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Novel-object place conditioning in adolescent and adult male and female rats: effects of social isolation
Physiology & Behavior 80 (2003) 317 – 325
Novel-object place conditioning in adolescent and adult male and female rats: effects of social isolation Lewis A. Douglas, Elena I. Varlinskaya, Linda P. Spear* Center for Developmental Psychobiology, Department of Psychology, Binghamton University, Binghamton, NY 13902-6000, USA Received 21 February 2003; received in revised form 30 July 2003; accepted 16 August 2003
Abstract Elevated levels of novelty seeking are often seen during adolescence. Recent studies using a conditioned place preference (CPP) paradigm have shown that novelty may be rewarding for adult rats. The present study explored the impact of age, sex, and isolation stress on novelty seeking and novelty reward by assessing novel object-induced CPP in adolescent and adult male and female Sprague – Dawley rats housed either socially or in isolation. Responding to the novel objects during conditioning was higher in adolescent animals than adults, and was suppressed by social isolation only in adulthood, particularly among males. Novel object CPP was strong among adolescent males, whereas only socially isolated adult males demonstrated preference for the compartment paired with the novel objects. This age difference was not evident in females, with both adolescent and adult group-housed females, but not their isolated counterparts, showing novel-object place conditioning. These dissociations between novelty-directed behaviors during conditioning and novelty reward in the CPP paradigm support the suggestion that mechanisms underlying novelty seeking are separable from those involved in the rewarding effects of novelty. High levels of novelty seeking demonstrated by adolescents do not necessarily predict high rewarding properties of novelty, with the latter also being influenced by environmental and gender-related factors. D 2003 Elsevier Inc. All rights reserved. Keywords: Novelty; Conditioned place preference; Adolescence; Sex differences; Social isolation; Rats
1. Introduction Relative to adults and children, human adolescents exhibit an increased level of risk-taking behavior, associated with the motivation to experience multiple new and intense stimuli to attain potential rewards [1,2]. The motivation to seek out new experiences, i.e., novelty seeking behavior, has been identified as a significant contributor to current and future drug use, multiple drug use, and later abuse [3 –6]. Given that adolescence is a time of acquiring new skills for survival away from parents, enhanced novelty seeking may have been evolutionally conserved for its adaptive value during this developmental period, contributing to exploration of novel areas and providing the opportunity to find new sources of food, water, and mates (see Ref. [7]). Although little explored to date, there is some experimental evidence that age may play a role in responding to novelty, with * Corresponding author. Tel.: +1-607-777-2825; fax: +1-607-7776418. E-mail address: [email protected] (L.P. Spear). 0031-9384/$ – see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2003.08.003
adolescent rodents being more responsive to novel stimuli than their adult counterparts. For instance, adolescent mice spent more time in a novel environment and had higher levels of novelty-induced activity than adults [8]. In a similar way, adolescent rats have been reported to be more active and to engage in more exploratory behaviors in novel situations than adults [9,10]. Responding to novelty may also be influenced by social factors. In humans, a number of social variables, including social conformity, peer deviance, and social support influence novelty seeking behavior in adolescence [11]. Likewise, the presence of peers before testing enhanced responding to novelty in nonhuman primates, increasing exploration of novel objects relative to their socially deprived counterparts [12]. In contrast, rats reared in social isolation were more responsive to novelty relative to rats housed socially (see Ref. [13] for references and review), as indexed by increased locomotor activity [14,15], enhanced preference for novel environments [16], and elevated levels of object exploration [17]. Such isolation-induced increases in object exploration were found to be age-specific, emerging only in animals
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socially deprived between 25 and 45 days of age and not by isolation initiated after P45 [18]. These findings suggest that responding to novelty of adolescent animals may be influenced by social factors in a way different from that of their adult counterparts. Conditioned place preference (CPP), a widely used behavioral paradigm to measure drug reward in laboratory animals, has been employed recently for assessment of the rewarding properties of novel environmental stimuli. In these studies, socially isolated adult male rats received access to novel objects in one distinct environment (e.g., one compartment of a CPP apparatus containing distinct tactile and visual cues), while receiving exposure to a second distinct environment (the other compartment of the apparatus) in the absence of any novel objects. In a choice test, when exposed to the entire apparatus without novel objects, rats showed preference for the environment that had been previously paired with novelty [19 –21]. Novelty of the objects is an important issue in this paradigm, since animals that received access to familiar objects in one of the compartments of the CPP apparatus did not show preference for that environment later on, and during conditioning they spent significantly less time interacting with the familiar object than their counterparts exposed to novel objects [21]. These results suggest that access to novel but not familiar objects is rewarding, findings that validate the use of this procedure for the assessment of novelty reward. There are no reports as to whether novel stimuli would support place preference conditioning in adolescent animals, nor whether such conditioning would be influenced by sex and housing conditions in an age-specific manner. Therefore, the main objective of the present study was to evaluate how age, sex, and isolate housing influence the rewarding properties of novelty using the novel-object place conditioning paradigm developed by Bevins and Bardo [20].
2. General methods 2.1. Animals Animals were derived from Sprague – Dawley VAF rats bred and reared in our colony at Binghamton University. Pups were housed until weaning with their parents in standard maternity cages with pine shavings as bedding material. All animals were housed in a temperature-controlled (22 jC) vivarium maintained on a 12-h/12-h illumination cycle (lights on at 0700 h) with ad libitum access to food (Purina Rat Chow, Lowell, MA) and water. Litters were culled to 10 pups (with a sex ratio of four pups of one sex and six of the other) within 24 h after birth. Rats were weaned on P21 and pair-housed in a standard hanging cage with a samesex littermate. Animals in the isolate housing condition were placed in individual cages 10 days before conditioning procedure (P23 if tested as adolescents and P55 if tested as adults), where they remained housed until the end of the
experiment. In all respects, maintenance and treatment of the animals were in accord with guidelines for animal care established by the National Institutes of Health [22]. 2.2. Procedure Conditioning and testing were conducted in Plexiglas chambers divided into two equal-sized compartments by a guillotine door. One compartment had horizontal black and white stripes on the walls and a wire mesh floor, while the other had vertical stripes and a metal rod floor. The chambers for adult conditioning and testing were 54 cm long, 40 cm wide, and 45 cm high, while adolescents were conditioned and tested in proportionally size-adjusted chambers (40 cm long, 30.5 cm wide, and 45 cm high). Four boxes were used at each age, allowing training and testing of up to four animals simultaneously. The behavior of the animals was recorded by video cameras mounted above each chamber. The chambers were located in a testing room maintained under dim illumination (20 –25 lx in the center of each chamber). Novel objects used for conditioning included wood blocks (9 cm long, 3.5 cm wide, and 2.5 cm high), white PVC pipe (5 cm long with a 2.8 cm inner diameter), rolls of cotton (10 cm long, 5 cm wide, and 2 cm high), balls of tin foil (about 5 cm in diameter), and hard plastic PEZ dispensers (with approximate diameters of 4 cm). Adolescent animals were conditioned starting at P33 and were tested on P38. For adults, conditioning was initiated on P65, with testing on P70. All experimental procedures were conducted within the first 5 h of the animal’s light cycle. On all conditioning days, each experimental animal was exposed to both sides of the apparatus for 15 min as well as to a third environment distinct from the apparatus (a clear breeder tub with pine shavings), also for 15 min. In one of the compartments of the CPP apparatus a new object was placed daily (paired side), whereas no objects were placed in the second compartment (unpaired side). There was a 1-h interval between the exposure to each of these three contexts, during which each animal was returned to its home cage. Order of exposure to the paired chamber, unpaired chamber, and third environment was counterbalanced within each group, as was the order in which the novel objects were provided to the animals across days. For experimental animals, the side of the chamber (horizontal versus vertical) paired with the novel objects was also counterbalanced. Animals in the control groups were placed into each side of the chamber daily, but in the absence of novel objects. Instead, these animals were exposed to the same novel objects as experimental subjects in the third environment –the breeder tub. As for experimental animals, order of exposure was counterbalanced for controls. These groups were included in the experimental design to control for possible handling/experience-induced alterations in intrinsic preference following repeated exposures to the compartments during conditioning. Animals were conditioned for 5 days, and the test session was conducted 24 h after the last conditioning trial. On the
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test day, no objects were placed in the CPP chambers. At the time of test, the door separating the two chambers was removed, and each subject was placed in the center of the CPP apparatus and allowed to freely locomote between the two compartments for 10 min. Time spent in each of the compartments was scored and analyzed. An animal was considered in a compartment if all four paws were located in that compartment (compartment entry). Animals spent some amount of time exploring (sniffing) the central area with paws located in both compartments or on the central line. An observer without knowledge of the treatment condition of any animal scored the videotape records by recording the data using a real-time event recording program on a Macintosh computer; reliability measures determined between highly experienced experimenters were greater than 90% agreement. 2.3. Experimental design and data analysis A total of 128 rats were used as experimental (n = 64) and control (n = 64) subjects. Animals were randomly assigned to the testing conditions with an equal number of males and females placed into each treatment group to allow analysis of sex effects. Animals were tested either as adolescents or as adults and were housed either in pairs or in social isolation. Therefore, the experimental design was a 2 (Conditioning Treatment: Experimental or Control) 2 (Age) 2 (Sex) 2 (Housing) factorial, with 8 animals tested in each of the 16 groups. To eliminate the possible confounding of litter with treatment effects, no more than one animal from a given litter was assigned to any of the 16 experimental groups [23]. The first 10 min of the conditioning sessions on Days 1 and 5 for the experimental groups (i.e., animals exposed to the novel objects in the CPP chambers) were videotaped for analysis of behavior directed toward the novel object as well as locomotor activity. Latency to contact the novel object with the nose or forepaws, time spent exploring the object, and time spent in forward locomotion were analyzed using separate 2 (Age) 2 (Sex) 2 (Housing) 2 (Day of Conditioning) mixed-factor ANOVAs, with day of conditioning treated as a repeated measure. Object exploration was defined as sniffing of the object and contact with the object with paws, nose, or mouth [21]. Time spent locomoting was defined as a period of forward-directed, longitudinal movement by the animal. Our preliminary results showed that during a 15-min observation session a dramatic decline in object exploration and locomotor activity occurred during the last 5-min bin, with animals regardless of age and housing conditions spending almost all the time resting or grooming. Based on these preliminary results, only first 10 min of the conditioning sessions were scored and analyzed. Time spent in each compartment on the test day was converted into a preference coefficient percentage. For experimental animals this was defined as (Time on the Paired Side Time on the Unpaired Side)/(Time on the Paired
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Side + Time on the Unpaired Side) 100; for control animals, the coefficient (%) was conservatively defined as (Time on the Preferred Side Time on the Nonpreferred Side)/(Time on the Preferred Side + Time on the Nonpreferred Side) 100. Thus, time spent near the central line with paws in both compartments was not included in either formula. To assess CPP, preference coefficients were analyzed using a 2 (Conditioning Treatment) 2 (Age) 2 (Sex) 2 (Housing) ANOVA. Novel-object CPP was defined as experimental animals spending significantly more time in the paired compartment than was spent by the control counterparts on their preferred side (i.e., main and interaction effects of conditioning treatment). Tukey/Kramer tests were used for post hoc comparisons. A significance level of P < .05 was used for all analyses and comparisons. Correlation analyses using Z tests were performed to determine whether behavior during conditioning (latency to contact the novel object and time spent exploring the object) significantly influenced novelty reward as indexed by the preference coefficient.
3. Results 3.1. Behavior during conditioning In general, responding to the novel objects was higher in adolescent animals than in their adult counterparts. Adolescents started to explore the novel objects more quickly and spent more time interacting with them than adults. Levels of locomotor activity were also elevated in adolescent rats. Social deprivation decreased responding to novelty in adult but not adolescent animals, and males were more affected than females. Latency to contact the novel objects did not differ as a function of day of conditioning, and data presented in Fig. 1 are collapsed across this variable. The overall ANOVA analysis of latency to contact a novel object during conditioning revealed significant main effects of age [ F(1,56) = 49.09, P < .001], sex [ F(1,56) = 5.75, P < .05], and housing [ F(1,56) = 7.28, P < .01], which were tempered by a significant three-way interaction of age, sex, and housing [ F(1,56) = 5.67, P < .05] (see Fig. 1). Adolescent males and females, both group-housed and socially isolated, had significantly shorter latencies to contact a novel object than adults from the same experimental conditions. Latency to contact the object for the adolescents did not differ as a function of sex or housing, whereas socially isolated adult male rats demonstrated significantly longer latencies to contact the novel object than did animals in all other conditions (see Fig. 1). Analysis of time spent with a novel object during conditioning revealed a significant Day Housing interaction [ F(1,56) = 4.96, P < .05]. Time spent with the object decreased markedly from Day 1 (133.3 F 6.4 s) to Day 5 (111.5 F 6.0 s) of conditioning in isolated animals, but not
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Fig. 1. Mean latency to contact the novel object for adolescent and adult male (left panel) and female (right panel) rats housed either in groups of two or in social isolation. Data are collapsed across day of conditioning due to the absence of significant main effects or interactions involving this variable. Error bars represent S.E.M. Adolescent animals had significantly shorter latencies to explore a novel object than adults, and latency to explore the object for the adolescents did not differ as a function of sex or housing, whereas socially isolated adult males demonstrated significantly longer latencies to explore the novel object than did animals in all other conditions.
in socially housed animals (Day 1: 135.3 F 4.2 s; Day 5: 134.8 F 5.1 s). The ANOVA comparisons also revealed significant main effects of age [ F(1,56) = 92.16, P < .001] and housing [ F(1,56) = 10.89, P < .01], as well as a significant Age Sex Housing interaction [ F(1,56) = 5.72, P < .05] (see Fig. 2 with data collapsed across day of conditioning). Adolescents spent more time interacting with the novel object than their adult counterparts regardless of sex and under both housing conditions. For the adolescents, time with the object did not differ as a function of sex or housing, whereas isolated adult males spent less time interacting with the novel object than group-housed adult male rats. The ANOVA comparing time spent in forward locomotion on Days 1 and 5 of conditioning showed significant main effects of age [ F(1,56) = 12.44, P < .001] and day [ F(1,56) = 8.82, P < .01], tempered by a significant Age Sex Day interaction [ F(1,56) = 6.84, P < .05]. On Day 1, time spent in forward locomotion was comparable in adolescent and adult male and female rats. On Day 5, however, age-related differences in locomotor activity became evident but only in males (see Fig. 3). Adult males, but not females, showed a significant decrease in locomotor activity from the first to the fifth conditioning day, with time spent in forward locomotion on Day 5 significantly lower than that of adolescents. 3.2. Place conditioning Generally, both adolescent and adult animals showed preference for the compartment paired with the novel
Fig. 2. Mean time spent with the novel object for adolescent and adult male (left panel) and female (right panel) group-housed and socially isolated rats, with data collapsed across day of conditioning. Error bars represent S.E.M. Adolescents spent more time interacting with the novel object than their adult counterparts regardless of sex and under both housing conditions. For the adolescents, time with the object did not differ as a function of sex or housing, whereas isolated adult males spent less time interacting with the novel object than group-housed adult male rats.
objects. The rewarding properties of novelty, however, differed as a function of age, sex, and housing. Adolescent males, both group-housed and socially deprived, showed reliable place preference, whereas only socially isolated adult males demonstrated preference for the compartment paired with the novel objects. This age difference was not
Fig. 3. Mean time spent in forward locomotion on Days 1 and 5 of conditioning for adolescent and adult male (left panel) and female (right panel) rats (collapsed across housing condition). Error bars represent S.E.M. On Day 1, time spent in forward locomotion was comparable in adolescent and adult male and female rats. On Day 5, however, age-related differences in locomotor activity became evident in males. Adult males, but not females, showed a significant decrease in locomotor activity from the first to the fifth conditioning day, with time spent in forward locomotion on Day 5 significantly lower than that of adolescents.
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Table 1 Time (mean F S.E.M.) spent in each of the compartments on the test day for adolescent and adult male and female rats housed either in pairs or in social isolation Age
Sex
Housing
Control preferred (s)
Experimental paired (s)
Control nonpreferred (s)
Experimental unpaired (s)
Adolescent Adolescent Adult Adult Adolescent Adolescent Adult Adult
male male male male female female female female
group isolate group isolate group isolate group isolate
233 F 7 271 F 9 256 F 7 268 F 7 255 F 9 265 F 11 262 F 12 255 F 14
344 F 10 356 F 10 236 F 5 311 F 26 355 F 11 276 F 13 351 F 28 286 F 7
211 F 7 202 F 12 233 F 5 206 F 10 194 F 10 222 F 15 213 F 15 209 F 11
156 F 16 133 F 7 212 F 8 151 F 18 151 F 11 218 F 15 148 F 19 195 F 9
evident in females, with both adolescent and adult grouphoused females, but not their isolated counterparts, showing novel-object place conditioning. Average time spent in each of the compartments for adolescent and adult male and female animals housed either socially or in social isolation are presented in Table 1. The overall ANOVA examining coefficients of preference revealed a significant main effect of conditioning treatment [ F(1,112) = 85.83, P < .001], as well as Age Sex Conditioning Treatment [ F(1,112) = 9.44, P < .01] and Sex Housing Conditioning Treatment interactions [ F(1,112) = 13.50, P < .001] (see Fig. 4). Since the multiple interactions involving sex suggested sex-dependent peculiarities in responding to the rewarding properties of novelty, ANOVAs were performed separately for males and females. The ANOVA comparing values of the coefficient for males revealed a significant Sex Housing Conditioning Treatment interaction [ F(1,56) = 4.40, P < .05] (see Fig. 4, left panel). Post hoc comparisons showed that the values of the coefficient were significantly greater in the experimental than control group for adolescent males, both group-housed and
isolated, as well as for isolated adult males. Coefficients of preference in group-housed adult males, however, did not differ between the paired and control conditions. This pattern of results suggests that regardless of their previous social history, adolescent males showed reliable place preference, whereas in adult males the rewarding properties of novelty emerged only under the social deprivation condition. No such age effects emerged in females. The ANOVA comparing the values of the coefficient for females showed a significant Housing Conditioning Treatment interaction [ F(1,56) = 11.83, P < .01] (see Fig. 4, right panel). Grouphoused females, but not their isolated counterparts, demonstrated novelty-induced CPP, as indexed by the significantly higher values of the coefficient for the paired condition compared to the controls. The results suggested that for females, regardless of age, social isolation disrupted expression of the rewarding properties of novelty in the CPP paradigm. Time spent in forward locomotion during the CPP test differed as a function of age [ F(1,112) = 6.07, P < .05] and sex [ F(1,112) = 5.36, P < .05]. Adolescent animals spent
Fig. 4. Values of the preference/avoidance coefficient for male (left panel) and female (right panel) adolescent and adult group-housed and isolated rats. Novelobject conditioned place preference was defined as animals in the paired group spending significantly more time in the paired compartment than was spent by the control counterparts on their preferred side. Adolescent males, both group-housed and socially deprived, showed reliable place preference, whereas only socially isolated adult males demonstrated preference for the compartment paired with the novel objects. This age difference was not evident in females, with both adolescent and adult group-housed females, but not their isolated counterparts, showing novel-object place conditioning.
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more time in forward locomotion than their adult counterparts (mean = 84.6 F 3.5 s for adolescents; mean = 72.4 F 3.5 s for adults), and females were more active than males (mean = 84.3 F 3.9 s for females; mean = 72.8 F 3.2 for males). The factors of age and sex, however, did not interact with conditioning treatment. 3.3. Correlations between novelty-directed behavior during conditioning and place preference No statistically significant correlations between indices of novelty seeking behavior (latency to contact the novel object, time spent in object exploration) and the coefficient of preference were found for males (r = .04 for latency to contact and r =.22 for exploration time) and females (r =.19 and r =.32, respectively) (all P’s>.05). These results suggest dissociations between novelty-directed behaviors evident during conditioning and novelty reward as indexed by preference coefficients in the CPP paradigm.
4. Discussion The results of the present study demonstrated that in general responding to novelty was higher in adolescent animals than in their adult counterparts. During conditioning, adolescents showed shorter latencies to contact the novel objects and spent more time interacting with them than adults. Age-related differences in responding to novelty were observed not only in males but also in female rats. High responding to the novel objects demonstrated by adolescents in the present study is in concordance with previously described enhanced activity and elevated levels of exploratory behavior of adolescent rats in novel situations [9,10]. This enhanced responding to novelty observed during adolescence may reflect an ontogenetic adaptation [24] that helps adolescents acquire new information and skills for the transition from parental dependence to independence. Adolescent-associated increases in novelty seeking also provide the motivation to explore novel areas away from home, a trait that may have been evolutionally conserved to foster emigration at the time of sexual emergence, a strategy useful for avoiding inbreeding and commonly seen among mammalian species [7]. It is possible that age differences in novel-object exploration may be related in part to characteristics of the testing situation. In the present study, the chambers of the CPP apparatus were proportionally size-adjusted for adolescent and adult animals, with adolescents being conditioned in smaller chambers than adults. The size of the objects, however, was not adjusted across age. Therefore, the probability of object contact might be higher for adolescents than for adults, given that the proportional size of the object relative to the size of the chamber and the rat was larger for adolescents than for their older counterparts. Although this factor could potentially contribute to the enhanced novelty
seeking observed in adolescent rats in the present study, it is unlikely to do so completely given that object contact was defined as active contact with the novel stimulus, specifically eliminating from consideration any passive contact that might result merely from proximity. Responding to novelty during adolescence was not affected by previous social deprivation, with both grouphoused and isolated adolescent rats demonstrating equivalently enhanced exploration of the novel objects during conditioning relative to their adult counterparts. These results contrast with other reports that rearing in social isolation increases object exploration [17,18]. For instance, Einon and Morgan [18] found that isolation rearing between P25 and P45 resulted in increased levels of object exploration. In this study, animals were tested after a larger deprivation period (20 days) and at an older age (P45 or P90) than in the present study (10 –15 days of deprivation, testing from P33– P37). Either of these variables may contribute to the lack of isolation effects on responding among adolescents in the present study. Of particular suspect is the issue of test age. Adolescents tested on P35 –36 show higher levels of exploration than older (P47 – 48) or younger (P23 – 24) rats [25], and hence, in the present study, novel object exploration of adolescent animals was assessed at its peak, when this behavior is the most pronounced. Responding to the novel objects during conditioning was lower in adults than in adolescents, and was even more attenuated in socially isolated adult males, as indexed by longer exploration latencies and reduced time with the novel objects. These findings are reminiscent of other studies that have shown elevated levels of environmental neophobia in socially deprived male rats [26]. In a variety of experimental paradigms, social isolation of males has been consistently associated with an increased anxiety as indicated by a longer latency to enter a novel environment [27,28] and a reduction in the time spent on the open arms of an elevated plus maze [29,30]. Interestingly, this social isolation effect was not evident in adult females or in adolescents of either gender. From the patterns of responding to novelty during conditioning, it might have been expected that interaction with the novel objects should be more rewarding for adolescent animals (regardless of their sex and housing conditions) than for adults, and the least rewarding for isolated adult males. The results of CPP testing, however, did not support this expectation. Age-related differences in novel object-induced CPP were apparent only in group-housed males. Adolescent males from both housing conditions showed reliable CPP on test day. In contrast, evidence for rewarding effects of the exposure to the novel objects emerged only in adult males who were socially deprived—animals that spent the least amount of time interacting with the objects during conditioning. These findings are reminiscent of those of Hall et al. [31] who observed in a non-anxiogenic situation that isolated male rats exhibited a stronger preference than group-housed rats for the novel than the familiar side of a CPP apparatus. It is likely that socially deprived male rats, although spending
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less time with the novel objects as a result of increased anxiety, nevertheless found these relatively restrained interactions with the novel stimulus to be especially rewarding. These speculations aside, what is clear at this point is that time spent with a novel object during conditioning does not necessarily reflect its rewarding value as assessed via CPP, a conclusion that was further supported by the lack of correlation between time spent with a novel object and the CPP preference coefficient. Stronger conditioning among adolescents than adults in the novel-object CPP paradigm was evident only for grouphoused male rats and was eliminated by social deprivation, with the rewarding properties of novelty being comparable in socially deprived males of both ages. This impact of housing on place preference conditioning could contribute to patterns of age differences in CPP observed in other studies. For instance, in a study using group-housed rats, age-related differences in nicotine-induced CPP were observed, with adolescent animals being more sensitive to the rewarding properties of nicotine than adults [32]. In contrast, in a study using isolate-housed animals, adolescents and adults were observed to exhibit similar magnitude CPP when conditioned with either cocaine or morphine [33]. The rewarding properties of both novelty and drugs of abuse are modulated by mesolimbic and mesocortical dopamine (DA) pathways (see Ref. [3] for references and review), whose activity is altered by stressors including social isolation [13] and which undergo considerable ontogenetic modifications during adolescence (see Ref. [7] for review). Thus, age- and housingrelated neural alterations in mesolimbic and mesocortical DA systems may contribute to the differential sensitivity to the rewarding effects of novelty between group-housed adolescent and adult male rats, age effects not apparent in their isolated counterparts. Although results of isolation-induced changes in DA systems of adult animals are not consistent (see Ref. [13] for references and review), some of the findings suggest a similar pattern of an imbalance between the mesocortical and mesolimbic DA systems in adolescent animals and socially deprived adults. Indeed, levels of basal DA synthesis and turnover in the prefrontal cortex (PFC) are considerably elevated in rats during adolescence [34,35], and in the same way socially deprived adults have been reported to have higher DA and DA metabolite levels in the PFC ([36 – 38]; but see also Ref. [39]). Likewise, basal DA synthesis in the nucleus accumbens is lower in adolescent animals than in their adult counterparts [34], and individually housed male mice also show enhanced mesocortical and reduced mesoaccumbens DA response to stress challenge compared to their group-housed counterparts [40]. Taken together, such findings may eventually clarify age-related differences in sensitivity to novelty reward in group-housed males and a lack of these differences in socially deprived males. It is recognized, however, that most of the literature addressing social deprivation effects has examined isolation rearing for an extended period of time (e.g., 10– 14 weeks), whereas in
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the present study animals were socially deprived only for 10 days before conditioning was initiated. No age-related difference in responding to the rewarding effects of novelty emerged in females, with both adolescent and adult group-housed females, but not their isolated counterparts, showing novel-object place conditioning. The effects of social isolation was opposite for males and females, allowing expression of rewarding effects of the novel objects in adult male rats but virtually eliminating object-induced CPP in females of both ages. Other studies have likewise observed gender effects in the consequences of social isolation, often viewed as an animal model of stress [13] and/or depression [41]. For instance, individual housing for a relatively short period of time (1 week) has been reported to induce a depression-like effect in female but not male mice [42]. To the extent that isolate housing reflects a stressor effect, these data are reminiscent of other findings that female rodents may be more responsive to stressors than males. The corticosterone response of male rats habituated to a chronic stressor, whereas females showed little hormonal habituation [43]. The suppression of activity in the open field initially seen following a stressor returns more quickly to basal, nonstress levels in male than female rats [44,45]. Probably the most striking finding is that exposure to an inescapable stressor facilitates classical conditioning in males [46,47] but impairs it in females [48]. In the same way, the results of the present study show that social isolation stress enhances CPP—a learning paradigm based on classical conditioning [49]—in adult males and attenuates it in female rats. Our results are also in agreement with reports that social isolation of males facilitates Pavlovian conditioning [50] as well as learning in a Morris water maze [51]. A strong dissociation between novelty seeking, as indexed by time spent with the novel objects, and novelty reward in the CPP paradigm was observed in adult males and females of both ages in the present study. Only adolescent males regardless of their housing conditions showed enhanced novelty seeking and strong novel object-induced CPP. Adult group-housed males were relatively high novelty responders, at least when compared with their isolated counterparts, but did not respond to the rewarding properties of novelty as indexed by the CPP paradigm. In contrast, socially deprived adult males spent less time exploring the novel objects during conditioning but showed strong preference for the compartment associated with novelty. Housing conditions did not affect novelty seeking in females with adolescent females being higher novelty responders than their adult counterparts. However, novelty was equally rewarding for females of both ages when they were grouphoused, but these rewarding effects of novelty were virtually eliminated by social isolation. Together these results suggest that mechanisms underlying novelty-directed behaviors and those involved in the rewarding effects of novelty are different. And, indeed, a dissociation between the DA receptor subtypes that mediate novel object exploration and those
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involved in novelty-associated reward has been suggested by recent data [19,21,52]. The D1 receptor antagonist SCH23390 blocked the increase in preference conditioned by access to novel objects at doses that did not affect object interaction [19], suggesting an involvement of D1 receptors in novelty reward but not in novelty exploration. Enhanced sensitivity to novelty reward in adolescent males regardless of their housing conditions may stem from their elevated levels of D1 receptors, with the overproduction and pruning of DA receptors reported in striatum and nucleus accumbens being more pronounced in males than females during adolescence [53]. Taken together, the results of the present paper demonstrate that, whereas adolescents show higher levels of novelty seeking than adults, their high levels of novelty seeking do not necessarily predict high rewarding properties of novelty. Novelty reward, unlike novelty seeking, is strongly influenced by environmental and gender-related factors. Further experiments are needed to understand determinants of novelty seeking and novelty reward as well as their underlying neural mechanisms in adolescence.
[16] [17] [18] [19]
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L.A. Douglas et al. / Physiology & Behavior 80 (2003) 317–325 [38] Jones GH, Hernandez TD, Kendall DA, Marsden CA, Robbins TW. Dopaminergic and serotonergic function following isolation rearing in rats: study of behavioural responses and postmortem and in vivo neurochemistry. Pharmacol Biochem Behav 1992;43:17 – 35. [39] Heidbreder CA, Weiss IC, Domeney AM, Pryce C, Homberg J, Hedou G, et al. Behavioral, neurochemical and endocrinological characterization of the early social isolation syndrome. Neuroscience 2000; 100:749 – 68. [40] Cabib S, Ventura R, Puglisi-Allegra S. Opposite imbalances between mesocortical and mesoaccumbens dopamine responses to stress by the same genotype depending on living conditions. Behav Brain Res 2002;129:179 – 85. [41] McKinney WT. Animal models of depression: an overview. Psychiatr Dev 1984;2:77 – 96. [42] Polanza P. Animal models of anxiety and depression: how are females different? Neurosci Biobehav Rev 2001;25:219 – 33. [43] Galea LA, McEwen BS, Tanapat P, Deak T, Spencer RL, Dhabhar FS. Sex differences in dendritic atrophy of CA3 pyramidal neurons in response to chronic restraint stress. Neuroscience 1997;81:689 – 97. [44] Haleem D, Kennett G, Curzon G. Adaptation of female rats to stress: shift to male pattern by inhibition of corticosterone synthesis. Brain Res 1988;458:339 – 47. [45] Kennett G, Chaouloff F, Marcou M, Curzon G. Female rats are more
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Novel-object place conditioning in adolescent and adult male and female rats: effects of social isolation Lewis A. Douglas, Elena I. Varlinskaya, Linda P. Spear* Center for Developmental Psychobiology, Department of Psychology, Binghamton University, Binghamton, NY 13902-6000, USA Received 21 February 2003; received in revised form 30 July 2003; accepted 16 August 2003
Abstract Elevated levels of novelty seeking are often seen during adolescence. Recent studies using a conditioned place preference (CPP) paradigm have shown that novelty may be rewarding for adult rats. The present study explored the impact of age, sex, and isolation stress on novelty seeking and novelty reward by assessing novel object-induced CPP in adolescent and adult male and female Sprague – Dawley rats housed either socially or in isolation. Responding to the novel objects during conditioning was higher in adolescent animals than adults, and was suppressed by social isolation only in adulthood, particularly among males. Novel object CPP was strong among adolescent males, whereas only socially isolated adult males demonstrated preference for the compartment paired with the novel objects. This age difference was not evident in females, with both adolescent and adult group-housed females, but not their isolated counterparts, showing novel-object place conditioning. These dissociations between novelty-directed behaviors during conditioning and novelty reward in the CPP paradigm support the suggestion that mechanisms underlying novelty seeking are separable from those involved in the rewarding effects of novelty. High levels of novelty seeking demonstrated by adolescents do not necessarily predict high rewarding properties of novelty, with the latter also being influenced by environmental and gender-related factors. D 2003 Elsevier Inc. All rights reserved. Keywords: Novelty; Conditioned place preference; Adolescence; Sex differences; Social isolation; Rats
1. Introduction Relative to adults and children, human adolescents exhibit an increased level of risk-taking behavior, associated with the motivation to experience multiple new and intense stimuli to attain potential rewards [1,2]. The motivation to seek out new experiences, i.e., novelty seeking behavior, has been identified as a significant contributor to current and future drug use, multiple drug use, and later abuse [3 –6]. Given that adolescence is a time of acquiring new skills for survival away from parents, enhanced novelty seeking may have been evolutionally conserved for its adaptive value during this developmental period, contributing to exploration of novel areas and providing the opportunity to find new sources of food, water, and mates (see Ref. [7]). Although little explored to date, there is some experimental evidence that age may play a role in responding to novelty, with * Corresponding author. Tel.: +1-607-777-2825; fax: +1-607-7776418. E-mail address: [email protected] (L.P. Spear). 0031-9384/$ – see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2003.08.003
adolescent rodents being more responsive to novel stimuli than their adult counterparts. For instance, adolescent mice spent more time in a novel environment and had higher levels of novelty-induced activity than adults [8]. In a similar way, adolescent rats have been reported to be more active and to engage in more exploratory behaviors in novel situations than adults [9,10]. Responding to novelty may also be influenced by social factors. In humans, a number of social variables, including social conformity, peer deviance, and social support influence novelty seeking behavior in adolescence [11]. Likewise, the presence of peers before testing enhanced responding to novelty in nonhuman primates, increasing exploration of novel objects relative to their socially deprived counterparts [12]. In contrast, rats reared in social isolation were more responsive to novelty relative to rats housed socially (see Ref. [13] for references and review), as indexed by increased locomotor activity [14,15], enhanced preference for novel environments [16], and elevated levels of object exploration [17]. Such isolation-induced increases in object exploration were found to be age-specific, emerging only in animals
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socially deprived between 25 and 45 days of age and not by isolation initiated after P45 [18]. These findings suggest that responding to novelty of adolescent animals may be influenced by social factors in a way different from that of their adult counterparts. Conditioned place preference (CPP), a widely used behavioral paradigm to measure drug reward in laboratory animals, has been employed recently for assessment of the rewarding properties of novel environmental stimuli. In these studies, socially isolated adult male rats received access to novel objects in one distinct environment (e.g., one compartment of a CPP apparatus containing distinct tactile and visual cues), while receiving exposure to a second distinct environment (the other compartment of the apparatus) in the absence of any novel objects. In a choice test, when exposed to the entire apparatus without novel objects, rats showed preference for the environment that had been previously paired with novelty [19 –21]. Novelty of the objects is an important issue in this paradigm, since animals that received access to familiar objects in one of the compartments of the CPP apparatus did not show preference for that environment later on, and during conditioning they spent significantly less time interacting with the familiar object than their counterparts exposed to novel objects [21]. These results suggest that access to novel but not familiar objects is rewarding, findings that validate the use of this procedure for the assessment of novelty reward. There are no reports as to whether novel stimuli would support place preference conditioning in adolescent animals, nor whether such conditioning would be influenced by sex and housing conditions in an age-specific manner. Therefore, the main objective of the present study was to evaluate how age, sex, and isolate housing influence the rewarding properties of novelty using the novel-object place conditioning paradigm developed by Bevins and Bardo [20].
2. General methods 2.1. Animals Animals were derived from Sprague – Dawley VAF rats bred and reared in our colony at Binghamton University. Pups were housed until weaning with their parents in standard maternity cages with pine shavings as bedding material. All animals were housed in a temperature-controlled (22 jC) vivarium maintained on a 12-h/12-h illumination cycle (lights on at 0700 h) with ad libitum access to food (Purina Rat Chow, Lowell, MA) and water. Litters were culled to 10 pups (with a sex ratio of four pups of one sex and six of the other) within 24 h after birth. Rats were weaned on P21 and pair-housed in a standard hanging cage with a samesex littermate. Animals in the isolate housing condition were placed in individual cages 10 days before conditioning procedure (P23 if tested as adolescents and P55 if tested as adults), where they remained housed until the end of the
experiment. In all respects, maintenance and treatment of the animals were in accord with guidelines for animal care established by the National Institutes of Health [22]. 2.2. Procedure Conditioning and testing were conducted in Plexiglas chambers divided into two equal-sized compartments by a guillotine door. One compartment had horizontal black and white stripes on the walls and a wire mesh floor, while the other had vertical stripes and a metal rod floor. The chambers for adult conditioning and testing were 54 cm long, 40 cm wide, and 45 cm high, while adolescents were conditioned and tested in proportionally size-adjusted chambers (40 cm long, 30.5 cm wide, and 45 cm high). Four boxes were used at each age, allowing training and testing of up to four animals simultaneously. The behavior of the animals was recorded by video cameras mounted above each chamber. The chambers were located in a testing room maintained under dim illumination (20 –25 lx in the center of each chamber). Novel objects used for conditioning included wood blocks (9 cm long, 3.5 cm wide, and 2.5 cm high), white PVC pipe (5 cm long with a 2.8 cm inner diameter), rolls of cotton (10 cm long, 5 cm wide, and 2 cm high), balls of tin foil (about 5 cm in diameter), and hard plastic PEZ dispensers (with approximate diameters of 4 cm). Adolescent animals were conditioned starting at P33 and were tested on P38. For adults, conditioning was initiated on P65, with testing on P70. All experimental procedures were conducted within the first 5 h of the animal’s light cycle. On all conditioning days, each experimental animal was exposed to both sides of the apparatus for 15 min as well as to a third environment distinct from the apparatus (a clear breeder tub with pine shavings), also for 15 min. In one of the compartments of the CPP apparatus a new object was placed daily (paired side), whereas no objects were placed in the second compartment (unpaired side). There was a 1-h interval between the exposure to each of these three contexts, during which each animal was returned to its home cage. Order of exposure to the paired chamber, unpaired chamber, and third environment was counterbalanced within each group, as was the order in which the novel objects were provided to the animals across days. For experimental animals, the side of the chamber (horizontal versus vertical) paired with the novel objects was also counterbalanced. Animals in the control groups were placed into each side of the chamber daily, but in the absence of novel objects. Instead, these animals were exposed to the same novel objects as experimental subjects in the third environment –the breeder tub. As for experimental animals, order of exposure was counterbalanced for controls. These groups were included in the experimental design to control for possible handling/experience-induced alterations in intrinsic preference following repeated exposures to the compartments during conditioning. Animals were conditioned for 5 days, and the test session was conducted 24 h after the last conditioning trial. On the
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test day, no objects were placed in the CPP chambers. At the time of test, the door separating the two chambers was removed, and each subject was placed in the center of the CPP apparatus and allowed to freely locomote between the two compartments for 10 min. Time spent in each of the compartments was scored and analyzed. An animal was considered in a compartment if all four paws were located in that compartment (compartment entry). Animals spent some amount of time exploring (sniffing) the central area with paws located in both compartments or on the central line. An observer without knowledge of the treatment condition of any animal scored the videotape records by recording the data using a real-time event recording program on a Macintosh computer; reliability measures determined between highly experienced experimenters were greater than 90% agreement. 2.3. Experimental design and data analysis A total of 128 rats were used as experimental (n = 64) and control (n = 64) subjects. Animals were randomly assigned to the testing conditions with an equal number of males and females placed into each treatment group to allow analysis of sex effects. Animals were tested either as adolescents or as adults and were housed either in pairs or in social isolation. Therefore, the experimental design was a 2 (Conditioning Treatment: Experimental or Control) 2 (Age) 2 (Sex) 2 (Housing) factorial, with 8 animals tested in each of the 16 groups. To eliminate the possible confounding of litter with treatment effects, no more than one animal from a given litter was assigned to any of the 16 experimental groups [23]. The first 10 min of the conditioning sessions on Days 1 and 5 for the experimental groups (i.e., animals exposed to the novel objects in the CPP chambers) were videotaped for analysis of behavior directed toward the novel object as well as locomotor activity. Latency to contact the novel object with the nose or forepaws, time spent exploring the object, and time spent in forward locomotion were analyzed using separate 2 (Age) 2 (Sex) 2 (Housing) 2 (Day of Conditioning) mixed-factor ANOVAs, with day of conditioning treated as a repeated measure. Object exploration was defined as sniffing of the object and contact with the object with paws, nose, or mouth [21]. Time spent locomoting was defined as a period of forward-directed, longitudinal movement by the animal. Our preliminary results showed that during a 15-min observation session a dramatic decline in object exploration and locomotor activity occurred during the last 5-min bin, with animals regardless of age and housing conditions spending almost all the time resting or grooming. Based on these preliminary results, only first 10 min of the conditioning sessions were scored and analyzed. Time spent in each compartment on the test day was converted into a preference coefficient percentage. For experimental animals this was defined as (Time on the Paired Side Time on the Unpaired Side)/(Time on the Paired
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Side + Time on the Unpaired Side) 100; for control animals, the coefficient (%) was conservatively defined as (Time on the Preferred Side Time on the Nonpreferred Side)/(Time on the Preferred Side + Time on the Nonpreferred Side) 100. Thus, time spent near the central line with paws in both compartments was not included in either formula. To assess CPP, preference coefficients were analyzed using a 2 (Conditioning Treatment) 2 (Age) 2 (Sex) 2 (Housing) ANOVA. Novel-object CPP was defined as experimental animals spending significantly more time in the paired compartment than was spent by the control counterparts on their preferred side (i.e., main and interaction effects of conditioning treatment). Tukey/Kramer tests were used for post hoc comparisons. A significance level of P < .05 was used for all analyses and comparisons. Correlation analyses using Z tests were performed to determine whether behavior during conditioning (latency to contact the novel object and time spent exploring the object) significantly influenced novelty reward as indexed by the preference coefficient.
3. Results 3.1. Behavior during conditioning In general, responding to the novel objects was higher in adolescent animals than in their adult counterparts. Adolescents started to explore the novel objects more quickly and spent more time interacting with them than adults. Levels of locomotor activity were also elevated in adolescent rats. Social deprivation decreased responding to novelty in adult but not adolescent animals, and males were more affected than females. Latency to contact the novel objects did not differ as a function of day of conditioning, and data presented in Fig. 1 are collapsed across this variable. The overall ANOVA analysis of latency to contact a novel object during conditioning revealed significant main effects of age [ F(1,56) = 49.09, P < .001], sex [ F(1,56) = 5.75, P < .05], and housing [ F(1,56) = 7.28, P < .01], which were tempered by a significant three-way interaction of age, sex, and housing [ F(1,56) = 5.67, P < .05] (see Fig. 1). Adolescent males and females, both group-housed and socially isolated, had significantly shorter latencies to contact a novel object than adults from the same experimental conditions. Latency to contact the object for the adolescents did not differ as a function of sex or housing, whereas socially isolated adult male rats demonstrated significantly longer latencies to contact the novel object than did animals in all other conditions (see Fig. 1). Analysis of time spent with a novel object during conditioning revealed a significant Day Housing interaction [ F(1,56) = 4.96, P < .05]. Time spent with the object decreased markedly from Day 1 (133.3 F 6.4 s) to Day 5 (111.5 F 6.0 s) of conditioning in isolated animals, but not
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Fig. 1. Mean latency to contact the novel object for adolescent and adult male (left panel) and female (right panel) rats housed either in groups of two or in social isolation. Data are collapsed across day of conditioning due to the absence of significant main effects or interactions involving this variable. Error bars represent S.E.M. Adolescent animals had significantly shorter latencies to explore a novel object than adults, and latency to explore the object for the adolescents did not differ as a function of sex or housing, whereas socially isolated adult males demonstrated significantly longer latencies to explore the novel object than did animals in all other conditions.
in socially housed animals (Day 1: 135.3 F 4.2 s; Day 5: 134.8 F 5.1 s). The ANOVA comparisons also revealed significant main effects of age [ F(1,56) = 92.16, P < .001] and housing [ F(1,56) = 10.89, P < .01], as well as a significant Age Sex Housing interaction [ F(1,56) = 5.72, P < .05] (see Fig. 2 with data collapsed across day of conditioning). Adolescents spent more time interacting with the novel object than their adult counterparts regardless of sex and under both housing conditions. For the adolescents, time with the object did not differ as a function of sex or housing, whereas isolated adult males spent less time interacting with the novel object than group-housed adult male rats. The ANOVA comparing time spent in forward locomotion on Days 1 and 5 of conditioning showed significant main effects of age [ F(1,56) = 12.44, P < .001] and day [ F(1,56) = 8.82, P < .01], tempered by a significant Age Sex Day interaction [ F(1,56) = 6.84, P < .05]. On Day 1, time spent in forward locomotion was comparable in adolescent and adult male and female rats. On Day 5, however, age-related differences in locomotor activity became evident but only in males (see Fig. 3). Adult males, but not females, showed a significant decrease in locomotor activity from the first to the fifth conditioning day, with time spent in forward locomotion on Day 5 significantly lower than that of adolescents. 3.2. Place conditioning Generally, both adolescent and adult animals showed preference for the compartment paired with the novel
Fig. 2. Mean time spent with the novel object for adolescent and adult male (left panel) and female (right panel) group-housed and socially isolated rats, with data collapsed across day of conditioning. Error bars represent S.E.M. Adolescents spent more time interacting with the novel object than their adult counterparts regardless of sex and under both housing conditions. For the adolescents, time with the object did not differ as a function of sex or housing, whereas isolated adult males spent less time interacting with the novel object than group-housed adult male rats.
objects. The rewarding properties of novelty, however, differed as a function of age, sex, and housing. Adolescent males, both group-housed and socially deprived, showed reliable place preference, whereas only socially isolated adult males demonstrated preference for the compartment paired with the novel objects. This age difference was not
Fig. 3. Mean time spent in forward locomotion on Days 1 and 5 of conditioning for adolescent and adult male (left panel) and female (right panel) rats (collapsed across housing condition). Error bars represent S.E.M. On Day 1, time spent in forward locomotion was comparable in adolescent and adult male and female rats. On Day 5, however, age-related differences in locomotor activity became evident in males. Adult males, but not females, showed a significant decrease in locomotor activity from the first to the fifth conditioning day, with time spent in forward locomotion on Day 5 significantly lower than that of adolescents.
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Table 1 Time (mean F S.E.M.) spent in each of the compartments on the test day for adolescent and adult male and female rats housed either in pairs or in social isolation Age
Sex
Housing
Control preferred (s)
Experimental paired (s)
Control nonpreferred (s)
Experimental unpaired (s)
Adolescent Adolescent Adult Adult Adolescent Adolescent Adult Adult
male male male male female female female female
group isolate group isolate group isolate group isolate
233 F 7 271 F 9 256 F 7 268 F 7 255 F 9 265 F 11 262 F 12 255 F 14
344 F 10 356 F 10 236 F 5 311 F 26 355 F 11 276 F 13 351 F 28 286 F 7
211 F 7 202 F 12 233 F 5 206 F 10 194 F 10 222 F 15 213 F 15 209 F 11
156 F 16 133 F 7 212 F 8 151 F 18 151 F 11 218 F 15 148 F 19 195 F 9
evident in females, with both adolescent and adult grouphoused females, but not their isolated counterparts, showing novel-object place conditioning. Average time spent in each of the compartments for adolescent and adult male and female animals housed either socially or in social isolation are presented in Table 1. The overall ANOVA examining coefficients of preference revealed a significant main effect of conditioning treatment [ F(1,112) = 85.83, P < .001], as well as Age Sex Conditioning Treatment [ F(1,112) = 9.44, P < .01] and Sex Housing Conditioning Treatment interactions [ F(1,112) = 13.50, P < .001] (see Fig. 4). Since the multiple interactions involving sex suggested sex-dependent peculiarities in responding to the rewarding properties of novelty, ANOVAs were performed separately for males and females. The ANOVA comparing values of the coefficient for males revealed a significant Sex Housing Conditioning Treatment interaction [ F(1,56) = 4.40, P < .05] (see Fig. 4, left panel). Post hoc comparisons showed that the values of the coefficient were significantly greater in the experimental than control group for adolescent males, both group-housed and
isolated, as well as for isolated adult males. Coefficients of preference in group-housed adult males, however, did not differ between the paired and control conditions. This pattern of results suggests that regardless of their previous social history, adolescent males showed reliable place preference, whereas in adult males the rewarding properties of novelty emerged only under the social deprivation condition. No such age effects emerged in females. The ANOVA comparing the values of the coefficient for females showed a significant Housing Conditioning Treatment interaction [ F(1,56) = 11.83, P < .01] (see Fig. 4, right panel). Grouphoused females, but not their isolated counterparts, demonstrated novelty-induced CPP, as indexed by the significantly higher values of the coefficient for the paired condition compared to the controls. The results suggested that for females, regardless of age, social isolation disrupted expression of the rewarding properties of novelty in the CPP paradigm. Time spent in forward locomotion during the CPP test differed as a function of age [ F(1,112) = 6.07, P < .05] and sex [ F(1,112) = 5.36, P < .05]. Adolescent animals spent
Fig. 4. Values of the preference/avoidance coefficient for male (left panel) and female (right panel) adolescent and adult group-housed and isolated rats. Novelobject conditioned place preference was defined as animals in the paired group spending significantly more time in the paired compartment than was spent by the control counterparts on their preferred side. Adolescent males, both group-housed and socially deprived, showed reliable place preference, whereas only socially isolated adult males demonstrated preference for the compartment paired with the novel objects. This age difference was not evident in females, with both adolescent and adult group-housed females, but not their isolated counterparts, showing novel-object place conditioning.
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more time in forward locomotion than their adult counterparts (mean = 84.6 F 3.5 s for adolescents; mean = 72.4 F 3.5 s for adults), and females were more active than males (mean = 84.3 F 3.9 s for females; mean = 72.8 F 3.2 for males). The factors of age and sex, however, did not interact with conditioning treatment. 3.3. Correlations between novelty-directed behavior during conditioning and place preference No statistically significant correlations between indices of novelty seeking behavior (latency to contact the novel object, time spent in object exploration) and the coefficient of preference were found for males (r = .04 for latency to contact and r =.22 for exploration time) and females (r =.19 and r =.32, respectively) (all P’s>.05). These results suggest dissociations between novelty-directed behaviors evident during conditioning and novelty reward as indexed by preference coefficients in the CPP paradigm.
4. Discussion The results of the present study demonstrated that in general responding to novelty was higher in adolescent animals than in their adult counterparts. During conditioning, adolescents showed shorter latencies to contact the novel objects and spent more time interacting with them than adults. Age-related differences in responding to novelty were observed not only in males but also in female rats. High responding to the novel objects demonstrated by adolescents in the present study is in concordance with previously described enhanced activity and elevated levels of exploratory behavior of adolescent rats in novel situations [9,10]. This enhanced responding to novelty observed during adolescence may reflect an ontogenetic adaptation [24] that helps adolescents acquire new information and skills for the transition from parental dependence to independence. Adolescent-associated increases in novelty seeking also provide the motivation to explore novel areas away from home, a trait that may have been evolutionally conserved to foster emigration at the time of sexual emergence, a strategy useful for avoiding inbreeding and commonly seen among mammalian species [7]. It is possible that age differences in novel-object exploration may be related in part to characteristics of the testing situation. In the present study, the chambers of the CPP apparatus were proportionally size-adjusted for adolescent and adult animals, with adolescents being conditioned in smaller chambers than adults. The size of the objects, however, was not adjusted across age. Therefore, the probability of object contact might be higher for adolescents than for adults, given that the proportional size of the object relative to the size of the chamber and the rat was larger for adolescents than for their older counterparts. Although this factor could potentially contribute to the enhanced novelty
seeking observed in adolescent rats in the present study, it is unlikely to do so completely given that object contact was defined as active contact with the novel stimulus, specifically eliminating from consideration any passive contact that might result merely from proximity. Responding to novelty during adolescence was not affected by previous social deprivation, with both grouphoused and isolated adolescent rats demonstrating equivalently enhanced exploration of the novel objects during conditioning relative to their adult counterparts. These results contrast with other reports that rearing in social isolation increases object exploration [17,18]. For instance, Einon and Morgan [18] found that isolation rearing between P25 and P45 resulted in increased levels of object exploration. In this study, animals were tested after a larger deprivation period (20 days) and at an older age (P45 or P90) than in the present study (10 –15 days of deprivation, testing from P33– P37). Either of these variables may contribute to the lack of isolation effects on responding among adolescents in the present study. Of particular suspect is the issue of test age. Adolescents tested on P35 –36 show higher levels of exploration than older (P47 – 48) or younger (P23 – 24) rats [25], and hence, in the present study, novel object exploration of adolescent animals was assessed at its peak, when this behavior is the most pronounced. Responding to the novel objects during conditioning was lower in adults than in adolescents, and was even more attenuated in socially isolated adult males, as indexed by longer exploration latencies and reduced time with the novel objects. These findings are reminiscent of other studies that have shown elevated levels of environmental neophobia in socially deprived male rats [26]. In a variety of experimental paradigms, social isolation of males has been consistently associated with an increased anxiety as indicated by a longer latency to enter a novel environment [27,28] and a reduction in the time spent on the open arms of an elevated plus maze [29,30]. Interestingly, this social isolation effect was not evident in adult females or in adolescents of either gender. From the patterns of responding to novelty during conditioning, it might have been expected that interaction with the novel objects should be more rewarding for adolescent animals (regardless of their sex and housing conditions) than for adults, and the least rewarding for isolated adult males. The results of CPP testing, however, did not support this expectation. Age-related differences in novel object-induced CPP were apparent only in group-housed males. Adolescent males from both housing conditions showed reliable CPP on test day. In contrast, evidence for rewarding effects of the exposure to the novel objects emerged only in adult males who were socially deprived—animals that spent the least amount of time interacting with the objects during conditioning. These findings are reminiscent of those of Hall et al. [31] who observed in a non-anxiogenic situation that isolated male rats exhibited a stronger preference than group-housed rats for the novel than the familiar side of a CPP apparatus. It is likely that socially deprived male rats, although spending
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less time with the novel objects as a result of increased anxiety, nevertheless found these relatively restrained interactions with the novel stimulus to be especially rewarding. These speculations aside, what is clear at this point is that time spent with a novel object during conditioning does not necessarily reflect its rewarding value as assessed via CPP, a conclusion that was further supported by the lack of correlation between time spent with a novel object and the CPP preference coefficient. Stronger conditioning among adolescents than adults in the novel-object CPP paradigm was evident only for grouphoused male rats and was eliminated by social deprivation, with the rewarding properties of novelty being comparable in socially deprived males of both ages. This impact of housing on place preference conditioning could contribute to patterns of age differences in CPP observed in other studies. For instance, in a study using group-housed rats, age-related differences in nicotine-induced CPP were observed, with adolescent animals being more sensitive to the rewarding properties of nicotine than adults [32]. In contrast, in a study using isolate-housed animals, adolescents and adults were observed to exhibit similar magnitude CPP when conditioned with either cocaine or morphine [33]. The rewarding properties of both novelty and drugs of abuse are modulated by mesolimbic and mesocortical dopamine (DA) pathways (see Ref. [3] for references and review), whose activity is altered by stressors including social isolation [13] and which undergo considerable ontogenetic modifications during adolescence (see Ref. [7] for review). Thus, age- and housingrelated neural alterations in mesolimbic and mesocortical DA systems may contribute to the differential sensitivity to the rewarding effects of novelty between group-housed adolescent and adult male rats, age effects not apparent in their isolated counterparts. Although results of isolation-induced changes in DA systems of adult animals are not consistent (see Ref. [13] for references and review), some of the findings suggest a similar pattern of an imbalance between the mesocortical and mesolimbic DA systems in adolescent animals and socially deprived adults. Indeed, levels of basal DA synthesis and turnover in the prefrontal cortex (PFC) are considerably elevated in rats during adolescence [34,35], and in the same way socially deprived adults have been reported to have higher DA and DA metabolite levels in the PFC ([36 – 38]; but see also Ref. [39]). Likewise, basal DA synthesis in the nucleus accumbens is lower in adolescent animals than in their adult counterparts [34], and individually housed male mice also show enhanced mesocortical and reduced mesoaccumbens DA response to stress challenge compared to their group-housed counterparts [40]. Taken together, such findings may eventually clarify age-related differences in sensitivity to novelty reward in group-housed males and a lack of these differences in socially deprived males. It is recognized, however, that most of the literature addressing social deprivation effects has examined isolation rearing for an extended period of time (e.g., 10– 14 weeks), whereas in
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the present study animals were socially deprived only for 10 days before conditioning was initiated. No age-related difference in responding to the rewarding effects of novelty emerged in females, with both adolescent and adult group-housed females, but not their isolated counterparts, showing novel-object place conditioning. The effects of social isolation was opposite for males and females, allowing expression of rewarding effects of the novel objects in adult male rats but virtually eliminating object-induced CPP in females of both ages. Other studies have likewise observed gender effects in the consequences of social isolation, often viewed as an animal model of stress [13] and/or depression [41]. For instance, individual housing for a relatively short period of time (1 week) has been reported to induce a depression-like effect in female but not male mice [42]. To the extent that isolate housing reflects a stressor effect, these data are reminiscent of other findings that female rodents may be more responsive to stressors than males. The corticosterone response of male rats habituated to a chronic stressor, whereas females showed little hormonal habituation [43]. The suppression of activity in the open field initially seen following a stressor returns more quickly to basal, nonstress levels in male than female rats [44,45]. Probably the most striking finding is that exposure to an inescapable stressor facilitates classical conditioning in males [46,47] but impairs it in females [48]. In the same way, the results of the present study show that social isolation stress enhances CPP—a learning paradigm based on classical conditioning [49]—in adult males and attenuates it in female rats. Our results are also in agreement with reports that social isolation of males facilitates Pavlovian conditioning [50] as well as learning in a Morris water maze [51]. A strong dissociation between novelty seeking, as indexed by time spent with the novel objects, and novelty reward in the CPP paradigm was observed in adult males and females of both ages in the present study. Only adolescent males regardless of their housing conditions showed enhanced novelty seeking and strong novel object-induced CPP. Adult group-housed males were relatively high novelty responders, at least when compared with their isolated counterparts, but did not respond to the rewarding properties of novelty as indexed by the CPP paradigm. In contrast, socially deprived adult males spent less time exploring the novel objects during conditioning but showed strong preference for the compartment associated with novelty. Housing conditions did not affect novelty seeking in females with adolescent females being higher novelty responders than their adult counterparts. However, novelty was equally rewarding for females of both ages when they were grouphoused, but these rewarding effects of novelty were virtually eliminated by social isolation. Together these results suggest that mechanisms underlying novelty-directed behaviors and those involved in the rewarding effects of novelty are different. And, indeed, a dissociation between the DA receptor subtypes that mediate novel object exploration and those
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involved in novelty-associated reward has been suggested by recent data [19,21,52]. The D1 receptor antagonist SCH23390 blocked the increase in preference conditioned by access to novel objects at doses that did not affect object interaction [19], suggesting an involvement of D1 receptors in novelty reward but not in novelty exploration. Enhanced sensitivity to novelty reward in adolescent males regardless of their housing conditions may stem from their elevated levels of D1 receptors, with the overproduction and pruning of DA receptors reported in striatum and nucleus accumbens being more pronounced in males than females during adolescence [53]. Taken together, the results of the present paper demonstrate that, whereas adolescents show higher levels of novelty seeking than adults, their high levels of novelty seeking do not necessarily predict high rewarding properties of novelty. Novelty reward, unlike novelty seeking, is strongly influenced by environmental and gender-related factors. Further experiments are needed to understand determinants of novelty seeking and novelty reward as well as their underlying neural mechanisms in adolescence.
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