Vulnerability to chronic subordination stress-induced

Progress in Neuro-Psychopharmacology & Biological Psychiatry 35 (2011) 1461–1471

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Progress in Neuro-Psychopharmacology & Biological Psychiatry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p n p

Vulnerability to chronic subordination stress-induced depression-like disorders in adult 129SvEv male mice Harold Dadomo a, Valentina Sanghez a, Luisana Di Cristo a, Andrea Lori a, Graziano Ceresini b, Isabelle Malinge c, Stefano Parmigiani a, Paola Palanza a, Malcolm Sheardown c, Alessandro Bartolomucci a,d,⁎ a

Department of Evolutionary and Functional Biology, University of Parma, Italy Department of Internal Medicine and Biomedical Sciences, University of Parma, Italy Takeda Cambridge Ltd, Cambridge, UK d Department of Integrative Biology and Physiology, University of Minnesota, Minneapolis, MN 55455, USA b c

a r t i c l e

i n f o

Article history: Received 15 July 2010 Received in revised form 4 November 2010 Accepted 5 November 2010 Available online 17 November 2010 Keywords: Anxiety Anhedonia Knockout mice Major depression Strain comparison Stress

a b s t r a c t Exposure to stressful life events is intimately linked with vulnerability to neuropsychiatric disorders such as major depression. Pre-clinical animal models offer an effective tool to disentangle the underlying molecular mechanisms. In particular, the 129SvEv strain is often used to develop transgenic mouse models but poorly characterized as far as behavior and neuroendocrine functions are concerned. Here we present a comprehensive characterization of 129SvEv male mice's vulnerability to social stress-induced depressionlike disorders and physiological comorbidities. We employed a well characterized mouse model of chronic social stress based on social defeat and subordination. Subordinate 129SvEv mice showed body weight gain, hyperphagia, increased adipose fat pads weight and basal plasma corticosterone. Home cage phenotyping revealed a suppression of spontaneous locomotor activity and transient hyperthermia. Subordinate 129SvEv mice also showed marked fearfulness, anhedonic-like response toward a novel but palatable food, increased anxiety in the elevated plus maze and social avoidance of an unfamiliar male mouse. A direct measured effect of the stressfulness of the living environment, i.e. the amount of daily aggression received, predicted the degree of corticosterone level and locomotor activity but not of the other parameters. This is the first study validating a chronic subordination stress paradigm in 129SvEv male mice. Results demonstrated remarkable stress vulnerability and establish the validity to use this mouse strain as a model for depression-like disorders. © 2010 Elsevier Inc. All rights reserved.

1. Introduction Major depression is a severe, life-threatening, and widespread psychiatric disorder. The link between exposure to stressful life events and increased risk for major depression or depression-like disorders has been confirmed in several studies (McEwen 2007; de Kloet et al 2005; Caspi et al., 2003; Kendler et al., 1999; Bartolomucci and Leopardi, 2009; Schmidt et al., 2008). Maladaptive mechanisms by which stressor exposure could increase risk for depression have been proposed, including deficient neuromodulatory homeostasis, alterations of neuronal structure and function, hyperactive hypothalamus-pituitary-adrenocortical (HPA) axis and hyperactive immune system (Krishnan and Nestler 2008; McEwen,

Abbreviations: HPA, Hypothalamus-pituitary-adrenocortical; EPM, Elevated plus maze; NPF, Novel/palatable food test; ACTH, Adrenocorticotropic hormone; HA, highaggression-received; LA, low-aggression-received). ⁎ Corresponding author. Department of Integrative Biology and Physiology, University of Minnesota, 321 Church St SE, Minneapolis, MN 55455, USA. Tel.: +1 612 626 7006; fax: +1 612 625 5149. E-mail address: [email protected] (A. Bartolomucci). 0278-5846/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2010.11.016

1999, 2007; Lupien et al., 2009; Sapolsky et al., 2000; Dantzer et al., 2008). It has been shown that certain individuals are better able to cope with stressful life events by engaging protective psychological strategies (Feder et al., 2009; Kaufman et al., 2004). Identifying individual susceptibility to stress, or inability to engage successful coping strategies, have the potential to uncover the molecular mechanisms involved in stress vulnerability and could suggest novel therapeutic approaches to neuropsychiatric disorders (Caspi et al., 2010). Genetic basis of individual vulnerability has been addressed by comparing genetically homogeneous inbred strains of mice (Fuller 1960; Crawley et al., 1997; Parmigiani et al., 1999). This approach has now been paralleled by direct genetic manipulation, e.g. gene knockout, inserting point mutations, etc., which are usually performed by means of homologous recombination in embryonic stem cells often obtained from various substrains of the murine 129 strain lineage (Thomas and Capecchi, 1987; Gerlai 2001; Capecchi 2005). The 129 strain behavioral and neuroendocrine phenotype, however, is poorly characterized partly because of experimental evidence of abnormal phenotypic features (Wahlsten 1982; Koike et al. 2006; Wolfer et al. 1997). Recent studies conducted on different 129 substrains reported high trait anxiety (Rodgers et al., 2002; Dulawa

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et al., 2004; Salomons et al., 2010; Chourbaji et al., 2010), impaired fear extinction (Hefner et al., 2008; Camp et al., 2009), and altered prepulse inhibition (Ralph et al., 2001). Of main interest, Chourbaji et al. (2010) recently demonstrated that 129SvEv mice (both males and females) show a remarkable behavioral despair in the learned helplessness model of depression and a passive coping style in the forced swim and tail suspension tests for antidepressant drug screening. Overall, recent studies established a potential depressionvulnerable phenotype for the 129SvEv strain. Aim of the present study was to investigate behavioral and physiological vulnerability of adult 129SvEv male mice to a validated naturalistic model of chronic subordination stress-induced depression-like disorders (Bartolomucci et al., 2001, 2004, 2009, 2010). Results of the present study demonstrated that 129SvEv male are highly vulnerable to social stress, showing physiological and behavioral alterations such as body weight gain, increased adiposity and plasma corticosterone, psychomotor disturbances, increased fearfulness, anxiety and social avoidance. 2. Methods 2.1. Animals 129SvEv were derived from breeders supplied by Takeda Cambridge (U.K.). Swiss CD-1 mice were derived from an outbred stock originally obtained from Charles River Italia (Lecco, Italy). Mice were born and reared at the University of Parma in a 12-hr light–dark cycle (lights on at 7:00) and maintained at 22± 2 °C. After weaning at postnatal day 25 (28 for CD-1) mice were housed in same-sex groups of siblings (3–6 per cage) in plexiglas cages (38 × 20 × 18 cm) with wood shaving bedding changed weekly. All efforts were made to minimize the number of animals used and their suffering. All animal experimentation was conducted in accordance with the European Communities Council Directive of November 24, 1986 (86/EEC) and approved by the ethical committees of the University of Parma and the Italian Institute of Health.

displaying upright posture flight behavior and squeaking vocalization was the ‘Subordinate’ (129SvEv, N = 23 and CD1, N = 8, see Results section). To give a rough estimate of mice aggressive behavior, the number of attack bouts performed by each animal was quantified by direct observations by a trained observer (Bartolomucci et al., 2001, 2009, in press). The numbers of attack bouts performed by each animal were quantified during the first four days than again at day 10 and 20 by direct observation. For ethical reasons, and to prevent injuries, the mice were separated by the partition if fighting escalated, i.e. when the dominant persistently bit the opponent on the back or tail. If the fight was interrupted, the number of attack bouts was computed according to the following formula: # of attacks received = (# of attacks received before mice were separated * 600) / time until mice were separated). Age-matched 129SvEv mice housed in groups of 3 littermates were included as the non-stressed control group (n = 30). This choice was based on previous experiments showing that grouped sibling mice maintain normal social behavior and social hierarchy while showing no metabolic, immune, endocrine, or behavioral evidences of stress activation or anxiety (see Bartolomucci et al., 2001, 2003, 2004, for details). Control mice were re-housed in group of 3 (from pre-existing groups of 4–6 animals per cage as described above) the same day in which the chronic stress procedure started and were manipulated exactly as the animals under stress as far as environmental factors are considered. Stressed and control males were littermates to reduce possible confounds of inter-litter variability. A schematic overview of the experimental procedure is depicted in Fig. 1A (see below for full procedural details). Novel/palatable food (NPF) test was performed on days 12–15, the elevated plus maze (EPM) test was performed on day 17 and the social interaction test was performed on day 20. The order of testing was based on intrinsic stressfulness of each test: NPF b EPM b social avoidance. Mice were sacrificed by decapitation (preceded by brief CO2 inhalation) on day 22 at 9:00 to obtain nadir value of corticosterone and ACTH (organ harvesting is scheduled accordingly). 2.3. Home cage phenotyping

2.2. Chronic subordination stress paradigm The procedure used in the present experiment is a modified version of our standard Chronic Subordination Stress Paradigm (Bartolomucci et al., 2001; 2004). The modified experimental procedure has recently been introduced to investigate stress vulnerability of inbred and/or transgenic mouse strains (Bartolomucci et al., 2010) which are often characterized by a lower aggressive phenotype when compared to males of the outbred CD1 strain (Parmigiani et al., 1999; Bartolomucci et al., 2010). The modified procedure requires that the experimental mouse is housed in a home cage to establish territory ownership (resident) and is subsequently subordinated in his home cage by an intruder CD1 male (Bartolomucci et al., 2010). Resident mice experience thus loss of territory ownership and lowering of social status which has been demonstrated to induce the deepest effects of chronic stress exposure in mice (Bartolomucci et al., 2005; Bartolomucci 2005, 2007 for review). Three-month old 129SvEv males (n = 31) were individually housed in Plexiglas cages (38 × 20 × 18 cm) for a 7 days baseline period. The three week stress procedure started after baseline, when each resident mouse received in its home cage a same-age CD-1 male mice as intruder (n = 31). After ten minutes of interaction, the two animals were separated by means of a wire mesh partition, which allowed continuous sensory contact but no physical interaction. Between 10:00 and 12:00 the partition was removed daily for a maximum of 10 min. Each 129SvEv-CD1 dyad was stable across the entire stress procedure. The social status was determined as follows: the chasing and biting animal was defined as ‘Dominant’ (129SvEv, N = 8 and CD1, N = 23; see Results section), while the mouse

Locomotor activity was monitored continuously while core body temperature was determined on a daily base. Body temperature was recorded always at 10:00 by using temperature-sensing subcutaneous transponders (Bio Medic Data Systems, Seaford, DE, USA). Sensors were implanted at least 15 days before the beginning of the experiment according to manufacturer instructions and previous experiments (Bartolomucci et al., 2009). Locomotor activity in the home cage was monitored by means of an automated system that uses small passive infrared sensors positioned on the top of each cage (TechnoSmart, Rome, Italy) as previously described (Bartolomucci et al., 2009). 2.4. Novel/palatable food test (NPF) Mice were presented in the home cage with a novel but highly palatable food, i.e. half a peanut, on a petri dish once a day for 4 consecutive days (days 12–15) starting at 9:00. On day 4, when the latency to ingest the palatable food was expected to be decreased because of habituation to novelty and hedonic response to the palatable food, the peanuts were presented in a normal housing cage in which bedding was changed (following the rationale previously established for the novelty induced suppression of feeding test for anxiety (Merali et al., 2003)). Latency to eat the peanut was recorded with a cut-off time of 600 s. If mice did not eat the peanut after 600 s the peanut was left in the cage. NPF test was scheduled daily at 9:00 to avoid a direct effect of aggression received and far away from circadian maximum of food intake which occurs before lights off in mice.


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Fig. 1. Experimental protocol and aggressive behavior. (A) A schematic overview of the experimental procedure of chronic psychosocial stress as well as the timetable and list of parameters assessed. (B) Attacks on day 1 when CD1 are introduced in 129SVEv home cage. On day one, 129SvEv males showed higher aggressive score when compared to intruder CD1 mice (N = 31). (C). Aggressive behavior along the entire stress phase. By day 4 all dyads established a stable social hierarchy and no subordinate 129SvEv mice (N = 23) showed any aggressive behavior toward their CD1 intruder. ** p b 0.01, § p b 0.06.

2.5. Elevated Plus Maze test The test was conducted at day 17 between 17:00 and 19:00 PM. A mouse was removed from the home cage and placed in the centre of the apparatus for a 5 min test. Conventional behaviors were scored by means of The Observer (Noldus, The Netherlands). These comprised the frequency of open- and closed-arm entries (arm entry defined as all four paws into an arm), total arm entries, and the amount of time spent by the animals in open, central, and closed parts of the maze. These data were used to additionally calculate percent arm entries ((frequency location / total) × 100) and percent time spent in different maze sections ((time location / 300) × 100). 2.6. Social approach test The social approach test was performed between 16:00 and 19:00 on day 20 of the stress procedure. The experimental mouse was introduced into a standard housing cage (38 × 20 × 18 cm) which was

unfamiliar to the experimental mouse, for two consecutive sessions of 2.5 min each (adapted from Berton et al., 2006; Bartolomucci et al., 2010. See Fig. 7A). During the first session (T1, “No target”) the open field contained an empty wire mesh cage (10 cm diameter) located at one end of the field. During the second session (T2, “Target”), a same-age unfamiliar CD-1 male mouse was introduced into the cage. Between sessions the experimental mouse was placed back into its home cage for approximately one minute. Locomotion was quantified with a video tracking system (Ethovision, Noldus, Wageningen, Netherlands) by dividing the arena into 3 zones (see Fig. 7A): the interaction zone (region 5 cm from the target wire mesh cage), the avoidance zone (square regions on the side opposite to the target measuring 5 × 20 cm) and the neutral zone (space not included in either the avoidance or the interaction zones). Total dwell time, frequency of entries and latency to enter were recorded. A social preference ratio was computed according to the following formula, x = time in interaction zone / (time in interaction zone + time in avoidance zone). The choice to use a standard housing cage vs. an

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open field test (Berton et al., 2006; Bartolomucci et al., 2010) is based on the observation that 129SvEv males show a persistent freezing behavior in an open field arena (Dadomo, Bartolomucci, personal communication).

doc test. Daily aggression score, body weight, food intake, body temperature, locomotor activity and NPF test were analyzed by means of 2 ways ANOVA for repeated measures followed by Tukey's HSD post doc test. Correlations were performed with Pearson's test. Data are shown as mean± SEM.

2.7. Metabolic parameters 3. Results Body weight was monitored weekly while food intake was monitored every other day. Energy efficiency was calculated by means of the following formula, x = grams of body weight gained along the entire stress phase / total amount of food (in grams) ingested. At autopsy perigonadal fat pad was dissected and weighted. 2.8. Hormonal analysis On day 22 of the stress procedure, mice were not exposed to social defeat and were sacrificed between 10:00 and 12:00. Trunk blood was collected in heparinized tubes, centrifuged at 4000 rpm for 10 min and plasma was frozen at −20 °C for later analysis. Levels of circulating corticosterone were measured in duplicate with a commercially available RIA kit (Diagnostic Systems Laboratories, Inc., USA) with a sensitivity of 0.06 ng/ml. The intra-assay variability was 3.4%. Levels of circulating ACTH were measured in duplicate by a commercially available RIA kit (MP Biomedical, USA) with a sensitivity of 5.7 pg/ml. The intra-assay variability was 4.5%. To avoid the inter-assay variability all samples were run in a single assay. 2.9. Statistical analysis Food efficiency, perigonadal fat pad weight, EPM and social approach tests related parameters were analyzed by means of independent Student's t-test. Attacks on day 1 and hormone concentrations were analyzed by means of 1 way ANOVA followed by Tukey's HSD post

3.1. Aggressive behavior On day 1, resident 129SvEv males showed remarkable aggression toward the intruder CD1 male (F(3,58) = 10.2, p = 0.00002; Fig. 1B). Despite higher aggressive scores on day 1, 23 out of 31 129SvEv males became subordinate in subsequent days (F(15,290) = 6.8, p = 0.000001). A small proportion (8/31) of 129SvEv males was able to achieve and maintain a dominant social rank. Overall, dominant 129SvEv mice were more aggressive than dominant CD1 mice on day 1 while the reverse occurred in subsequent days (Fig. 1C). The present paper aims at establishing vulnerability to chronic subordination stress in 129SvEv mice, therefore further analyses will focus on subordinate 129SvEv and control groups only. 3.2. Chronic subordination stress increases weight gain, food intake, adiposity, plasma corticosterone and body temperature Physiological phenotyping of experimental mice included body weight gain, food intake, weight of perigonadal adipose fat pad, plasma HPA-axis hormones concentration and body temperature. Exposure to chronic stress determined a significant increase in body weight when compared to the baseline phase and to the control group (Fig. 2A; F(1, 51) = 27.7, p b 0.00001). Subordinate 129SvEv mice also showed hyperphagia (Fig. 2B; F(1, 31) = 39.2, p b 0.00001), higher energy efficiency (Fig. 2C; t23,30 = 3.9, p b 0.001) and a slight

Fig. 2. Metabolic parameters. Subordinate 129SvEv mice showed increased weight gain (A; determined at day 21) and food intake (B) across the entire experimental phase when compared to baseline phase or controls. Overall 21-days energy efficiency (C) and perigonadal fat pad weight (D) determined at sacrifice were significantly increased in subordinate 129SvEV mice when compared to controls. Subordinate, N = 23, Controls N = 30. ***p b 0.0001, *p b 0.05 vs. controls.


but significant increase in perigonadal adipose fat pad weight when compared to controls (Fig. 2D; t23,30 = 1.8, p b 0.05). Body weight, food intake and adipose fat pad weight were not affected by the amount of aggression received. On the contrary, basal corticosterone but not ACTH was significantly associated with the amount of aggression received by subordinate males along the experimental phase (Fig. 3A; r = 0–77, p b 0.00001). Therefore, we performed a median split according to the amount of attacks received and distinguished high-aggressionreceived (HA, black dots in Fig. 3A) and low-aggression-received (LA, empty dots in Fig. 3A) groups. Corticosterone but not ACTH was significantly increased in HA but not in LA stressed mice (Fig. 3B,C; F(2, 28) = 8.1, p b 0.01). Exposure to chronic stress also determined a transient but significant increase in core body temperature in the first week of the experimental phase when compared to the baseline phase and the control group (Fig. 4; F(3,87) = 4.2, p b 0.01). Hyperthermia in the first week was positively correlated with amount of aggression received in days 1–4 (r = 0.7, p b 0.001) but not with the amount of attacks performed in the same days (r = 0.1, NS). This suggest that hyperthermia was not determined by the individual coping style but a direct consequence of aggression received and overall stressfulness of the experimental procedure. 3.3. Chronic subordination stress depresses locomotor activity Home cage phenotyping encompassed 22 h (time required for daily aggression excluded) continuous scoring of locomotor activity to assess psychomotor disturbance (Fuchs, 2005; Bartolomucci et al.,

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Fig. 4. Body temperature. Subordinate 129SvEv mice showed a transient but significant stress-induced hyperthermia during the first week of the experimental procedure. Subordinate, N = 23, Control N = 30. **p b 0.01 vs. Baseline and same week of Control group.

2005). Depression of locomotor activity in the dark phase was dependent upon the amount of aggression received (r = −0.51, p b 0.05) which suggests that the greater the stressfulness of the environment, the greater the behavioral and motivational consequences. Indeed, subordinate 129SvEv mice exposed to high level of aggression but not mice exposed to low level of aggression showed long lasting reduction of home cage locomotor activity in the dark phase (Fig. 5AB; F(3, 57) = 10.0, p = 0.00002). Amplitude of circadian

Fig. 3. Hormonal analysis. (A) There was a positive correlation between basal plasma corticosterone and mean daily number of aggression received. No correlation emerged between plasma ACTH and aggression. (B) Basal plasma corticosterone was increased in subordinate 129SvEv mice receiving high aggression but not in mice receiving low aggression. (C) ACTH was not affected by chronic stress. Hormones level was determined at sacrifice. Subordinate, N = 22, Controls N = 11. **p b 0.01 vs. controls.

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Fig. 5. Home cage locomotor activity. Stress-induced suppression of locomotor activity was dependent upon the amount of daily aggression received. (A, higher panel) Subordinated mice exposed to low level of aggression showed no change in locomotor activity. (B, higher panel) Subordinate mice exposed to high level of aggression showed a significant and long lasting reduction of locomotor activity in the dark phase (A, the active phase for mice, when compared to baseline (Bas). shows a daily rhythm of locomotor activity in baseline and stress phase for subordinate mice receiving Low or High aggression. Panel B shows mean. (C) Subordinated mice exposed to high level of aggression but not to low level of aggression showed a transient reduction in circadian rhythmicity of activity (Dark/light phase) that did not reached statistical significance. Activity score = frequency of infrared brakes. N = 23, **p b 0.01, *p b 0.05 vs. Baseline.

rhythm of locomotor activity (dark/light) was also significantly reduced in subordinate 129SvEv mice (Fig. 5C). 3.4. Chronic psychosocial stress increases anxiety, social withdrawal and anhedonic-like response to a palatable food Behavioral phenotyping encompassed fear related parameters such as anxiety-like state (Kalueff et al., 2007), behavioral response to

a palatable but novel food (Anisman & Matheson, 2005) and social avoidance (Berton et al., 2006; Stein et al., 2001). Anxiety-like state was determined by means of the standard EPM test. Exposure to chronic stress determined a significative reduction of frequency of entries and time spent in the aversive open arms of the EPM (Fig. 6AB; frequency: t23,30 = 2.1, p b 0.05; duration: t23,30 = 2.2, p b 0.05), which is conventionally interpreted in favor of increased anxiety-like state (Rodgers and Dalvi, 1997). Aggression received did not affect anxiety


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Fig. 6. Elevated plus maze and novel/palatable food test. (A,B) Subordinate 129SvEv mice showed increased anxiety in the EPM as demonstrated by a significant decrease in entries and time spent into open arms. Subordinate, N = 23, Controls N = 30 (C) Schematic overview of the NPF test. Subordinate 129SvEv mice showed a persistent and not habituating higher latency to eat the peanut in the NPF test. Subordinate, N = 11, Controls N = 15. * p b 0.05 vs. controls.

in the EPM. Frequency of closed-arm entry (Fig. 6A) or total frequency of arms entry (Control = 8 ± 2, Subordinates = 10 ± 2, NS) were not affected by stress exposure. Response to a novel but palatable food (NPF test) was determined in the home cage (Fig. 6C). Exposure to chronic stress determined a long lasting and not-habituating inhibition to eat the peanut across the 4 days of the experimental procedure (Fig. 6C; F(1,26) = 8.2, p b 0.01). We interpret this finding in favor of fear-induced anhedoniclike response toward a palatable food. In the social approach test, mice were first exposed for 2.5 min to a home cage sized arena containing an empty wire mesh cage (T1). After 1 min interval, mice were placed again in the apparatus for 2.5 min (T2). The wire mesh cage now contains an unfamiliar CD1 male (Fig. 7A). Subordinate 129SvEv mice showed avoidance of the CD1 male as demonstrated by a higher latency to reach the interaction zone in T2 (Fig. 7B; t18,11 = −2.9, p b 0.01), increased time in the avoidance zone (Fig. 7C; t18,11 = 4.2, p b 0.001), a tendency for lower time in the interaction zone (Fig. 7C; t18,11 = 1.8, p = 0.07) and a lower avoidance/preference ratio in T2 (Fig. 7D; t18,11 = 2.4, p b 0.05). Time spent (Control = 119.9 ± 5.0 s, Subordinates = 110.1 ± 11.2 s, NS) and entries (Control = 7.3 ± 0.7, Subordinates = 6.0 ± 0.8, NS) into the interaction zone during T1 were not affected by stress exposure. This allows ruling out a primary effect of locomotion and the use of space in T1 (preference vs avoidance of the interaction zone) on measures of social avoidance in T2. Aggression received and avoidance/preference ratio in T2 showed a tendency for negative association (r = 0.46, p b 0.1). This finding can be interpreted as increased social anxiety and social withdrawal induced by chronic social subordination, which might be particularly severe in mice exposed to high level of aggression (Bartolomucci et al., 2010). Finally, total distance moved in

T1 and T2 was significantly lower for Subordinate mice when compared to Controls (T1, Control = 567 ± 31 cm, Subordinate = 397 ± 70 cm, NS; T2, Control = 400 ± 30 cm, Subordinate = 243 ± 45, NS), thus confirming the stress-induced psychomotor disturbance observed in the home cage phenotyping. 4. Discussion The main finding of the present study is that 129SvEv male mice are remarkably vulnerable to chronic subordination stress-induced behavioral and physiological alterations. Specifically, subordinate 129SvEv males showed depression of home cage locomotor activity, increased anxiety-like state, avoidance of palatable food and increased social avoidance of an unfamiliar same-sex mouse. Subordinate 129SvEv mice also showed increased body weight gain and basal plasma corticosterone as well as hyperphagia. Overall, subordinate 129SvEv mice showed similar or greater stress-induced behavioral and physiological consequences when compared to CD1 and C57BL/6J mice under the same experimental conditions (Bartolomucci et al., 2001, 2004, 2005, 2009, in press; Table 1). CD1, C57 and 129SvEv strains exposed to chronic subordination stress showed similar weight gain and hyperphagia when fed a standard diet. However, the 129SvEv was the only strain to show a slight but significant hyperplasia of the perigonadal adipose fat pad under standard diet (Bartolomucci et al., 2009; Cero et al., unpublished). This result is in apparent contrast with previous reports showing that 129 mice were resistant to obesity induced by several weeks of high fat diet when compared to C57BL/6 or mixed 129 × C57 strains (Almind and Kahn, 2004; Almind et al., 2007; Su et al., 2008; Donovan et al., 2009; Mori et al., 2010). On the other hand, C57BL6/J mice were reported to have higher autonomic

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Fig. 7. Social approach test. (A) Schematic overview of the experimental apparatus and procedure. Subordinate 129SvEv mice showed increased social avoidance of the intruder CD1 in the T2 as demonstrated by (B) increased latency to enter the interaction zone, increased time spent in the avoidance zone (C) and a tendency for lower time spent in the interaction zone (C). Subordinate 129SvEV mice showed a significant decrease of the social approach/avoidance parameter, which take into account time spent in the interaction and avoidance zone of the arena (implicating increased avoidance of the interaction zone) in T2 when the intruder CD1 was present in the wire mesh cage. Subordinate, N = 11, Controls N = 18. **p b 0.01, *p b 0.05, §p b 0.07 vs. controls and T1 in panel D.

stress responsivity when compared to the 129SvEv strain (van Bogaert et al., 2006), which is usually associated with shrinkage of adipose organ (Almind and Kahn, 2004; van Bogaert et al., 2006; Morrison et al., 2008; Bartolomucci et al., 2009). Therefore, while C57BL6/J might be more vulnerable than 129 strains under basal condition, stress and autonomic activation will reverse this phenotype by making “the resilient” 129SvEv strain vulnerable to obesity and “the vulnerable” C57BL6/J resilient. Further studies should validate this

hypothesis by comparing the vulnerability to obesity of 129SvEv and C57BL6/J mice exposed to chronic subordination stress and high fat diet (Bartolomucci et al., 2009). In the present study, 129SvEv mice exposed to high level of aggression showed increased basal plasma corticosterone. Specifically, there was a strong positive correlation between the average number of attack bouts received and the basal plasma corticosterone level with a minimal overlap between values from mice receiving high and

Table 1 Chronic subordination stress-induced effects in male mice fed standard chow diet: comparison between 129SvEV, C57BL/6J and Swiss CD1. Strain 129 SvEv Low aggression received High aggression received C57BL6/Ja Low aggression received High aggression received Swiss CD1c,d

Body weight gain

Fat pad weight

Feeding

Body T

HPA-axis

Locomotor activity

Anxiety (EPM)

Social avoidance

Anhedonia

↑ ↑

↑ ↑

↑ ↑

↑ ↑

↔ ↑

↔ ↓

↑ ↑

↑ ↑

↑ ↑

↑ ↑ ↑

↓b ↓b ↔

↑ ↑ ↑

↑ ↑ ↑

↑ ↑ ↑

↔ ↓ ↓

NA NA ↔

↔ ↔ NA

NA NA NA

↓ decreased, ↑ increased, ↔ not affected, when compared to controls. NA, not available. a These data refers to wild type C57BL/6J from Bartolomucci et al., 2010. b Unpublished, from Cero et al., 2010. c Data from Bartolomucci et al., 2001, 2003; 2004, 2009. d Level of aggression as a potential modulating factor, has not been established for CD1 male mice.


low level of daily aggression (Fig. 3A). Therefore, increased plasma corticosterone can be considered a direct measure of perceived stress for 129SvEv males. The lack of increase in plasma ACTH might be interpreted in favor of an exaggerated adrenocortical sensitivity to ACTH previously demonstrated for the 129 strain (Peinado et al., 2005). The 129 strains lineage is characterized by marked fear and high trait anxiety (Rodgers et al., 2002; Dulawa et al., 2004; Salomons et al., 2010; Chourbaji et al., 2010). Chronic subordination stress clearly aggravated anxiety-like state as demonstrated by the lower time spent in the aversive open arms of the EPM (e.g. Rodgers and Dalvi, 1997). Support to the conclusion of a stress-induced anxiogenic effect also derives from the other behavioral tests we used in the present study which are both characterized by a clear fear motivated anxiety component, i.e. the NPF test and the social approach test (Berton et al., 2006; Anisman and Matheson, 2005). Indeed, subordinate 129SvEv mice showed a persistent avoidance of a novel but palatable food. Anhedonia is a hallmark of major depressive disorders and several animal models have been established so far (Willner et al., 1992; Anisman & Matheson, 2005; Rygula et al., 2005; Schweizer et al., 2009). Here we used a novel, minimally invasive and procedurally simple test based on the rationale that rodents have a clear preference for lipid-rich foods, such as peanuts (e.g. Gaillard et al., 2008). Control mice showed a consistent daily reduction in the latency to eat the peanut. The latency increases again when the peanut is presented in a clean bedding-filled cage in the T4 (Fig. 6C; Merali, et al., 2003; Anisman and Matheson, 2005; Dulawa and Hen, 2005). On the contrary, subordinate 129SvEv mice showed minimal or no habituation over time and persistently (~70% of mice tested) failed to ingest the peanuts within the cut-off time of 600 seconds. Unpublished observations confirmed that stress-induced effects in the NPF test might be specific to the 129SvEv strain. Indeed subordinate CD1 or C57BL6/J mice showed no or minor food avoidance under the same experimental conditions (Dadomo et al., 2011). In the same unpublished study, we also showed that while subordinate 129SvEv mice showed food avoidance in the NPF test, they showed no avoidance of a sucrose solution in the sucrose preference test (Dadomo et al., 2011). Further pharmacological studies should demonstrate predictive validity for the NPF test as a test of depression associated anhedoniclike state. Finally, subordinate 129SvEv mice also showed a remarkable avoidance of a social partner in the social approach test, which is considered a direct measure of increased social anxiety and social withdrawal (Berton et al., 2006; Krishnan et al., 2007; Covington et al., 2009; Bartolomucci et al., 2010). Social avoidance exhibited by stressed 129SvEv males may be related to decreased motivation for social interaction or increased fear of social threat (Hefner et al., 2008 ) seen as a syndromal dimension of depression (Berton et al., 2006; Stein et al., 2001). Subordinate 129SvEv mice exposed to high level of aggression also showed suppression of home cage spontaneous activity and a reduction of the amplitude of the circadian rhythm of activity which are considered an equivalent of psychomotor disturbances seen in depressed patients (Willner et al., 1992; Fuchs et al., 1996; Meerlo et al., 1996), while mice exposed to low aggression were fully resilient and showed no reduction of activity. This finding fully replicate previous data obtained in C57BL/6J mice (Bartolomucci et al., 2010), thus confirming that high level of aggression received is necessary to induce psychomotor disturbances in subordinate mice. Anxiety and cognitive functions have been widely investigated in the 129SvEv lineage (Rodgers et al., 2002; Dulawa et al., 2004; Salomons et al., 2010; Chourbaji et al., 2010; Hefner et al., 2008; Camp et al., 2009; Ralph et al., 2001). On the contrary, social behavior and stress vulnerability received limited attention. In the present study we demonstrated that adult 129SvEv male mice show a remarkable territorial aggression against intruder males of the highly aggressive and physically heavier (~40% larger body weight) CD1

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strain (Parmigiani et al., 1999). Specifically, on day one resident 129SvEv males showed higher aggressive behavior when compared to intruder CD1 males. In addition, 8 out 31 resident 129SvEv males became overtly dominant against their intruder CD1. These results are in striking contrast with the phenotype previously observed for C57BL6/J mice. C57BL6/J males did not show higher aggressive score on day 1 than intruder CD1, nor become dominant in the subordination stress protocol (Bartolomucci et al., 2010). Previous work from our laboratory established that sustained defense and subsequent loss of territory ownership and hierarchical rank lowering, is a precipitating factor for individual vulnerability to stress-induced disorders (Bartolomucci 2005; Bartolomucci et al., 2005). Accordingly, we hypothesize that the remarkable vulnerability showed by 129SvEv males might be due to their inability to maintain territorial ownership toward same-sex intruders and consequent lowering of social status. It is remarkable that high level of daily aggression received, a direct measure of perceived stress (Bartolomucci et al., 2010), only affected a few selected parameters in subordinate 129SvEv male mice, i.e. plasma corticosterone level and locomotor activity, while leaving the other parameters unaffected. Accordingly, while 129SvEv mice exposed to high level of aggression showed a stress-vulnerable phenotype, 129SvEv mice exposed to low level of aggression were at least in part stress-resilient. This conclusion is partially at variance with our previous study in C57BL/6J mice. Irrespective of aggression received, C57BL/6J mice showed persistent stress-induced physiological changes. On the contrary, stress-induced behavioral effects (depressed locomotor activity and social avoidance) were only evident in C57BL/6J mice exposed to high level of aggression but not in mice exposed to low level of aggression (Table 1; Bartolomucci et al., 2010). Overall, this indirect strain comparison established a previously unrecognized difference in stress vulnerability between 129SvEv and C57BL/6J strains. Present knowledge does not allow interpreting this strain-dependent sensitivity to chronic subordination stress but highlights a novel experimental tool to disentangle the complex mechanisms of individual resilience to stress (Krishnan et al., 2007; Schmidt et al., 2008; 2010; Feder et al., 2009). 5. Conclusion The 129 strain lineage has a long, complex and confusing history after it has been first established by Leslie Dunn at Columbia University in 1928 (Simpson et al., 1997; Threadgill et al., 1997). In the vast majority of gene targeting experiments, embryonic stem cells are derived from any of the 129 mouse substrains (Simpson et al., 1997; Scheel et al., 2003). However, the targeted gene deletion obtained in 129 stem cells is often subsequently backcrossed in other strains, e.g. C57BL6/J, which is better phonotypically characterized. It would be of great advantage to investigate the effect of the gene targeting directly in the stem cells donor strain, i.e. the 129 (see also Chourbaji et al., 2010 for additional discussion). The present study is the first to establish vulnerability to chronic social stress. Overall, present data demonstrate that 129SvEv male mice are remarkably vulnerable to subordination stress-induced depression-like disorders and physiological comorbidities and might represent an ideal animal model to investigate underlying psychological and molecular mechanisms. Our psychosocial stress procedure in which a mouse is housed in continuous sensory contact with a dominant male interspersed by direct daily social defeats is likely to tap into similar social stress mechanisms as those implicated in human depression (Bartolomucci 2007; Sapolsky 2005). Remarkably, sustained suppression of locomotor activity, increased anxiety, anhedonia, body weight gain, corticosterone level and body temperature are all found both in animals exposed to chronic psychosocial stress and individuals with major depression (Malkesman et al., 2009; Cryan and Mombereau, 2004; Avgustinovich et al., 2005; Koolhaas et al., 1997; Fuchs, 2005; Sapolsky 2005; Bartolomucci 2005, 2007; Caspi et al., 2003; Kalueff et al., 2007;

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