Adrenocortical activity during conditions of brief social separation in preweaning rats

BEHAVIORAL AND NEURAL BIOLOGY 54, 42--55

(1990)

Adrenocortical Activity during Conditions of Brief Social Separation in Preweaning Rats MICHAEL B. HENNESSY Department of Psychology, Wright State University, Dayton, Ohio 45435 AND JOANNE WEINBERG l

Department of Anatomy, The University of British Columbia, Vancouver, British Columbia, Canada V6T 1W5 Rat pups (18 days of age) placed into a novel test cage for 30 min exhibited greater concentrations of plasma corticosterone if alone than if with the biological mother regardless of whether she was conscious or anesthetized. Pups tested with a conscious nonlactating female had higher corticosterone levels than did pups tested with their own conscious mother. Anesthetizing the adult stimulus females eliminated this differential effect. Further, pups isolated in the home cage for 30 min exhibited corticosterone elevations as great as those of pups placed into a novel environment. However, pups left in the home cage with the mother for 30 min displayed corticosterone elevations similar to those of pups isolated in the home cage. In contrast, if pups were left in the home cage with both the mother and the littermates corticosteroid levels were reduced. These results indicate that, under certain conditions, brief separation from the mother can elevate plasma corticosterone levels in the 18-day-old rat. The differential effect of the biological mother and the nonlactating female appears attributable to differences in the behavior of the adult females. Finally, brief isolation from all littermates was also found to evoke a plasma corticosterone response. © 1990 AcademicPress, Inc.

The authors thank Anne Tamborski, Susan Suchy, Kevin Fung, and Gretta D'Alquen for technical assistance, and Sally Mendoza for her helpful comments on an earlier draft of this manuscript. The work was supported by Grants BNS-8701620 and 8822240 from the National Science Foundation and a Biomedical Sciences Seed Grant from Wright State University to M.B.H., and by Grants MA-8040 and MA-9730 from the Medical Research Council of Canada to J.W. Portions of these data were presented at the 1988 meeting of the Midwest Psychological Association. Address correspondence and reprint requests to Michael B. Hennessy, Ph.D., Department of Psychology, Wright State University, Dayton, OH 45435. 42 0163-1047/90 $3.00 Copyright© 1990by AcademicPress. Inc. All rights of reproductionin any formreserved.

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43

In the preweaning rat, a variety of physiological and behavioral changes have been observed during maternal separation. Lowered heart rate, reduced oxygen consumption, and a number of other physiological and behavioral responses appear to result from the removal of specific regulatory influences exerted on the pup by particular features of maternal stimulation (Hofer, 1973; Schanberg, Evoniuk, & Kuhn, 1984; Stanton, Gutierrez, & Levine, 1988; Stone, Bonnet, & Hofer, 1976). These responses typically emerge only after a prolonged period of separation (Hofer, 1987). On the other hand, increased heart rate, increased emission of ultrasonic vocalizations, and other behavioral changes occur almost immediately following removal of the mother and littermates in 2-weekold rat pups and appear to be due to the separation from social partners per se, rather than to the withdrawal of specific physiological regulatory influences (Hofer & Shair, 1980, 1987). Circulating levels of adrenal corticosteroids are elevated by a brief period of maternal separation in some primate species (Coe, Mendoza, Smotherman, & Levine, 1978; Smotherman, Hunt, McGinnis, & Levine, 1979) as well as in guinea pigs (Hennessy & Ritchey, 1987). However, the effect of brief maternal separation on the adrenocortical activity of rat pups is unclear. Kuhn, Butler, and Schanberg (1978) observed no change in the circulating corticosterone levels of 10-day-old rat pups that were removed from the home cage and isolated for either 90 or 120 min in a test cage. Other investigators have found that 14- to 20-day-old rat pups exhibit significant increases in plasma concentrations of both corticosterone and ACTH when taken from the nest and isolated in a novel environment for 10 to 30 min (Stanton et al., 1988; Takahashi, Kalin, Barksdale, Vanden Burgt, & Brownfield, 1988). Although this manipulation clearly increased pituitary-adrenal activity, the responses may have been elicited by several factors including novelty and handling, as well as by maternal separation. In a study that controlled for the effects of handling and the novelty of the test environment, Stanton and Levine (1988) found that 20- and 24-day-old rat pups removed from the nest to a test cage for 30 min exhibited significantly greater plasma concentrations of corticosterone if alone than if with an anesthetized, lactating female. However, because of the particular focus of that study, the lactating female with which the pups were tested was an unfamiliar one rather than the pup's biological mother. Thus, all pups were actually separated from their own mothers. This precludes an unambiguous interpretation of maternal separation effects, particularly in light of recent findings suggesting that rat pups may be able to discriminate their own mother from other lactating females (Hepper, 1986). Thus, the question of whether brief maternal separation, in and of itself, evokes an increase in pituitary-adrenal activity in the preweaning rat remains unresolved. The present experiments were designed primarily to assess the effect

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HENNESSY AND WEINBERG

of brief maternal separation on plasma corticosterone levels of preweaning rats. Pups were placed into a novel test cage alone, with the biological mother, or with a nonlactating adult female. To examine the effect of the adult females' behavior on the endocrine responses of the pups, tests were conducted with both conscious and anesthetized adult females. In addition, the effect of separation on the corticosteroid response of pups remaining in the familiar home cage was investigated. That is, corticosterone levels of pups left in the home cage alone, with the mother only, or with the mother and littermates were examined. Pups were 18 days old at the time of testing. At this age, pups are clearly past the stress hyporesponsive period, and so exhibit reliable corticosterone responses to stressors (Levine, 1968; Sapolsky & Meaney, 1986); yet they are several days from weaning, and thus are still subject to maternal modulation of physiological and behavioral activity. EXPERIMENT 1

This experiment employed procedures similar to those found to produce plasma corticosteroid elevations to maternal separation in infant guinea pigs (Hennessy & Moorman, 1989; Hennessy & Ritchey, 1987). The plasma corticosterone levels of rat pups isolated for 30 min in a test cage were compared to those of pups tested in the same manner, but with their mother present. Because the test conditions differed only in the presence/absence of the mother, differences in corticosterone levels between conditions could be attributed to maternal separation. Additional pups were tested in the presence of a nonlactating adult female. Because albino rat pups of this age can distinguish a lactating from a nonlactating female on the basis of olfactory cues (Leon & Moltz, 1971), this condition assessed whether a discriminable adult female would have the same effect on corticosterone levels as would the pup's own mother. Method Animals. Virgin female and male Sprague-Dawley albino rats (Harlan Sprague-Dawley, Inc., Indianapolis, IN) were bred. Forty-eight resulting offspring (24 males, 24 females) from 13 litters were used in this experiment. Each litter was maintained with its biological mother in a clear polycarbonate cage (45.7 × 23.8 x 20.0 cm) with wood-chip bedding. In most litters, some pups were removed for testing in another experiment on Days 5 and 6 (day of birth = Day 0). The litters, which then ranged from 3 to 10 pups, were not subsequently disturbed, except for routine cage cleaning, until testing in the present experiment on Day 18. No cages were cleaned after Day 16. The colony room was maintained on a ll-h on/13-h off cycle (lights on at 0630). Procedures. Six male and six female pups were assigned to each of four conditions: Base, Mother, Nonlactator, and Alone. In the Base

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45

condition, the pup was removed from the litter prior to any disturbance and taken to a nearby room where a blood sample was immediately collected for estimation of resting corticosterone levels. In the Mother condition, the pup was placed together with its mother into an empty polycarbonate cage of the same dimensions as the home cage for 30 min. The cage was situated on a heating pad to maintain the floor of the cage at about 32-34°C. The Nonlactator condition was identical to the Mother condition with the exception that the pup was exposed to the test cage together with a nonlactating female rather than with the mother. The nonlactating female was of the same age as the mother. The behavior of the nonlactating female was monitored to ensure that she did not act aggressively toward the pups. The Alone condition differed from the previous two conditions only in that the pup was placed into the test cage alone. At the end of the 30-rain test session, blood samples were collected from pups in the three latter test conditions. No more than one male and one female from a litter were assigned to a particular condition (no more than one pup total from a litter was assigned to the Mother condition). For each litter, any pups assigned to the Base condition were the first pups tested. Then at 2-min intervals, pups assigned to the Alone, Nonlactator, and Mother conditions were placed into their respective test cages in order. This order of testing was chosen so that (a) pups in the Base condition were the first pups of their litters to be tested and (b) no pups were left in the home cage without the mother prior to testing. Further, this order was considered to be conservative in that if removal of pups produced some increase in corticosterone levels of pups remaining in the home cage, this influence would tend to diminish, rather than enhance, any effect of maternal separation. That is, pups in the Mother condition were exposed to more potential disturbance due to the removal of other pups than were pups in the Alone condition. All testing was performed between 0830 and 1100 h. Blood collection and corticosterone determination. Blood was collected in heparinized tubes following decapitation under ether anesthesia. This procedure took less than 2 min to complete from the time the pup was removed from its home (Base condition) or test (Mother, Nonlactator, and Alone conditions) cage, which is rapid enough to ensure that the sampling procedure itself did not influence corticosterone levels in the blood collected (Coover, Heybach, Lenz, & Miller, 1979; Davidson, Jones, & Levine, 1968). Samples were centrifuged to separate plasma which was then frozen until assayed for corticosterone using standard radioimmunoassay procedures. Duplicate aliquots were assayed using antisera, tritiated corticosterone, and general protocol provided by Radioassay Systems Laboratories (Carson, CA).

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Results

A 2 (Gender) × 4 (Condition) analysis of variance (ANOVA) on plasma corticosterone values yielded main effects of Gender [F(1, 40) = 6.38, p < .05] and Condition [F(3, 40) = 39.31, p < .01]. Females had slightly higher plasma corticosterone levels than did males (collapsed across test conditions, female • = 14.37 /~g/100 ml, male ~ = 11.75 /zg/100 ml). In addition, corticosterone levels of pups in all three test conditions (Mother, Nonlactator, Alone) were elevated over resting levels (Newman-Keuls comparisons, all ps < .01, Fig. 1). Importantly, corticosterone levels of pups in the Nonlactator and Alone conditions were also significantly higher than those of pups tested with their mother (ps < .01). The difference between the Nonlactator and Alone conditions was of marginal significance (p < .10). No instances of overt aggression occurred in the Nonlactator condition. Instead, these females sometimes licked the pups and often remained in physical contact with them. Although systematic comparisons were not made, it appeared that the behavior of the nonlactating females was generally similar to that of the lactating females during testing.

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EXPERIMENT 2 The finding that pups in the Alone condition had significantly higher plasma corticosterone levels than did pups in the Mother condition demonstrates that brief maternal separation can elevate plasma corticosteroid levels in 18-day-old rat pups. The finding that pups in the Nonlactator condition had significantly higher levels of corticosterone than did pups in the Mother condition might be explained by differences in the behavior of the adult females. Although we did not observe obvious differences in behavior, the treatment of the pups by the mothers may still have been more effective than that of the nonlactators in reducing the arousal of the pups (or less likely to further increase the arousal of the pups), and thus prevented further plasma corticosterone elevations. Alternatively, pups might recognize the mother in part through cues provided by her behavior. The second experiment assessed the importance of the behavior of the adult females. Pups were again placed into the test cage with either a nonlactating adult female or their own mother. However, this time both the nonlactating female and the mother were anesthetized. If the results of the first experiment were due to differential behavior by the adult females, then anesthetizing the females, and thus eliminating differences in their behavior, should also eliminate the differential corticosterone responses of pups in these conditions. To determine if there was a relationship between corticosterone levels and the time pups spent in physical contact with the two types of adult females, we monitored the amount of time that pups spent in contact with adults. Finally, we included a condition in which the pup was isolated in the home cage for 30 rain. Hennessy and Moorman (1989) observed that guinea pig pups display plasma corticosteroid elevations to maternal separation under conditions like those of Experiment 1, but show no corticosteroid increase following 30 or 180 rain of isolation in the familiar home cage. Experiment 2 provided an opportunity to examine the generalizability of these effects to another rodent species. Method Animals. Sixty Sprague-Dawley albino rat pups (30 males, 30 females) were obtained from 12 litters as described in Experiment 1. At birth, litters were culled to 10 pups (5 males, 5 females when possible). The litters were not further disturbed, except for routine cage cleaning, until testing at Day 18. Procedures. Six male and six female pups were assigned to each of five conditions: Base, Mother, Nonlactator, Alone-Novel, and AloneHome. Each of the 12 litters contributed one pup to each condition. The Base, Mother, and Nonlactator conditions were the same as the similarly

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named conditions of the first experiment, with the exception that in both the Mother and the Nonlactator conditions, the adult female was anesthetized with sodium pentobarbital (47.5 mg/kg body wt, IP). The mother was taken from the home cage to be anesthetized after the pup assigned to the Base condition had been removed for blood sampling. After the mother and a nonlactating female had been anesthetized, they were each placed at one end of separate test cages with their ventrums exposed. Five to 10 min following the mother's removal, pups were placed into the test cages at 2-rain intervals. Pups assigned to the Mother and Nonlactator conditions were positioned so that they were in contact with the test females. Pups in the Alone-Novel condition were placed into an empty test cage as was done in the Alone condition of Experiment 1. The order of testing in the Mother, Nonlactator, and Alone-Novel conditions was counterbalanced among litters. After the pups designated for the Base, Mother, Nonlactator, and Alone-Novel conditions were taken for testing, all remaining pups, except one, were removed. The one remaining pup thus constituted the subject for the Alone-Home condition. In the Mother and Nonlactator conditions, an instantaneous observation was made every 30 s to note whether the pup was in physical contact with the adult female (excluding her tail). All other methodological details were as described for Experiment 1. Results A 2 (Gender) x 5 (Condition) ANOVA on corticosterone levels yielded a significant main effect of Condition [F(4, 50) = 27.11, p < .01]. No other effects were significant. Plasma corticosterone was elevated over resting levels in pups tested in the Mother, Nonlactator, Alone-Novel, and Alone-Home conditions (Newman-Keuls comparisons, all ps < .01, Fig. 2). Once again, the presence of the mother in the test cage resulted in lower plasma corticosterone levels than observed in pups tested alone (p < .05). Unlike the previous experiment, however, the corticosterone levels of pups tested with their own mother were not significantly lower than those of pups tested with a nonlactating female. The difference betwen the Nonlactator and Alone conditions was once again of marginal significance (p < .10). In both the Mother and the Nonlactator conditions, pups spent virtually the entire test session in contact with the adult female. In the Mother condition, 10 of 12 pups were in contact with the mother during all 60 instantaneous observations, while in the Nonlactator condition, 9 of 12 pups were in contact during all 60 observations. No pup in either condition was out of physical contact with the adult female during more than 3 of the instantaneous observations.

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EXPERIMENT 3 Experiment 3 assessed the influence of disturbance involved in removing animals from the home cage on the corticosterone levels of the remaining pups. This question arises because in the earlier experiments, multiple animals were taken from the same home cage on the test day. Therefore, disturbance involved in serially removing animals for testing may have affected corticosterone levels of subsequently tested pups. For pups examined in the novel test cage, the influence of disturbance was controlled by using either a sequence of testing in which these effects would tend to diminish, rather than enhance, effects attributed to separation (Experiment 1) or a counterbalancing procedure (Experiment 2). Nonetheless, disturbance may still have influenced the corticosterone levels of the pups removed to the novel test cage. Experiment 3 examined this issue by measuring corticosterone levels of pups as they were serially removed from the home cage. The influence of disturbance is particularly pertinent for an understanding of the high corticosterone levels obtained in the Alone-Home condition of Experiment 2. Here the available comparison group (Base condition) was composed of nonmanipulated pups, rather than of pups exposed to the same manipulation as the Alone-Home pups, except for

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separation from the mother. Therefore, in the present experiment a group of pups remaining with the mother, but exposed to the same manipulation as was the A l o n e - H o m e group, including the removal of all littermates, was tested. Additional pups were treated in a comparable manner, but left with littermates as well as the mother during the 30-min test period, so that the specific effect of isolation from littermates could be evaluated.

Method Animals. Sixty-five rat pups (32 males, 33 females) from 17 litters were tested. As in Experiment 2, litters were generally culled to 10 pups (5 males, 5 females when possible), although 3 litters consisted of 9 pups and 1 litter of 8 pups at the time of testing. Procedure. For 12 litters, one pup was removed from the home cage prior to any disturbance, and a blood sample was collected to estimate basal plasma corticosterone levels. Just following blood sample collection, the lactating female was lifted from the home cage as had been done in Experiment 2 when the mother was removed for anesthetization. But in this case, she was immediately returned to the home cage. The litter was then left undisturbed for 7.5 min. This interval was chosen because, in Experiment 2, there was a 5- to 10-min delay between the removal of the mother and the testing of the next pup. At 7.5, 9.5, and 11.5 min following the disturbance of the adult female, an individual pup was removed from the home cage for collection of a blood sample. These samples were used to estimate the effect of disturbance on the corticosterone levels of pups at the time they were removed to the novel test cage in the earlier two experiments. Following collection of the last of these blood samples, all pups except one were removed from the home cage. The remaining pup stayed with its mother for an additional 30 min, at which time a blood sample was collected. This group of pups thus received the same manipulation, including isolation from littermates, as pups in the A l one-H om e group of Experiment 2, with the exception that the mother was present. An additional five litters were treated in the exact fashion just described except that prior to the final 30-min delay, the experimenter lifted all pups but the designated test pup from the home cage, and then immediately returned them to it. Blood samples were collected only from the designated test pup of each litter. Corticosterone levels of pups remaining with their mothers and littermates were compared to those of pups left with only the mother for the final 30 min to examine the effect attributable specifically to isolation from littermates. All other methodological details were as described for Experiment 2. Results and Discussion Mean plasma corticosterone levels of pups in all conditions are shown in Table 1. A 2 (Gender) x 4 (Condition) ANOVA was used to analyze

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TABLE 1 Mean (_+ Standard Error) Plasma Corticosterone Concentrations in the Six Conditions of Experiment 3

Base 5.0 (0.6)

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9.5 a

11.5°

With mother only

With mother and littermates

5.3 (0.6)

6.8 (0.9)

5.5 (0.5)

16.1 (1.8)

10.0 (1.7)

" Pups removed from the cage 7.5, 9.5, and 11.5 rain following disturbance of the mother.

the corticosterone levels of pups in the Base condition and those sampled 7.5, 9.5, and 11.5 min following disturbance of the mother. The ANOVA yielded no significant effects. Because the pups sampled at 7.5, 9.5, and 11.5 min were exposed to an experimental manipulation that was the same as that received by pups prior to testing in the Mother, Nonlactator, and Alone-Novel conditions of Experiment 2, and similar to that received by pups in Experiment 1, our results suggest that home cage disturbance did not affect circulating corticosterone levels of pups in Experiments 1 and 2 at the time they were removed to the test cage. Further, as Table 1 illustrates, there was not even a clear trend toward increasing corticosterone levels with increasing disturbance (and increasing time since the initiation of disturbance) across the four conditions. In contrast, pups that remained in the home cage with only the mother for 30 min exhibited plasma corticosterone concentrations (y = 16.1 _+ 1.8/xg/100 ml) that were about as great as those shown by pups isolated in the home cage for 30 min in Experiment 2 (2 = 19.4 + 1.4/xg/100 ml). The heightened corticosteroid concentrations of pups remaining with only the mother can be accounted for in part by isolation from littermates. That is, pups remaining with only the mother had significantly higher plasma corticosterone levels than did pups remaining with the mother and littermates [~ = 10.0 + 1.7/xg/100 ml; t(15) = 2.05, p < .05, onetailed test].

GENERAL DISCUSSION In Experiments 1 and 2, we found that pups placed into a novel test cage alone exhibited greater concentrations of plasma corticosterone than did pups placed into the test cage together with the biological mother. This effect was evident regardless of whether the mother was conscious or anesthetized. It should be noted, however, that all pups placed into the novel test cage evinced marked plasma corticosterone elevations. Clearly, the presence of the biological mother did not prevent other factors (e.g., exposure to the novel environment, handling, and/or isolation from littermates) from elevating plasma corticosteroid levels. Rather, separation from the mother produced a further increment in the

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plasma corticosterone response. The present results demonstrate that brief maternal separation can evoke plasma corticosterone elevations in 18-day-old rat pups. Interestingly, the results of a study conducted at about the same time as the present experiments indicate that separated 10-day-old pups begin to exhibit plasma corticosterone elevations when separations reach about 120 min in duration (Kuhn & Schanberg, 1988). Thus, the length of maternal separation sufficient to evoke hypothalamopituitary-adrenal activation may diminish between 10 and 18 days of age, a period during which the rat pup assumes primary responsibility for maintenance of contact with the mother. In contrast to the present findings, Stanton et al. (1988) found that during prolonged maternal separation (at least 8 h, and particularly after 24 h) preweaning pups showed a general potentiation of the magnitude of the corticosteroid response to subsequent novelty exposure. Thus, the hypothalamo-pituitary-adrenal system of the rat pup exhibits distinct effects of short-term and long-term maternal separation procedures. These effects appear to correspond to the two general classes of separation effects described by Hofer (1987) and discussed in the introduction. In the first experiment, pups tested with a conscious nonlactating female had higher plasma corticosterone levels than did pups tested with their own conscious mother. The results of Experiment 2 suggest this difference was due to differences in the behavior of these females. When the females were anesthetized, which eliminated differences in their behavior, the corticosterone levels of pups in the Mother and Nonlactator conditions no longer differed. Statistical comparison of absolute hormone levels across experiments is not appropriate. Yet, inspection of Figs. 1 and 2 certainly suggests that anesthetizing the adult females eliminated this differential effect on pup corticosterone levels primarily by increasing levels in the Mother condition. That is, some aspect of the mother's behavior that tended to moderate pup corticosterone levels, rather than some aspect of the behavior of the nonlactating female that tended to elevate pup corticosterone levels, seems to have been eliminated by the anesthetization procedure. We feel that it is most likely that the mother's behavior calmed the pups or reduced their arousal, and thereby moderated the plasma corticosterone response. However, it is also possible that the mother's behavior provided discriminable stimuli which, presumably together with odors, and possibly other cues, specified her presence to the pups (i.e., provided information that separation from the mother had not occurred). One question that arises concerns the stimulus properties of the mother that underlie the separation effect. That is, what is it about the mother that the pups must be separated from in order to elicit plasma corticosteroid elevations? In the guinea pig, both contact-derived and distal maternal cues contribute to the effect (Hennessy, 1988). Results of stud-

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ies with rats suggest that cues of several modalities mediate separation effects, but that tactile/thermal cues may be of particular importance. In a brief separation paradigm, Hofer and Shair (1980) found that exposure to fur-like tactile properties significantly reduced the ultrasonic vocalizing of isolated pups. Similarly, Siegel, Richardson, and Campbell (1988) found that fur-like tactile as well as thermal cues, but not olfactory stimulation associated with the mother, moderated the increased heart rate exhibited by 16-day-old pups isolated in a novel environment. In pups that had been deprived of their mother for 24 h, subsequent contact with an anesthetized lactating female in the absence of suckling was sufficient to reduce corticosterone elevations in a novel environment (Stanton, Wallstrom, & Levine, 1987). Odor cues did not appear capable of accounting for this effect. Our finding that 18-day-old pups spent virtually the entire test session in physical contact with the anesthetized lactating and nonlactating females suggests that tactile/thermal cues may have been especially important in the present study as well. However, although thermal cues may have helped mediate the separation effect, it should be emphasized that neither hypothermia nor metabolic thermal challenge was likely to have affected pups during maternal separation. The fact that the test cage was warmed, the relatively advanced age of the pups, and the brief duration of the separation all argue against this possibility. The presence of littermates markedly reduces behavioral responses shown by maternally separated 14-day-old rats (Hofer & Shair, 1987). The results of Experiment 3 indicate that the presence of littermates is also an important determinant of the hypothalamic-pituitary-adrenal activity of pups of about this age. Pups disturbed and left with only the mother exhibited significantly higher plasma corticosterone levels than did pups disturbed and left with the mother and littermates. Further, because pups left with only the mother displayed corticosteroid levels similar to those seen in pups isolated in the home cage in Experiment 2, one might conclude that maternal separation has no effect in the home cage. However, such a conclusion may be premature. Informal observation indicated that the experimental manipulations of Experiment 3 increased the locomotor activity of the maternal females. Therefore, pups may have still experienced some physical separation from the mother as she moved about the cage. For those pups left with littermates, contact with siblings may have compensated for this maternal separation to some extent. We feel that this issue needs to be resolved before conclusive statements can be made regarding the effects of maternal separation in the home cage. In any event, the results of Experiment 3 do indicate that under appropriate conditions, brief separation from all littermates, like brief separation from the mother, can elevate plasma corticosterone levels in the preweaning rat.

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