Developmental Psychobiology

Mark J. Cumming1 Madison A. Thompson1 Cheryl M. McCormick1,2 1

Centre for Neuroscience Brock University 500 Glenridge Avenue, St. Catharines, Ontario, Canada, L2S 3A1 2

Department of Psychology Brock University 500 Glenridge Avenue, St. Catharines, Ontario, Canada, L2S 3A1 E-mail: [email protected]

Adolescent Social Instability Stress Increases Aggression in a Food Competition Task in Adult Male Long-Evans Rats ABSTRACT: Adolescent social instability stress (SS; daily 1 hr isolation þ new cage partners postnatal days 30–45; thereafter with original cage partner, also in the SS condition) and control (CTL) rats competed for access to a preferred food in five sessions against their cage partner. In the first session, SS pairs displayed more aggression (face whacks, p ¼ .02; rear attacks, p ¼ .03), were less likely to relinquish access to the food voluntarily (p ¼ .03), spent more time at the feeder than CTL pairs (p ¼ .06), but did not differ in latency to access the feeder (p ¼ .41). Pairs were considered in dominant–submissive relationships (DSR) if one rat spent significantly more time at the feeder than the other; 8 of 12 SS and 8 of 12 CTL pairs displayed DSRs (remaining: no-DSR). Aggression increased from the 1st to 5th session (p < .001), was greater in no-DSR than DSR pairs (p ¼ .04; consistent with the proposed function of DSRs to be the reduction of aggression in groups), and was higher in SS than CTL pairs (p ¼ .05). Because the increased aggression of SS compared with CTL pairs did not result in a significant increase in their time at the feeder, the increased aggression may be considered maladaptive, and may reflect an increased motivation for food reward. These results add to evidence that SS in adolescence modifies the adult social repertoire of rats and highlight the importance of adolescent social experiences for adult behavior. ß 2014 Wiley Periodicals, Inc. Dev Psychobiol 56: 1575–1588, 2014. Keywords: adolescence; early experiences; social; stress; rats; dominance; aggression

INTRODUCTION Adolescence is the life stage that involves the transition from childhood to adulthood and is a time of heightened maturation and development of many systems,

Manuscript Received: 11 June 2014 Manuscript Accepted: 5 August 2014 Correspondence to: Cheryl M. McCormick, Canada Research Chair in Neuroscience, Department of Psychology and Centre for Neuroscience, Brock University, 500 Glenridge Ave, St. Catharines, Ontario, Canada L2S 3A1. Contract grant sponsor: Natural Sciences and Engineering Research Council (NSERC) of Canada Contract grant sponsor: Canadian Foundation for Innovation (CFI) (PI: CMM) Article first published online in Wiley Online Library (wileyonlinelibrary.com): 1 September 2014 DOI 10.1002/dev.21252  ß 2014 Wiley Periodicals, Inc.

including the CNS (Blakemore, 2012; Sturman & Moghaddam, 2011). Parallels in the behavioral characteristics of adolescent rats and humans such as increased novelty-seeking, heightened emotional reactivity, risk-taking, and impulsivity, point to the value of animal models in generating hypotheses of relevance for human development (McCormick, Hodges, & Simone, 2014). Adolescence is an important time of social learning and of social restructuring in both rats and humans. For example, in humans, adolescence involves a shift away from family relationships toward peer relationships (Nelson, Leibenluft, McClure, & Pine, 2005), and interactions with peers increase in adolescence in rats as they expand their territorial range beyond the nest (Trezza, Baarendse, & Vanderschuren, 2010). Further, the reward value of social interactions in rats is greater in adolescence than in adulthood (e.g.,

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Calcagnetti & Schechter, 1992; Douglas, Varlinskaya, & Spear, 2004). Rats engage in more social play in adolescence than in adulthood, with the peak of social play occurring between postnatal days 30 and 42 (early to midadolescence; pre-puberty) (Pellis, Field, Smith, & Field, 1997). Social play in adolescence rehearses skills necessary for reciprocal social exchanges in adulthood (Pellis & Pellis, 2007). Deprivation of social interactions, and particularly of social play, in adolescence impairs adult social behavior (Bulygina, Amstislavskaia, & Maslova, 2005; Hol, Van Den Berg, van Ree, & Spruijt, 1999; Meng, Li, Han, Shao, & Wang, 2010; van den Berg et al., 1999), although there is one report of better sexual performance in isolated-housing male rats than in pair-housed male rats (Swanson & van de Poll, 1983). The loss of social experiences disrupts the development of structures critical for social interactions such as the medial prefrontal cortex and hippocampus (Baarendse, Counotte, O’Donnell, & Vanderschuren, 2013; Bell, Pellis, & Kolb, 2010; Bianchi et al., 2006; Fone & Porkness, 2008; van Kerkhof, Damsteegt, Trezza, Voorn, & Vanderschuren, 2013). Although social isolation is not without consequences when experienced in adulthood, the effects of such deprivation are longer lasting and/or more severe when experienced in adolescence (e.g., Einon & Morgan, 1977; Hall, 1998; Lukkes, Mokin, Scholl, & Forster, 2009). Further, adolescents may be particularly sensitive to social experiences because of ongoing maturation and heightened plasticity of the nervous system at this stage of development compared with adulthood (Buwalda, Geerdink, Vidal, & Koolhaas, 2011). Most studies of the consequences for adult social behavior of adolescent social experiences have involved social deprivation by housing rats in isolation for lengthy periods. Although there are numerous studies that have manipulated, rather than eliminated, adolescent social experiences, most of these have focussed on the lasting effects on emotional and cognitive behavior instead of social behavior, and most have cast the manipulation as a form of social stress (see reviews by Green & McCormick, 2013b; McCormick & Green, 2013). Nevertheless, the available evidence suggests that the quality, and not just the presence or absence, of social experiences in adolescence also affects social development. For example, the experience of social defeat by adolescent males through interactions with an aggressive, larger adult, increased play (but in a more submissive form) with the adolescent’s cage partner, and rendered the adolescent better able to cope with aggressive encounters in adulthood (Buwalda, Stubbendorff, Zickert, & Koolhaas, 2013). Although these findings suggest resilience was conferred by such

Developmental Psychobiology

adolescent experiences, such a result is likely situationspecific; the same rats may fare less well in adulthood than control rats on other measures. The results, however, may be attributed to isolation, which typically occurs after the social defeat experience (e.g., de Jong, van der Vegt, Buwalda, & Koolhaas, 2005). We have manipulated social experiences in adolescence by briefly isolating rats for 1 hr followed by a pairing with a new cage partner that also is undergoing the isolation procedure daily from postnatal Day 30 to postnatal Day 45 (mid-adolescence). Although this social instability stress (SS) procedure might be interpreted as a form of enrichment based on the high reward value of social interactions in adolescence, the finding of reduced performance on hippocampal-dependent tasks of SS rats when tested long after the termination of the SS procedure (Green & McCormick, 2013b; McCormick, Nixon, Thomas, Lowie, & Dyck, 2010; McCormick et al., 2012) suggests vulnerability rather than resilience. SS rats also differed from control rats in their social behavior in adulthood. Compared with controls, SS rats showed deficits in mating behavior (specifically, reduced copulatory efficiency) and were more likely to fit the definition in the literature of “sexually sluggish” (McCormick et al., 2013). SS rats were less likely than control rats to initiate social interactions with an unfamiliar peer in an arena to which they have been habituated, especially when the unfamiliar peer was another SS male (Green, Barnes, & McCormick, 2013). When the unfamiliar peer was behind wire mesh thereby limiting physical interactions, SS rats spent about 70% of the time in proximity to the peer, as did control rats, suggesting that social instability stress in adolescence did not make rats socially avoidant. Instead, social instability stress may have altered the development of an appropriate social repertoire. To date, our investigations of social behavior in adulthood after social instability stress involved interactions with unfamiliar conspecifics outside the housing cage, although we have examined behavior of pairs in the housing cage during the SS procedure in adolescence. The behavior of rats returning to a new cage partner after each daily, 1 hr isolation did not differ markedly from that of rats returning to a familiar partner after isolation, and aggressive interactions between either familiar or unfamiliar cage partners were a rarity (McCormick, Merrick, Secen, & Helmreich, 2007). Nevertheless, differences in behavior between SS and control rats might emerge under conditions of competition. To further investigate the hypothesis that social instability stress in adolescence alters the development of the adult social repertoire, we investigated behavior

Developmental Psychobiology

in a food competition task against a familiar cage mate. Competitions for food or water are used as measures of social dominance in rodents (e.g., Barnum, Blandino, & Deak, 2008; Hoshaw, Evans, Mueller, Valentino, & Lucki, 2006; Parent, Del Corpo, Cameron, & Meaney, 2013). Social dominance is common in social species and involves the formation of a dominant–submissive relationship (DSR) between a dyadic pair. The definition of dominance varies, but most definitions are based in recognition of a stable asymmetrical social relationship, whereby one animal in the pair consistently wins competitive interactions and/or has priority of access to resources (see reviews by Chase & Seitz, 2011; Drews, 1993). Behavioral observations of wild rats under seminatural conditions suggest there is ecological validity to food competition tasks; higher status rats have priority access to foods, and rats of lower status suffer more than higher status rats from the negative consequences of food shortage (reviewed in Berdoy, Smith, & MacDonald, 1995). When food is not scarce, subordinate rats’ access to food involves different behavioral strategies (e.g., reduced duration of feeding bouts, approach food at times in the day that are less preferred) than those of dominant rats, which serves to minimize aggressive interactions that might otherwise be required for food access (Berdoy, 1994). DSRs emerge after puberty in rats; social interactions tend to be symmetrical before puberty (Adams & Boice, 1989; Pellis & Pellis, 1990). Although the SS procedure ends around the time that DSRs typically begin to form, the previous social instability stress may alter the formation of DSRs. There is evidence that experiences at earlier stages of life than adolescence alter dominance behavior in adulthood. For example, in a water competition test in adulthood, rats exposed to daily mild stress exposures during the first 2 weeks of life had greater access to the water spout than did control rats (Gonza´lez Jatuff, Bera´stegui, Rodrı´guez, & Rodrı´guez Echandı´a, 1999), and rats that received less maternal licking and grooming had greater access to the water spout than did rats that received more maternal licking and grooming (Parent et al., 2013). A study of social instability restricted to the post-pubertal period (weekly changes in cage partners of triads of rats for 10 weeks beginning on postnatal Day 45) found that rats that underwent social instability had increased aggressive behavior toward an intruder compared to rats from a stable social group, but the increased aggression in rats from the social instability groups was only in comparison to the subordinate rats from stable groups, not the dominant rats from stable groups (Lore & Stipo-Flaherty, 1984). These studies, however, involved competitions against either unfamiliar rats (Gonza´lez Jatuff et al., 1999; Lore & Stipo-Flaherty,

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1984) or against cage partners that were grouped together in adulthood (Parent et al., 2013). We instead focused our initial study on testing cage partners that had been together since postnatal Day 22 onward, with the proviso that for SS rats, the time with their original cage partner was interrupted between postnatal days 30 and 45 to introduce new cage partners daily. This focus was because dominant versus submissive status may be an important moderator of the effects of social instability stress in adolescence (e.g., dominant SS rats may be more resilient to, or recover more readily from, the procedure). For example, dominant versus submissive status within cage partners was associated with immune function and physiological responses to stressors (Barnum et al., 2008). Thus, knowledge of dominance-submissive status of pairs of cage mates may explain variability in the consequences of social instability stress. The first step, however, is to investigate the extent to which DSRs form and how they are manifested as a consequence of the social instability stress procedure. We predicted that SS pairs of rats would show more aggression during the food competition than would control pairs based on our hypothesis of an impoverished social repertoire after social instability stress. A posited function of DSR relationships is to reduce overt aggression and thereby minimize the energy expenditure necessary to acquire resources, especially when limited (e.g., Hillman & Bilkey, 2012; Weiss, King, & Enns, 2002); thus increased aggression between pairs during a competition might be considered maladaptive.

METHODS Animals Male Long-Evans rats (n ¼ 48) (Charles River, St. Constant, Quebec, Canada) were shipped to the Brock University colony at postnatal Day 21 (PND 21). Animals were shipped at the time of weaning when they would be undergoing the stress of change in housing that occurs at weaning [e.g., movement of cage to another housing room is sufficient to elevate HPA function for at least 24 hr in adults (Olfe, Domanska, Schuett, & Kiank, 2010)] to minimize effects of shipping. In addition, lasting effects of shipping stress on reproductive function in mice occurred when shipped postpubertally in late-adolescence (PND 42 or 56), but not when shipped at younger (PND 21 or 28 days of age) or older (70 days, adults) ages (Ismail, Garas, Blaustein, 2011; Laroche, Gasbarro, Herman, & Blaustein, 2009a,b). In rats, weaning at 30 days of age had a greater effect on adult HPA function than did weaning at 21 days of age (Cook, 1999). There was no difference in behavioral responses to amphetamine in mid-adolescent rats reared in our colony compared with those obtained from a breeder at PND 21 (Mathews, Kelly, & McCormick, 2011).

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Rats were pair-housed in a 30  33  18 cm home cage with free access to rat chow and water. The colony room was on a 12:12 hr light cycle (lights on at 08:00 am). From PND 45 to 59, males were gradually habituated to a 12:12 hr reverse light cycle (lights off at 10:00 am) and remained housed under the reverse light cycle throughout behavioral testing so that behavioral testing could be conducted during the dark phase of the rats’ while allowing experimenters to test during the daytime. All experimental procedures were in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85–23, revised 1996) and guidelines established by the Canadian Council on Animal Care, and were approved by the Brock University Animal Care and Use Committee. Figure 1 provides a timeline of all experimental procedures.

Developmental Psychobiology ventilated plastic container, daily from PND 30 to 45 (midadolescence). Isolation occurred at a variable time each day during the light phase to reduce habituation to the procedure. After each daily isolation, SS males were pair-housed with a new cage partner that also was undergoing the SS procedure. After the final isolation on PND 45, SS males returned to their original cage partner. From PND 30 to PND 45, CTL males remained with their original cage partner and were not handled outside of regular cage maintenance and to be weighed on PND 30, PND 45, and PND 59. SS rats were always pair-housed with another SS rat, and CTL rats were pair-housed with another CTL rat. Felt markers were used to colour the tails to identify individuals within a cage. After the last SS procedure on PND 45, cages were dummy coded so that behavioral testing and analysis of test sessions would be conducted blind to experimental condition.

Social Instability Stress (SS) Procedure Rats were randomly assigned to the adolescent social instability stress (SS, n ¼ 24) group or non-stressed control (CTL, n ¼ 24) group. The SS procedure was as described previously (reviewed in McCormick, 2010; McCormick et al., 2014) and involved isolation for 1 hr in a 12  10 cm

Food Competition Sessions

Apparatus. The food competition test apparatus and procedure was based on that of Malatynska and colleagues (Malatynska & Knapp, 2005; Malatynska, Pinhasov, Crooke, Smith-Swintosky, & Brenneman, 2007). The apparatus was

FIGURE 1 A timeline of the procedures across postnatal age (PND ¼ postnatal day) for the two experimental groups.

Developmental Psychobiology constructed of transparent acrylic and consisted of two equally sized 20 cm  16 cm  16 cm chambers connected by a 40 cm  10 cm  10 cm hallway. At the centre of the hallway was a self-refilling feeder filled with sweetened, condensed milk, a highly palatable substance for rats. The feeder had a 1 cm opening—wide enough to provide access to only one rat at a time. Two grooves were cut into the side walls of the hallway for insertion of clear acrylic plates, which allowed rats to be isolated in the chambers before testing and ensured that each male began at an equal distance from the feeder at the start of a testing session. A testing session began upon removal of the acrylic plates.

Procedure. All testing was performed under dim red light conditions and began 1 hr into the dark phase. Rats were habituated individually to the test apparatus in three daily 10 min exploration sessions in the apparatus from PND 59 to 61. The food competition tests began on PND 62 and consisted of five sessions (5 min sessions daily for 5 days). To increase motivation for the sweetened, condensed milk in the feeder, rats were food restricted for 23 hr before each test session. Food access was provided for 1 hr after each session, and water access was provided at all times after sessions. Test sessions were recorded with an overhead camera, and digital copies of all recorded sessions were used for behavioral analysis. Before the start of each test session, opposing males (cage partners) were placed in opposite chambers, enclosed by acrylic plates. At the start of each test session, the acrylic plates were lifted, allowing both males access to the feeder. A pair was considered to have a Dominant–Submissive Relationship (DSR) if there was a significant difference in mean drinking time between the two rats across the five sessions or across the last four sessions (p < .05, one-tailed, pairedsamples t-test). In a DSR pair, the rat with the longer mean drinking time across sessions was labeled the dominant male and the rat the shorter mean drinking time across sessions was labeled the submissive male. If the competing cage partners did not differ in mean drinking time across sessions, they were considered not to have a DSR, and the pair was labeled no-DSR. For the purpose of pair-wise statistical analyses, the no-DSR rat that spent more time drinking at the feeder in the 5th test session (irrespective of statistical significance) was labeled as EQ1 (Equal 1) and the other male as EQ2.

Behavioral Measures. The digital recordings of the 1st and 5th sessions were scored twice, and the 2nd, 3rd, and 4th sessions once. In the first viewing, only time spent drinking at the feeder of each rat was measured, which provided the opportunity to develop a sense of the range of behaviors exhibited so that a scoring system of competitive behavior could be developed for the second viewing. Three main offensive behaviors were evident in the recordings: pins, face whacks, and rear attacks. Pins (one rat is lying on its back while the other rat is above it) are typically characterized as a play behavior, but it also occurs in the context of an attack (Pellis & Pellis, 1987). Pins, however, were infrequent and

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thus were not included in statistical analyses. We use the terms face whacks and rear attacks to describe offensive behaviors used by rats against each other to gain or maintain access to the feeder, and their prominence was likely because of the restricted dimensions of the alley in which the feeder was placed (for example, rats were unable to stand upright). A face whack was defined as one of the pair using its forepaw forcefully to strike the facial region of the other rat. We used the term face whack to differentiate the behavior from paw strikes, which have been defined as the simultaneous use of both forepaws to force a rat away from a feeder by striking in the facial or shoulder region of the drinking rat (e.g., Albert, Dyson, Walsh, & Wong, 1988). An offensive face whack was when a rat struck a rat drinking at the feeder, a defensive face whack was when the drinking rat struck the other rat, and a neutral face whack was all other instances of strikes at the facial region (neither rat was at the feeder). A rear attack was when a rat would approach the rat drinking at the feeder from behind, wedge its snout under the mid-region of the drinking rat, and use its snout to shove the drinking rat away from the feeder. We also counted the number of times rats relinquished access to the feeder either voluntarily (voluntary retreat: drinking bout that terminated in the absence of intervention by the other rat) or involuntary (involuntary retreat: termination of the drinking bout was preceded by a face whack or rear attack). The durations of drinking bouts were calculated as the total time spent drinking at the feeder divided by the number of voluntary and involuntary retreats. Statistical Analyses Analyses were performed with SPSS version 20 for Macintosh and involved t-tests and mixed-factor analysis of variance (ANOVA). Post hoc analyses involved F tests for simple effects and paired and independent-samples t-tests, with alpha set at p < .05, two-tailed.

RESULTS Weight The interaction of Age and Stress Group was significant F1,92 ¼ 3.32, p ¼ .04, although there was no difference between SS and CTL rats at PND 30 (beginning of stress procedure, p ¼ .71), PND 45 (end of stress procedure, p ¼ .08), or PND 59 (before testing in adulthood, p ¼ .61). SS rats gained less weight than did controls from PND 30 to PND 45 (p ¼ .03), but did not differ from CTL in weight gain from PND 45 to 59 (p ¼ .41) (see Fig. 2).

Behavior of SS and CTL Pairs in 1st Session CTL and SS pairs did not differ in latency of the first of the pair to reach the feeder (t22 ¼ .84, p ¼ .41), in drink time at the feeder (p ¼ .06), or in mean duration

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Developmental Psychobiology

Assessment of Dominant-Submissive Relationships (DSR)

FIGURE 2 Mean (SEM) weight at postnatal day (PND) ages before and after the social instability stress (SS) procedure for SS and control (CTL) rats. The inset shows the significant difference in weight gain between the groups from the start to the end of the SS procedure ( , p < .05).

of a drinking bout (p ¼ .82). SS pairs were more aggressive than CTL pairs, with a higher number of offensive face whacks (p ¼ .01) and rear attacks (p ¼ .03) than CTL pairs. SS and CTL pairs did not differ in the number of defensive face whacks (p ¼ .63), neutral face whacks (p ¼ .50) (all there types of face whacks combined, SS > CTL, p < .02), or voluntary retreats from the feeder (p ¼ .45). SS pairs made more involuntary retreats from the feeder than did CTL pairs (p ¼ .03) (see Fig. 3).

Pairs were considered to have formed a dominant– submissive relationship (DSR) if one rat spent significantly more time at the feeder than the other across the five sessions or in the last four sessions. A similar number of SS and CTL pairs displayed significant DSRs (8 of 12 pairs each). Status within Pair was either dominant or submissive for DSR pairs and EQ1 and EQ2 for no-DSR pairs, with EQ1 being the rat that drank more than the other in the fifth session. Dominant rats spent 18.9% (S.D. ¼ 3.6) more time drinking than the submissive rat for SS pairs, and dominant rats spent 19.0% (S.D. ¼ 8.3) more time drinking than the submissive rat for CTL pairs. The difference in time spent drinking of non-DSR pairs was 5.6% (S.D. ¼ 4.5). Larger rats within a pair were not more likely to be dominant: An ANOVA showed no effect of DSR status, of Status within Pair, or of Stress Group on weight at PND 59 (all ps > .19) (see Tab. 1). Analyses were then conducted to investigate change in behavior from the 1st to 5th session in SS and CTL rats based on DSR status (DSR pair, no-DSR pair). A Session X DSR X Status within Pair X Stress Group ANOVA on drink time at the feeder revealed a significant increase in time spent at the feeder from the 1st to the 5th session (F1,20 ¼ 35.42, p < .0001) and a main effect of Status within Pair (p ¼ .002), which is simply a reflection of the relationship between the dependent and independent variables (i.e., drink time was the basis for determining Status within Pair), as were interactions of DSR X Status within Pair (p ¼ .03)

FIGURE 3 Mean (SEM) of the behavioral measures in the 1st food competition session.  higher in pairs that underwent the social instability stress (SS) procedure than in control (CTL) pairs; p < .05). The white asterisk indicates that although the total number of face whacks was greater in SS than in CTL pairs, the difference was significant only for offensive face whacks when analysed separately.

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Table 1. Mean (SEM) Weight in Grams at the Start of Food Competition Sessions in Pairs of Rats DSR Pairs

CTL rats SS rats

No-DSR Pairs

Dominant

Submissive

EQ1

EQ2

362.1 (11.9) 342.5 (14.4)

360.5 (10.5) 365.9 (12.0)

341.0 (2.7) 337.8 (11.1)

344.5 (19.9) 348.5 (4.7)

Notes: DSR, dominant–submissive relationship. For DSR pairs, the rat in the pair that spent more time at feeder than the other across sessions was designated dominant. EQ1 and EQ2 applies to rats of no-DSR pairs (EQ, equal; no difference in drink time). For statistical analyses, the rat that spent more time at the feeder (irrespective of statistical significance) in the 5th session was designated EQ1. There was no relationship between status within the pair and weight gain for either pairs that underwent the social instability stress (SS) procedure or for control (CTL) pairs.

and of Session by Status within Pair (p ¼ .05) (see Fig. 4a). The only effect of Stress Group was a near significant interaction with Session (p ¼ .06; main effect of Stress Group and other interactions all p > .12). Latency to the feeder decreased from the 1st to the 5th session (F1,20 ¼ 21.50, p < .001; mean 1st session ¼ 64.4 s, SEM ¼ 12.87; mean 5th session ¼ 4.25 s, SEM ¼ .69), but no other main effect or interaction was significant (all p > .20). For the mean length of drinking bouts, the effect of Session (p ¼ .001), of Status within Pair (p ¼ .007), the interaction of Status within Pair X DSR Status (p ¼ .03), and the interaction of Status within Pair X Group X Session (p ¼ .05) were significant; all other main effects and interactions were p > .35). For CTL pairs, the interaction of Status within Pair X Session was significant (p ¼ .04), with dominant/EQ1 rats having longer drinking bouts than submissive/EQ2 rats in both the 1st (p ¼ .006) and the 5th (p ¼ .004) session, and with dominant/EQ1 rats (p ¼ .02) and not submissive/EQ2 rats (p ¼ .12) decreasing the length of drinking bouts from the 1st to the 5th session. For SS pairs, the interaction of Status within Pair X Session was not significant (p ¼ .28). In the 5th Session, SS and CTL rats did not differ in length of drinking bout for either the Dominant/EQ1 (p ¼ .68) or the Submissive/EQ2 (p ¼ .29) groups (see Fig. 4b). A Session X DSR X Status within Pair X Stress Group ANOVA on total number of face whacks revealed that SS pairs were more aggressive than CTL pairs (p ¼ .05), that no-DSR pairs were more aggressive than DSR pairs (p ¼ .03), and that aggressive behavior increased from the 1st to the 5th session (p < .001) (the main effect of Status within Pairs, p ¼ .43; all interactions p > .05) (see Fig. 4c). When the three types of face whacks were analyzed separately (see Fig. 5), the results were in the same direction for offensive face whacks as for total face whacks (SS > CTL, p ¼ .03; no-DSR > DSR, p ¼ .02; 5th > 1st, p < .001; all other ps > .08). For defensive face whacks: SS > CTL, p ¼ .02; 5th > 1st, p < .003; and a Group X Session

interaction, p ¼ .03, with SS > CTL in 5th session only; all other ps > .20). For neutral face whacks: no-DSR > DSR, p ¼ .03; 5th > 1st, p < .001; and a Stress Group X Session interaction, p ¼ .03, with SS > CTL in 5th session only; all other ps > .10). For rear attacks, only the effect of Session (p < .001) and of Status within Pair (p ¼ .05) were significant (all other main effects and interactions p > 10), with more rear attacks in the 5th session than in the 1st, and with Submissive/EQ2 rats making more rear attacks than Dominant/EQ1 rats. For voluntary retreats, the Session X Stress Group interaction was significant (p ¼ .02), with CTL pairs increasing the number of voluntary retreats from the from the 1st to the 5th session (p ¼ .01) and no change across sessions in SS pairs (p ¼ .76) (main effect of Session, p ¼ .04; all other ps > 15). For involuntary retreats, the Session X Stress Group X Status within Pair interaction was significant (p ¼ .03). Whereas in the 1st session, only the effect of Stress Group was significant (more involuntary retreats in SS than in CTL, p ¼ .02), in the 5th session, the interaction of Stress Group and Status within Pair was significant (p ¼ .03), with CTL pairs showing no difference in involuntary retreats on the basis of Status (p ¼ .79), and with the Dominant/EQ1 rats making more involuntary retreats than Submissive/EQ2 (p ¼ .003) among SS pairs, and no difference between SS and CTLs for either the Dominant/EQ1 (p ¼ .12) or the Submissive/EQ2 (p ¼ .89) groups.

DISCUSSION Increased Aggression in Food Competition Tests After Social Instability Stress Pairs of rats that had undergone the social instability stress (SS) procedure in adolescence were more aggressive as adults than were control (CTL) pairs during food competition sessions, as indicated by a higher number of offensive face whacks and rear attacks,

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FIGURE 4

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Developmental Psychobiology

FIGURE 5 Mean (SEM) of each type of face whack made in the 5th session for pairs that underwent the social instability stress (SS) procedure and for control (CTL) pairs in the two dominant–submissive relationship groups (DSR; the rat in the pair that spent more time at feeder than the other across sessions was designated dominant) and those that did not (no-DSR).

which resulted in more involuntary retreats from the feeder in SS pairs than in CTL pairs in the 1st session. Although there was an overall increase in face whacks and in time at the feeder from the 1st to the 5th session, the higher number of offensive face whacks in SS pairs than in CTL pairs remained evident in the 5th session, and SS pairs also displayed more defensive and neutral face whacks than did CTL pairs in the 5th session. SS and CTL pairs, however, did not differ in number of involuntary retreats, length of drinking bouts, or in total time spent drinking in the 5th session, which suggests that more face whacks were required to displace the

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opponent from the feeder for SS pairs than for CTL pairs. Further, CTL pairs increased the number of voluntary retreats from the feeder from the 1st to the 5th session whereas the SS did not, which suggests that CTL pairs may have been avoiding aggressive interactions. Because the increased aggressive behavior of SS pairs compared with CTL pairs did not result in a significant increase in the time at the feeder for the SS pairs, the increased aggression may be considered maladaptive. There are two, non-mutually exclusive, possibilities for the increased aggression in SS pairs. The increased aggression may be another manifestation of an impoverished social repertoire in SS rats; we previously observed reduced social interactions in SS rats interacting with a novel male (Green et al., 2013) and reduced copulatory efficiency in sexual encounters with females (McCormick et al., 2013). Because SS rats show deficits as adults on hippocampal-dependent behaviors and differ from CTL rats on measures of hippocampal plasticity (Green & McCormick, 2013a; McCormick et al., 2012), and because of the role of the hippocampus in social behaviors such as behavioral sequencing in male rats (the predictability of behavior of one rat by the behavior of its partner) (Maaswinkel, Gispen, & Spruijt, 1997) and social anxiety (e.g., File & Seth, 2003), we have proposed that deficits in social behavior in SS rats may be related to the effects on hippocampal development (McCormick et al., 2014). There is growing evidence for the role of hippocampal signaling in aggressive behavior (e.g., Pagani et al., 2014). Further, chronic stress in adult rats increased aggression and decreased sociability, which was associated with reduced neuroligins in the hippocampus (van der Kooij et al., 2014). Stressor exposures in adolescence using a different set of stressors (e.g., bright light, predator scents) (Marquez et al., 2013) or administration of corticosterone to mimic stressinduced elevations (Veenit, Cordero, Tzanoulinou, & Sandi, 2013) led to increased aggression in resident versus intruder tests in adulthood, several weeks after the stress exposures. A second possibility is that SS pairs were more aggressive than were CTL pairs because of a heightened

FIGURE 4 Mean (SEM) of the behavioral measures in the 1st and 5th session based on dominant–submissive relationship (DSR; the rat in the pair that spent more time at feeder than the other across sessions was designated dominant) and those that did not (no-DSR). EQ1 and EQ2 refers to rats of no-DSR pairs (EQ ¼ equal; no difference in drink time). For statistical analyses, the rat that spent more time at the feeder (irrespective of statistical significance) in the 5th session was designated EQ1. Bar graphs to the right indicate the statistically significant main effects and/or interactions of mixed factor ANOVAs (Stress Group  DSR Status  Status within Pair  Session). For drink time at feeder, significant interactions are a reflection of the relationship between the independent and dependent measure (drink time used to designate status of pair and status within pair).  p < .05 for comparison between groups illustrated with different coloured bars; #p < .05 for comparison between groups illustrated with bars of the same colour.

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motivation for the sweetened condensed milk. The mesolimbic dopamine system is implicated in motivation for both “natural” (food, sex) and drug-related rewards (Baik, 2013), and we have some modest evidence for altered mesolimbic dopamine function after SS in rats based on increased sensitivity to the locomotor-activating effects of amphetamine and reduced conditioned place preference to amphetamine in adulthood (Mathews, Mills, & McCormick, 2008; McCormick, Robarts, Kopeikina, & Kelsey, 2005). SS and CTL rats, however, did not differ in latency to the sweetened condensed milk in either session; both groups increased time at the feeder from the 1st to the 5th session, and the modestly higher (p ¼ .06) time at the feeder of SS pairs compared with CTL pairs was limited to the 1st session. Nevertheless, the increased face whacks in SS rats could be a reflection of a heightened motivation for the food. A limitation of the present study was that we did not test the extent to which the effects of the social instability stress procedure depended on the adolescent age of exposure. Nevertheless, when we have included a comparison group of rats that underwent the procedure as adults, we did not find the long-lasting effects of adult-exposure that we found after adolescent exposure, which suggests the effects of the social instability may be specific to the adolescent period in males (Morrissey, Mathews, & McCormick, 2011; McCormick et al., 2005). These, studies, however did not involve measures of social behavior. A second limitation is that we did not include females, for which the effects likely differ considering the vast sex differences in the social repertoire, although females do form dominant–submissive relationships (e.g., Parent et al., 2013). We have described age and sex-specific effects of social instability stress in other studies with other behavioral endpoints (McCormick, Robarts, Gleason, & Kelsey, 2004; McCormick et al., 2005), but the effects of adolescent social instability stress on social behavior in females remain to be determined. A third limitation is that we did not include an experimental group of cage partners that involved an SS male housed with a control male from PND 45 until the time of behavioral testing. We have found that the reduction in social interactions in SS males is greater when the interaction involves an unfamiliar SS male than when the interaction involves an unfamiliar control male, and that pairs of control males had the highest social interactions (Green et al., 2013). Thus, SS rats that had been housed for a long time with a control rat would likely be different than when housed with another SS rat given that the behavior of one in the pair depends on that of the other.

Developmental Psychobiology

The Food Competition Test as an Indicator of Dominant-Submissive Status SS and CTL pairs were equally likely to form DSRs, with 67% of pairs of both groups designated as a DSR based on the difference in drinking of sweetened milk across sessions. In other food competition tasks involving dyads between cage partners or unfamiliar rats, DSR relationships were observed in 50% to 88% of pairs (Askew, Gonzalez, Stahl, & Karom, 2006; Barnum et al., 2008; Hoshaw et al., 2006). In some studies, stricter criteria are used than a significant difference in time at the feeder (e.g., one rat must spend more than 40% more time at the feeder than the other) and only about 25% of pairs were designated as having a DSR (Malatynska & Knapp, 2005). There was no difference, however, between rats in the present study in behavior on the basis of their status within the pair other than the amount of time spent at the feeder. Further, rats designated as dominant did not weigh more than their submissive partner, which might suggest the criteria we used were too lax. Although there are reports that dominant rats weighed more than subordinates (e.g., Kozorovitskiy & Gould, 2004; Lore & Stipo-Flaherty, 1984), and pairing a much smaller rat with a larger rat has been used as a means of ensuring a DSR relationship in a dyad (e.g., Arakawa, 2006), weight did not predict dominance status in other studies (e.g., Baenninger, 1970; Masur & Benedito, 1974; Pellis & Pellis, 1991). The designation of pairs as DSR or noDSR, however, was a significant factor: DSR pairs displayed fewer aggressive face whacks than did noDSR pairs, which is in keeping with the posited function of DSRs to be a mechanism to reduce aggression in groups (e.g., Hillman & Bilkey, 2012; Weiss et al., 2002). In studies of newly formed rat colonies that establish dominance hierarchies, aggression decreased over time (Blanchard, Hori, Tom, & Blanchard, 1988). We cannot determine the extent to which DSRs formed during food competition or captured a DSR established previously in the home cage. Others, however, have shown that there are behavioral differences between dominant and submissive rats before the status is determined; in studies of formation of dominance hierarchies in new colonies, rats that showed more risk-taking behavior and higher motivation for food reward (Davis, Krause, Melhorn, Sakai, & Benoit, 2009) or were previously rated as highly aggressive (Blanchard et al., 1988) were more likely to become dominant. Further, dominant and submissive rats have been found to differ on various physiological and neural parameters. For example, dominant rats

Developmental Psychobiology

had higher V1A vasopressin receptor binding than submissive rats in several neural regions, but did not differ from submissive rats in plasma testosterone or corticosterone concentrations (Askew et al., 2006). Indications of higher stress are often reported in the submissive rats of the DSR pair compared with the dominant rats (Blanchard, Sakai, McEwen, Weiss, & Blanchard, 1993; Hoshaw et al., 2006). In addition, glucocorticoids are important in the formation and retention of DSRs (reviewed in van der Kooij & Sandi, 2012). For example, when one of the dyad had been recently exposed to a stressor, it was more likely to become submissive, and those dyads were more stable than those of pairs in which neither had been stressed (Cordero & Sandi, 2007). It may be that differences between the dominant and submissive rat in dyads in the present study are negligible because of the long-standing relationship between the two rats in the pair before the food competition task. DSRs formed in the juvenile period are considered to differ from ones that form in adulthood. Further, the behavior of dominant and submissive rats was indistinguishable after the initial period of status determination (Lore & Stipo-Flaherty, 1984), thus the lack of difference between dominant and submissive rats in the present study may be based in their formation at an early stage of life and longstanding. Nevertheless, others have reported that longstanding cage partners of colony-reared rats that were designated as dominant or submissive on the basis of a food competition task in adulthood differed in hypothalamic interleukin-1 concentrations and in hyperthermic responses to stressors (Barnum et al., 2008). Thus, differences between rats in a DSR pair might emerge using measures of physiology rather than behavior during the food competition test and weight.

CONCLUSION SS and CTL rats had a similar prevalence of DSRs among cage partners as indicated by asymmetries within pairs in access to the feeder in the food competition sessions, but competition involved more aggressive behavior in SS pairs than in CTL pairs. These findings are consistent with the hypothesis that social instability stress in adolescence results in an impoverished social repertoire in adulthood, and adds to growing evidence of the influence of social relationships in adolescence on the trajectory of development into adulthood.

ACKNOWLEDGMENTS The research was conducted as part of the requirements of the BSc, Honours, in Neuroscience, Brock University (MJC,

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MAT). The research was funded by the Natural Sciences and Engineering Research Council (NSERC) of Canada and the Canadian Foundation for Innovation (CFI) (PI: CMM). NSERC and CFI had no role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. We thank Travis Hodges for assistance in data collection. The authors have no conflicts of interest to declare.

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