- Email: [email protected]
Chronic stress alters pituitary-adrenal function in prepubertal male rats
0306-4530/87 $3.00 + 0.00 PergamonJournalsLtd.
Psychoneuroendocrinology,Vol. 12, No. 5, pp. 393-398, 1987. Printed in Great Britain.
CHRONIC STRESS ALTERS P I T U I T A R Y - A D R E N A L FUNCTION IN PREPUBERTAL MALE RATS C. RESTREPO and A. ARMARIO* Departamento de Biologia Celular y Fisiologfa, Facultad de Ciencias, Universidad Aut6noma de Barcelona, Bellaterra, Spain
(Received 18 August 1986; in final form 9 March 1987) SUMMARY The effect of exposure to chronic stress on the pituitary-adrenal axis was studied in prepubertal rats. The chronic stress consisted of exposing the animals 6 days a week for 32 days to one stressor randomly chosen among several each day, Chronic stress did not alter either food intake or body weight gain. However, it was stressful for the rats, as they had increased adrenal weights. Chronically stressed rats showed normal pituitary-adrenal basal activity and normal ACTH responses to a novel, acutely applied stressor. However, this treatment resulted in higher corticosterone response to an acute stressor, as a consequence of enhanced adrenocortical response to ACTH. In addition, chronic stress induced increasing sensitivity of the adrenal to the inhibitory action of dexamethasone. Exploratory activity and defecation rate in a novel environment were not affected by the chronic stress. All these data indicate that behavioral and endocrine changes induced by chronic stress were somewhat different from those previously found in adult rats. Prepubertal rats appear to be less sensitive than adult rats to the chronic stress we used. INTRODUCTION
previous chronic stress on pituitary-adrenal (PA) response to a novel acute stressor remains controversial. This is most likely due to: (a) the use of corticosteroids as a measure of adrenocorticotropin (ACTH) release, since it has been reported that chronic stress can alter adrenocortical responsiveness to ACTH (Riegle, 1973; Armario et al., 1985), and the adrenal cortex is not sensitive to high circulating ACTH levels because of maximal adrenocortical secretion; Co) the chronic stress model used; for instance, a chronic continuous stress such as cold increased PA response to a novel acute stressor (Vernikos et al., 1982), whereas chronic intermittent stresses did not alter or decrease it (Riegle, 1973; Armario et al., 1984a,b). We found that chronic intermittent stresses did not alter the ACTH response to novel acute stressors in adult male rats (Armario et al., 1984b, 1985), which suggests that a history of chronic exposure to stress does not increase emotional reactivity in adult animals. The post-weaning period appears to be critical for the normal development of the animals. In addition, it has been reported that the corticosterone response to acute stress was higher in prepubertal than in adult male rats (Goldman et al., 1973; Lescoat et al., 1978). Therefore, chronic stress could exert a more marked effect on emotional reactivity in the post-weaning period than in adulthood. However, no studies have been carried out on this subject. The present work investigates the influence of chronic stress on emotional reactivity in prepubertal rats as measured by behavioral response to a novel environment and PA activity. In order to reduce the possibility of adaptation, the animals were subjected every day to one stressor randomly chosen among several (Katz et al., 1981). T H E EFFECT o f
*To whom correspondence should be addressed. 393
394
C. RESTREPOand A. ARMAR10 MATERIALS AND METHODS
Animals and stress protocol Male Sprague-Dawley rats 22 - 2 4 days old were used. They were kept in a controlled environment (lights on from 0700 hr to 1900 hr, temperature 23°C). Food and water were always freely available. The animals were randomly allocated among control and chronic stress groups. Those rats assigned to the chronic stress groups were exposed, 6 days a week for 32 days, to one stressor randomly chosen each day among the following: intraperitoneal (i.p.) saline injection (twice a day), ether anesthesia, 4 hr of continuous noise caused by an alarm bell (85 db), and 15 min of forced swimming in a water tank (diameter = 40 cm, height = 60 cm, temperature = 18°C). The experimental period lasted for 32 days. The day after the last exposure of the chronically stressed rats, the animals from the two experimental groups were subjected to several acute manipulations (see below). Noise was the stressor applied to the chronically stressed animals the day before the animals were subjected to the acute treatments. We have found that the stress of noise by itself, applied to the rats the day before, altered neither the exploratory activity of the rats, basal levels of PA hormones, nor the PA response to an acute stressor (unpublished data).
Experiment 1 This was designed to study: (a) the effects of previous chronic stress on emotional reactivity as measured by PA activation caused by a novel acute stressor; (b) the influence of chronic stress on the behavioral response to a novel exploratory box. Two days before the end of the chronic treatment, 10 animals from each group (control and chronically stressed) were tested in a hole-board apparatus for 4 min. The hole-board is an exploratory box with four holes in the bottom (File & Wardill, 1975) which was situated in a room provided with the same illumination and background noise as the animal house. The number of boluses defecated, rears and head-dips were measured manually. Time spent headdipping was recorded using a stop-watch. A dip was considered to have taken place when the introduction of the head into the holes was at least to the level of the eyes. After each test the apparatus was cleaned. The day before the holeboard test, the stressor applied to the chronically stressed rats was noise, for the reasons explained above. Three days later, approximately 20 hr after the last stress sessions for the chronically stressed animals, the rats were subjected to one of two acute treatments. Some animals were killed by decapitation within 20 sec after being taken from the animal house. This procedure allowed measurement of the basal hormone levels. Others were subjected to restraint stress in plastic tubes (4.5 cm diameter, 18 cm length) for 10 or 30 min before being quickly killed. The adrenals were removed, trimmed of fat and weighed. Body weight gain and food intake were measured throughout all the experiments.
Experiment 2 The aim of this experiment was to study the effects of chronic stress on adrenocortical responsiveness to ACTH and the sensitivity of the PA axis to feedback inhibition by dexamethasone. On day 33 after the beginning of the experimental phase, the animals were injected i.p. with either saline or three doses of dexamethasone phosphate (3, 10 or 25 gg/100 g body wt). Two hours later, those injected with saline or 3 or 10 gg dexamethasone were subjected to restraint stress in the same tubes already describe. Those injected with 25 gg dexamethasone were s.c. injected with 0.5 IU/100 g body wt of porcine ACTH (Sigma). All the animals were killed 20 min after the onset of stress or ACTH administration.
Hormone assays The trunk blood was collected into plastic tubes and maintained in ice-cold water to prevent ACTH degradation. The blood was centrifuged at the same temperature, and the serum was frozen at - 2 0 ° C . Corticosterone and ACTH were analyzed by radioimmunoassay as previously described (Armario & Castellanos, 1984; Armario et al., 1984c). All samples to be statistically compared were processed within the same assay to avoid inter-assay variations. Intraassay coefficients of variation averaged 10% for ACTH and 5% for corticosterone.
Statistical analysis The results were analyzed with Student's t-test or with two-way analysis of variance (ANOVA). Data were log transformed before their statistical evaluation to achieve homogeneity of variance. RESULTS
Experiment 1 Chronic stress did not alter either body weight gain or food intake. However, (p <
a significant
0 . 0 1 ) i n c r e a s e in t h e r e l a t i v e a d r e n a l w e i g h t w a s o b s e r v e d in t h e c h r o n i c a l l y s t r e s s e d
c o m p a r e d to t h e c o n t r o l r a t s ( T a b l e I). F i g . 1 s h o w s t h e P A r e s p o n s e s to a n o v e l a c u t e s t r e s s o r in t h e c o n t r o l a n d c h r o n i c a l l y s t r e s s e d r a t s . A N O V A
r e v e a l e d a s i g n i f i c a n t (p < 0 . 0 0 1 ) e f f e c t
of acute stress, but not of previous chronic treatment, on serum ACTH.
For corticosterone a
395
PITUITARY-ADRENAL FUNCTION AND CHRONIC STRESS TABLE I. EFFECT OF CHRONIC STRESS ON SOME PHYSIOLOGICALVARIABLES
Group Control Chronic stress
Initial body weight (g)
Body weight gain (g)
Food intake (g/rat/day)
84.8 + 4.2 (11) 81.5 :t: 2.0 (11)
239.2 -4- 5.1 (11) 239.0 ± 6.8 (11)
23.2 :t: 0.5 (8) 23.5 ± 0.3 (8)
Adrenal weight (g/100 g/body wt) 11.9 ± 0.4 (9) 14.9 + 0.6* (8)
Means + S.E.M. are presented. Numbersof animalsper group are indicatedin parentheses except for food intake column, in which number of cages controlledare indicated. * p < 0.05.
200"
~ 30
~150-=
~'0
1oo.
E
"r
-I
•#J 1 0 " 4
Iolt 0
10
30
0
10
30
min
FIG. 1. The effect o f chronic stress on p i t u i t a r y - a d r e n a l response to a novel acute stressor (restraint). Means and S.E.M.
are presented. Numbersof animalsper group are inside bars. Open bars indicatecontrol, and closed bars chronically stressed, rats. Numbersunder bars indicatethe period of exposure to restraint. Significantdifferencesbetween acutely stressed rats and their respectivebasal groups are not indicated(see text). *p < 0.01 betweencontrol and chronically stressed rats under the same acute treatment.
significant (p < 0.001) effect of acute stress, but not of previous chronic treatment, was found. However the interaction between the two main factors was significant (p < 0.01). Comparisons between means indicated that the corticosterone response to acute stress was higher in the chronically stressed rats than in the controls (p < 0.01) at 10 and 30 min. Chronic stress did not modify either the defecation rate or the exploratory activity in a novel environment (Table I).
Experiment 2 In the dexamethasone-treated rats, ACTH administration induced a higher (p < 0.05) corticosterone response in the chronically stressed rats than in the controls (Fig. 2). Fig. 3 depicts the effect of dexamethasone on the PA response to restraint stress. Previous chronic stress significantly (p < 0.01) increased the corticosterone, but not the ACTH response, to acute stress, which is in accordance with the results of Experiment 1. Administration of dexamethasone prior to stress significantly inhibited the ACTH response, to the same extent in the two experimental groups. In contrast, the higher dose of dexamethasone significantly (p < 0.05) inhibited the corticosterone response to stress in the chronically stressed rats but not in the controls, compared with their respective basal values.
C. RESTREPO and A. ARMARIO
396
,25t i i|
tl L
d.ALI;L
S
A
FIG. 2. Adrenocortical responsiveness to ACTH (A) or saline (S) administration in control and chronically stressed rats. The dose of ACTH administered was 0.5 IU/100 g body wt. Means and S.E.M. are presented. Numbers of animals per group are inside bars. Open bars indicate control, and closed bars chronically stressed, rats. * p < 0.05 vs the respective control group.
600-
'•
o
c o
400
Q.
"I" 2 0 0 I0 <
03
10
k
o w
uo o
0 310
0
310
0 310
FIG 3. Effectiveness of dexamethasone in suppressing serum ACTH and corticosterone responses to 20 min restraint stress. Open bars represent control, and closed bars chronically stressed, rats. Means and S.E.M. are presented. Numbers of animals per group are inside bars. Numbers under bars indicate dose of dexamethasone administered 2 hr before stress. * p < 0.05, * p < 0.01 vs their respective non-dexamethasone treated values.
TABLE II. EFFECT OF CHRONIC STRESS ON THE HOLE-BOARDPERFORMANCES OF PREPUBERTALMALE RATS
Group
Defecation
Rearing
Head-dips
Head-dipping (sec)
Control (10) Chronic stress (10)
2.3 + 0.8 3.5 + 0.9
14.0 + 2.5 14.7 + 1.8
7.1 + 1.5 4.0 ± 0.9
9.6 + 2.2 5.8 + 1.7
Means -4- S.E.M. are presented. Numbers of animals per group are indicated in parentheses.
PITUITARY-ADRENALFUNCTIONAND CHRONICSTRESS
397
DISCUSSION
The model of chronic stress used in the present experiment did not alter either food intake or body weight gain in prepubertal male rats. However, this procedure was stressful for the rats, as evidenced by the higher adrenal weights of the chronically stressed animals. Neither the defecation rate nor exploratory activity in a novel environment were altered by chronic stress. The present data are not in agreement with those obtained in adult male rats (Armario et al., 1985), since the latter animals showed normal defecation rates but reduced exploratory activity in a novel environment. It therefore appears that previous chronic stress does not alter emotional reactivity in adult or in prepubertal animals, but the former appear to be more sensitive to chronic stress in terms of exploratory activity. It seems likely that reduced exploratory activity of the adult rats is linked to motivational rather than emotional mechanisms. If this were the case adult rats would be more sensitive than prepubertal ones to behavioral depression caused by chronic stress. Although ACTH responses to a novel acute stressor were normal in the chronically stressed animals, corticosterone secretion was higher, most likely due to the increased response of the adrenal cortex to ACTH, which is in accordance with the results obtained in adult rats (Armario et al., 1985). However, it appears that chronic stress caused some changes in the PA axis of the prepubertal rats. The inhibition of pituitary ACTH release by dexamethasone was similar in the control and chronically stressed rats, but synthetic corticosteroid significantly inhibited corticosterone secretion only in the chronically stressed rats. These data indicate that corticosterone secretion was inhibited by a mechanism independent of ACTH. A dissociation between the two hormones has been reported by others (Dempsher & Gann, 1983; Sherman et al., 1984; Lypka & Szczudlik, 1985). It may be that the adrenal cortex of chronically stressed rats is more sensitive to the direct inhibitory action of corticosteroids on their own secretion (Trung et al., 1984). Alternatively, a more marked inhibition by dexamethasone of some unknown factors able to potentiate the adrenocortical response to ACTH, such as endogenous peptides from the anterior pituitary (A1-Dujaili et al., 1981; Sharp & Sowers, 1983; Jornot et al., 1985), might occur in chronically stressed rats. It has been reported that adult rats chronically subjected to cold showed reduced PA sensitivity to the inhibitory action of another synthetic corticosteroid (prednisolone) (Vernikos et al., 1982). Whether the discrepancies between our results and those of Vernikos et al. (1982) are due to the different ages of the animals or to the different chronic stress models is not known at present. Although the PA axis of young rats differs from that of adult rats in its sensitivity to feedback inhibition by dexamethasone (Goldman e t a l . , 1973), it appears that the model of chronic stress might be the most relevant factor, since adult rats chronically subjected to a stress similar to that used in the present experiment showed increased rather than decreased responsiveness to the inhibitory action of dexamethasone (unpublished data). The effect of the chronic stress we used on the exploratory activity of prepubertal rats appeared to be milder than its effect on adult rats. However, the chronically stressed prepubertal rats showed increased corticosterone responses to a novel, acutely applied stressor, increased adrenocortical responses to ACTH, and enhanced sensitivity of the adrenal to the direct inhibitory action of dexamethasone on corticosterone secretion. REFERENCES Al-Dujaili EAS, Hope J, Estivariz FE, Lowry PJ, Edwards CRW (1981) Circulating human pituitary pro-melanotropin
398
C. RESTREPOand A. ARMARIO
enhances the adrenal response to ACTH. Nature 291: 156-159. Armario A, Castellanos JM (1984) A simple procedure for direct corticosterone radioimmunoassay in the rat. Rev Esp Fisiol 40: 437-442. Armario A, Castellanos JM, Balasch J (1984a) Dissociation between corticosterone and growth hormone adaptation to chronic stress in the rat. Horm Metab Res 16: 142-145. Armario A, CasteUanos JM, Balasch J (1984b) Effect of chronic noise on corticotropin function and on emotional reactivity in adult rats. Neuroendocrinol Lett 6:121 - 127. Armario A, CasteUanos JM, Balasch J (1984c) Effect of crowding on emotional reactivity in male rats. Neuroendocrinology 39: 330-333. Amario A, Restrepo C, Castellanos JM, Balasch J (1985) Dissociation between adrenocorticotropin and corticosterone responses to restraint stress after previous chronic exposure to stress. Life Sci 36: 2085-2092. Dempsher DP, Gann DS (1983) Increased cortisol secretion after small hemorrhage is not attributable to changes in adrenocorticotropin. Endocrinology 113: 86-93. File SE, Wardill AG (1975) Validity of head-dipping as a measure of exploration in a modified hole-board. Psychopharmacologia 44: 53-59. Goldman L, Winget C, Hollingshead GW, Levine S (1973) Postweaning development of negative feedback in the pituitary - adrenal system of the rat. Neuroendocrinology 12:199 - 211. Jornot LH, Capponi AM, Vallotton MB (1985) Responses of rat adrenal glomerulosa and inner zone cells to synthetic ACTH analogs and proopiomelanocortin derived peptides. J Steroid Biochem 2 2 : 2 2 1 - 2 2 5 . Katz RJ, Roth KA, Carroll BJ (1981) Acute and chronic stress effects on open field activity in the rat: implications for a model of depression. Neurosci Biobehav Rev 5: 247-251. Lescoat G, Feliot J, Maniey J (1978) Evolution de la concentration de la corticost6rone plasmatique dans les conditions basales ou apr~s agression psychique au cours de la croissance chez le rat. Influence de la gonadectomie n6onatale. J Physiol (Paris) 74" 591-599. Lypka A, Szczudlik A (1985) Dexamethasone suppresses cortisol but not ACTH and beta-endorphin plasma concentration in healthy man. Horm Metab Res 17" 547. Riegle GD (1973) Chronic stress effects on adrenocortical responsiveness in young and aged rats. Neuroendocrinology 11: 1 - 1 0 . Sharp B, Sowers JR (1983) Adrenocortical response to corticotropin is inhibited by 73-MSH antisera in normotensive and spontaneously hypertensive rats. Biochem Biophys Res Comm 110: 357-363. Sherman BM, Schlechte JA, Pfohl BM (1984) Dissociation of plasma cortisol and ACTH responses to dexamethasone in healthy subjects. Hormone Res 20: 157-165. Trung MTPH, de Smitter N, Bogyo A, Girard F (1984) Inhibition of cortisol production in isolated guinea-pig adrenal cells. J Steroid Biochem 21: 9 3 - 9 9 . Vernikos J, Dallman MF, Bonner C, Katzen A, Shinsako J (1982) Pituitary-adrenal function in rats chronically exposed to cold. Endocrinology 110: 413-420.
Psychoneuroendocrinology,Vol. 12, No. 5, pp. 393-398, 1987. Printed in Great Britain.
CHRONIC STRESS ALTERS P I T U I T A R Y - A D R E N A L FUNCTION IN PREPUBERTAL MALE RATS C. RESTREPO and A. ARMARIO* Departamento de Biologia Celular y Fisiologfa, Facultad de Ciencias, Universidad Aut6noma de Barcelona, Bellaterra, Spain
(Received 18 August 1986; in final form 9 March 1987) SUMMARY The effect of exposure to chronic stress on the pituitary-adrenal axis was studied in prepubertal rats. The chronic stress consisted of exposing the animals 6 days a week for 32 days to one stressor randomly chosen among several each day, Chronic stress did not alter either food intake or body weight gain. However, it was stressful for the rats, as they had increased adrenal weights. Chronically stressed rats showed normal pituitary-adrenal basal activity and normal ACTH responses to a novel, acutely applied stressor. However, this treatment resulted in higher corticosterone response to an acute stressor, as a consequence of enhanced adrenocortical response to ACTH. In addition, chronic stress induced increasing sensitivity of the adrenal to the inhibitory action of dexamethasone. Exploratory activity and defecation rate in a novel environment were not affected by the chronic stress. All these data indicate that behavioral and endocrine changes induced by chronic stress were somewhat different from those previously found in adult rats. Prepubertal rats appear to be less sensitive than adult rats to the chronic stress we used. INTRODUCTION
previous chronic stress on pituitary-adrenal (PA) response to a novel acute stressor remains controversial. This is most likely due to: (a) the use of corticosteroids as a measure of adrenocorticotropin (ACTH) release, since it has been reported that chronic stress can alter adrenocortical responsiveness to ACTH (Riegle, 1973; Armario et al., 1985), and the adrenal cortex is not sensitive to high circulating ACTH levels because of maximal adrenocortical secretion; Co) the chronic stress model used; for instance, a chronic continuous stress such as cold increased PA response to a novel acute stressor (Vernikos et al., 1982), whereas chronic intermittent stresses did not alter or decrease it (Riegle, 1973; Armario et al., 1984a,b). We found that chronic intermittent stresses did not alter the ACTH response to novel acute stressors in adult male rats (Armario et al., 1984b, 1985), which suggests that a history of chronic exposure to stress does not increase emotional reactivity in adult animals. The post-weaning period appears to be critical for the normal development of the animals. In addition, it has been reported that the corticosterone response to acute stress was higher in prepubertal than in adult male rats (Goldman et al., 1973; Lescoat et al., 1978). Therefore, chronic stress could exert a more marked effect on emotional reactivity in the post-weaning period than in adulthood. However, no studies have been carried out on this subject. The present work investigates the influence of chronic stress on emotional reactivity in prepubertal rats as measured by behavioral response to a novel environment and PA activity. In order to reduce the possibility of adaptation, the animals were subjected every day to one stressor randomly chosen among several (Katz et al., 1981). T H E EFFECT o f
*To whom correspondence should be addressed. 393
394
C. RESTREPOand A. ARMAR10 MATERIALS AND METHODS
Animals and stress protocol Male Sprague-Dawley rats 22 - 2 4 days old were used. They were kept in a controlled environment (lights on from 0700 hr to 1900 hr, temperature 23°C). Food and water were always freely available. The animals were randomly allocated among control and chronic stress groups. Those rats assigned to the chronic stress groups were exposed, 6 days a week for 32 days, to one stressor randomly chosen each day among the following: intraperitoneal (i.p.) saline injection (twice a day), ether anesthesia, 4 hr of continuous noise caused by an alarm bell (85 db), and 15 min of forced swimming in a water tank (diameter = 40 cm, height = 60 cm, temperature = 18°C). The experimental period lasted for 32 days. The day after the last exposure of the chronically stressed rats, the animals from the two experimental groups were subjected to several acute manipulations (see below). Noise was the stressor applied to the chronically stressed animals the day before the animals were subjected to the acute treatments. We have found that the stress of noise by itself, applied to the rats the day before, altered neither the exploratory activity of the rats, basal levels of PA hormones, nor the PA response to an acute stressor (unpublished data).
Experiment 1 This was designed to study: (a) the effects of previous chronic stress on emotional reactivity as measured by PA activation caused by a novel acute stressor; (b) the influence of chronic stress on the behavioral response to a novel exploratory box. Two days before the end of the chronic treatment, 10 animals from each group (control and chronically stressed) were tested in a hole-board apparatus for 4 min. The hole-board is an exploratory box with four holes in the bottom (File & Wardill, 1975) which was situated in a room provided with the same illumination and background noise as the animal house. The number of boluses defecated, rears and head-dips were measured manually. Time spent headdipping was recorded using a stop-watch. A dip was considered to have taken place when the introduction of the head into the holes was at least to the level of the eyes. After each test the apparatus was cleaned. The day before the holeboard test, the stressor applied to the chronically stressed rats was noise, for the reasons explained above. Three days later, approximately 20 hr after the last stress sessions for the chronically stressed animals, the rats were subjected to one of two acute treatments. Some animals were killed by decapitation within 20 sec after being taken from the animal house. This procedure allowed measurement of the basal hormone levels. Others were subjected to restraint stress in plastic tubes (4.5 cm diameter, 18 cm length) for 10 or 30 min before being quickly killed. The adrenals were removed, trimmed of fat and weighed. Body weight gain and food intake were measured throughout all the experiments.
Experiment 2 The aim of this experiment was to study the effects of chronic stress on adrenocortical responsiveness to ACTH and the sensitivity of the PA axis to feedback inhibition by dexamethasone. On day 33 after the beginning of the experimental phase, the animals were injected i.p. with either saline or three doses of dexamethasone phosphate (3, 10 or 25 gg/100 g body wt). Two hours later, those injected with saline or 3 or 10 gg dexamethasone were subjected to restraint stress in the same tubes already describe. Those injected with 25 gg dexamethasone were s.c. injected with 0.5 IU/100 g body wt of porcine ACTH (Sigma). All the animals were killed 20 min after the onset of stress or ACTH administration.
Hormone assays The trunk blood was collected into plastic tubes and maintained in ice-cold water to prevent ACTH degradation. The blood was centrifuged at the same temperature, and the serum was frozen at - 2 0 ° C . Corticosterone and ACTH were analyzed by radioimmunoassay as previously described (Armario & Castellanos, 1984; Armario et al., 1984c). All samples to be statistically compared were processed within the same assay to avoid inter-assay variations. Intraassay coefficients of variation averaged 10% for ACTH and 5% for corticosterone.
Statistical analysis The results were analyzed with Student's t-test or with two-way analysis of variance (ANOVA). Data were log transformed before their statistical evaluation to achieve homogeneity of variance. RESULTS
Experiment 1 Chronic stress did not alter either body weight gain or food intake. However, (p <
a significant
0 . 0 1 ) i n c r e a s e in t h e r e l a t i v e a d r e n a l w e i g h t w a s o b s e r v e d in t h e c h r o n i c a l l y s t r e s s e d
c o m p a r e d to t h e c o n t r o l r a t s ( T a b l e I). F i g . 1 s h o w s t h e P A r e s p o n s e s to a n o v e l a c u t e s t r e s s o r in t h e c o n t r o l a n d c h r o n i c a l l y s t r e s s e d r a t s . A N O V A
r e v e a l e d a s i g n i f i c a n t (p < 0 . 0 0 1 ) e f f e c t
of acute stress, but not of previous chronic treatment, on serum ACTH.
For corticosterone a
395
PITUITARY-ADRENAL FUNCTION AND CHRONIC STRESS TABLE I. EFFECT OF CHRONIC STRESS ON SOME PHYSIOLOGICALVARIABLES
Group Control Chronic stress
Initial body weight (g)
Body weight gain (g)
Food intake (g/rat/day)
84.8 + 4.2 (11) 81.5 :t: 2.0 (11)
239.2 -4- 5.1 (11) 239.0 ± 6.8 (11)
23.2 :t: 0.5 (8) 23.5 ± 0.3 (8)
Adrenal weight (g/100 g/body wt) 11.9 ± 0.4 (9) 14.9 + 0.6* (8)
Means + S.E.M. are presented. Numbersof animalsper group are indicatedin parentheses except for food intake column, in which number of cages controlledare indicated. * p < 0.05.
200"
~ 30
~150-=
~'0
1oo.
E
"r
-I
•#J 1 0 " 4
Iolt 0
10
30
0
10
30
min
FIG. 1. The effect o f chronic stress on p i t u i t a r y - a d r e n a l response to a novel acute stressor (restraint). Means and S.E.M.
are presented. Numbersof animalsper group are inside bars. Open bars indicatecontrol, and closed bars chronically stressed, rats. Numbersunder bars indicatethe period of exposure to restraint. Significantdifferencesbetween acutely stressed rats and their respectivebasal groups are not indicated(see text). *p < 0.01 betweencontrol and chronically stressed rats under the same acute treatment.
significant (p < 0.001) effect of acute stress, but not of previous chronic treatment, was found. However the interaction between the two main factors was significant (p < 0.01). Comparisons between means indicated that the corticosterone response to acute stress was higher in the chronically stressed rats than in the controls (p < 0.01) at 10 and 30 min. Chronic stress did not modify either the defecation rate or the exploratory activity in a novel environment (Table I).
Experiment 2 In the dexamethasone-treated rats, ACTH administration induced a higher (p < 0.05) corticosterone response in the chronically stressed rats than in the controls (Fig. 2). Fig. 3 depicts the effect of dexamethasone on the PA response to restraint stress. Previous chronic stress significantly (p < 0.01) increased the corticosterone, but not the ACTH response, to acute stress, which is in accordance with the results of Experiment 1. Administration of dexamethasone prior to stress significantly inhibited the ACTH response, to the same extent in the two experimental groups. In contrast, the higher dose of dexamethasone significantly (p < 0.05) inhibited the corticosterone response to stress in the chronically stressed rats but not in the controls, compared with their respective basal values.
C. RESTREPO and A. ARMARIO
396
,25t i i|
tl L
d.ALI;L
S
A
FIG. 2. Adrenocortical responsiveness to ACTH (A) or saline (S) administration in control and chronically stressed rats. The dose of ACTH administered was 0.5 IU/100 g body wt. Means and S.E.M. are presented. Numbers of animals per group are inside bars. Open bars indicate control, and closed bars chronically stressed, rats. * p < 0.05 vs the respective control group.
600-
'•
o
c o
400
Q.
"I" 2 0 0 I0 <
03
10
k
o w
uo o
0 310
0
310
0 310
FIG 3. Effectiveness of dexamethasone in suppressing serum ACTH and corticosterone responses to 20 min restraint stress. Open bars represent control, and closed bars chronically stressed, rats. Means and S.E.M. are presented. Numbers of animals per group are inside bars. Numbers under bars indicate dose of dexamethasone administered 2 hr before stress. * p < 0.05, * p < 0.01 vs their respective non-dexamethasone treated values.
TABLE II. EFFECT OF CHRONIC STRESS ON THE HOLE-BOARDPERFORMANCES OF PREPUBERTALMALE RATS
Group
Defecation
Rearing
Head-dips
Head-dipping (sec)
Control (10) Chronic stress (10)
2.3 + 0.8 3.5 + 0.9
14.0 + 2.5 14.7 + 1.8
7.1 + 1.5 4.0 ± 0.9
9.6 + 2.2 5.8 + 1.7
Means -4- S.E.M. are presented. Numbers of animals per group are indicated in parentheses.
PITUITARY-ADRENALFUNCTIONAND CHRONICSTRESS
397
DISCUSSION
The model of chronic stress used in the present experiment did not alter either food intake or body weight gain in prepubertal male rats. However, this procedure was stressful for the rats, as evidenced by the higher adrenal weights of the chronically stressed animals. Neither the defecation rate nor exploratory activity in a novel environment were altered by chronic stress. The present data are not in agreement with those obtained in adult male rats (Armario et al., 1985), since the latter animals showed normal defecation rates but reduced exploratory activity in a novel environment. It therefore appears that previous chronic stress does not alter emotional reactivity in adult or in prepubertal animals, but the former appear to be more sensitive to chronic stress in terms of exploratory activity. It seems likely that reduced exploratory activity of the adult rats is linked to motivational rather than emotional mechanisms. If this were the case adult rats would be more sensitive than prepubertal ones to behavioral depression caused by chronic stress. Although ACTH responses to a novel acute stressor were normal in the chronically stressed animals, corticosterone secretion was higher, most likely due to the increased response of the adrenal cortex to ACTH, which is in accordance with the results obtained in adult rats (Armario et al., 1985). However, it appears that chronic stress caused some changes in the PA axis of the prepubertal rats. The inhibition of pituitary ACTH release by dexamethasone was similar in the control and chronically stressed rats, but synthetic corticosteroid significantly inhibited corticosterone secretion only in the chronically stressed rats. These data indicate that corticosterone secretion was inhibited by a mechanism independent of ACTH. A dissociation between the two hormones has been reported by others (Dempsher & Gann, 1983; Sherman et al., 1984; Lypka & Szczudlik, 1985). It may be that the adrenal cortex of chronically stressed rats is more sensitive to the direct inhibitory action of corticosteroids on their own secretion (Trung et al., 1984). Alternatively, a more marked inhibition by dexamethasone of some unknown factors able to potentiate the adrenocortical response to ACTH, such as endogenous peptides from the anterior pituitary (A1-Dujaili et al., 1981; Sharp & Sowers, 1983; Jornot et al., 1985), might occur in chronically stressed rats. It has been reported that adult rats chronically subjected to cold showed reduced PA sensitivity to the inhibitory action of another synthetic corticosteroid (prednisolone) (Vernikos et al., 1982). Whether the discrepancies between our results and those of Vernikos et al. (1982) are due to the different ages of the animals or to the different chronic stress models is not known at present. Although the PA axis of young rats differs from that of adult rats in its sensitivity to feedback inhibition by dexamethasone (Goldman e t a l . , 1973), it appears that the model of chronic stress might be the most relevant factor, since adult rats chronically subjected to a stress similar to that used in the present experiment showed increased rather than decreased responsiveness to the inhibitory action of dexamethasone (unpublished data). The effect of the chronic stress we used on the exploratory activity of prepubertal rats appeared to be milder than its effect on adult rats. However, the chronically stressed prepubertal rats showed increased corticosterone responses to a novel, acutely applied stressor, increased adrenocortical responses to ACTH, and enhanced sensitivity of the adrenal to the direct inhibitory action of dexamethasone on corticosterone secretion. REFERENCES Al-Dujaili EAS, Hope J, Estivariz FE, Lowry PJ, Edwards CRW (1981) Circulating human pituitary pro-melanotropin
398
C. RESTREPOand A. ARMARIO
enhances the adrenal response to ACTH. Nature 291: 156-159. Armario A, Castellanos JM (1984) A simple procedure for direct corticosterone radioimmunoassay in the rat. Rev Esp Fisiol 40: 437-442. Armario A, Castellanos JM, Balasch J (1984a) Dissociation between corticosterone and growth hormone adaptation to chronic stress in the rat. Horm Metab Res 16: 142-145. Armario A, CasteUanos JM, Balasch J (1984b) Effect of chronic noise on corticotropin function and on emotional reactivity in adult rats. Neuroendocrinol Lett 6:121 - 127. Armario A, CasteUanos JM, Balasch J (1984c) Effect of crowding on emotional reactivity in male rats. Neuroendocrinology 39: 330-333. Amario A, Restrepo C, Castellanos JM, Balasch J (1985) Dissociation between adrenocorticotropin and corticosterone responses to restraint stress after previous chronic exposure to stress. Life Sci 36: 2085-2092. Dempsher DP, Gann DS (1983) Increased cortisol secretion after small hemorrhage is not attributable to changes in adrenocorticotropin. Endocrinology 113: 86-93. File SE, Wardill AG (1975) Validity of head-dipping as a measure of exploration in a modified hole-board. Psychopharmacologia 44: 53-59. Goldman L, Winget C, Hollingshead GW, Levine S (1973) Postweaning development of negative feedback in the pituitary - adrenal system of the rat. Neuroendocrinology 12:199 - 211. Jornot LH, Capponi AM, Vallotton MB (1985) Responses of rat adrenal glomerulosa and inner zone cells to synthetic ACTH analogs and proopiomelanocortin derived peptides. J Steroid Biochem 2 2 : 2 2 1 - 2 2 5 . Katz RJ, Roth KA, Carroll BJ (1981) Acute and chronic stress effects on open field activity in the rat: implications for a model of depression. Neurosci Biobehav Rev 5: 247-251. Lescoat G, Feliot J, Maniey J (1978) Evolution de la concentration de la corticost6rone plasmatique dans les conditions basales ou apr~s agression psychique au cours de la croissance chez le rat. Influence de la gonadectomie n6onatale. J Physiol (Paris) 74" 591-599. Lypka A, Szczudlik A (1985) Dexamethasone suppresses cortisol but not ACTH and beta-endorphin plasma concentration in healthy man. Horm Metab Res 17" 547. Riegle GD (1973) Chronic stress effects on adrenocortical responsiveness in young and aged rats. Neuroendocrinology 11: 1 - 1 0 . Sharp B, Sowers JR (1983) Adrenocortical response to corticotropin is inhibited by 73-MSH antisera in normotensive and spontaneously hypertensive rats. Biochem Biophys Res Comm 110: 357-363. Sherman BM, Schlechte JA, Pfohl BM (1984) Dissociation of plasma cortisol and ACTH responses to dexamethasone in healthy subjects. Hormone Res 20: 157-165. Trung MTPH, de Smitter N, Bogyo A, Girard F (1984) Inhibition of cortisol production in isolated guinea-pig adrenal cells. J Steroid Biochem 21: 9 3 - 9 9 . Vernikos J, Dallman MF, Bonner C, Katzen A, Shinsako J (1982) Pituitary-adrenal function in rats chronically exposed to cold. Endocrinology 110: 413-420.