C until assayed for corticosterone (CORT) using a radioimmunoassay kit (ImmuChemTM Double Antibody Corticosterone ¹²⁵I RIA KIT, MP biomedicals, NY, USA).

4.1 Manual measurement of food intake

CSS and home cage female mice were isolated for 8 days, and chow was weighed once a day in order to assess food intake.

4.2 Metabolic cages

Calorimetry, food and water intake, and locomotor activity were measured using the Labmaster system (TSE Systems). The LabMaster instrument consists of a combination of sensitive feeding and drinking sensors for automated online measurement. The calorimetry system is an open-circuit system that determines O₂ consumption, CO₂ production and respiratory exchange rate (RER). A photobeam-based activity monitoring system detects and records ambulatory movements, including rearing and climbing, in every cage. All the parameters are measured continuously and simultaneously. Data were collected after 48 h of adaptation in acclimated singly housed mice.

4.3 Blood glucose-level measurements

Glucose-tolerance tests (GTTs) were performed as previously described (Kuperman et al., 2010). Briefly, following 4 h of fasting, glucose (2 g/kg of body weight [BW]) was injected i.p., and whole venous blood obtained from the tail vein at 0, 15, 30, 60, 90 and 120 min after injection, and was measured for glucose by using an automatic glucometer (One Touch; Lifescan). Glucose levels in fed animals were measured using the same glucometer.

4.4 Body composition

Body composition was assessed using Echo-MRI (Echo Medical Systems).

4.5 Statistical methods

Data are expressed as mean ± SEM. As stress protocols were comparable but not identical for male and female mice, we did not employ statistical models directly comparing male and female mice but compared each stressed group to its control group. All dataset distributions were assessed for normality using the Kolmogorov–Smirnov test to determine which statistical tests should be employed. When comparing normally distributed data, the independent Student's t test was used; analysis of weight changes, reflecting within-animal comparisons necessary to address individual variability, employed repeated measured analysis of variance (RM-ANOVA), followed by post hoc comparisons corrected by Tukey test. When data departed from normality, the Mann–Whitney U test was applied. An alpha level of .05 was determined significant.

5.1 Changes in BW

As expected, both CSS paradigms affected BW gain. However, female mice were affected for a longer period of time than male mice (Figure 2a,b). Stressed female mice (n = 27) gained significantly less weight than home cage female mice (n = 23) during Weeks 2–4 of monitoring (including stress protocol and 1-week post-stress). RM-ANOVA revealed significant main effects for time (Weeks 2–9) (F_[7,336] = 150.6; P < .0001) and for group (F_[1,48] = 12.47; P < .001); the interaction time × group was also significant (F_[7,336] = 5.3; P < .0001]. Follow-up comparisons were significant for the third to fifth weeks of weight monitoring (Week 3: P < .0001; Week 4: P < .0001; Week 5: P < .0005; Figure 2a). Male stressed mice (n = 13) significantly differed from home cage control mice (n = 12) during Week 3. RM-ANOVA indicated significant main effects for time (Weeks 2–9) (F_[7,161] = 147.6; P < .0001) but not of group (F_[1,23] = 1.96; P = .17); the interaction time × group was significant (F_[7,161] = 7.54; P < .0001), and follow-up comparisons indicated a difference only during the third week of weight monitoring (Week 3: P < .005, Figure 2b).

At the end of the experiment (after ASR, some 2 months after CSS), we conducted explorative metabolic analysis of female mice, described in Figure S3. At this time point, there was no difference in BW between CSS and home cage female mice (Figure S3f), which limits the ability of this dataset to provide evidence on mechanisms at work. Briefly, In order to test food intake, we isolated CSS and home cage female mice for 8 days, and we weighed once a day the chow in order to assess food intake. We found no differences between groups (Figure S3a). At a later time point, female CSS and home cage mice were tested in metabolic cages using the Labmaster system (TSE Systems). Data were collected after 48 h of adaptation in acclimated singly housed mice (Kuperman et al., 2010). We found no differences between groups in food intake (Figure S3b) or water intake (Figure S3c); no difference in energy expenditure (Figure S3d,e), RER (Figure S3f), BW (Figure S3g) or mobility (Figure S3h). Next, we measured body composition, but no difference was found in lean mass (Figure S3i) or fat mass (Figure S3j). Finally, we measure blood glucose in fed animals, where we found significantly reduced glucose level in CSS animals (unpaired two-tailed t test, t[23] = 2.39, P < .05, Figure S3k). Follow up analysis using the Glucose Tolerance Test did not reveal differences between groups (Figure S3l).

5.2 Social interaction test

It was previously shown that CSDS causes long-term reduction in social interaction, measured 24 hr after the last stress session (Tsankova et al., 2006; Berton et al., 2006). During SIT, most control mice spent more time interacting with a social target than with an empty mesh. Therefore, we compared the interaction ratio between mice undergoing CSS (Stress F, n = 26; Stress M, n = 22) and the control group (Home F, n = 22; Home M, n = 10) and found significantly reduced interaction ratio 24 h after the last stress session in stressed mice of both sexes (Mann–Whitney U test, females U = 161; P < .01, males U = 13; P < .0001, Figure 2c,d). An additional chi-square test was performed on the proportion of mice categorized as ‘susceptible’ to the stress protocol (mice with interaction scores < 1) or ‘unsusceptible’ to stress (mice with interaction scores > 1). In male mice, the proportion of susceptible mice was significantly higher in the stressed versus the control group (χ²[1,N = 32] = 12.37, P < .0005), and the vast majority of stressed mice (21 out of 22) were categorized as susceptible, as opposed to 50% in standard CSDS protocols (Krishnan et al., 2007). In female mice, there was no difference between ratio of susceptible stressed versus control mice (χ²[1,N = 48] = 0.01, N.S.), and most stressed mice (25 out of 26) had an interaction ratio higher than 1 (Figure 2c).

5.3 DLT test

The DLT test took place 1 week after the last social stress session, in order to measure long-term effects of CSS on anxiety and neophobia. We were surprised to find that stressed female mice spent more time in the light (t_[49] = 2.16; P < .05, Figure 3a), travelled greater distance in the light (t_[49] = 4.26; P < .0001, Figure 3c), made more dark–light crossings (t_[49] = 3.53; P < .001, Figure 3e) and entered the light more readily (Mann–Whitney U = 182.5; P < .01, Figure 3g), than female control mice. There were no significant differences between CSS male mice and their respective control group (time in light: t_[33] = 0.75; P = .45, Figure 3b; distance in light: t_[33] = 0.8; P = .42, Figure 3d; dark–light crossing: t_[32] = 1.05; P = .29, Figure 3f; time to enter light: Mann–Whitney U = 90; P = .24, Figure 3h). However, stressed females (Figure 3i), but not males (Figure 3j), exhibited increased average velocity during the test relative to control group mice (females: t_[49] = 4.9; P < .0001; males: t_[33] = 1.35; P = .18).

5.4 Forced swim test

In order to measure long-term effects of CSS on depressive like symptoms, we tested the mice in the FST 3–4 weeks after the end of the stress protocol. No differences in total immobility time were observed between the CSS and control groups in either female or male mice (females: Mann–Whitney U = 232; P = .26, Figure S1c; males: t_[23] = 0.44; P = .44, Figure S1d). Similarly, no differences were found in latency to first immobility session in chronically stressed groups relative to their controls (females: Mann–Whitney U = 270; P = .74, Figure S1a; males: t_[23] = 0.88; P = .38, Figure S1b).

5.5 Fear conditioning

No differences in freezing duration were observed between stress and control groups during FC and extinction (Table S1). However, in the cue test, that assessed spontaneous recovery of fear, stressed males, but not stressed females, exhibited increased freezing relative to their control group (male: t_[21] = 2.09; P < .05; female: t_[48] = 0.53; NS; Figure 5b,c). Specifically, stressed male mice showed more freezing than controls upon initial entry into the experimental chamber, before hearing any tones, and also during later presentations of tones (Figure 5c, right panel). There were no differences in the context test that took place 2 h after the cue test, and similar freezing levels were observed between stressed and control groups, both female and male (Figure 5d,e, Table S1).

5.6 Acoustic startle response test

We used the ASR test as a measure of long-term anxiety following CSS. We did not find significant differences between stressed and control groups, in neither male nor female mice, in any of the examined indices (see Supplementary Information S1 and Figure S2).

5.7 Corticosterone

HPA axis activity was assessed by measuring basal CORT levels 2 months after the last stress session. Only male mice showed a significant alteration in HPA activity following CSS: RM-ANOVA indicated significant main effect of group (F_[1,22] = 5.18; P < .05), significant main effect of sample time (F_[1,22] = 46.03; P < .0001) but nonsignificant interaction (F_[1,22] = 1.37; P = .25). CSS mice Female mice showed a significant main effect of sample time (F_[1,22] = 48.79; P < .0001), but there was no significant group effect (F_[1,22] = 0.67; P = .41) or interaction (F_[1,22] = 0.4; P = .52).