Neuroendocrine correlates of sustained stress: The activity-stress paradigm

Brain Research Bulletin, Vol. 20, pp. 407-414. Pergamon Press plc, 1988.Printed in the U.S.A.

0361-9230/88$3.00 + 80

Neuroendocrine Correlates of Sustained Stress: The Activity-Stress Paradigm G. JEAN KANT, SALLY M. ANDERSON, GURPREET S. DHILLON’ AND EDWARD H. MOUGEY Department

of Medical Neurosciences, Washington, Received

Walter Reed Army Institute of Research DC 20307-51002*3

8 September 1987

G. S. DHILLON AND E. H. MOUGEY. Neuroendocrine correlates of sustained BRAIN RES BULL 20(3) 407-414, 1988.-Rats sacrificed after 4 days in the activity-stress paradigm or after 4 days of food restriction had significantly elevated levels of plasma corticosterone as compared to control rats. The approximately 5 fold increase in corticosterone in the stressed treatment groups was consistently found in all experiments. ACTH levels were elevated in activity-stress and food-restricted groups in some experiments but these increases were not statistically significant. Prolactin levels were significantly elevated in foodrestricted group rats as compared to controls or activity-stress group animals in one experiment but this finding was not repeated in further experiments. In a second series of experiments, rats from activity-stressed and food-restricted treatment groups and controls were exposed to an acute stressor for 1.5 min prior to sacrifice to assess the effects of prior sustained stress on hormonal responses to an acute stressor. Exposure to 15 min of immobilization or intermittent footshock immediately prior to sacrifice increased plasma levels of corticosterone, ACTH and prolactin in control, foodrestricted and activity-stressed rats. Generally, hormonal responses to the acute stress were similar in all treatment groups. However, in two experiments where the resting levels of corticosterone were especially elevated in the activity-stress group, the acute stress-induced rise in corticosterone was less than that seen for the other two treatment groups. In another experiment, administration of dexamethasone suppressed acute stress-evoked levels of ACTH and corticosterone in control, activity-stressed and food-restricted rats. Thus, rats exposed to 4 days of sustained stress were found to have consistently elevated resting levels of corticosterone. Hormonal responses to acute stress or dexamethasone administration, however, were generally similar in activity-stressed, food-restricted and control rats. KANT, G. J., S. M. ANDERSON,

stress:

The activity-stress

Activity-stress

paradigm

paradigm.

Corticosterone

Frolactin

ACTH

stress is thought to be a significant factor in the development of various diseases including hypertension, gastrointestinal disorders and depression [l, 3, 4, 8, 12, 13, 24, 32, 381. Although animal models of chronic stress would be useful in determining the factors involved in the development of these diseases and in the design of disease treatment or prevention strategies, relatively few well-characterized animal models are available for the study of chronic stress. Most animal models of stress are either acute models where stressors of short duration are utilized or repeated stress models in which animals are exposed to acute stressors for a short period each day over a period of days [5, 7, 14-16, 18-20, 22, 27, 281. True chronic or sustained stress models where exposure to stressors is relatively continuous include intermittent footshock, food restriction or deprivation, cold exposure and the activity-stress paradigm [2, 17, 25, 29, 30, 33-35, 371. In the activity-stress paradigm, rats allowed unlimited 24

Dexamethasone

hr per day access to a running wheel and suddenly restricted to 1 hr per day feeding, increase running and gradually decrease food intake until death ensues. This model has been reported to produce gastric ulceration and also to increase brain turnover of norepinephrine, two commonly accepted indices of stress [lo, 11, 25, 29-31, 361. Our laboratory has been studying the effects of repeated or sustained stress on neuroendocrine function [2, 14, 15, 171. We have found that rats exposed around-the-clock to an intermittent footshock escape/avoidance paradigm have elevated levels of plasma corticosterone for at least the first seven days in this environment, but that their levels of plasma prolactin and ACTH are similar to those of controls after 24 hr of intermittent footshock [ 171. The objectives of the present series of experiments were first to determine whether similar neuroendocrine effects were observed in a different type of continuously stressful environment, and, second to examine neuroendocrine re-

CHRONIC

This work was done while G. S. Dhillon held a National Research Council-WRAIR Associateship. The views of the author(s) do not purport to reflect the position of the Department of the Army or the Department of Defense, (para 4-3, AR 360-5). Research was conducted in compliance with the Animal Welfare Act, and other Federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guidefor the Care and Use ofLaboratory Animals, NIH publication 85-23. All procedures were reviewed and approved by the WRAIR Animal Use Review Committee.

407

408

KANT ET AL.

sponses to pharmacological and physiological challenge in the stressed animals. The stress model selected was the activity-stress paradigm.

4000 1

METHOD

Animals

5

Male Sprague Dawley rats (2755.50 grams; approx. 8 weeks of age) were used in these experiments. Rats were purchased from Zivic-Miller and maintained in individual hanging cages prior to the experiment. Food and water were freely available. Lights were on from 0600 to 1800 hr. Rats were housed under these conditions for at least two weeks prior to the experiment. Groups of weight matched rats were then assigned to control, food-restricted or activity-stress groups.

Y 0

3000 -

3

2000 -

2 ti g

lOOO-

LI General Experimental

-9

Procedures

Activity-stress group rats were transferred to individual metal grid cages attached to activity wheels for five days prior to food restriction. Rats had free access to the running wheel via an open door between the wheel and grid cage. Wheel revolutions were recorded daily. Rats in the control and food-restricted groups continued to live in their original home cages. Following this habituation period to the wheel during which all rats had free access to food, food was withdrawn from both the activity-stress and food-restricted groups and subsequently made available only for one hour per day between 1400 and 1500 hours. Rats were not individually matched for food intake between activity-stressed and food-restricted groups. Rats in each group were simply allowed 1 hr food availability. The average food intake was similar; however, between the two groups (see data below). Wheel revolutions, food consumption and body weight were recorded daily. Activity-Stress and Plasma Corticosterone, Prolactin

ACTH and

In the first series of experiments, four similar studies were performed with six activity-stress, six food-restricted, and six control animals run in each study. Rats from all three groups were sacrificed by decapitation between 0800 and 1100 hr on the morning corresponding to 4 days of foodrestriction. Rats were sacrificed immediately upon removal from their home cage (~1 min) to avoid handling-induced increases in plasma hormones. Following decapitation, trunk blood was collected and processed as described below. Plasma was stored and later assayed for ACTH, corticosterone and prolactin by radioimmunoassay.

I -8

I -7

I1 -6

-5

OAYSPRECEDING

I, -4

-3

I -2

I, -1

0

SACRIFICE

FIG. I. Effects of food restriction on running wheel activity. Food was suddenly restricted to 1hr per day availability between 1400 and 1500 hours, 4 days prior to sacrifice, as indicated by the arrow. Values represent the mean of 24 rats ?SEM. with a 30 set average

intershock

interval,

i.e., approximately

30 shocks of 5 set each delivered in the 15 min session). Stressed rats were sacrificed immediately following the acute stress exposure. Response of Plasma Corticosterone Challenge

to Dexamethasone

Since both activity-stress and food-restricted groups demonstrated similar hormonal levels in the preceding experiments and since the number of activity wheels was insufficient to provide an adequate number of subjects for the design of the experiment to be described, only food-restricted (Cday) and control groups were used in this experiment. Sixteen rats from the control group and 16 rats from the food-restricted group were injected with 0.4 mg/kg dexamethasone twenty-four hours prior to sacrifice (3 days into the activity-stress paradigm) and 16 rats from each group were injected with vehicle 24 hr prior to sacrifice. Two hours prior to sacrifice, rats received a second injection of dexamethasone (0.2 mg/kg) or vehicle. Half of the rats from each treatment group (e.g., food-restricted-dexamethasoneinjected) were sacrificed immediately upon removal from their home cage and the other eight animals from the group were sacrificed immediately following 15 min of intermittent footshock as described above. Blood Collection and Assay Procedures

Response

of Plasma Hormones

to Stress Challenge

In another series of experiments, twelve rats were assigned to each of control, food-restricted and activity-stress groups. Following four days of activity-stress and/or food restriction, six rats from each treatment group were sacrificed by decapitation and six rats from each group were individually challenged with an acute stressor for 15 min and then immediately sacrificed by decapitation. In one experiment, the acute stressor was immobilization in a plastic cylinder (5.7 cm dia.). In two other separate experiments, the acute stressor was exposure to 15 min of intermittent footshock (1.6 mA, 5 set duration, variable interval schedule

Rats were sacrificed using a guillotine and trunk blood was collected in a heparinized beaker. Trasylol was added to inhibit peptidases. Following centrifugation, the plasma was stored at -40°C until assayed for plasma hormones. Corticosterone was measured by radioimmunoassay using an antibody produced in our laboratory against corticosterone-21-hemisuccinate:BSA [26]. Somogyi reagents were used to separate free from bound ligand. Corticosterone[ 1,2-H(N), specific activity 50 Ci/mmol, New England Nuclear] was the labelled ligand. Assay sensitivity was 0.6 pg%. The intraassay and interassay coefficients of variance were 6% and 12% respectively.

ACTIVITY-STRESS

409

AND HORMONES TABLE

1

EFFECTS OF ACTIVITY-STRESS OR FOOD-RESTRICTION AND BODY WEIGHT

Food Intake (grams/day) Body Weight (g) (+- during 4 days)

ON FOOD INTAKE

Controls

FoodRestricted

ActivityStress

28.0 ” 0.8

10.8 f 0.6*

10.4 ? 0.6*

+14.3 * 3.9

-20.5 f 3.9*

-24.6 f 2.8*

Values represent the mean f SEM. N=24. Food intake and body weight changes for the 4 days immediately prior to sacrifice, i.e., the food-restricted days for the experimental groups. *Significantly different from control, ~~0.05, Student’s r-test (two-tailed).

TABLE 2 EFFECTS OF ACTIVITY-STRESS OR FOOD-RESTRICTION ON PLASMA ACTH, CORTICOSTERONE AND PROLACTIN

ACTH (Pg/ml) Corticosterone (~$100 ml) Prolactin (@ml)

Controls

FoodRestricted

ActivityStress

28.2 rf: 5.0

41.2 2 5.9

41.3 ? 6.7

2.7 -t 0.5

11.8 -+ 1.8*

13.1 f 2.0*

8.6 2 1.0

18.8 + 4.6*

8.6 t 1.5

Values represent the mean + SEM following sacrifice after 4 days of food restriction for the experimental groups. N=24. Analysis of variance results: ACTH (F=l.6, p>O.OS); corticosterone (F=ll.l, p
Materials for the prolactin assay were provided by the National Institute of Health through the Rat Pituitary Hormone Distribution Program. Prolactin was radioiodinated as previously described [22]. Within assay variation was <8% and between assay variation <12%. ACTH was measured using a radioimmunoassay kit (Immuno Nuclear Cot-p). The antiserum supplied with the kit was prepared against the 14-24 ACTH sequence common to most mammalian species including rat and human ACTH. This antiserum has been shown to cross-react 100% with porcine ACTHI-SI) and human1-e4 and is assumed to react 100% with rat ACTH which shares the human 1-24sequence. Human ACTH was used as a standard in this assay since rat ACTH is not commercially available. ACTH values reported are thus human ACTH equivalents. The assay was performed in 12x75 polypropylene tubes using an overnight incubation at 4°C. Assay sensitivity was approximately 10 pg/ml. The intraassay coefficient of variation was 2S%at 380 pg/ml and the interassay coefficient of variation was <5%. Statistics

All data were analyzed using analysis of variance (ANOVA) procedures for main effects of treatment group (control, food-restricted or activity-stress) and, where appropriate, main effects of acute stress or drug administration and the interactions among these main effects. Following

findings of a significant F score, preselected followup comparisons were made using Student’s t-test (two-tailed).

RESULTS

Effect of Activity-Stress and Prolactin

on Plasma

Corticosterone,

ACTH

In the fist series of experiments, four similar studies were performed with six activity-stress, six food-restricted and six control animals run in each of the four studies. Animals were sacrificed following 4 days of food restriction or activity-stress. Data from all four studies were combined and are shown in Fig. 1 and Tables 1 and 2. As shown in Fig. 1, rats in the activity-stress group increased running when food restriction was imposed. Rats consumed less food and lost weight in the food-restricted and activity-stress groups as compared to home-caged controls (Table 1). Levels of plasma corticosterone were markedly elevated in both the food-restricted and the activity-stress groups as compared to controls but were not different from each other (Table 2). Plasma ACTH levels were higher in both stress groups as compared to controls but this difference was not statistically significant. Plasma prolactin levels were significantly higher in the food-restricted group as compared to controls. However, this finding was not replicated in further experiments (see below).

KANT ET AL.

410

TABLE

3

EFFECTS OF ACUTE NOVEL STRESS ON PLASMA CORTICOSTERONE

(~g/lOOml)

Treatment Group Acute Stress

Controls

Food-Restricted

Activity-Stress

Experiment 1 No Stress Immobilization

1.9 -c 0.3 15.8 ? 3.7t

13.6 + 0.7* 25.6 t 1.4t

16.5 2 2.8* 22.3 t 4.5

Experiment 2 No Stress Footshock

1.8 + 0.3 24.5 2 2.2t

11.3 t 2.9* 31.7 2 1.2t

17.5 t 4.2* 33.2 t 2.lt

Experiment 3 No Stress Footshock

3.7 + 1.7 12.8 it 1.7t

8.8 5 2.2* 24.4 2 2.8t

16 + 4.0* 22.7 + 2.9

Values represent the mean + SEM. N=5 or 6. Overall F score and probability for (a) effects of treatment group: experiment 1 (F=lO, pO.O5); experiment 2 (F=0.99,p>O.O5); and experiment 3 (F=1.5, p>O.OS). *Significantly different from control group, p-cO.05, Student’s f-test (two-tailed). Wigniticantly different from treatment-matched no acute stress group, p ~0.05, Student’s t-test (two-tailed). TABLE 4 EFFECTS OF ACUTE NOVEL STRESS ON PLASMA ACTH

(pg/ml)

Treatment Group Acute Stress

Controls

Food-Restricted

Activity-Stress

Experiment 1 No Stress Immobilization

25.7 2 8.2 230 ‘- 63*

54.2 ? 10.6 186 * 53*

77.5 ir 13.9 132 2 67

Experiment 2 No Stress Footshock

47.8 _t 22.7 414 _f 82*

35.1 ? 7.0 437 -+ 38*

49.2 t 10.3 345 2 76*

Experiment 3 No Stress Footshock

6.3 + 4.3 296 -+ 44*

10.4 _’ 5.2 250 t 43*

60.3 2 39.8 247 t 74*

Values represent the mean t SEM. N=5 or 6. Overall F score and probability for (a) effects of treatment group: experiment 1 (F=0.14, p>O.O5); experiment 2 (F=0.36, p>O.OS); experiment 3 (F=0.18, p>O.O5); (b) effects of acute stress: experiment 1 (F=11.6, pcO.01); experiment 2 (F=77, pO.OS); experiment 2 (F=0.59,p>0.05); and experiment 3 (F=0.73, p>O.OS). *Significantly different from treatment-matched no acute stress group, pcO.05, Student’s t-test (two-tailed).

Response

of Plasma Hormones

to Stress Challenge

These experiments replicated and extended the studies described above. Some rats in each of the following three experiments were sacrificed after 4 days of sustained stress exactly as in the above studies. However, other rats in each treatment group were exposed to 15 min of acute stress immediately prior to sacrifice in order to assess the responsiv-

ity of continuously stressed animals to an acute stress challenge. In the non-acutely stressed animals in each of the three experiments, plasma corticosterone levels were higher in both the food-restricted and activity-stress groups as compared to controls (Table 3), as seen in the first studies (above). Corticosterone levels were higher in the activity-

ACTIVITY-STRESS

AND HORMONES

411

TABLE 5 EFFECTS OF ACUTE NOVEL STRESS ON PLASMA PROLACTIN (n&l) Treatment Group Controls

Acute Stress

Food-Restricted

Activity-Stress

Experiment 1 No Stress Immobilization

4.7 +- 1.2 21.9 ? 5.9*

4.1 + 1.2 37.6 f 13.6*

2.5 If: 0.7 16.9 2 6.7

Experiment 2 No Stress Footshock

4.4 f 0.9 46.0 ” 8.5*

8.1 f 2.9 57.4 f 11.2*

3.4 f 0.6 38.6 f 7.9*

Experiment 3 No Stress Footshock

3.0 2 0.5 43.0 ? 6.3*

4.8 + 47.2 2

4.8 + 2.0 34.6 2 7.3*

1.2 6.7*

Values represent the mean f SEM. N=5 or 6. Overall F score and probability for (a) effects of treatment group: experiment 1 (F= 1.5, p>O.O5); experiment 2 (F=1.4, ~~0.05); experiment 3 (F=0.84, p>O.O5); (b) effects of acute stress: experiment 1 (F=16.6, pO.OS); experiment 2 (F=0.5l,p>0.05); and experiment 3 (F=0.93, p>O.OS). *Significantly different from treatment-matched no acute stress group, ~~0.05, Student’s t-test (two-tailed).

stress than in the food-restricted rats in this series of experiments, but this difference was not statistically significant. Either acute immobilization or footshock elevated levels of plasma corticosterone, ACTH and prolactin (Tables 3-S)

in treatment control, activity-stress, and food-restricted rats. The hormonal responses to acute stress were generally similar in all groups. However, plasma corticosterone responses to acute stress in experiments 1 and 3 in the activity-stress group (Table 3) were small compared to control or foodrestricted group responses. This attenuated corticosterone response to stress may be due to the particularly high resting corticosterone level, since corticosterone response to stress is not linear over a broad range [20]. Response of Plasma Corticosterone Challenge

to Dexamethasone

This experiment was similar to some aspects of the above experiments but differed in that all animals were injected with either vehicle or dexamethasone. Injections were given 24 hr and 2 hr prior to sacrifice. Only food-restricted and control groups were tested in this experiment. Resting (nonacutely-stressed) levels of corticosterone and ACTH in vehicle-injected animals were higher in the food-restricted as compared to treatment control rats (Table 6). Acute stress increased levels of corticosterone and ACTH in both treatment controls and food-restricted groups. Dexamethasone pretreatment lowered both resting and acute stress-evoked levels of plasma corticosterone and ACTH in rats in the food-restricted group compared to vehicle-pretreated food-restricted rats (Table 6). Dexamethasone also lowered corticosterone and ACTH levels in the acutely-stressed treatment control group as compared to vehicle-injected treatment controls. However, while dexamethasone also decreased resting levels of ACTH and corticosterone in the non-acutely stressed control group, this decrease was not statistically significant possibly because

the vehicle-pretreated, non-acutely stressed values in the controls were already very low. As shown in Table 7, dexamethasone administration had no effect on resting or acute-stress evoked levels of plasma prolactin. In this experiment, resting levels of prolactin were similar in control and food-restricted groups. Acute stress increased levels of prolactin in both control and foodrestricted rats to a similar level. DISCUSSION

Our laboratory is interested in utilizing animal models of chronic stress for the purpose of studying neuroendocrine and neurochemical adaption and maladaption to stressful environments. We have previously studied the effects of around-the-clock intermittent footshock on escapelavoidance performance, circadian patterning of lever pressing for food, body weight, organ weights and levels of plasma ACTH, corticosterone and prolactin [17,23]. Animals were sacrificed after 1, 2, 3, 4, 7, or 14 days in this environment. Weight gain was decreased, lever pressing for food was decreased and levels of plasma corticosterone were increased in stressed as compared to control animals. Levels of ACTH and prolactin were similar in controls and stressed animals at all time points measured, suggesting a very rapid habituation of these hormones to the stressful environment since acute stress markedly elevates these hormones [19]. The present experiments were performed to compare the effects of a different type of sustained stressor with those from the previous studies of intermittent footshock. Two stress models were examined, i.e., food restriction and activity-stress (which includes food restriction). The foodrestricted group was included as a type of control group, since food-restricted rats have been reported to not develop ulcers or die as do animals placed into the activity-stress paradigm. Thus, the food-restricted group is possibly less physiologically stressed than the activity-stress group. In

KANT ET AL.

412

TABLE 6 DEXAMETHASONE

SUPPRESSION

OF PLASMA

CORTICOSTERONE

Corticosterone

AND ACTH

ACTH (pg/mU

(~Lg/100ml) Treatment Group Acute Stress

Controls

Food-Rest.

Controls

Food-Rest.

Vehicle No Stress Stress

7.0 t 3.9 19.6 + 2.5

15.7 2 2.1 31.7 k 2.0

7.2 2 4.3 244 2 41

34.2 f 7.3 190 + 37

Dexamethasone No Stress Stress

2.2 k 0.7 7.7 + 2.7*

3.1 + 0.8* 11.0 + 2.1*

5.5 c 3.0 21.1 + 11.3*

6.8 + 3.2* 30.1 _t 12.6*

Values represent the mean t SEM. N=8. Three way analysis of variance for main effects of treatment group, drug, and acute stress found significant effects of treatment on corticosterone (F=14.3, p
TABLE 7 both the activity-stress model and the food-restricted model, we again find elevated levels of plasma corticosterone in stressed rats as compared to controls. While there was a tendency for ACTH levels to be higher in the two stress groups as compared to controls, this increase was not statistically significant. Since ACTH regulates corticosterone release, it may appear surprising that no significant change in ACTH levels was observed concurrent with the large increases in corticosterone. However, this is not an uncommon finding. For example, large circadian variations in corticosterone are not accompanied by significant ACTH changes [19, 37, 391. One explanation for this phenomenon is that small changes in ACTH (not always statistically significant, given the magnitude of the change and the variability of ACTH levels) appear to be sufficient to cause large changes in corticosterone release. The released corticosterone rapidly feeds back to shut off further ACTH release, limiting the range of ACTH levels. In one set of experiments, prolactin levels were elevated in the food-restricted group. The increase in prolactin was not seen in the other stressed group (activity-stress) within the same experiment or in food-restricted groups in other experiments. Thus, the importance of this isolated finding is not clear at present. Immobilization or footshock markedly elevated prolactin levels in plasma. However, acute stressinduced levels of prolactin were similar in controls, foodrestricted and activity-stressed groups. Since corticosterone feeds back and negatively affects secretion of ACTH, we had hypothesized that the elevated levels of corticosterone seen in the food-restricted and activity-stress groups might inhibit the ACTH and/or corticosterone responses to a novel stress challenge. However, the ACTH responses to immobilization or footshock shown in Tables 4 and 6 were similar in controls and continuously stressed groups. In two of three experiments (shown in Table 3), the corticosterone response to a 15 min stressor was

DEXAMETHASONE

AND PLASMA

PROLACTIN

Prolactin (&ml) Treatment Group Acute Stress

Controls

“chicle No Stress Stress

3.3 t 0.5 25.2 + 4.7

2.6 + 0.4 22.7 k 4.4

Dexamethasone No Stress Stress

1.5 k 0.1 25.1 % 8.8

2.1 + 0.4 28.6 + 6.4

Food-Restricted

Values represent the mean _f SEM. N=8. Three way analysis of variance found significant effects of acute stress (F=53, p
smaller in the activity-stressed group as compared to control groups. This attenuated response is most likely a reflection of the lack of proportional corticosterone response to graded increases in stress [20]. Corticosterone response is curvilinear and falls off fairly rapidly. Thus, less additional corticosterone response may be evoked when the resting level is substantially elevated as is the case for the activity-stressed animals in those experiments. It has been reported that some depressed patients exhibit elevated levels of cortisol and that some patients do not suppress cortisol levels following administration of the synthetic glucocorticoid dexamethasone [9,21]. Cortisol levels have also been reported to be elevated in patients diagnosed with anorexia nervosa and non-suppression of cortisol levels following dexamethasone is also found in these patients [6]. In the present study, stress did cause elevations in levels of or food-restricted

ACTIVITY-STRESS

AND HORMONES

413

corticosterone. Food restriction alone was sufficient to elevate corticosterone levels, which lends support to the suggestion by Fichter et al. that the endocrine dysfunctions observed in anorexia nervosa are mainly due to nutritional abnormalities and can be reversed following feeding and weight gain. Although non-suppression of cortisol by dexamethasone has been found in both the above patient groups, dexamethasone was effective in the stressed animals in the present study. This may indicate that stress of 4 days duration is insufficient to cause dexamethasone non-suppression. Three rat models of sustained stress (intermittent footshock, food restriction and activity-stress) have now been shown to elevate plasma corticosterone levels during the first 4 to 7 days of stress, while plasma prolactin and ACTH levels have been shown to be unchanged by these paradigms. However, none of these models has yet been shown to maintain elevated corticosterone levels over a period of several

weeks to months as may be the case for humans suffering from depression or anorexia nervosa. Corticosterone levels return to control levels after two weeks in the intermittent footshock model and rats were only tested in the activitystress paradigm in the present study for 4 days to avoid the severe debilitation (leading to death) that has been reported in rats exposed to this paradigm for longer periods. Animal paradigms incorporating longer periods of chronic stress are necessary to model the effects of chronic stress on neuroendocrine function in humans.

ACKNOWLEDGEMENTS

The authors wish to thank Clyde Kenion, Golden Driver, Hugh Jarrard, Angela Brown, SGT Geraldine Davis, SGT Terence Eggleston, Paul D’Angelo and Willie Gamble for excellent technical assistance and Brenda Messer and Karen Daniels for secretarial support.

REFERENCES 1. Ader, R. Psychoneuroimmunology. New York: Academic Press, 1981. 2. Anderson, S. M., J. R. Leu and G. J. Kant. Effect of stress on [Hlcyclohexyladenosine binding to rat brain membranes. Pharmacol Biochem Behav 26: 829-833, 1987. 3. Anisman, H. and J. M. Zacharko. Depression: The predisposing intluence of stress. Behav Brain Sci 5: 89-137, 1982. 4. Brady, J. V., R. W. Porter, D. J. Conrad and J. W. Mason.

Avoidance behavior and the development of gastroduodenal ulcers. J Exp Analy Behav 1: 69-72, i958. _ 5. Du Ruisseau. P.. Y. Tache. P. Brazeau and R. Collu. Pattern of adenohypophyseal hormone changes induced by various stressors in female and male rats. Neuroendocrinology 27: 257-271, 1978. 6. Fichter, M. M., P. Doerr, K. M. Pirke and R. Lund. Behavior, attitude, nutrition and endocrinology in anorexia nervosa. Acta Psychiatr &and 66: 429-444, 1982. 7. Friedman, S. B., R. Ader, L. J. Grotta and T. Larson. Plasma

corticosterone response to parameters of electric shock stimulation in the rat. Psychosom Med 29: 323-328, 1967. 8. Galosv. R. A.. L. K. Clarke. M. R. Vasko and I. L. Crawford. Neurophysiology and neuropharmacology of cardiovascular regulation and stress. Neurosci Biobehav Rev 5: 137-175, 1981. 9. Gold, P. W., L. Loriaux, A. Roy, M. A. Kling, J. R. Calabrese, C. H. Kellner. L. K. Nieman, R. M. Post, D. Pickar, W. Gallucci, P. Avgerinos, S. Paul, E. H. Oldtield, G. B. Cutler and G. P. Chrousos. Responses to corticotropin-releasing hormone in the hypercortisolism of depression and Cushings disease. N Engl J Med 314: 1329-1334, 1986. 10. Hellhammer, D. H., J. Hingtgen, S. Wade, P. Shea and M. Aprison. Serotonergic changes in specific areas of rat brain associated with activity-stress gastric lesions. Psychosom Med 45: 115-122, 1983. 11. Hellhammer, D. H., M. A. Rea, M. Bell and L. Belkien. Activity wheel stress: Effects on brain monoamines and the pituitary-gonadal axis. Neuropsychobiology 11:251-254, 1984. 12. Henry, J. P., J. P. Meehan and P. M. Stephens. The use of psychosocial stimuli to induce prolonged systolic hypertension in mice. Psychosom Med 29: 4OU32, 1967. 13. Herd, J. A., W. H. Morse, R. T. Kelleher and L. G. Jones. Arterial hypertension in the squirrel monkey during behavioral experiments. Am J Physiol217: 24-29, 1969. 14. Kant, G. J., B. N. Bunnell, E. H. Mougey, L. L. Pennington and J. L. Meyerhoff. Effects of repeated stress on pituitary cyclic AMP, and plasma prolactin, corticosterone and growth hormone in male rats. Pharmacol Biochem Behav 18: 967971, 1983.

15. Kant, G. J., T. Eggleston, L. Landman-Roberts, C. C. Kenion, G. C. Driver and J. L. Meyerhoff. Habituation to repeated stress is stressor specific. Pharmacol Biochem Behav 22: 631634, 1985. 16. Kant, G. J., R. H. Lenox, B. N. Bunnell, E. H. Mougey, L. L. Pennington and J. L. Meverhoff. Comnarison of stress response in male and female rats: pituitary cyclic AMP and piasma prolactin, growth hormone and corticosterone. Psychoneuroendocrinology

8: 421-428,

1983.

17. Kant, G. J., J. R. Leu, S. M. Anderson and E. H. Mougey Effects of chronic stress on plasma corticosterone, ACTH and prolactin. Physiol Behav 40: 775-779, 1987. 18. Kant, G. J., J. L. Meyerhoff, B. N. Bunnell and R. H. Lenox. Cyclic AMP and cyclic GMP response to stress in brain and pituitary: Stress elevates pituitary cyclic AMP. Pharmacol Biochem Behav 17: 1067-1072, 1982. 19. Kant, G. J., E. H. Mougey and J. L. Meyerhoff. Diurnal variation in neuroendocrine response to stress in rats: Plasma ACTH, P-endorphin, /3-LPH, corticosterone and prolactin and pituitary cyclic AMP responses. Neuroendocrinology 43: 38s 390, 1986.

20. Kant, G. J., E. H. Mougey, L. L. Pennington and J. L. Meyerhoff. Graded footshock stress elevates pituitary cyclic AMP and plasma /3-endorphin, P-LPH, corticosterone and prolactin. Life Sci 33: 2657-2663, 1983. 21. Lamberts, S. W. J. Neuro-endocrine aspects of the dexamethasone suppression test in psychiatry. Life Sci 39: 91-95, 1986. 77 Lenox, R. H., G. J. Kant, G. R. Sessions, L. L. Pennington, E. H. Mougev and J. L. Meverhoff. Snecific hormonal and neurochemical responses to different stressors. Neuroendocrinology A&.

30: 300-308, 1980. 23. Leu, J. R. and G. J. Kant. Effects of chronic stress on physiol-

ogy and behavior: eating, drinking, avoidance/escape performance and organ weights. Submitted. 24. Maier, S. F. and M. E. P. Seligman. Learned helplessness: theory and evidence. J Exp Psycho1 [Gen] 105: 3-46, 1976. 25. Manning, F. J., G. W. Henry, C. A. Montgomery, Jr., C. 0. Simmons and G. R. Sessions. Microscopic examination of the activity-stress ulcer in the rat. Physiol Behav 21: 269-274, 1978. 26. Mougey, E. H. A radioimmunoassay for tetrahydrocortisol. Anal Biochem 91: 566-582, 1978. 27. Mueller, G. P. Beta-endorphin immunoreactivity

in rat plasma: variations in response to different physical stimuli. Life Sci 29:

1669-1674, 1981. 28. Natelson, B. H., W. N. Tapp, J. E. Adamus, J. C. Mittler and B. E. Levin. Humoral indices of stress in rats. Physiol Behav 26: 1049-1054. 1981.

414

KANT ET AL.

29. Pare, W. P. The influence

35. Tache, Y., P. Du Ruisseau, J. R. Ducharme and R. Collu. Pat-

30.

tern of adenohypophyseal changes in male rats following chronic stress. Neuroendocrinology 26: 201219, 1978. 36. Tsuda, A., M. Tanaka, Y. Kohno, Y. Ida, Y. Hoaki, K. Iimori, R. Nakagawa, T. Nishikawa and N. Nagasaki. Daily increase in noradrenaline turnover in brain regions of activity-stress rats.

31.

32. 33.

of food consumption and running activity on the activity-stress ulcer in a rat. Dig Dis 20: 262273, 1975. Pare, W. P. Organ weights in rats with activity-stress ulcers. Bull Psychon Sot 9: 11-13, 1977. Rea, M. A. and D. H. Hellhammer. Activity wheel stress: Changes in brain norepinephrine turnover and the occurrence of gastric lesions. Psychother Psychosom 42: 218-223, 1984. Selye, H. Stress in Health and Disease. Boston: Butterworths, 1976. Stone, E. A. Reduction in cortical beta adrenergic receptor density after chronic intermittent food deprivation. Neurosci

Lett 40: 33-37, 1983. 34. Tache, Y., P. Du Ruisseau,

J. Tache, H. Selye and R. Collu. Shift in adenohypophyseal activity during chronic intermittent immobilization of rats. Neuroendocrinology 22: 325-336, 1976.

Pharmacol Biochem Behav 19: 393-396, 1983. 37. Vemikos, J., M. F. Dallman, C. Bonner, A. Katzen

and J. Shin&o. Pituitary-adrenal function in rats chronically exposed to cold. Endocrinology 110: 413-420, 1982. 38. Weiss, J. M., P. A. Goodman, B. J. Losito, S. Corrigan, J. M. Charry and W. H. Bailey. Behavioral depression produced by an uncontrollable stressor: relationship to norepinephrine, dopamine and serotonin levels in various regions of rat brain. Brain Res Rev 3: 167-205, 1981. 39. Wilkinson, C. W., J. Shin&o and M. F. Dallman. Return of

pituitary-adrenal function after adrenal enucleation or transplantation: diurnal rhythms and responses to ether. Endocrinology 109: 162-169, 1981.