A chronic stress state in rats: Effects of repeated stress on basal corticosterone and behavior

Physiology&Behavior,Vol. 51, pp. 689-698, 1992

0031-9384/92 $5.00 + .00 1992 Pergamon Press Ltd.

Printed in the USA.

A Chronic Stress State in Rats: Effects of Repeated Stress on Basal Corticosterone and Behavior JOHN

E. O T T E N W E L L E R , 1 R I C H A R D J. S E R V A T I U S , W A L T E R N. T A P P , S U S A N M I C H A E L T. B E R G E N A N D B E N J A M I N H. N A T E L S O N

D. D R A S T A L ,

Neurobehavioral Unit 127A, VA Medical Center, East Orange, N J 07019 and Department of Neurosciences, New Jersey Medical School, Newark, N J R e c e i v e d 6 J u n e 1991 OTTENWELLER, J. E., R. J. SERVATIUS, W. N. TAPP. S. D. DRASTAL, M. T. BERGEN AND B. H. NATELSON. A chronic stress state in rats. Effects of repeated stress on basal corticosterone and behavior. PHYSIOL BEHAV 51(4) 689-698, 1992.--The chronic stress state has previously been defined as persistent visceral arousal coupled with behavioral abnormalities. To determine the number of stressor exposures necessary to induce a chronic stress state, male rats were given 2 hours of inescapable shock on 10, 7, 4, or 3 consecutive days. The 3-day stress group had the most pervasive changes in the variables measured: persistently elevated basal plasma corticosterone (CORT), continued weight loss in the post-stressor period, and abnormal behavior. More exposures to the stress regimen did not produce higher CORT levels or greater behavioral changes. Acutely stressed rats, exposed to 1 day of inescapable shock, had l~ersistent CORT elevations without the other changes seen in the 3-day stress group. The data suggest that 3 days of our stress regimen are sufficient to produce a state of chronic stress and that some signs of this state begin to appear as early as the first exposure to our inescapable stress regimen. Stress

Corticosterone

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Behavior

W H I L E it is generally believed by the public and health professionals that chronic exposure to stressful situations has deleterious effects, there is not much evidence to support this belief (5). This is mainly due to the difficulty in objectively evaluating how much stress people experience in different situations. Thus, researchers have had to use animal models to discover the pathophysiological consequences of repeated exposures to stressors (2,4,8-10,19,20,22,23). But it is difficult to judge the relevance of these models to the condition of chronic stress in people because no chronic stress syndrome has been explicitly defined in the clinical psychiatric literature. However, post-traumatic stress disorder (PTSD) is a syndrome that clearly reflects a condition in people that is chronically stressful (7). Therefore, it seems appropriate to use this syndrome as a guide for developing an animal model of a chronic stress state. PTSD patients show sustained visceral arousal with elevated glucocorticoids (21) and catecholamines (11). However, in animal models of repeated stress, the acute response to a stressor often habituates when the stressor is presented repeatedly (3,12,17). This raises the question of whether an animal is chronically stressed when its acute response to a stressor is diminishing. To circumvent this problem, some researchers have

given a series of different or heterotypic stressors over many days (10,18). Katz et al. reasoned that unpredictability in the sequence of stressor presentation would prevent habituation and thus maintain sustained visceral arousal in response to the stressors (10). This paradigm can sustain adrenal activation over 3 weeks, but it has an inherent weakness. While it guards against stressor predictability, it also makes it difficult to manipulate other stressor parameters like intensity. This is particularly important because we have previously shown that stressor intensity is a critical variable for determining whether basal C O R T levels are elevated during repeated stressor presentation (14-17). Another issue in modelling a chronic stress state is that sustained adrenal activation, in and of itself, is not sufficient to define this state because other conditions, such as Cushing's disease, can produce persistent adrenal activation. In addition to sustained visceral arousal, the diagnostic criteria for PTSD include several types of behavioral disruption including numbing, hyperalertness, and sleep disturbances (7). Because of these clinical criteria and our own work on behavioral responses to single presentations of stressors (13), we have suggested that sustained behavioral disruption must accompany visceral arousal for an adequate model of a chronic stress state (15).

This research was supported by VA Medical Research funds. Requests for reprints should be addressed to John E. Ottenweller, Ph.D. Neurobehavioral Unit (127A), VA Medical Center, East Orange, NJ 07019.

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Chronic stress research utilizing the heterotypic stress paradigm has yielded various behavioral abnormalities (2,10,18). But homotypic chronic stress can also produce abnormal behavior as measured by activity in a running wheel (6,15) and body weight changes (15). We include body weight as an index of behavior because we have previously found that stress-induced body weight losses were largely due to suppression of feeding (15). Thus changes in body weight are indirectly monitoring feeding behavior. We have also reported changes in circadian activity patterns during and after chronic stress (l 5), which may be analogous to the sleep disturbances seen in PTSD patients. Finally, we have found an increased latency to drop from a suspended wire as a consequence of homotypic chronic stress (15). The increased hanging wire latencies we reported were observed both on the last day of stressor presentation and 4 days post-stressor (15). This persistent behavioral alteration lead us to examine the post-stressor period in more detail. We sought to determine (a) how long basal CORT levels remained elevated after the last stressor, and (b) whether the number of stress sessions affected basal CORT levels or the behavior observed during the post-stressor period. Thus, the aim of the present experiments was to determine the minimum number of stress sessions necessary and sufficient to produce a maximal and stable state of chronic stress and to learn how long such a state persisted after the last exposure to the stressor. EXPERIMENT 1 METHOD Young adult male Sprague-Dawley rats were obtained from Charles River (Wilmington, DE) and were housed individually in shoe-box type cages with free access to Purina Rodent Laboratory Chow and tap water. They were maintained on a 12 h photoperiod with the onset of light at 0700 h. The rats were kept under these conditions for 8 weeks before the experiment began,

At the start of the experiment, rats were stratified in terms of body weight (body weights ranged from 330-400 g) and then randomly assigned to receive 10, 7, 4, 3, or 0 days of our chronic stress regimen (n = 8 for all groups). This regimen has been described in detail previously (15). Briefly, stress sessions began at 0900 h when the rats were transferred to a another room. Here, they were immobilized in hardware cloth tubes, tail electrodes were attached, and then 40 2-mA shocks (on for 166 out of 200 ms during each 3-s shock) were given over 2 hours using a constant current shocker. The amperage of the shocks was verified throughout the stress session. After the last shock, the tail electrodes were removed, the rats were placed back in their cages, and then they were returned to the room in which they were housed. The rats were placed in the chronic stress regimen in a staggered fashion such that the 10-day stress group was stressed alone for the first 3 days of the study. On the fourth day, the rats in the 7-day group began receiving stress along with the 10-day group. Similarly, on the seventh day, rats in the 4-day group began receiving stress. The 3-day group began the stress regimen on the eighth day, and all stressed rats were stressed together for the final 3 days. Control rats remained undisturbed in their home cages during this period. This stress regimen was reviewed and approved by the Animal Welfare Subcommittee of the East Orange VAMC. Rats were weighed throughout the experiment in the morning prior to any of the experimental manipulations except blood sampling. The schedule of body weight measurements can be found in Fig. 1 and is reported relative to the last day of stressor exposure. On the afternoon of the last stressor day and the next 3 days, rats were behaviorally tested for their latency to drop from a horizontal wire suspended 45 cm off the ground (15). Blood samples were drawn by tail-clip at 0900 h for the first 3 days after the last stress session. The plasma was separated by centrifugation and stored frozen at - 7 0 ° C until assayed for CORT. Plasma CORT was assayed using a double-antibody radioimmunoassay kit (RSL 125t Corticosterone Kit #07-120102,

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ICN Biomedicals Inc, Carson, CA). The assay was performed as described in the instructions except that 5 #1 plasma samples were diluted 1:80 before being assayed, instead of 1:200. The minimum detectable doses for the assays reported in this paper were less than 0.06/~g/dl. For the high pool (49.2/~g/dl), interassay variability was 6.9% and intraassay variability was 6.1%. For the low pool (7.6 ug/dl), interassay variability was 8.1% and intraassay variability was 9.9%. RIA data reduction was performed on an IBM PC using a four-parameter log-logit curvefit (M. L. Jaffe and Associates, Jupiter, FL).

We also examined the changes in body weight after the last stressor exposure that all stressed groups received on the same day. In this paper, we have designated the 24-h period after the last stressor presentation as the 1st day post-stressor, with subsequent days indexed accordingly. The changes in body weight that occurred over the first complete day post-stressor indicated that the initial post-stressor weight gain in the 10-day group was greater than that in the other groups, p < 0.05 [Fig. I(B)]. Also, the 3-day stress group continued to lose weight over the first day post-stressor. These data again suggest that more than 3 days of stressor exposure resulted in some habituation.

RESULTS

Body Weight Body weight data were analyzed with a 5 X 14 (stress group X weighing day) mixed analysis of variance (ANOVA). There was a significant main effect of days, F(13,455) = 148.3, and a group X days interaction, F(52,455) = 39.4, p < 0.05. This interaction is illustrated in Fig. I(A). Large decreases in body weight were found on the day after the initial exposure to the stress regimen. The rate of weight loss over the first 3 days of stress was the same for all the stressed groups. The changes in body weight that occurred over the last 3 days of stressor exposure were also examined to determine whether there might have been habituation to the stressor [Fig. I(B)]. Note that this was the entire stressor period for the 3-day stress group. Dunn's tests for a priori comparisons between experimental groups indicated that weight loss in the 3-day stress group was greater than that in the other groups over the same time period. The weight loss in the 4-day group was significantly less than that for the 3-day group, but greater than that in the remaining groups. The 10- and 7-day groups lost similar amounts of weight over this time period, and this weight loss was significantly greater than the weight changes seen in controls, all p < 0.05. These data indicate that prior exposures to the stressor reduced the loss of body weight over the last 3 stressor days and thus that there may have been some habituation to the stressor.

COR T The basal CORT levels were analyzed with a 5 X 3 (stress group x sampling day) ANOVA. The main effects of group, F(4,39) = 7.8, and days, F(2,70) = 22.9, were significant, p < 0.05. Dunnetts' tests for a priori comparisons between experimental groups and controls indicated that basal CORT levels were significantly elevated in all the stress groups on each of the 3 days during the post-stressor period, p < 0.05 (Fig. 2).

Hanging Wire Due to heterogeneity of variance, the hanging-wire latencies were log transformed before being subjected to a 5 X 4 (stress group X testing day) ANOVA. Repeated exposure to the stress regimen resulted in longer latencies to drop compared with the latencies in controls, F(4,120) = 2.54, p < 0.05. Dunnett's tests were used to analyze the changes that occurred in latency over days, and these changes differed among the stressed groups (Fig. 3). Rats in the 10-, 7-, and 4-day stress groups initially had longer hanging wire latencies than controls, but the latencies in these stress groups recovered to control values over the 3-day poststressor period. In contrast, the hanging wire latencies for the 3day stress group did not recover during the post-stressor period.

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DISCUSSION

Repeated exposure to our stress regimen produced elevated basal plasma CORT levels which were similar to those reported earlier by ourselves ( 14-16) and others ( 10,18). In addition, it resulted in loss of body weight and longer hanging wire latencies, findings that were again similar to those in our earlier work (15). The primary goal of the present research was to determine the minimum number of exposures to our stress regimen that produced both persistent CORT elevations and behavioral abnormalities. The stressed groups in Experiment 1 had similarly elevated basal CORT for 3 days post-stressor. This was somewhat surprising because we expected greater elevations in basal CORT with more exposures to the stress regimen. Not only were the elevations in basal CORT similar on the first day post-stressor, but the rates at which basal CORT returned toward control levels were also similar among the stress groups. Thus, in this experiment, stress-induced basal CORT elevations were insensitive to the number of times the rats were exposed to the stress regimen. However, the pattern of the behavioral changes was different among the stress groups. As measured by hanging wire latency, rats in the 10-, 7-, and 4-day stress groups showed behavioral recovery in the post-stressor period. Rats in the 3-day group did not. These data were in contrast to Ottenweller et al. 1989 (15), in which 10-day stressed rats exhibited longer hanging wire latencies than controls 4 days post-stressor. However, in that study, rats were not repeatedly exposed to the hanging wire apparatus. Thus, the behavioral recovery of the 10-, 7-, and 4-day stress groups in the current study may suggest retest habituation to the hanging wire apparatus. Given this interpretation, the 3-day stress group exhibited resistance to the retest habituation. With respect to the initial body weight changes after the last stress session, the 10-, 7-, and 4-day stress groups began gaining weight the first day post-stressor. The 3-day stress group did not begin gaining weight until the second day post-stressor. We have previously shown that body weight losses that occur during this type of stress are largely due to reduced feeding (15). Therefore,

the fact that the 3-day stress group had a delay betbre they began to gain body weight may be evidence for a suppression of feeding beyond the last stress session. The two behaviors measured in this experiment showed consistent patterns of recovery in that rats exposed to 3 days of the stress regimen exhibited delayed or absent recovery while rats having more exposures recovered quickly. These behavioral data may suggest that the 10-, 7-, and 4-day stress groups habituated somewhat to the stress regimen, even though it was relatively intense. In summary, the 3-day stress group had elevated basal CORT levels that were similar to those in the stress groups receiving more exposures to the stress regimen, and these levels returned toward control levels at the same rate as those in the other stress groups. The 3-day stress group also had a consistent set of behavioral abnormalities. For these reasons, we would propose that 3 days of exposure to our stress regimen provides a useful model of a chronic stress state. EXPERIMENT 2 The first experiment demonstrated that giving more than 3 days of our stress regimen produced no demonstrably greater degree of stress, even when we went as long as 10 days. The next step was to determine the effects of a single exposure to our stress regimen. In both Experiment l and previous work (15), stressed rats were compared to nonshocked controls. It remained unclear whether the last exposure to the stress regimen by itself might have produced the observed differences between the stressed rats and controls, particularly since the responses in the stressed groups were so similar. To begin to address this issue, we measured basal CORT levels and behavior in rats exposed to 1 day of our chronic stress regimen and in nonshocked controls. METHOD

The rats were of the same strain, of similar weight, and were maintained in similar conditions as those described above for

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Experiment 1. These rats were divided into two experimental groups (n = 8): those exposed to one stress session of tail-shocks and those maintained as nonshocked controls. Blood was sampied, handled, and assayed for plasma CORT as described above. Hanging wire latencies were determined at the same times as described in Experiment 1. The days on which body weights were measured can be found in Fig. 4. RESULTS

Body Weight Body weight data were analyzed with a 2 X 6 (stress group x weighing day) ANOVA. The main effect of days, F(5,80) = 32.8, and the group X days interaction, F(5,75) = 9.7, were significant, p < 0.05. As seen in Fig. 4, the stressor produced a loss of body weight for one day, after which body weight increased at the same rate as in controls.

COR T Plasma CORT values were subjected to a 2 X 4 (stress group X sampling day) ANOVA. The main effects of group, F(I,15) = 30.72, sampling day, F(3,42) = 8.6, and the group x sampling day interaction, F(3,42) = 10.0, were significant, all p < 0.05. Basal plasma CORT levels were elevated by a single exposure to the stress regimen, the elevated levels recovered toward control levels over the post-stressor period, and yet the stressed rats continued to have elevated CORT levels even 5 days post-stressor, p < 0.05 (Fig. 5).

Hanging Wire The latencies to drop from a hanging wire were analyzed by a 2 X 3 (stress group × testing day) ANOVA, and there were no significant effects. The latencies were similar in stressed rats (mean = 3.83 s) and control rats (mean = 3.66 s) over the 3

days of testing (Fig. 6). The control latencies for this experiment were very similar to those reported in Experiment 1. DISCUSSION

One exposure to our stress regimen produced elevated basal CORT levels for a surprisingly long time, i.e., at least 5 days post-stressor. As mentioned above, we expected the degree of elevation in basal CORT levels and/or the rate at which CORT levels returned toward control levels to be related to the number of stressor exposures. Inasmuch as the degree of CORT elevation and the rate it declined post-stressor were similar to those in Experiment 1, again we would conclude that the elevations in basal CORT levels were independent of the number of exposures to our stress regimen. One possible explanation for these unexpected results was that a nonstress environmental factor common to the stressed groups was responsible for their similar elevations in basal CORT. The blood sampling procedure would be an obvious possibility, but this was rejected for two reasons: (a) sampling in a given individual was over before samplinginduced CORT elevations could have occurred, and (b) the control groups exhibited extremely low CORT levels even after repeated sampling. It remains to be determined whether the elevations in basal CORT caused by our stress regimen represent elevations that could be found across the entire day, or a shift in the peak of the circadian CORT rhythm that normally occurs late in the afternoon, or possibly even the development of a secondary CORT peak in the morning. The hanging wire latencies were not affected by a single exposure to the stress regimen, and body weight gains were seen immediately post-stressor. Thus a single exposure to the stress regimen did not produce both persistent adrenocortical activation and behavioral abnormalities, our operational definition of a chronic stress state. EXPERIMENT 3 Taken together, Experiments 1 and 2 suggested that exposure to our chronic stress regimen for 3 days, but not 1 day, produced

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a condition we have operationally defined as a chronic stress state. But because the 1-day stress group did have elevated basal C O R T levels, a direct comparison between the 3- and 1-day stress groups was necessary to establish that the 3-day stress group was the more appropriate model for a chronic stress state. It was possible that the rats, the conditions, or the assays varied between Experiments 1 and 2 that were performed at different times. Moreover, there are several possible choices of control groups for our chronic stress model, e.g., unshocked rats, or stressed rats exposed to either the same number of weaker stressors or

fewer stressors of the same intensity neither of which would be stressful enough to produce a chronic stress state. We have chosen to study the latter group because we were interested in examining the differences between acute exposure and chronic exposure to a stressful situation, both of which could potentially induce the chronic stress state we were interested in studying. Thus, Experiment 3 was undertaken to directly compare unshocked, lday stress, and 3-day stress groups. Another goal of Experiment 3 was to further characterize the behavior of the 3-day stress group. Again using the diagnostic

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criteria for PTSD as a guide, the open-field test was selected. It seemed that open-field activity might reveal behavioral abnormalities that were analogous to some of those seen in PTSD patients (i.e., numbing or hyperalertness). In addition, heterotypic stressor paradigms have been reported to suppress openfield activity (10). Thus, our use of an open-field test would facilitate a comparison of the behavioral consequences of heterotypic and homotypic chronic stress paradigms. METHOD

The rats and their conditions were similar to those in Experiments 1 and 2. Open-field activity was assessed in all rats 3 days before the first stress session to control for any preexisting differences in open-field activity. Rats were then stratified in terms of open-field activity and randomly assigned to groups receiving 3 days or 1 day of the stress regimen, or they were placed in one of two control groups (n = 8). As in Experiments 1 and 2, one control group was kept in an identical, but separate, housing unit in the same room as the experimental rats (different box, DB-control). A second control group, housed with the stressed rats (same box, SB-control), was added to determine whether simply housing unstressed rats with stressed rats would elevate basal CORT levels. Before stress, the mean squares crossed in the open-field by the four groups were: 3-day = 46.0, 1-day = 45.5, DB-control = 46.1, SB-control = 46.1. ANOVA indicated that the four groups had similar open-field activity, F(3,28) = 0.005, and body weight, F(3,28) = 0.496. The schedules for weighing, blood sampling, and open-field testing can be found in Figs. 7-9. RESULTS

Body Weight As in Experiments 1 and 2, body weight declined when rats were stressed (Fig. 7). A 4 X 8 ANOVA (stress group X weighing day) revealed a significant main effect of days, F(7,196) = 213.2,

and a group X days interaction, F(21,196) = 8.19, allp < 0.05. The 3-day stress group, as in Experiment 1, continued to lose weight post-stressor, while the l-day stress group did not.

COR T A one-way (stress group) ANOVA showed that basal CORT levels differed among the stress groups, F(3,28) = 7.4, p < 0.05. Both stress groups had similarly elevated basal CORT levels the day after the last stress session compared with either control group (Dunn's tests, p < 0.05, Fig. 8). The control groups did not differ. Although they seem somewhat higher, the CORT levels in this experiment were probably comparable to those in Experiments 1 and 2 inasmuch as the pools for this assay ran 30% higher than those for the assays in the first two experiments.

Open-Field Activity A 4 X 3 (stress group X testing day) ANOVA revealed a significant main effect of days, F(2,56) = 38.26, and a significant group x days interaction, F(6,56) = 5.63. On the first day poststressor, open-field activity was suppressed in both stress groups relative to that in the SB-control group, but it recovered by the third day post-stressor (Fig. 9). The DB-control group had less open-field activity than the SB-control group, and this also recovered by the third day post-stressor, all p < 0.05. DISCUSSION

A direct comparison between repeated and single exposures to our stress regimen largely replicated the findings in Experiments 1 and 2. As observed previously, the 3-day stress group continued to lose weight in the post-stressor period, while this was not seen in the 1-day group. In addition, basal CORT levels in both stress groups were elevated on the first day post-stressor. Exposure to the stress regimen suppressed open-field activity in both the 3- and l-day stress groups which recovered by 3 days post-stressor. Others have also found that repeated exposure to

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stressors will suppress open-field activity (10,22). Contrary to expectations, chronic and acute stressor exposure had similar effects on open-field activity when testing began 1 day poststressor. The severity of the acute stressor may account for this similarity. In the Katz et al. study (10), a single shock was used for an acute stressor, whereas we used multiple shocks in a single exposure as our stress regimen. This probably explains why they reported an increase in open-field activity after an acute stressor and a difference between acute and chronic stressor exposure that we did not find in the current experiment. Unexpectedly, the DB-controls were less active in the openfield than the SB-controls. This may have been due to the blood

sampling in the morning before the open-field test that was the first novel experience for the DB-controls in some time. On the other hand, the SB-controls had been exposed to having the stressed rats removed and then having them returned after the stress sessions for the 3 days before the sampling and open-field testing that occurred on the first day post-stressor. This may have inoculated the SB-controls to the mild stress caused by blood sampling. This interpretation would be consistent with our hypothesis that an intensity continuum describes the development of the chronic stress state (15). Blood sampling may represent a mild stressor that only affects behavior. Exposure to stressed animals

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during their stress sessions [(15) bucket controls] may be a relatively moderate psychological stressor causing elevated basal C O R T levels but no behavioral abnormalities. The 1-day stress group would be even more stressed because this group had elevated basal C O R T and some behavioral abnormalities, without persistent weight loss. We would suggest that the 3-day stress group was in a severe chronic stress state because they had elevated basal C O R T levels, abnormal behavior, and continued weight loss in the post-stressor period, all the hallmarks of our model for a chronic stress state. A goal of the present research was to parametrically explore the number of sessions of inescapable shock necessary and sufficient to produce a chronic stress state. The 3-day stress group compared with the other stress groups: 1. did not show signs of habituation to the stressor through a decreased rate of weight loss during stressor exposure; 2. continued to lose weight in the post-stressor period while the other groups gained weight; 3. had elevations in basal plasma C O R T levels that were as great and as long as those in any of the other stress groups; and 4. had longer hanging wire latencies that did not recover over the post-stressor period that we examined.

These data suggest that habituation to the stressor and enhanced recovery may have occurred in the repeatedly stressed rats that received more than 3 days of our stress regimen. Thus, more than 3 days of our stress regimen is not needed to produce a chronic stress state, and may in fact enable the process of habituation to begin. The next steps in the development of our model of a chronic stress state would be: 1. to extend the post-stressor basal sampling to other hormones, especially those of the adrenal axis; 2. to examine C O R T levels around the clock in the post-stressor period; 3. to determine the pharmacological relevance of this model to anxiety and depression; and 4. to determine the role of learning in the development of the physiological and behavioral changes that occur in our model of a chronic stress state.

ACKNOWLEDGEMENTS The authors wish to acknowledge the technical assistance of Nathaniel McCafferty and the computer support of Thomas Pritzel.

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