Physiology & Behavior 116–117 (2013) 13–22

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Physiology & Behavior

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Ameliorative effects of brief daily periods of social interaction on isolation-inducedbehavioral and hormonal alterations

Sivan Raz ⁎Psychology Department, The Center for Psychobiological Research, The Max Stern Yezreel Valley College, IsraelPsychology Department, Tel Hai College, Israel

H I G H L I G H T S

• Social isolation in adulthood exerts major effects on rats' physiology and behavior.• Brief daily periods of social interaction abolish or ameliorate isolation-induced behavioral alterations.• Brief daily periods of social interaction ameliorate isolation-induced alterations in corticosterone levels.• Partial isolation may be used as preventive treatment protecting rats from the deleterious effects of isolation.

⁎ Yezreel Valley College, 19300, Israel. Tel.: +972 546E-mail address: sivanr@yvc.ac.il.

0031-9384/$ – see front matter © 2013 Elsevier Inc. Allhttp://dx.doi.org/10.1016/j.physbeh.2013.03.009

a b s t r a c t

a r t i c l e i n f o

Article history:Received 31 October 2012Received in revised form 24 February 2013Accepted 13 March 2013

Keywords:Social isolationBrief social interactionBehavioral alterationsCorticosterone

The present study investigated the effects of brief daily periods of social interaction on social-isolation-inducedbehavioral and hormonal alterations and deficits. Adult maleWistar rats were allocated to one of three housingconditions: 1) social housing (two per cage); 2) social isolation (one per cage); or partial social isolation (one percagewith access to anothermale rat for 60 min/day). After 14 days in these different housing conditions, the an-imals were subjected to various behavioral tests, including sucrose preference test, acoustic startle response,two-way active shuttle avoidance, pre-pulse inhibition, open field, cooperation learning task, and levels of corti-costerone. Results revealed that social isolation had a substantial impact on rats' performance onmost behavioraltests aswell as on their corticosterone levels. Importantly, however, the results clearly demonstrate that allowingotherwise isolated animals to have a brief (60 min) daily social contact with another rat to a great extent abol-ishes or ameliorates most of the isolation-induced behavioral and hormonal alterations. Hence, providing isolat-ed animals with brief daily periods of social contact may be used as a “preventive treatment” in order to protectthem from the deleterious effects of isolation.

© 2013 Elsevier Inc. All rights reserved.

1. Introduction

Social isolation in laboratory rats has been used extensively in an at-tempt to model various symptoms of human psychopathologies, such asanxiety [1,2], depression [3], schizophrenia [4], and substance abuse [5].Preventing rats from normal interaction and communication has beenfound to exert major effects on both physiological and behavioral pro-cesses [6–8]. The specific effects of social isolation upon rat behaviorare highly dependent on the age at onset of isolation, the type of isola-tion, the degree of any concurrent physical deprivation, the length of iso-lation and so forth [7]. For example,mostmodels to date have focused onadult behaviors after isolation rearing early in life, either as pups or ado-lescents (e.g. [9–18]), which is a very different model than social isola-tion in adulthood [7,19]. Rats housed in isolation at adulthood tend tobe more irritable, restless and hyperactive compared with rats housed

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in groups [20,21]. These rats also show patterns of hypersensitivity,novelty-seeking, anxiety-like phenotype, stress and depressive-like be-havior [20–28]. Isolation housing also increases aggression and interfereswith the performance of a cooperation task in male rats [29–35]. Isola-tion also affects physiological parameters such as alterations in basallevels of plasma corticosterone and ACTH, changes in HPA axis and adre-nocortical function in response to stress stimuli, heavier adrenal glandsand neurochemical changes [7,19,22,23,36–41]. Some reports on adultisolation also provide evidence for increased drug intake andmodulationof responses to rewarding stimuli [25,26,42–46].

We have reported that adult rats housed in isolation self-administersignificantly higher amounts of morphine solution (but not water) com-pared with rats housed in pairs. Interestingly, we also found that as littleas 60 min/day of social interaction from the onset of housing allocation(partial isolation) completely abolished the isolation-induced increasein morphine intake [47]. To the best of our knowledge, this was thefirst report that brief daily periods of social interaction neutralize the in-crease in morphine intake among isolated adult rats. In juvenile rats,

14 S. Raz / Physiology & Behavior 116–117 (2013) 13–22

brief periods of socialization have been shown to ameliorate some ofthe effects of isolation rearing on later behavior [48]. This “partial isola-tion” reversal effect has also been observed in “isolation-inducedaggression” and in an operant cooperation task [49,50,54]. In thesestudies, 60 min/day of social contact were sufficient to reverse the ef-fects of 24 h of social restriction daily. Notwithstanding, the hypothe-sized ameliorative effect of brief periods of social interaction duringthe isolation phase has rarely been studied.

The present study aimed at further exploring the effects of short-term daily social interaction (partial isolation) in otherwise socially iso-lated adult rats. Specifically, we examined behavioral and hormonal dif-ferences between rats housed at adulthood in complete social isolation,partial isolation (60 min/day of social interaction) or pairs. We used arelatively short period (14 days) of social restriction without limitingauditory, olfactory, or visual contact, and examined the influence ofsuch social isolation in various behavioral laboratory settings. Weemployed a set of well-established behavioral measures, including su-crose preference test, acoustic startle response, two-way active shuttleavoidance, pre-pulse inhibition, open field and cooperation learningtask. In addition, we examined between-group differences in serumlevels of corticosterone.

In light of our previous findings [47], we expected that isolation-induced behavioral and hormonal deficits would be prevented, at leastto some extent, by brief daily periods of social physical contact. Behavior-al and hormonal patterns of partially isolated rats were expected to besimilar to those of paired rats or at some intermediate level betweenthe patterns of completely isolated rats and those of paired rats.

2. Materials and methods

2.1. Subjects

Subjects were adult male Wistar rats (Harlan). Their age at the be-ginning of the experiments was 49 days, and their average weightwas 190 g. Throughout the study, subjects were kept in a roomcontrolled for temperature (22 ± 1 °C) and humidity (33%) andmaintained on a 12-hour light/dark cycle (lights on— 07:00 h) in stan-dard plastic cages (30W × 30L × 18H cm) with transparent walls andsawdust bedding. Standard rat food and tap water (two bottles percage) were available ad libitum. All experimental procedures wereconducted in accordance with the NIH Guidelines for the Care and Useof Laboratory Animals and approved by the National Council for AnimalResearch in Israel.

2.2. Experimental procedures

Upon arrival, animals were housed five to six per cage and allowed toadapt to the animal facility for one week. Rats were then randomlyassigned to the different experimental groups. In both experiments, sub-jectsweredivided into three groups of differinghousing conditions: socialhousing: two animals per 30W × 30L × 18H cm cage (Exp. 1 n = 10;Exp. 2 n = 16); isolated housing: one animal per 30W × 30L × 18Hcm cage (Exp. 1 n = 11; Exp. 2 n = 16); and partial isolation: one ratper 30W × 30L × 18H cm (Exp. 1 n = 12; Exp. 2 n = 16) with accessto anothermale rat (always the sameone,matchedbyweight, in aneutralcage) for 60 min only per day (between 9:00 and 10:00). During thattime the rats had access to unlimited physical interaction. Since all cageswere kept in the same animal room, isolationwas not on a visual, olfacto-ry or auditory basis.

2.2.1. Stage I — adaptation (days 1–14)Animals in both experiments were maintained under these differ-

ent housing conditions for 14 days, with ad libitum access to food andwater. Water intake was measured during the last three days of thisphase (days 12–14).

Behavioral tests began on day 15. Animals remained under the differ-ent housing conditions during the behavioral testing days. The order ofbehavioral tests was not randomized. Any potential confounding effectsof orderwere equal between the groups as all animals were tested in thesame order.

2.3. Experiment 1

2.3.1. Stage II — sucrose preference (days 15–16)Subjects were given access to both water and sucrose solution

(10%), in their home cages, in a two-bottle choice test that lasted twodays. Water and sucrose solution intakes were measured every 24 hbyweighing the bottles. To obtain an estimate of the fluid consumptionfor a single animal in the social housing (paired) group, the total intakeof water and sucrose solution was divided in half.

2.3.2. Stage III — acoustic startle response (day 17)The acoustic startle response test is used to assess emotional reactiv-

ity and sensory-motor gating. The acoustic startle response was mea-sured using two automated, ventilated, sound-attenuated startleboxes (40W × 40L × 50H cm; Campden, UK) that were positioned ina dimly lit room. The startle box consisted of a Plexiglas chamber(W18 × 9L × 8H cm) mounted on a piezoelectric accelerometer.Movements of the rats inside the chamber resulted in changes in thevoltage output of the accelerometer. These signals were amplified, dig-itized, and fed into a computer data-acquisition board for further anal-ysis. Rats were familiarized with the startle test room for 30 minbefore being placed in the chamber. The startle session began with a5 minute acclimatization period, with a background noise level of57 dB thatwasmaintained throughout the session. Rats were subjectedto 10 tones (40 ms, 120 dB noise stimuli), with inter-trial intervals of1 min. The maximum startle response for each trial was measured byNewton units (N). For each animal, the average of the ten responseswas taken as an index of the intensity of its startle reflex response.

2.3.3. Stage IV — two-way active shuttle avoidance (day 18)The two-way active shuttle avoidance test is used for studying cog-

nitive/learning function. An animal activity monitor (Campden, UK)used has two automated compartments (20W × 25L × 22H cm), andaccess between the compartments is provided by a 7.5 × 10 cm pas-sageway. Rats were placed individually in the test chamber and weregiven 5 min to acclimate. Each rat was tested in a 60-trial session com-prising a conditioned stimulus (80 dB, 10 s tone) followed by anuncon-ditioned stimulus (10 s, 0.8 mA foot shock) delivered through the gridfloor, with an ITI of 1 min. Performance was scored as avoidance(shuttling to the adjacent compartment while the tone is on), escape(shuttling to the adjacent compartment during shock application) orfreezing (no shuttling to the other compartment until after theshock). Onset latencies to behavior were measured automatically byKinder Scientific software (Campden, UK).

2.3.4. Stage V — serum levels of corticosterone (day 19)Rats were sacrificed by decapitation approximately 24 h after the

last behavioral test. Trunk blood was collected in chilled test tubes,kept at room temperature for 30 min and then centrifuged at 1000 ×gfor 10 min at 4 °C. The serum was separated and stored at −80 °Cuntil assayed. Levels of corticosterone in blood serum were evaluatedphotometrically by a Multiskan FC Microplate Reader, w/Incubator,using a commercial Enzyme-Linked Immunosorbent Assay (ELISA) kit(AssayPro, St. Charles, MO, USA) according to manufacturer's protocol.

2.4. Experiment 2

2.4.1. Stage II — pre-pulse inhibition (PPI) (day 15)Startle response and PPI were measured with the same apparatus

as described above (Section 2.3.2). The session (a total of 90 trials)

15S. Raz / Physiology & Behavior 116–117 (2013) 13–22

started with a five minute acclimatization period, with a backgroundnoise level of 57 dB that was maintained throughout the session. Forevaluating the startle response, the first ten trials consisted of single40 ms 120 dB “pulse-alone” startle stimuli (with ITI of 1 min).These trials were used to obtain a measure of habituation in responseto repeated delivery of the startle stimuli. The rest of the 80 trials(10 s ITI) consisted of random delivery of ten “no” stimuli trials, dur-ing which no stimuli were delivered, 14 “pre-stimuli” trials (at 59, 61,65, 69, 73, 78 or 85 dB), and 56 “pre-pulse” trials that consisted of asingle 120 dB pulse preceded (80 ms interval) by a 20 ms “pre-pulse”of 2, 4, 8, 12, 16, 21 or 28 dB above background (i.e., 59, 61, 65, 69, 73,78 or 85 dB) [55]. The level of PPI (percentage PPI) was determinedaccording to the following formula: 100 − ([startle magnitude onacoustic pre-pulse trials / startle magnitude on “120 dB pulse alone”trials] × 100), such that a 0% value indicated no difference betweenthe responses to pre-pulse trials and pulse alone trials (i.e., no PPI).

2.4.2. Stage III — open field (day 16)The open field test is used in assessment of exploratory behaviors

and generalized motor activity in rodents and considered a model ofanxiety-like behavior. The open field is made of a black lusterlessPerspex box (100L × 100W × 40H cm) placed in a dimly lit room(50 lx). A single rat is placed in the corner of the open field (facingthe wall) and given 5 min of free exploration. The apparatus wascleaned with alcohol and water after each test session. The behaviorwas videotaped by a CC TV Panasonic camera, and post-recording anal-ysis was conducted with Ethovision XT software (Noldus, The Nether-lands). A virtual square center (60 × 60 cm) was defined in the openfield arena. Measurements included the following: locomotor activity(total distance in cm traveled overall in the open field); immobility(total seconds of lack of movement); percent of distance traveled inthe center zone (calculated as the (distance traveled (cm) in the centerzone divided by the total distance) × 100); and the percent of timespent in the center zone (calculated as the (time (s) in center zone di-vided by 300) × 100).

2.4.3. Stage IV — cooperation learning task (days 17–21)The cooperation learning task is designed to investigate cooperative

learning ability, since two rats have to perform a complex coordinatedshuttling behavior [34,49–53]. The apparatus consists of a rectangularblack lusterless Perspex chamber, divided vertically into two tracks bya transparent partition with halls (1 cm in diameter with 10 cm inter-spaces) in it. The length of the test cage is 100 cm, the width of everytrack is 20 cm and the height of the walls is 40 cm. At one end of eachtrack is a feeder for delivery of 0.5 ml sucrose solution (10%), controlledby Ethovision xt 7.0 software (Noldus, TheNetherlands). The chamber'sfloor was virtually divided into three sections: ‘N’ (30 cm), ‘M’ (40 cm),and ‘D’ (30 cm) (‘N’, ‘M’ and ‘D’ indicate proximity to the reinforcementdispenser: near, middle and distant, respectively). For the animals tobecome familiarized with the arena and the task, they are first (day17) placed individually in the chamber for 10 min maximum andgiven the chance to obtain up to ten sucrose rewards. To obtain the su-crose reward, the animal had to move (shuttle) from one end of thetrack (D-floor) to the other end, where the sucrose dispenser is located(N-floor), and back. In the second stage, pairs of animals were placedin the chamber together, each animal in a different track (eachpair consisted of two rats from the same experimental group;i.e. cage-mates in the social group andplay-mates in the partial isolationgroup; alwayswith the same partner). They had to perform coordinatedback-and-forth shuttling in close proximity in order to obtain the su-crose reward. Specifically, pairs of rats had to synchronize their behav-ior so that they are first standing together on the D-floor for aminimumof 0.5 s, then shuttle to theM-floor and remain together on thatfloor forat least 0.5 s, and then shuttle to the N-floor and remain there for atleast 0.5 s before the reinforcement was presented. If both animalsmoved in close proximity from one end to the other and back, both of

them received the reward. If not, neither of them received it (for exam-ple, if one partner ‘fails towait’ on the D-floor for its pair-partner to joinand shuttles alone back to the N-floor, this uncoordinated individualback and forth shuttle is not reinforced). Every training session lasted30 min, on four consecutive days (days 18–21). The questionwas to de-termine howmany rewards the pairs from the different housing condi-tions groups obtained on each day of the test. Trials were controlled bylive video using a CC TV Panasonic camera and analyzed via Ethovisionxt7.0 software (Noldus, The Netherlands).

2.5. Statistical analysis

2.5.1. Experiment 1Results of the sucrose preference test were analyzed by one-way

ANOVA, with housing conditions (social, partial or isolated) as the inde-pendent variable and the two-day average of sucrose solution or waterconsumption as the dependent variable. For the startle response test,data were analyzed by one-way ANOVA, with housing condition asthe independent variable and the average of the ten startle response tri-als as the dependent variable. The two-way shuttle avoidance datawereanalyzed by separate one-way ANOVAs for percentage of avoidance, es-cape and freezing responses. To assess the latency to respond in thetwo-way shuttle task, the 60 trials were divided by ten to create sixblocks. Results were analyzed by a 3 × 6 repeated measures ANOVA,with housing condition as the between-group factor and block as thewithin-subjects factor. Corticosterone level data were analyzed byone-way ANOVA, with housing conditions as the independent variableand corticosterone levels as the dependent variable.

2.5.2. Experiment 2One-wayANOVAwas used to analyze the between-groups differences

in response magnitude on pulse-alone trials. A 3 × 7 repeated-measuresANOVA, with housing conditions as the between-group factor andpre-pulse intensity as thewithin-subjects factor,was used to analyze star-tle magnitude and %PPI data on trials with pulse preceded by pre-pulse.For the open-field test, one-way ANOVAwas used to analyze overall loco-motor activity, immobility, percent of distance traveled in the center zone,and percent of time spent in the center zone. A 3 × 4 repeated-measuresANOVA, with housing conditions as the between-group factor and day asthewithin-subjects factor, was used to analyze the reward obtaining datafrom the cooperation learning task.

In both experiments 1 and 2, Tukey (HSD) was used as the post hoctest, when appropriate. Numeric results are presented as Mean ± SEM(in both text and figures), and considered significant for p-values lessthan 0.05.

3. Results

Throughout the experiment, no significant differences in bodyweightemerged as a function of housing conditions. Further, no between-groupdifferences were found in water base-line consumption.

3.1. Experiment 1

3.1.1. Sucrose preferenceThe rats' drinking data are presented as mean total volume of sucrose

solution and water consumed by subjects in the different groups. “Mean”represents the average fluid intake across two days of observation as themost representative estimate of average total consumption. Results re-vealed that 1 h/dayof social interaction reversed the isolation-induced in-crease in sucrose intake. Significant between-group differences in sucrosesolution intake were seen [F(2,25) = 18.22, p = 0.0001, η2

p = 0.59].Post hoc tests showed that isolated rats consumed more sucrose(92.0 ml ± 6.86) compared with the partial isolation group(59.05 ml ± 3.12) (p = 0.0001, Cohen's d = 1.85) and comparedwith the social housing group (45.41 ml ± 2.32) (p = 0.0001,

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Fig. 2.Mean startle response amplitude (in Newton units) among the different housingconditions. Error bars represent SEM.

16 S. Raz / Physiology & Behavior 116–117 (2013) 13–22

Cohen's d = 2.75). No significant differences were found between par-tial isolation and social housing conditions. No differences in water in-take were seen between the three groups (Fig. 1).

3.1.2. Startle responseSignificant differences inmean startle responsewere found between

groups [F(2,30) = 5.59, p = 0.009, η2p = 0.27]. Post hoc analysis re-

vealed that rats in isolated housing had a stronger startle response(2.39 N ± 0.22) relative to rats in partial isolation (1.82 N ± 0.12)(p = 0.05, Cohen's d = 0.97) and to rats in social housing (1.62 N ±0.14) (p = 0.009, Cohen's d = 1.47). Therewere no differences in star-tle response between the partial isolation and the social housing groups(Fig. 2).

3.1.3. Two-way active shuttle avoidance

3.1.3.1. Avoidance responses. Analysis revealed a significant effect ofgroup on percent of avoidance responses during the task [F(2,30) =6.63, p = 0.004, η2

p = 0.31]. Post-hoc testing indicated that percent ofavoidance responses of the isolated housing group was significantlylower (33.03% ± 4.42) than that of the partial isolation group(58.75% ± 6.58) (p = 0.01, Cohen's d = 1.34) and of the social housinggroup (59.83% ± 6.28) (p = 0.01, Cohen's d = 1.54). No differenceswere found between the partial isolation and the social housing groups(Fig. 3a).

3.1.3.2. Escape responses. Analysis revealed a significant effect ofgroup on percent of escape responses during the task [F(2,30) = 5,p = 0.013, η2

p = 0.25]. Post-hoc testing indicated that percent ofescape responses among the isolated housing group was significant-ly higher (63.64% ± 5.26) than among the partial isolation group(41.11% ± 6.62) (p = 0.032, Cohen's d = 1.11) and among the so-cial housing group (38.83% ± 6.22) (p = 0.023, Cohen's d = 1.33).No differences were found between the partial isolation and the so-cial housing groups (Fig. 3a).

3.1.3.3. No escape responses (freezing). There was no significant effectof group on percent of freezing responses during the task.

3.1.3.4. Latency. Significant differences in mean response latency werefound between groups [F(2,30) = 6.08, p = 0.006, η2

p = 0.29]. Posthoc analysis revealed that animals in isolated housing had significantlylonger latencies to respond (9.12 s ± 0.43) compared with animals inpartial isolation (6.04 s ± 0.71) (p = 0.009, Cohen's d = 1.53) andcompared with animals in social housing (6.29 s ± 0.74) (p = 0.02,Cohen's d = 1.48) (Fig. 3b). The partial isolation and the social housinggroups did not differ in their mean response latencies. There was also amain effect of block [F(5,150) = 33.72, p = 0.0001, η2

p = 0.53], such

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Fig. 1. Sucrose solution and water intake (ml) during 2 days of two-bottle test amongsocial housing, partial isolation and isolated housing groups. Error bars represent SEM.

that the mean response latencies of animals in all three groups becamegradually shorter (Fig. 3b). There was no group × block interactioneffect.

3.1.4. Serum levels of corticosteroneAnalysis revealed significant differences in corticosterone levels

between groups [F(2,29) = 10.06, p = 0.0001, η2p = 0.41].1 Post

hoc analysis showed that animals in isolated housing had significant-ly lower levels of corticosterone (79.44 ng/ml ± 4.18) comparedwith animals in partial isolation (121.6 ng/ml ± 13.45) (p = 0.02,Cohen's d = 1.22) and compared with those in social housing(150.42 ng/ml ± 12.44) (p = 0.0001, Cohen's d = 2.52). The partialisolation group had intermediate levels of corticosterone, betweenthe social and isolated housing groups. However, the difference be-tween the social housing and the partial isolation groups was not sta-tistically significant (p = 0.18) (Fig. 4).

3.2. Experiment 2

3.2.1. Pre-pulse inhibition

3.2.1.1. Startle magnitude. In line with Study 1, on trials with pulsealone the one-way ANOVA revealed a higher mean startle magnitudeamong isolated (2.46 N ± 0.18) compared with partially isolated rats(1.49 N ± 0.16) (p = 0.0001, Cohen's d = 1.55) and compared withrats in social housing (1.68 N ± 0.16) (p = 0.001, Cohen's d = 1.25)[F(2,45) = 12.21, p = 0.0001, η2

p = 0.35] (Fig. 5). There were nodifferences between the partial isolation and the social housinggroups.

In addition, the higher levels of startlemagnitude among isolated ratswere maintained through all pre-pulse intensities. A 3 × 7 repeated-measures ANOVA revealed a main effect of housing conditions[F(2,45) = 4.12, p = 0.02, η2

p = 0.16]. Post hoc Tukey testing showedthat this difference was significant only among isolated animals com-pared with partially isolated animals (p = 0.017, Cohen's d = 0.97).There was also a significant main effect of pre-pulse intensities[F(6,270) = 54.54, p = 0.0001,η2

p = 0.55] (Fig. 5). As pre-pulse inten-sity increased, the startle response magnitude decreased. However, theinteraction between pre-pulse intensity and housing conditions wasnot significant.

1 One sample from the social housing group was excluded from the statistical anal-ysis since corticosterone level exceeded three standard deviations above the groupmean.

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Fig. 5. Mean startle response amplitude (in Newton units) among animals housed indifferent housing conditions. ‘P’ means ‘pulse alone’ and ‘pp’ indicates pulse precededby pre-pulse. Error bars represent SEM.

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Fig. 3. (a) Mean percent of avoidance, escape and freezing responses and (b) mean re-sponse latencies (seconds) among the different housing conditions. Error bars representSEM.

17S. Raz / Physiology & Behavior 116–117 (2013) 13–22

3.2.1.2. PPI. There was a significant effect of pre-pulse intensity,reflecting the increasing effectiveness of more intense pre-pulses ininducing greater %PPI values [F(6,270) = 70.12, p = 0.0001, η2

p =0.61, data not shown] with no interaction or between-groups effects.

3.2.2. Open field

3.2.2.1. Locomotor activity in the open field arena. Significant differencesin overall locomotion were found between groups [F(2,45) = 5.03,p = 0.01, η2

p = 0.18]. Total distance (cm) moved by isolates wasgreater (3256.83 cm ± 102.75) compared with those in the partial iso-lation group (2525.28 cm ± 96.02) (p = 0.02, Cohen's d = 1.84) andcompared with those in the social housing group (2540.84 cm ±

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Fig. 4. Mean serum corticosterone levels (ng/ml) among rats of different housing condi-tions. Error bars represent SEM.

290.59) (p = 0.02, Cohen's d = 0.82). No differences were seen be-tween the partial isolation and the social housing groups (Fig. 6a).

3.2.2.2. Immobility. Significant differences in immobility (freezing)were seen between groups [F(2,45) = 4.36, p = 0.019, η2

p = 0.16].Isolated animals exhibited less freezing behavior (29.84 s ± 2.71)than did socially housed animals (68.77 s ± 15.51) (p = 0.014,Cohen's d = 0.87). Animals in partial isolation exhibited intermedi-ate levels of freezing (47.34 s ± 3.67), but the difference did notreach statistical significance (Fig. 6b).

3.2.2.3. Percent of locomotor activity in the center zone. Significant dif-ferences were found between groups [F(2,45) = 17.72, p = 0.0001,η2

p = 0.44]. Isolated animals traveled a greater distance (cm) in thecenter zone of the open field arena (28.01% ± 1.38) relative to partiallyisolated animals (9.89% ± 1.74) (p = 0.0001, Cohen's d = 2.88) andrelative to socially housed animals (15.43% ± 3.12) (p = 0.001,Cohen's d = 1.31). No statistical differences were seen between thepartial isolation and the social housing groups (Fig. 6c).

3.2.2.4. Percent of time spent in the center zone. Significant differenceswere found between groups [F(2,45) = 19.76, p = 0.0001, η2

p =0.47]. Isolated animals spent much more time in the center of the openfield arena (20.94% ± 0.99) relative to partially isolated animals(7.16% ± 1.29) (p = 0.0001, Cohen's d = 2.99) and relative to sociallyhoused animals (10.96% ± 2.24) (p = 0.0001, Cohen's d = 1.44). Nodifferences were seen between the partial isolation and the social hous-ing groups (Fig. 6d).

3.2.3. Cooperation learning taskOn the first day of training (individual learning), no between-group

differences were found in baseline level of reward gaining. On days 1–4of the cooperation task (paired training), analysis revealed amain effectof housing conditions [F(2,45) = 33.85, p = 0.0001, η2

p = 0.60], suchthat animals in partial isolation obtained more sucrose rewards(6.97 ± 0.45) than did isolated animals (3.47 ± 0.29) (p = 0.0001,Cohen's d = 2.88) and socially housed animals (3.81 ± 0.20) (p =0.0001, Cohen's d = 2.26) (Fig. 7). No significant interaction orwithin-subjects effects were found.

4. Discussion

Environmental and social stressors and the absence of positive so-cial interactions in social species are associated with behavioral andneuroendocrine dysfunction and increased vulnerability to affectivedisorders [56].

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Fig. 6. Mean levels of overall locomotor activity (a); immobility (b); percent of distance traveled in center zone (c); and percent of time spent in center zone (d) during anopen-field test, among animals housed in different housing conditions. Error bars represent SEM.

18 S. Raz / Physiology & Behavior 116–117 (2013) 13–22

The aim of the present study was to explore behavioral and endo-crine differences among adultmale rats housed in complete social isola-tion, partial isolation or social (paired) housing. As expected, socialisolation had a substantial impact on rats' performance onmost behav-ioral tests, aswell as on their corticosterone levels. Importantly, howev-er, and consistent with our hypotheses, the results clearly demonstratethat allowing isolated animals a brief (60 min) daily period of social in-teraction during the isolation phase counteracted most behavioral andhormonal alterations induced by isolation.

4.1. Experiment 1

In the sucrose preference test, in agreement with previous reports[7,57–59], the results show that isolation housing significantly increasessucrose intake, supporting the evidence regarding the enhanced

0

2

4

6

8

10

12

1 2 3 4

Rew

ards

Days

Social housing

Partial isolation

Isolated housing

***

***

* **

Fig. 7. Number of sucrose rewards obtained by animals housed in different housingconditions, during days 1–4 of cooperation learning task. Error bars represent SEM.

incentivemotivation and reward-sensitivity observed as a consequenceof this housing condition [57]. It should be noted that animals in thepartial isolation group consumed significantly less sucrose than animalsin the complete isolation group and amounts of sucrose similar to thosein the social housing condition. Social isolationmay change the sensitiv-ity and reactivity to various stimuli. Therefore, isolated rats may bemore reactive to the sweet taste of sucrose or to the novelty of thetaste. Itmay also be that social isolation somehow affects the rewardingconsequences of sucrose. Social isolation increases not only sucrosepreference but also a preference for rewarding drugs such as cocaine[60] amphetamine [61] and ethanol [62], as well as increased anticipa-tory behavior [63], suggesting isolation-induced increase in the suscep-tibility to the reinforcing properties of a variety of stimuli [64].However, it is difficult from our study alone to determine whether in-creased consumption after isolated housing is because of increased sen-sitivity, making the sucrosemore rewarding, orwhether it is an attemptto compensate for a possible decreased effectiveness of it as a rewardingstimulus. The fact that there were no differences in water intake be-tween the groups suggests that this finding cannot be explained by ageneral enhancement of fluid intake by isolates, but rather due to selec-tive enhancement of sucrose intake. Moreover, since there were no sig-nificant differences in body weight between isolated and pairedsubjects during the course of the experiments (see also [20,65,66]),these results cannot be explained by differences in body weight thatmight affect daily intake and/or sucrose reactivity. In order to betterevaluate sucrose preference and reward sensitivity, future studiesshould usemultiple sucrose concentrations (instead of a single 10% con-centration as in the present study).

In keeping with other studies [47], the results of the startle responsetest revealed a significant potentiation of acoustic startle in isolatedcompared with paired rats. However, short daily social interactioncounteracted this elevation in startle reactivity, since rats in partial iso-lation exhibited similar startle responses to those of paired rats. Startleresponse is a common behavioral test for assessment of emotional

19S. Raz / Physiology & Behavior 116–117 (2013) 13–22

reactivity in rodents, and is often used to assess the effects of anti-anxiety drugs [67–70]. It is important, however, to differentiate be-tween conditioned and unconditioned startle responses. Many studieshave focused on the fear-conditioning or fear-potentiated startle para-digmwhichmay not provide an idealmodel formore general and dura-ble states of stress, anxiety, discomfort and apprehension [71–74]. Inthe present study, no conditioning was used, so the startle reflex wasbasically an unconditioned response.

Results of the two-way shuttle avoidance task revealed that isolatedrats had impaired acquisition of the avoidance response. They exhibitedsignificantly more escape responses and fewer avoidance responsescompared with paired and partially isolated rats. Similarly, throughoutthe six blocks of the task, isolated rats had longer latencies to respondcomparedwith paired and partially isolated rats. These results are in ac-cordance with previous findings of impaired acquisition of the avoid-ance response as a result of isolation rearing [75] and of postoperativeisolation housing [76]. It is possible that rats in isolation have poorer ca-pability for forming an association between the shock (US) and cue (CS)in the apparatus, and subsequently, learning the instrumental responseto avoid the shock. Choi et al. [77] suggested that deficits in avoidancelearning may be related to alterations in the function of the lateral andbasal nuclei of the amygdala. The current results may suggest thatshort daily interaction with another rat may have some protective ef-fects against the development of such brain and behavioral alterations.

Another explanation for the reduced avoidance among isolated ratsmay be that they were less sensitive to the aversive/painful shock. Inthat sense, perhaps the “fear” aroused by the presence of the CS failedto motivate learning of the instrumental response in isolates to thesame degree that it did among paired and partially isolated rats.Conflicting results have been reported concerning isolation-induced sen-sitivity to painful stimuli. While some studies report isolation-inducedreduction in pain sensitivity (i.e. hypoalgesia) [78,79], others have failedto demonstrate such effect [80]. Further studies are required in order todetermine isolation effects on pain/shock sensitivity, especially withrespect to isolation in adult. This is important for the interpretation ofisolation impact on many paradigms utilized to assess conditioned be-havioral responses [64]. Whether reduced avoidance in isolates reflectslearning impairment or reduced sensitivity to the shock, the importantand novel finding of this experiment is that this effect was preventedin the partially isolated animals.

The present results also showmodifications of the HPA axis functionas reflected in serum levels of corticosterone. Abnormalities in the behav-ioral response of isolated rats have been associated with functionalchanges in the endocrine response, although differences in social isola-tion procedures, age, or test environments among studies yielded incon-clusive results [81]. The basal level of corticosterone in plasmawas foundto be either unchanged [82–84], increased [85–87] or decreased [88,89]in socially isolated animals. In the current study, corticosterone levels(measured about 24 h after the last behavioral test) were lowest in iso-lated animals and highest in paired animals, with intermediate levelsof corticosterone among partially isolated animals (although corticoste-rone levels of paired and partially isolated animals did not differ statisti-cally). A similar reduction in corticosterone among isolated adult malerodents has been reported in other studies (e.g. [88–91]). According toMiachon et al. [88], possible explanations for lower levels of corticoste-rone among isolates may include either exhaustion of corticosteronestores or a defect in the positive feedforward effect of ACTH on cor-ticosterone. However, while Miachon et al. used a prolonged period(13 weeks) of isolation, the current study used only two weeks of isola-tion, perhaps making the second hypothesis more plausible. Futurestudies should attempt to differentiate between these two possible ex-planations. Another interpretation of the between-group differences incorticosterone levels may be that pair-housed and partially isolated ani-mals (rather than isolated animals) are stressed by social encounters, es-pecially intraspecific aggression, and this is reflected by higher baselinecorticosterone [91]. Nonetheless, in the context of the current study,

the most important finding is that 60 min/day of social interactioncounteracted the effects of social isolation not only at the overt behavior-al level but also at the more covert endocrine/hormonal level.

4.2. Experiment 2

Results of the pre-pulse-inhibition (PPI) test replicated and extend-ed the results of the startle response test from experiment 1, sincerats in isolation exhibited significantly higher amplitudes of startlenot only on pulse-alone trials but also on trials where the startlingstimulus was preceded by pre-pulse. Higher average levels of startlemagnitude among isolated rats relative to paired and partially isolat-ed rats were maintained consistently through all pre-pulse intensi-ties. These results provide further support for the hypothesizedemotional state associated with an anxiogenic profile produced byisolation at young adulthood, and for the counteracting or anxiolyticeffects of short daily periods of social interaction. In the presentstudy, animals from the different housing conditions did not differin their average percent of startle response inhibition on pre-pulsetrials. This is in line with others who reported no PPI deficits amongWistar rats housed in isolation for 14 days at young adulthood [92].The majority of reports of the influence of isolation on PPI refer to iso-lation rearing. A significant PPI deficit is usually seen when rats arereared from weaning under conditions of isolation and tested atadult age [9,11,93–97]. Results obtained in the present study showthat isolation of rats for two weeks at adult age is not sufficient to in-duce changes in PPI. Future studies should explore the possible effectof partial isolation on isolation-induced alterations in PPI levelsamong rats reared from weaning in differential housing conditionsfor a relatively long period of time (e.g. [97]).

Results of the open field test indicated that isolated rats hadmore openfield activity than their socially housed andpartially isolated counterparts.This was reflected in higher rates of overall locomotion, less freezing be-havior, and greater distance traveled and time spent in the center zoneof the open field arena. Hyperactivity upon exposure to a novel environ-ment is one of the most prominent behavioral consequences of isolation,and may suggest increased reactivity of individually-housed animals to-wards environmental stimuli [20,78,98–100]. Isolated animals may beless sensitive than socially-housed controls to the novelty of the test envi-ronment and therefore exhibit higher levels of exploration. It has beensuggested that isolation-induced locomotor hyperactivity in an openfield represents anunfocused activity that does not facilitate but rather in-hibits an animal's proper recognition of the surroundings [99]. Increasedtime spent in the center of the open field is often interpreted as decreasein anxiety-like behavior. Hence, themost parsimonious explanationof thecurrent data is that the conditions of the behavioral testing, in isolatedrats, favored enhancements of exploration and/or novelty-seeking behav-iors rather than anxiety [100].

The current results with respect to open field provide a demon-stration of the counteracting effect of partial isolation on suchisolation-induced elevation in exploratory and hyperactive behaviors.

The hypothesis regarding between-group differences in cooperationlearning was only partly supported by the current results. According toprevious reports [50], cooperation learning among isolated rats wasexpected to be impaired in comparison to socially housed rats. Howev-er, the present results revealed no suchdifferences, as reflected in a sim-ilar average number of reinforcements obtained by animals from thetwo housing groups. Interestingly, animals from the partial isolationgroup exhibited superior performance in cooperative behavior andgained significantlymoremutual reinforcements on all days of training.This finding probably cannot be explained by a general enhancement inlocomotion activity or a differential sensitivity to the rewarding value ofthe sucrose reinforcement. First, no between-group differences wereseen on the individual-learning session that preceded the cooperation(paired) training sessions. Second, the results of open field and sucrosepreference tests did not imply heightened levels of locomotion or

20 S. Raz / Physiology & Behavior 116–117 (2013) 13–22

sucrose preference among partially isolated rats, but rather the con-trary. It is more plausible that partially isolated animals were indeedfaster to learn the cooperative shuttling in close proximity, which wasnecessary for gaining themutual rewards. Perhaps themost likely inter-pretation of these results is that rats housed in different conditionsdiffered in their motivation to engage in social interaction. Hence,partially-isolated rats may bemoremotivated to spend time in proxim-ity to one another and to coordinate their behavior. In the course of theexperiment, observation of the behavior of partially isolated rats duringthe 60-minute daily social interaction sessions revealed that they wereintensively and gregariously engaged in high-arousal social behaviors,among them fighting, a kind of rough and tumble play, social groomingand anal investigation. It is possible that this intensive but brief socialcontact may serve as a catalyst for neuronal, cognitive and emotionalprocesses that may account for the superior cooperation performanceof partially isolated animals. Despite this, caution should be takenwhen interpreting the present results, and they should be viewed aspreliminary, due to the lack of within-subject or interaction effectsreflected in the similar mean numbers of rewards obtained by animalsfrom all housing conditions on the four successive training sessions. Inother words, none of the groups exhibited improvement in cooperativebehavior as a result of recurrent experience. No “learning curve” couldbe drawn. It is highly possible that for such a complex learning task,four days of training is insufficient. Future studies are advised to applythis cooperation task for prolonged periods (at least 10–12 days),whichmay allowanimals to better acquire and perform the coordinatedbehavior. That way a “learning curve” is expected to emerge, perhapsaccompanied by within-subject or interaction effects.

In both experiments 1 and 2, the order of behavioral tests was notrandomized. However, any potential confounding effects of orderwere equal between the groups as all animals were tested in thesame order. Nevertheless, the possibility of interactions of multipletesting as a confounding factor should be taken into account in inter-pretation of the current results. Moreover, animals from all groups, inboth experiments 1 and 2, were subjected to only one behavioral testeach day. However, for the partial isolation group, the behavioral testswere always preceded by the daily session of social interaction. Thismight partly confound interpretation of the between-group differ-ences reported here. Future studies should further examine this by,for example, reversing the order of events by placing the social inter-action experience later in the day, after completeness of the behavior-al test.

4.3. Summary and conclusions

Taken together, the results of experiments 1 and 2 replicate andextend the literature concerning isolation-induced behavioral andhormonal alterations and deficits among adult rats. They show thatisolation may exert major impact upon rats' physiology and behavioreven under less strict conditions, i.e. isolation that takes place atadulthood (as opposed to infancy or post-weaning, which is muchmore common), relatively short duration of isolation (14 days), isola-tion that does not include restriction of visual, olfactory or auditorysigns, and a control (social housing) group consisting of only tworats per cage. Hence, our paradigm may be best understood as “socialrestriction in adulthood”. We thus attribute the behavioral alterationsseen in the isolated group to the lack of direct tactile contact/physicalsocial interaction between the animals. A commonly held view is thatisolated housing among rats produces physiological and behavioralchanges that may be cataloged under the general category of “stress”.However, some of the findings reported in the literature as well assome of the current data are not easily interpreted in this way. For ex-ample, lower basal corticosterone levels and higher exploration of thecenter of an open-field may be interpreted as reflecting lower ratherthan higher levels of stress among isolated rats. Therefore, it is notsuggested that the effects of social isolation are necessarily attributed

to chronic stress but rather to the chronic deprivation of stimuli crit-ical to maintenance of particular behavioral and endocrine mecha-nisms [59]. The present findings support the notion that socialisolation affects considerably the emotional reactivity of adult rats,perhaps making social isolates hyper-emotional and abnormally reac-tive and sensitive to various environmental stimuli [17].

Importantly, although most of the present behavioral findings,with respect to adult isolation, are not themselves novel (but rathera replication and validation of existing literature), the present resultsclearly demonstrate that allowing otherwise isolated animals brief(60-min) daily social contact with another rat may abolish or to agreat extent ameliorate most of the isolation-induced behavioraland hormonal alterations. To the best of our knowledge, this isamong the first systematic demonstrations of this “partial-isolation”reversal effect among adult rats. It is assumed that physiologicalchanges occurring in isolated animals during these brief daily periodsof social interaction are associated with and mediate the reversal ofthe effects of isolation. There may be a “time window” of 60 min (orless) in which these changes occur — providing a good opportunityto further investigate these physiological processes, especially usingbiochemical and electrophysiological methods. Although 60 min ofsocial interaction per day was sufficient to reverse the effects of24-h social restriction daily, it is possible that even shorter periods(e.g. 45 or 30 min) of social interaction would be sufficient. In thesame line, it would be interesting if fewer sessions or even a singlesession of interaction might be already sufficient to induce these ef-fects. Future studies may attempt to determine the minimum amountof interaction that is needed to reverse these effects of social isolation.Finally, the results reported here may also have important practicalimplications. In many studies in the field of biological psychologyand neuroscience, subjects are housed in isolation for convenienceor due to some technical constraints, even though the isolation itselfis not a direct variable investigated within the study. Furthermore,many such studies fail to consider or control for the confounding ef-fects of social isolation upon subjects' physiology and behavior,which might make their results hard to interpret. In light of this, pro-viding isolated animals with brief daily periods of social contact maybe used as a “preventive treatment” in order to protect subjects fromthe deleterious effects of isolation and reduce the risk of a possibleisolation-induced bias in result patterns.

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