Hormones and Behavior 60 (2011) 28–36

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Hormones and Behavior

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Post-weaning social isolation induces abnormal forms of aggression in conjunctionwith increased glucocorticoid and autonomic stress responses

Mate Toth, Eva Mikics, Aron Tulogdi, Mano Aliczki, Jozsef Haller ⁎Department of Behavioral Neuroscience, Institute of Experimental Medicine, Budapest, Hungary

⁎ Corresponding author at: Institute of ExperimenBehavioral Neuroscience, P.O. Box 67, H-1450 Budapest,

E-mail address: haller@koki.hu (J. Haller).

0018-506X/$ – see front matter © 2011 Elsevier Inc. Aldoi:10.1016/j.yhbeh.2011.02.003

a b s t r a c t

a r t i c l e i n f o

Article history:Received 4 November 2010Revised 14 January 2011Accepted 2 February 2011Available online 21 February 2011

Keywords:RatPost-weaning social isolationAggressionArousalStressHeart rateGlucocorticoid

We showed earlier that social isolation from weaning (a paradigm frequently used to model social neglect inchildren) induces abnormal forms of attack in rats, and assumed that these are associated with hyperarousal.To investigate this hypothesis, we deprived rats of social contacts from weaning and studied their behavior,glucocorticoid and autonomic stress responses in the resident-intruder paradigm at the age of 82 days. Socialisolation resulted in abnormal attack patterns characterized by attacks on vulnerable targets, deficient socialcommunication and increased defensive behaviors (defensive upright, flight, freezing). During aggressiveencounters, socially deprived rats rapidly switched from one behavior to another, i.e. showed an increasednumber of behavioral transitions as compared to controls. We tentatively term this behavioral feature“behavioral fragmentation” and considered it a form of behavioral arousal. Basal levels of plasmacorticosterone regularly assessed by radioimmunoassay between 27 and 78 days of age were not affected.In contrast, aggression-induced glucocorticoid responses were approximately doubled by socially isolation.Diurnal oscillations in heart rate assessed by in vivo biotelemetry were not affected by social isolation. Incontrast, the aggression-induced increase in heart rate was higher in socially isolated than in socially housedrats. Thus, post-weaning social isolation induced abnormal forms of aggression that developed on thebackground of increased behavioral, endocrine and autonomic arousal. We suggest that this paradigmmay beused to model aggression-related psychopathologies associated with hyperarousal, particularly those that aretriggered by adverse rearing conditions.

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Introduction

Brain mechanisms of aggression control are usually studied in theresident/intruder paradigm in rodents. In order to identify the braincenters involved in, and their role in aggressive behavior, thisparadigm was employed in conjunction with a variety of techniquesincluding brain lesions, electrophysiology, immunocytochemistry,systemic and local pharmacologic manipulations, transgenic techni-ques, in vivo microdialysis of neuropeptide release during aggression,etc. (Abrahams et al., 2005; Albert et al., 1989; Chiavegatto et al.,2001; Ferris et al., 2006; Gobrogge et al., 2009; Kruk et al., 1979;Lonstein and Stern, 1997; Veenema et al., 2010).

Recently, this basic model of aggression research was enriched bynovel behavioral approaches that allow the identification of abnormalpatterns of aggression (de Almeida and Miczek, 2002; de Boer et al.,2003; Haller and Kruk, 2006; Haller et al., 2001; Miczek et al., 2002;Natarajan et al., 2009). These approaches are based on the fact thataggressiveness in animals follows certain behavioral “rules” thatmitigate the conflict between potentially dangerous forms of behavior

and survival. It was suggested that animal aggression can beconsidered abnormal if there was a mismatch between provocationand response (i.e. the aggressive response surpassed species-typicallevels; de Almeida and Miczek, 2002; Miczek et al., 2002), if attackswere targeted on inappropriate partners (e.g. females; de Boer et al.,2003; Natarajan et al., 2009) or inappropriate body parts (i.e. thoseprone to serious injury like the head, throat and belly; Haller andKruk, 2006; Haller et al., 2005; Haller et al., 2001), if attacks were notsignaled by threats, and/or the submissive signals of opponents wereignored (de Boer et al., 2003; Haller and Kruk, 2006; Haller et al.,2001; Natarajan et al., 2009). In general, these criteria are similar toparticularities of human aggressiveness that are expressed in certainaggression-related psychopathologies (Haller and Kruk, 2006; Halleret al., 2005). Three laboratory models of abnormal aggression weredeveloped so far. The escalated aggression model involves aggres-siveness that surpasses species-typical levels and is induced byfrustration or provocation (de Almeida and Miczek, 2002; Miczeket al., 2002). This model is based on the attack priming phenomenondiscovered by Potegal (1992). The hypoarousal model involves thechronic limitation of glucocorticoid secretion, which mimics the lowglucocorticoid levels seen in violent, antisocially disordered people(Haller et al., 2001; Haller and Kruk, 2006). Finally, genetic modelsmake use of mice selected for high aggressiveness, rats selected for

29M. Toth et al. / Hormones and Behavior 60 (2011) 28–36

low anxiety, or selected subpopulations of feral rats (de Boer et al.,2003; Natarajan et al., 2009; Neumann et al., 2010). Abnormalfeatures of aggression were observed in all these models. Aggressivebehavior was increased on the long run by a variety of early adverseexperiences (maternal separation: Suomi, 1997; Veenema et al., 2006,2007; early defeat: Delville et al., 1998; Wommack et al., 2003).Although abnormal features of aggression were not investigated inthese models, they have the potential to become valuable novelmodels of abnormal aggression.

The models briefly reviewed above have translational value andallow the study of regulatory mechanisms that underlie abnormalresponses to provocation, the development of callous-unemotionaltraits, and the impact of genetic predispositions on aggressiveness.Neither of these models covers an important form of abnormalaggression, namely the one that is precipitated by exacerbatedemotional responses. It is generally believed that abnormal humanaggression is of two major types: emotional and callous-unemotionalviolence (Blair, 2001; Meloy, 2006; Scarpa and Raine, 1997; Vitielloet al., 1990). Emotional violence appears to be driven by strong stressresponses, which feature is believed to be causally related toaggressive traits (Chida and Hamer, 2008; Murray-Close et al., 2008;Smith and Gallo, 1999; Suls and Wan, 1993). Early determinants ofemotional aggression often are adverse experiences suffered duringchildhood, particularly social neglect (Chapple et al., 2005; Goldsteinet al., 2006; Heinrichs et al., 2003; Pesonen et al., 2010; Teicher et al.,2003; Uchino et al., 1996). Therefore, one can hypothesize thatmodels of early social isolation would mimic aspects of psychopathol-ogies that involve outbursts of emotional aggression.

In line with this assumption, adult aggressiveness was increased inanimals that were submitted to post-weaning social isolation (alsocalled social deprivation) (rats: Day et al., 1982; Potegal and Einon,1989; rhesus monkeys: Harlow et al., 1965; Kempes et al., 2008;guinea pigs: Sachser et al., 1994). Increased levels of aggressiveness,however, cannot be considered abnormal per se, at least not accordingto the criteria outlined above. We have recently shown that post-weaning social isolation not only increases attack counts, but leads toa dramatic increase in attacks aimed at vulnerable body parts ofopponents, and to a similarly dramatic decrease in the social signalingof attacks. In addition, the aggressiveness of socially deprived rats wasambiguous, as increased attack counts were associated with increaseddefensiveness. We hypothesized that this abnormal behavioralpattern is associated with aggression-induced hyperarousal (Toth etal., 2008). This assumption was based on literature findings, which,however, are conflicting to a certain extent. For example, post-weaning social isolation increased stress response under certainconditions, but decreased it or was without consequences under otherconditions (Gentsch et al., 1981; Sanchez et al., 1998; Schrijver et al.,2002; van den Berg et al., 1999; Weiss et al., 2004). Therefore, thepresent study aimed at studying glucocorticoid and autonomic stressresponses in socially reared and socially deprived rats that wereexposed to aggressive encounters, to assess whether abnormalfeatures of behavior develop under conditions of hyperarousal insocially deprived rats. Observations made during these experimentsprompted a third one, where the frequency and duration of behaviorswere compared to investigate the possibility of identifying symptomsof behavioral hyperarousal in socially deprived rats. Ultimately, thesestudies aimed at developing a model of hyperarousal-driven aggres-siveness that may be used in future studies to study the brainmechanisms underlying this specific form of abnormal aggression.

Materials and methods

Animals and housing conditions

Subjects were male Wistar rats (Charles-River) from the breedingfacility of our Institute. Pups were weaned on the 21th postnatal day

and housed socially or individually for 7–8 weeks in Makrolon cagesmeasuring 45 × 35 × 25 cm. Food and water were available ad libitumthroughout, while temperature and relative humidity were kept at22±2 °C and 60±10%, respectively. Rats were maintained in a lightcycle of 12:12 h with lights off at 1000 h. The weight of subjects was400–450 g during testing. The opponents used in aggressiveencounters came from the same source and weighed approximately300 g. These ratswere group housed but otherwisemaintained underconditions similar to the subjects. Each intruderwas used three timesin a random order (see below). The experiments were carried out inaccordance with the European Communities Council Directive of24 November 1986 (86/609/EEC) and were reviewed and approvedby the Animal Welfare Committee of the Institute of ExperimentalMedicine.

Experimental design

Experiment 1Rats were weaned at the age of 21 days, after which they were

housed either individually (social deprivation; n=17) or in groups of4 (social housing; n=16). Subjects were left undisturbed except forblood sampling for corticosterone measurements. Sampling wasperformed in three sessions: at the end of the first post-weaningweek (at the age of 27 and 29 days), during the play-fighting period(at the age of 34, 36 and 38 days), and when they reached adulthood(at the age of 76, 78 and 82 days; see below for the details of thissampling period). Noteworthy, blood sampling was performed by thetail incision method, which involves minimal disturbance for thesubjects and does not induce changes in basal corticosterone levels,not even after repeated sampling at short intervals (Fluttert et al.,2000). When rats reached 76 days of age, animals were transferred toindividual cages after blood sampling (60 × 40 × 50 cm). This stepwasnecessary as the resident-intruder test requires short-term socialisolation for the establishment of territorial behavior. Blood samplingwas repeated in the new cage at the age of 78 days, and rats werefaced with opponent at the age of 82 days. The resident/intruder testwas performed at the beginning of the dark period and lasted 20 min.Blood was sampled again at the end of the aggressive encounter. Tocontrol the effect of repeated blood sampling from weaning toadulthood, glucocorticoid measurements were repeated in a group ofrats that were not disturbed until the age of 76 days. In this groupstudied in parallel, blood was sampled immediately before transfer-ring them to individual cages (age 76 days), 2 days after the cageswitch (age 78 days) and after the aggressive encounter (age82 days). Sample size was 7–8 in these groups. The encounter wasvideo recorded and behavior was later analyzed by an experimenterblind to the treatments.

Experiment 2Weaning and housingwas performed as described for Experiment 1.

Sample size was 12 for both the socially housed and the sociallydeprived rats. Rats were left undisturbed till the 8th post-weaningweek when they were implanted with biotelemetry e-mitters (seebelow for details). After surgery, all rats were transferred toindividual telemetric recording cages (40 × 30 × 25 cm). Rats wereallowed 1 week recovery; this period also served for the induction ofterritorial behavior. As telemetric signals become stable after about 3days, recordings obtained on the 5th, 6th, and 7th post-implantationdays were used to investigate changes in daily rhythms. On the 8thday of individual housing, subjects were faced with an intruder ofsmaller size for 20 min in the early hours of the dark period. Theencounter was video recorded and behavior was later analyzed by anexperimenter blind to the treatment. Biotelemetric data samplingwas performed throughout. Sampling interval was 1 min during therecovery period and 10 s on the day of the aggressive encounter.However, experimental findings are presented for 1-min-long

30 M. Toth et al. / Hormones and Behavior 60 (2011) 28–36

intervals in the Results session. To check for the temporal consistency ofheart rate responses, dyadic encounters were repeated 2 and 4 dayslater.

Experiment 3This experiment was performed to investigate the behavior of

subjects in detail and to study the impact of cage size on behavioralchanges induced by post-weaning social isolation. Note that the sizeof biotelemetric receivers limited the size of the resident-intrudercages used in Experiment 2. Weaning and housing was performed asdescribed for Experiment 1. At the age of 76 days, both socially housedand socially deprived rats were introduced into resident/intrudercages that either measured 60 × 40 × 50 cm or 40 × 30 × 25 cm. Fourdays later, subjects were faced with an intruder of smaller size for20 min in the early hours of the dark period. The encounter was videorecorded and behavior was later analyzed by an experimenter blind tothe treatment.

Litter handling and group assignment

Litters were not disturbed before weaning, i.e. dams were allowedto raise pups under normal conditions. Litter sizes were 6–10; 40–60%of the pups were males. The number of litters used was 9, 8 and 8 inExperiments 1, 2, and 3, respectively. Only males were studied. Ratsbelonging to the various litters were randomly assigned to social andisolation housing. As a result, social groups were formed from malesthat came from different litters, whereas isolated rats were taken fromall litters, i.e. all litters were represented in this group.

The resident-intruder test

Resident-intruder tests were run in the early hours of the darkperiod under dim red illumination provided by two 40 W red bulbsplaced on the ceiling of the experimental room, i.e. at a height of2.5 m. Encounters lasted 20 min in all experiments. Analysis focusedon behaviors that showed abnormal features in an earlier study,namely on attack targeting and the relationship between offensivethreats and attacks (Toth et al., 2008). Attack episodes were analyzedat low speed (frame-by-frame if necessary) for identifying attacktargets and the context of attack. An attack was considered vulnerableif it targeted the head (areas anterior to the ears), throat (the ventralarea below the ears), the belly (ventral areas between legs) or thepaws. Dorsal and lateral areas (posterior to the ears and dorsal to thelegs) were considered non-vulnerable targets. An attack wasconsidered not signaled if it was not preceded by an offensive threat,and signaled if it was performed in the context of an offensive threat.Both vulnerable and not-signaled attacks were expressed as % of totalattacks according to the formulas “vulnerable attack counts/totalattack counts*100” and “not signaled attack counts/total attackcounts*100,” respectively. We also differentiated soft and hard bites.An attack was identified as “hard bite” when it involved kicking(clinch fights) or induced a strong startle response in the intruder(large jumps or immediate submission). “Soft bites” were notassociated with kicking and induced no response in the intruder orinduced mild quivering only. Similar discrimination was employedearlier (Toth et al., 2008; Lammers et al., 1988). Noteworthy, nobleeding wounds or visible injuries were noticed in this experiment. Adetailed behavioral analysis was performed in Experiment 3. Thefrequency and duration of the following behavioral variables wereassessed: exploration (sniffing directed toward the environment);social investigation (sniffing directed towards the opponent's flank,nasal, or anogenital region); grooming (self-grooming with forepawsand scratching with hind legs); offense (aggressive grooming, lateralthreat, offensive upright posture, mounting and chasing takentogether); defense (defensive upright, defensive kick, fleeing andfreezing taken together); dominant posture (keeping down the

opponent while he is laying on his back); submissive posture (layingon back while kept down by the opponent).

Biotelemetry

Biotelemetry e-mitters (Minimitter Co., Bend, OR, USA) wereimplanted into the abdominal cavity of rats through a midlineabdominal incision under ketamine–xylazine–pipolphen anesthesia.The negative and positive heart rate leads were attached to theanterior right side of the chest (near the clavicle) and to the posteriorchest wall (left to the sternum and anterior to the last rib),respectively. Recordings were made by means of the VitalViewsystem (Minimitter Co.) which provided body temperature, heartrate, and locomotion data around the clock.

Blood sampling and corticosterone measurements

Rats were transferred to the sampling room that was adjacent totheir maintenance room, and a small incision was applied 1 cm fromthe tip of their tail. Blood was sampled on ice-cold EDTA-containingtubes. After blood sampling, the subjects of social groups remained inthe sampling room until the last (fourth) blood sample was collected,to avoid stress exposure for cage mates. The whole procedure lastedless than 1 min for individual rats, and about 4 min in total for a socialgroup. This rapid blood sampling procedure allows blood samplingwithout measurable changes in basal corticosterone levels (Fluttertet al., 2000). The sampled blood volume was 0.1–0.2 ml between 27and 38 days of age and 0.3–0.5 ml in adult rats. Blood was centrifugedat 4 °C; plasmawas separated and kept at−20 °C till analyzed. Plasmacorticosterone concentrations were measured by radioimmunoassay.The assay was performed in duplicate. Prior to assay, corticosteronewas separated from CBG at low pH (200 μl 0.1 mol citric acid; 1-h incubation at room temperature). Antiserum was raised in rabbitsagainst corticosterone-3-carboxymethyloxime bovine serum albumin(prepared at the Institute of Experimental Medicine, Budapest), and125I-labeled carboxymethyloxime–tyrosine–methyl ester derivative(Isotope Institute Ltd., Budapest, Hungary) was used as tracer. Theinterference with plasma transcortin was eliminated by inactivatingtranscortin at low pH. The corticosterone antibody cross-reactivitywith other naturally occurring adrenal steroids was b0.05%, except fordesoxycorticosterone (1.5%) and progesterone (2.3%). Final dilution ofthe antibody was 1:40,000. Incubation time was 24 h at 4 °C, and asecond antibody (anti-rabbit from goat), for separation 6% polyeth-ylene glycol solution was used. A calibration curve was prepared fromcorticosterone (Calbiochem) and ranged from 0.27 to 40 pmol/tube.The sensitivity of the assay was 1 pmol/ml. Intra- and inter-assaycoefficient of variation was 9.6% and 16.6%, respectively. All samplesfrom an experiment were measured in the same assay.

Statistics

Data were expressed as mean±standard error of the mean (SEM).Data were analyzed by factorial ANOVA by using repeated measuresfactor were appropriate. Factors were identified in Results. Behavioraldata were square root transformed to fulfill ANOVA requirementswhere necessary. Post-hoc comparisons by the Fisher LSD test weremade only when the main effects were significant. The Spearman testwas used for the calculation of correlations.

Results

Experiment 1—endocrine arousal

To control for the effects of repeated blood sampling during thedevelopmental period, data were analyzed by two-factor ANOVA(Factor 1, social background; Factor 2, blood sampling history). In line

Fig. 1. Behavioral and endocrine changes following social isolation in Experiment 1.Upper panel: bite counts, and the percentage of bites that were aimed at vulnerabletargets (head, throat or belly), were not signaled, or were initiated from defensivepostures (e.g. escape, defense or submission). Middle panel: basal corticosterone levelsduring development and the response to aggression in the resident-intruder test. Lowerpanel: the impact of blood sampling history on the corticosterone response toaggression. PND, postnatal age in days; *, significant increase compared to sociallyhoused subjects (pb0.01 at least); #, significant increase compared to basal levels(pb0.01 at least).

31M. Toth et al. / Hormones and Behavior 60 (2011) 28–36

with earlier observations, social isolation increased the frequency ofhard attacks (Fsocial background(1,45)=4.8; pb0.04) without alteringthe number of soft bites (Fsocial background(1,45)=0.04; pN0.8). Totalattack counts showed a marginal increase (Fsocial background(1,45)=2.66; pb0.08). Blood sampling history had no effect on attack counts(hard bites: Fblood sampling(1,45)=0.63; pN0.4; soft bites: Fblood sampling

(1,45)=0.15; pN0.7; all bites: Fblood sampling(1,45)=0.46; pN0.4).Social deprivation also increased the percentage of attacks aimed atvulnerable targets (Fsocial background(1,34)=9.72, pb0.01) and thepercentage of non-signaled attacks (Fsocial background(1,34)=8,45;pb0.01). The history of blood sampling did not affect these measures(vulnerable attacks: Fblood sampling(1,34)=1.44; pN0.2; not signaledattacks: Fblood sampling(1,34)=0.32; pN0.5). Interestingly, sociallydeprived rats initiated a considerable number of attacks fromdefensive postures (e.g. escape, defense or submission) (F(1,36)=5.37, pb0.05). These data were summarized in the upper panels ofFig. 1. For clarity, data were collapsed over the factor blood samplingas this factor showed no statistically significant effects. In line withearlier data, the correlation between the percentage of vulnerableattacks and the percentage of non-signaled attacks was significant(Spearman R=0.647; pb0.0001). This suggests that the larger thenumber of vulnerable attacks, the larger the likelihood that attacks arenot signaled.

In the case of corticosterone measurements, the two factorsshowed a significant interaction demonstrating that plasma cortico-sterone levels depended on the interplay between time and housingconditions (Finteraction(7154)=6.13; pb0.0001). Basal levels (days27–78) showed no significant changes over time and did not dependon housing (Fig. 1, middle panel). Fighting significantly increasedplasma corticosterone in both groups (pb0.0001). However, theincrease was significantly larger in socially deprived rats (pb0.001).To control for the long history of blood sampling, the findingsobtained in these rats were compared to those obtained in subjectsthat were not submitted to repeated blood sampling duringdevelopment. Noteworthy, the four groups were run in parallel. Inthe last week of the experiment when blood was sampled from allanimals, corticosterone plasma levels depended on the interactionbetween blood sampling history and social background (Finteraction(2,72)=7.56; pN0.001). Pairwise comparisons along these factorsshowed that transfer to the resident/intruder cages did not, whileaggressive encounters did increase plasma corticosterone levels(Fig. 1, lower panel). The increase was significantly larger in sociallydeprived rats as compared to controls.

Experiment 2—autonomic arousal

In this experiment, subjects were exposed to aggressive encoun-ters three times. Therefore, behavioral data were analyzed by two-factor ANOVA (Factor 1: social background; Factor 2 (repeatedmeasures): encounter). The percentage of vulnerable attacks wasincreased by social deprivation, an effect that was not affected by day(successive encounters) (Fsocial background(1,60)=14.17; pb0.0005;Fencounter(2,60)=0.32; pN0.7; Finteraction(2,60)=0.89; pN0.4) (Fig. 2,upper panel). The same was true for the percentage of attacks thatwere not signaled by offensive threats (Fsocial background(1,60)=4.52;pb0.04; Fencounter(2,60)=0.26; pN0.7; Finteraction(2,60)=0.67;pN0.5). However, the visual inspection of data suggested that thepercentage of not signaled attacks changed over time in both sociallyhoused and socially deprived rats. Overall, values noticed in the twogroups appeared to approach each other over time. Even if thesechanges proved non-significant in this study, one cannot exclude thatthis difference gradually disappeared if rats were exposed toadditional encounters.

In the period preceding the aggressive encounter, heart rates, bodytemperatures and locomotion counts showed a strong daily rhythm.Fclock hour(17374) values were 130.6, 71.00 and 144.86 for heart rates,

body temperature and locomotion, respectively; p values were lowerthan 0.0001 in all cases. However, this rhythm was not affected bysocial background (F(1,22) values for this factor were 1.37 (pN0.2),0.40 (pN0.5) and 4.16 (pN0.05), while Finteraction(17374) values were0.73 (pN0.7), 0.01 (pN0.9) and 0.96 (pN0.4) for heart rates, bodytemperature and locomotion, respectively). The lower panel of Fig. 2exemplifies these diurnal variations by showing heart rates. Even ifgroup differences were not significant, the visual inspection of theheart rate rhythms (but not that of body temperature or locomotion)suggested that the two groups were different at one particular time-point, namely in the early light phase. To exclude that these putative

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Fig. 2. Behavioral and autonomic changes following social isolation in Experiment 2.Upper panel: the percentage of attacks aimed at vulnerable targets and the percentageof attacks without social signaling. Closed columns marked with continuous lines showthe overall means obtained in socially housed and socially deprived rats. Noteworthy,social background was the only factor that provided significant differences. Opencolumns marked with dashed lines show the means obtained during the 1st, 2nd and3rd encounter. For further details see text. Lower panel: diurnal variation of heart ratesduring the 3 days that preceded the aggressive encounter. The dark phase of the daywas indicated by grey bands. o, marginal increase compared to socially housed subjects(p=0.064); *, significant increase compared to socially housed subjects.

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Fig. 3. Heart rate changes during and after the three 20-min-long encounters that wereperformed 2 days apart. The upper, middle and lower panel shows encounters 1, 2 and3, respectively. The duration of the encounter was indicated on the lower panel. Thehorizontal gray bands show variation seen during baseline measurements. Thehorizontal lines indicate the time periods for which the asterisks apply. *, significantincrease compared to socially housed subjects.

32 M. Toth et al. / Hormones and Behavior 60 (2011) 28–36

group differences were masked by daily variations, we pooled dataacross days. This analysis revealed that heart rates in the sociallydeprived groupweremarginally decreased in the early light phase butnot at other time-points (F(1,22)=3.79; p=0.064). The relevance ofthis tendency was probably minor, as behavior was assessed in thedark phase.

Social deprivation increased the aggression-induced autonomicactivation (Fig. 3). In the case of the first resident/intruder test, thedifference was significant for the whole duration of the encounter andalso for the following 80 min (Fsocial background(1,20)=5.87; pb0.03;Ftime(99,2178)=6.30; pb0.0001; Finteraction(99,2178)=0.51; pN0.9)(Fig. 3, upper panel). In the case of encounters 2 and 3, there was asignificant interaction between factors (Finteraction(99,2178)=1.26(pb0.05) and Finteraction(99,2178)=1.29 (pb0.04), respectively). Inthese encounters, the difference between the two groups wassignificant for certain periods of the aggressive encounter (Fig. 3,middle and lower panels). Taken together, these data show that thefirst aggressive encounter increased autonomic activation massivelyin socially deprived rats, and this persisted long after the terminationof the aggressive encounter, i.e. in a period when no autonomicactivation was seen in socially housed rats. In subsequent encounters,social deprivation increased the peak of the autonomic response.

The changes seen in autonomic activation were not secondaryto changes in locomotion. Despite some group differences espe-cially at encounter 1, housing did not affect locomotion significantly(Fsocial background(1,22) values were 0.17 (pN0.6), 0.72 (pN0.4) and0.18 (pN0.6) in the case of encounters 1, 2 and 3, respectively)(Fig. 4). In contrast, time effects were highly significant, demon-

strating a significant increase in locomotion during aggressiveencounters (Ftime(99,2178) values were 10.31, 8.81 and 8.05 in thecase of encounters 1, 2 and 3, respectively; pwas lower than 0.0001 inall three cases). There was no interaction between factors (Finteraction(99,2178)=1.10 (pN0.2), 0.95 (pN0.5) and 1.11 (pN0.2) forencounters 1, 2 and 3, respectively).

Experiment 3—behavioral arousal

When analyzing behaviors in Experiments 1 and 2, we noticed thatsocially deprived rats switched from one behavior to another rapidly.This fitful pattern of behavior suggested that social deprivationincreases the rate of behavioral transitions (Fig. 5, upper panel).Experiment 3 examined this possibility in a different set of rats thatwere exposed to opponents in cages of different sizes to control forinherent cage size differences in Experiments 1 and 2 (see Materialsand methods for details). As expected, social deprivation affected the

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Fig. 4. Locomotor activity during and after the three 20-min-long encounters that wereperformed 2 days apart. The first, second and third encounters were shown in theupper, middle and lower panels, respectively. Social deprivation induced non-significant variations only. The horizontal gray bars show variation seen duringbaseline measurements.

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duration and frequency of behaviors in a different manner (Table 1).The frequency of exploration, social interactions, and total frequencyof aggressive interactions increased significantly. Marginal increaseswere noticed in grooming and offense. Thus, social deprivationincreased the frequency of most of the behaviors except for dominantpostures. As a consequence, the number of behaviors performed perunit time was significantly increased by social deprivation in the caseof all three non-social behaviors, social interactions, and aggressiveinteractions (F(1,26) values were 23.78, 14.49, and 8.86, respectively,while pwas smaller than 0.0001, 0.001, and 0.01, respectively) (Fig. 5,lower left-hand panel). The same significant difference was seenwhen all the behaviors were considered together (F(1,26)=28.85;pb0.0001; data not shown). The increase in the frequency ofbehaviors brought about a significant decrease in the average boutduration (total duration divided by frequency) of all three non-social,social and aggressive behaviors (F values for housing were 26.73, 7.24and 4.21; pb0.001, 0.02 and 0.05, respectively). Cage size had noimpact on bout duration in either case (F values for cage size andinteraction were between 0.01 and 3.22, while p values were above

0.1). A similar change was noticed when the average bout duration ofall behaviors was considered (F(1,26)=31.46; pb0.0001; Fig. 5,lower right-hand panel).

Noteworthy, the internal structure of aggressive interactionsshowed interesting changes in response to isolation rearing. Thetotal frequency of aggressive interactions increased; within thisoverall change, however, the frequency of defensive postures wasincreasedwhile the frequency of dominant postures was decreased bysocial deprivation. These changes support our earlier findings on theambiguity of aggressive behaviors in socially deprived rats.

Discussion

Social deprivation from weaning increased the percentage ofvulnerable attacks and decreased the signaling of attack intentions. Inaddition and surprisingly, socially deprived rats frequently attackedfrom unusual positions e.g. from submissive posture, defensiveupright or during escape. In the third experiment, where behaviorwas analyzed in detail, defensive behaviors were also increased bysocial deprivation. These findings replicate those published earlier(Toth et al., 2008), and support the notion that adult rats raised undersocial deprivation show violent attacks that are poorly associatedwithsocial signaling yet remain ambiguous as are paralleled by defensive-ness. These abnormal features of attack were associated with signs ofbehavioral, endocrine and autonomic hyperarousal. During aggressiveencounters, socially deprived rats rapidly switched from one behaviorto another, i.e. showed an increased number of behavioral transitions.We tentatively term this behavioral feature “behavioral fragmenta-tion.” Neither basal corticosterone levels nor basal heart rates wereaffected. In contrast, the endocrine and autonomic stress responseswere markedly increased by early social deprivation.

Since the pioneering work of Harlow et al. (1965), early socialisolation in animals was extensively used to model the psychiatricconsequences of early social adversities including social neglect. Itwas demonstrated that the vast variety of psychological disordersattributed to early social neglect have their behavioral counterparts insuch models including increased aggressiveness (Day et al., 1982;Fone and Porkess, 2008; Heim et al., 2004; Kempes et al., 2008;Potegal and Einon, 1989; Pryce et al., 2005; Sachser et al., 1994).Although virtually no children experience the degree of socialdeprivation as such models entail, these findings indicate that earlysocial deprivation still has high face validity. We recently showed thatthe heightened aggressiveness of socially deprived rats is associatedto a behavioral profile that renders the behavior of such subjectsmarkedly different from those usually shown in resident/intruderconflicts (Toth et al., 2008). Such rats targeted their attacks towardsvulnerable body parts of their opponents (head, throat and belly),showed deficient social signaling (less attacks were preceded byoffensive threats), and their behavior was ambiguous in the sense thatincreased attack frequencies were associated by increased defensivebehaviors. We suggested that these abnormal features render sociallydeprived rats similar to humans that show aggression-relatedpsychopathologies in conjunction with early social neglect, furtherenhancing the face validity of the social deprivation model. Wehypothesized that the abnormal attack patterns seen in sociallydeprived rats develop in conjunction with enhanced emotional/physiological stress responses, i.e. in conjunctionwith hyperarousal inaggressive contexts (Toth et al., 2008). This assumption putativelyrendered our model more similar to social neglect-induced, aggres-sion-related psychopathologies as increased stress responses (endo-crine and autonomic) are well-documented consequences of earlysocial disturbances in humans (Fries et al., 2008; Heinrichs et al.,2003; Marsman et al., 2008; Pesonen et al., 2010; Shirtcliff and Essex,2008); moreover, increased stress responses are believed to bestrongly related to aggressive traits (Chida and Hamer, 2008; Murray-Close et al., 2008; Smith and Gallo, 1999; Suls and Wan, 1993).

non-social behaviors social interactions aggressive interactions

Socially deprived

Socially housed

0

2

3

4

Non-socialbehaviors

Socialinteractions

Agonisticinteractions

0

1

2

3

4

5

6

All behaviors

1

Ave

rage

bou

t dur

atio

n(t

otal

dur

atio

n/fr

eque

ncy,

sec

)

Fre

quen

cy (

coun

ts /

1% ti

me)

Fig. 5. Behavioral fragmentation. Upper panel: the succession of behaviors in socially housed and socially isolated rats during aggressive encounters. Each behavioral category (non-social, social, and aggressive) was represented by columns of different color, the width of which shows the duration of the respective behavior. To easy reading, the first 5 min of theaggressive encounter was depicted. Socially isolated rats showed a larger number of behavioral transitions. This behavioral change was quantified and represented in the lowerpanels as the number of behaviors performed per unit time (left-hand panel) and average bout duration (right-hand panel). For further details see section Experiment 3—behavioralarousal. *, significant group difference (pb0.01 at least).

34 M. Toth et al. / Hormones and Behavior 60 (2011) 28–36

Earlier studies on the effect of social deprivation on the function ofthe HPA-axis were not entirely consistent. Van den Berg et al. (1999)found that social deprivation increases aggression-induced cortico-sterone and adrenaline stress responses. In line with this finding,Weiss et al. (2004) reported that socially deprived rats showenhanced glucocorticoid stress responses to startle, whereas Serraet al. (2005) found that social deprivation blunts the dexamethasone-induced suppression of corticosterone secretion. However, sociallydeprived rats showed decreased glucocorticoid stress responses toopen-field exposure and immobilization stress (Gentsch et al., 1981;Sanchez et al., 1998). In another study, immobilization-induced ACTHand corticosterone responses remained unaltered by post-weaningsocial isolation (Schrijver et al., 2002). Conflicting findings werereported in the case of basal ACTH and corticosterone levels as well.

Table 1The relationship between the duration and frequency of behaviors in socially housed and s

Housing Cage RES EXP SOC GRO

Duration of behaviorsSocially housed Small† 0.82±0.52 54.95±4.10 22.75±2.66 7.03±

Large‡ 2.25±1.11 62.28±3.37 17.20±4.16 8.80±Socially deprived Small† 0.28±0.15 52.21±4.24 20.09±1.48 10.70±

Large‡ 0.76±0.35 54.10±2.64 20.18±1.86 10.89±Hhousing(1,24) (p) 3.18 (0.09) 2.18 (0.15) 0.01 (0.90) 1.35 (0Hcage(1,24) (p) 2.85 (0.10) 1.55 (0.22) 1.16 (0.29) 0.15 (0Hinteraction(1,24) (p) 0.69 (0.41) 0.54 (0.46) 1.24 (0.27) 0.1 (0.

Frequency of behaviorsSocially housed Small† 2.67±1.98 133.00±8.08 103.83±5.08 11.67±

Large‡ 7.50±3.53 132.50±8.91 85.50±8.35 15.67±Socially deprived Small† 2.25±1.16 188.13±15.19 116.50±6.72 19.88±

Large‡ 2.88±0.69 230.38±14.55 143.38±10.36 24.13±Hhousing(1,24) (p) 1.76 (0.20) 33.07 (0.0001) 17.95 (0.0005) 3.50 (0Hcage(1,24) (p) 2.06 (0.16) 2.46 (0.13) 0.26 (0.61) 0.85 (0Hinteraction(1,24) (p) 1.22 (0.27) 2.58 (0.12) 7.37 (0.01) 0.01 (0

†, similar to that used in the biotelemetry study; ‡, similar to that used as resident/intruder cGRO, grooming; OFF, offense; DEF, defense, SUB, submissive posture; DOM, dominant postursignificant housing effect; printed in italic, marginal housing effect.

Heidbreder et al. (2000) and Schrijver et al. (2002) found no changes,whereas Sanchez et al. (1995; 1998) found decreased ACTH andcorticosterone basal levels in socially deprived subjects. It is importantto note here that changes in the stress response are stressor-specificand depend on a variety of local conditions that are often difficult toidentify (Pacak and Palkovits, 2001). The data shortly reviewed abovesuggest that glucocorticoid stress responses are increased by earlysocial deprivation in the case of many but not in all situations.

Regarding the effects of social deprivation on autonomic activa-tion, available data are unfortunately sparse. Rearing monkeys insocial isolation from weaning induced subtle changes in noise-induced heart rate changes, without a clear enhancement of responses(Martin et al., 1988). It is worth to note, however, that sociallydeprived rats showed increased adrenaline stress responses in

ocially deprived rats.

OFF DEF SUB DOM totAGO

0.97 1.87±0.81 0.72±0.60 4.08±0.76 1.25±0.25 11.62±2.812.78 1.10±0.45 0.00±0.00 2.70±1.62 1.00±0.34 7.10±2.523.08 5.05±1.13 1.46±0.61 1.34±0.45 1.81±0.32 12.99±1.841.95 3.74±0.97 0.44±0.18 0.86±0.28 1.34±0.38 10.44±1.91.25) 9.28 (0.006) 1.71 (0.20) 7.73 (0.01) 1.72 (0.20) 1.11 (0.30).69) 1.18 (0.28) 3.72 (0.07) 1.27 (0.27) 1.12 (0.30) 2.50 (0.12)75) 0.08 (0.77) 0.11 (0.73) 0.3 (0.58) 0.11 (0.74) 0.19 (0.66)

2.96 33.33±6.61 13.83±2.88 2.17±1.33 9.33±1.58 60.00±11.573.09 24.17±4.24 11.17±3.88 0.00±0.00 5.50±2.64 41.83±7.976.10 36.50±6.15 45.38±10.68 4.75±2.35 4.50±0.85 93.50±16.303.35 50.25±12.04 39.88±8.02 1.88±0.77 3.25±1.11 97.13±17.84.07) 2.91 (0.10) 13.97 (0.001) 2.06 (0.16) 5.23 (0.03) 8.39 (0.007).36) 0.07 (0.79) 0.25 (0.61) 2.64 (0.12) 2.69 (0.11) 0.22 (0.63).90) 1.78 (0.19) 0.03 (0.86) 0.05 (0.82) 0.69 (0.41) 0.5 (0.48)

ages in the corticosterone study; RES, resting; EXP, exploration; SOC, social interactions;e; totAGO, the sum of aggressive behaviors (OFF+DEF+SUB+DOM); printed in bold,

35M. Toth et al. / Hormones and Behavior 60 (2011) 28–36

aggressive situations, which may be relevant for aggression-inducedchanges in heart rates (Nishikawa and Tanaka, 1978; van den Berg et al.,1999). Our findings on basal corticosterone levels are in line with thoseof Heidbreder et al. (2000) and Schrijver et al. (2002), whereas theaggression-induced changes in glucocorticoid stress responses supportthose of van den Berg et al. (1999). Namely, we found that socialdeprivation does not affect basal levels of glucocorticoids but the above-mentioned abnormal features of aggression develop on the backgroundof an increased glucocorticoid stress response. In additionwe found thataggression-induced autonomic activation was considerably larger insocially deprived rats, a finding that is in line with enhanced adrenalinestress responses seen in such rats.

Post-weaning social isolation was also shown to induce symptomsof behavioral hyperarousal in non-social contexts. For example,socially deprived rats showed enhanced startle, disrupted prepulseinhibition, increased hyper-vigilance and novelty-induced locomo-tion (Domeney and Feldon, 1998; Gentsch et al., 1988; Gentsch et al.,1982; Heidbreder et al., 2000; Varty et al., 2000;Weiss et al., 1999). Insocial contexts, signs of behavioral hyperarousal were less evident.Increased aggressiveness may be interpreted in conjunction witharousal, yet aggression levels per se cannot be considered clear-cutevidence in this respect. Other behavioral features noticed in sociallydeprived animals (e.g. social anxiety, decreased submissiveness inaggressive confrontations, low social rank after re-socialization, etc.)are even less illustrative from the point of view of arousal levels(Bastian et al., 2003; Hol et al., 1999; Kempes et al., 2008; Luciano andLore, 1975; Sachser et al., 1994; van den Berg et al., 1999; Von Frijtaget al., 2002). We believe that the phenomenon called here “behavioralfragmentation” may be considered a behavioral sign of hyperarousalthat is expressed in a social context.

Taken together, we found that rats reared in social isolation showabnormal features of aggressiveness, augmented glucocorticoid andautonomic stress responses as well as behavioral fragmentation, aputative sign of behavioral arousal. As shown above, these behavioraland physiological features are shared by people grown up underadverse social conditions, which raise the possibility that post-weaning social isolation can be used as a laboratory model ofaggression-related psychopathologies that develop in response toadverse rearing conditions. This model appears to be especiallyrelevant to emotional violence which is believed to be driven bystrong, as opposed to callous-unemotional violence which is associ-atedwith diminished stress responses (see Haller and Kruk, 2006 for areview). Two of the abnormal aggression models developed so farwere shown to be associated with exacerbated stress responses:frustration-induced aggression by mice and, surprisingly aggressionshown by rats selected for low anxiety (de Almeida andMiczek, 2002;Miczek et al., 2002; Neumann et al., 2010). Aggressiveness induced byearly maternal separation may also belong to this type, althoughabnormal features of aggression were not yet shown in this model(Veenema et al., 2006). All three procedures can be consideredmodelsof emotional aggressiveness; yet, each addresses a specific subtype.The frustration model allows the study of the effects of acute stressorsin otherwise normal animals, whereas the selection model focuses ongenetic predispositions. In contrast, the social deprivation modelinvolves a constitutive hyperarousal that develops in response toenvironmental factors.

The resident/intruder model was used for decades to studyaggression. Recent models, however, focus on abnormal formscharacterized by a mismatch between provocation and response(i.e. on responses that surpasses species-typical levels), indifferencetowards species-specific rules (e.g. decreased signaling of intention,attacking females, or attacking vulnerable targets) and/or insensitiv-ity towards the social signals of the opponent (e.g. sustaining attacksdespite clear signs of submissiveness on the other side). Suchabnormal forms of aggression were induced by endocrine manipula-tions, frustration or instigation, genetic selection or the repeated

exposure of feral rodents to aggressive encounters (de Almeida andMiczek, 2002; de Boer et al., 2003; Haller and Kruk, 2006; Haller et al.,2001; Miczek et al., 2002; Natarajan et al., 2009; Neumann et al.,2010). All these models focus on particular aspects and mechanismsthat lead to the development of abnormal aggression in rodents withthe ultimate aim of understanding the development of similarlyabnormal forms of aggression in humans. We suggest here that post-weaning social isolation may be used to study the mechanisms thatunderlie aggression-related psychopathologies associated with hy-perarousal, particularly those that are triggered by adverse rearingconditions.

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgments

This work was supported by OTKA Grant No. 82069.

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