David L. Pitman

Plasma corticosterone levels during repeated presentation of two intensities of restraint stress: Chronic stress and habituation

This study measured plasma corticosterone levels in male rats during repeated daily presentations of two intensities of restraint stress. The corticosterone response to a stress session was defined as the change from pre-stress levels to levels after 60 minutes of restraint. With the relatively intense stress imposed by four limb prone restraint, the corticosterone response partially habituated over seven days due to increasing basal corticosterone levels. However, even on day 7, there was still a large corticosterone response. With the milder stress of immobilization in a tube, the corticosterone response did not habituate at all over 21 days of repeated stress despite rising basal levels. Stress levels of corticosterone did not show significant change over days in either of the two restraint groups. Further, rising basal corticosterone levels suggest that repeated restraint produced a chronic stress state in these rats which may vary in some qualitative way with stressor intensity. Control rats placed in the same room as the stressed rats during the two stresses initially had increased corticosterone levels that matched the levels achieved in the stressed rats. The responses in control rats for the intense stress did not habituate completely in 7 days, whereas those in the control rats for the mild stress habituated completely within 3 days. These data suggest intraspecific communication of the intensity of stress.

Effect of stressor intensity on habituation of the adrenocortical stress response

Although it is known that the number of presentations of a stressor can influence the adrenocortical stress response, relatively little information exists on how stressor intensity affects this process. To evaluate this, we repeatedly presented rats with stressors of 3 different intensities and sampled blood for corticosterone. The first major finding was that the rats initial adrenocortical responsiveness regardless of the stressor employed was a critical variable. Rats that showed a small corticosterone response showed no evidence of habituation or of differences due to stressor intensity. Rats that showed an initial robust response all showed partial habituation of their corticosterone response over time but the patterns varied with stressor intensity. Handled and prone restrained rats showed the same pattern but rats subjected to the more intense stressor of supine restraint showed delay in habituation and tonically elevated responses. These data indicate that individual differences in reactivity to stressors as well as stressor intensity can influence the pattern of the stress response over the course of repeated administration of the stressor.

Adrenocortical and behavioral responses to repeated stressors: toward an animal model of chronic stress and stress-related mental illness

Research in chronic stress has been hampered by the absence of an operational definition for that condition. To explore possible criteria for chronic stress, we repeatedly exposed rats to 2 hr of tail shock per day. After several days, we found elevated prestress corticosterone levels and abnormal behavior, including decreased food consumption, fear-like suppression of activity immediately before stress, greater hesitancy to drop from a suspended wire, and decreased exploratory behavior in a novel environment. A less intensely stressed group of rats also had elevated prestress corticosterone levels, but not the abnormal behaviors that persisted in shocked rats after the stress sessions were discontinued. We propose that abnormalities in both adrenocortical function and behavior are a better marker for chronic stress than abnormalities in either of these systems alone. The animal model we have described may be useful for studying factors that contribute to development of chronic stress or PTSD.

Adrenal hormonal indices of stress in laboratory rats

When individual rats were exposed to different intensities of a stressor, foot shock, plasma catecholamines were found to be sensitive and reliable indices of the stress. Plasma corticosterone did not perform as well. Similarly, levels of both plasma epinephrine and norepinephrine correlated highly significantly with a behavioral measure of the degree of stress--namely, the amount of movement about the cage seen during the 30 sec shock period. Importantly, this behavioral measure was as sensitive and reliable an index of stress as the catecholamines. However, use of either the catecholamines or this behavioral measure as a clinically useful measure of the level of stress was limited by the fact that their responses to the stressor were extremely short-lived. Nonetheless, because the catecholamines reliably and sensitively track the intensity of a stressor, they appear to be a good visceral measure of stress, perhaps the best currently available. But the behavioral concomitants of stress are quickly and easily quantifiable and present a wide range to study, starting with alerting, through a progression of more aroused motor activity, and ending with fight-flight. Because the behavioral concomitants of stress have not been as intensively studied as the endocrine ones, we believe that future efforts to find a clinically useful index of stress will be rewarded by a refocussing of attention away from the visceral respondent to the overt behavioral one.

Effects of repeated stress on T cell numbers and function in rats

Although stress has been reported to affect various functions of the immune system, the mechanism that mediate these effects remain unclear. Thus we examined the effects that 1, 7, and 14 days of stress could have on various aspects of immune and endocrine function in rats. Rats subjected to repeated stress (7 and 14 days) showed significant decreases in the total number of mononuclear cells, particularly suppressor/cytotoxic (CD8) T cells, in the spleen and blood. The mitogenic responses of T cells to phytohemagglutinin (PHA) and concanavalin-A (Con A) were also significantly diminished at these times, as well as after acute (1 day) stress in the case of PHA stimulation. The mechanisms of this impaired T cell mitogenesis were explored by assessing the effects of stress on T cell interleukin 2 (IL-2) production and T cell responsiveness to IL-2. T cells from repeatedly stressed rat showed a decreased production of IL-2 in response to PHA, although their proliferative response to exogenous IL-2 was normal. Repeated stress also decreased body weight and spleen weight, increased adrenal weight, and decreased plasma levels of triiodothyronine and testosterone. These results suggest that lower levels of IL-2 production during stress could be one reason for the decreased mitogen responsiveness of T cells, often seen with stress. This is important because defective IL-2 production could also lead to significant impairment of immunoregulatory T cell generation and thus a predisposition to malignancy or autoimmune disease that some have associated with stress.

Effects of exposure to stressors of varying predictability on adrenal function in rats.

For 5 days, rats were exposed to shocks that were signalled by a light 0, 33, 66, or 100% of the time. Basal hormone levels and responses to a light-shock pair were measured daily. Greater predictability was associated with higher basal plasma corticosterone and norepinephrine levels indicative of chronic stress. Habituation of the corticosterone response was also less in the groups with greater predictability. However, predictability did not affect plasma prolactin or epinephrine responses. Because the endocrine systems responded differently, it is unlikely that the changes were due to a unitary process. Greater predictability appeared to be more stressful in this paradigm. Both associative and nonassociative factors have major roles in determining the hormonal responses to repeated presentation of stressors.

An assessment of prolactin's value as an index of stress

On an every other day basis, chronically catheterized male rats were subjected to a 30 sec grid shock in either an ascending (0.0, 0.25, 1.0, 4.0 mA) or descending order. A third group was repeatedly subjected to 1.0 mA shock over the same time frame. In 85% of the shock trials, plasma prolactin increased from baseline levels, thus indicating that prolactin is a relatively reliable index of stress. However prolactin did not change in a step-wise fashion with stressor intensity for a significant number of rats. Data from the group given repeated exposure to the 1 mA stressor showed no evidence of habituation--a process which might have explained the findings. This study indicates that prolactin levels do not sensitively track stressor intensity for individual rats.

Methodological problems in the study of classical aversive conditioning of adrenocortical responses

The present studies were conducted to demonstrate classical aversive conditioning of a corticosterone stress response in male rats. In the first experiment animals exposed to an odor which had previously preceded tube restraint stress had significantly higher plasma corticosterone levels than animals which were probed with a novel odor, or animals in which stress was followed, rather than preceded, by the odor. Careful consideration of corticosterone responses obtained on different days during training made it impossible to conclude unequivocally that learning had occurred. A second experiment was designed specifically to deal with the problems raised by the first, but we were again unable to show rigorously that learning had taken place. However, with data and paradigms comparable to our own, other researchers have prematurely concluded that aversive visceral conditioning can occur. It is suggested that the methodological considerations raised in the present studies could also be important for interpreting other experiments. Given the classical importance ascribed to learning components in many psychosomatic pathologies, future experiments are necessary to determine if stress responses can be classically conditioned, but they must be more rigorously designed than in the past.

A methodological improvement for experimental control and blood sampling in rats

We have developed an apparatus and techniques that permit repeated, undisturbed blood sampling and concurrent assessment of activity in rats. The apparatus also allows experimental control of conditioning stimuli and contingencies without the need to transfer the rat to a separate chamber. In the experimental apparatus, body weights remain stable at approximately 90% of the original body weight. Plasma corticosterone and prolactin levels in the apparatus are comparable to levels obtained in undisturbed free-ranging rats and remain at basal levels for the 7 day duration of this experiment. Additionally, circadian activity rhythms in the apparatus are similar to those seen in free-ranging rats. This technique has potential applications for investigators who need to be able to control shock delivery, monitor activity, and sample blood without disturbing rats.

Classical aversive conditioning of catecholamine and corticosterone responses.

Some earlier work, not rigorously controlled, suggested that conditional increases in sympathoadrenal stress hormones could occur. The purpose of this experiment was to test this idea further using an appropriately controlled design. To do this, we subjected rats living in a tether-type apparatus to differential fear conditioning. Chronic catheterization allowed us to sample blood before and after conditional stimulus probes without having to touch the rats. No evidence for conditional changes in norepinephrine and corticosterone was found. In contrast, differential conditioning of epinephrine responses was found. The conditional response, however, was not a simple one in that conditional increases in epinephrine following CS+ probes were not always seen. These data support the idea that learned changes in hormonal stress respondents can occur. But they leave open the question of why clear cut conditional changes in these visceral systems are so difficult to obtain.