- Email: [email protected]
Behavioral and cardiac responses after intracerebroventricular corticotropin-releasing hormone (CRH) administration: Role of adrenal cortical hormones
HORMONES AND BEHAVIOR 26,
375-384 (1992)
Behavioral and Cardiac Responses after lntracerebroventricular Corticotropin-Releasing Hormone (CRH) Administration: Role of Adrenal Cortical Hormones S. M. KORTE,’ W. EISINGA, W. TIMMERMAN, C. NYAKAS, AND B. BOHUS Department of Animul Physialogy,
University of Groningen, The Netherlands
I’. 0. Box 14, 9750 AA Haren,
lntracerebroventricularly (icv) administered corticotropin-releasing hormone (CRH) produces a dose-dependent increase in heart rate in association with hehavioral activation. The present study was designed to investigate whether these CRH-induced responses are dependent on adrenal function. The effects of adrenalectomy (ADX) and subsequent corticosterone replacement were studied. Administration icv of 300 ng of CRH failed to produce behavioral activation and tachycardia in ADX rats. Corticosterone replacement restored the CRH-induced behavioral response to preoperative levels, whereas the CRH-induced tachycardia was partially restored. This latter result may be related to the fact that the baseline heart rate of ADX animals appeared to be significantly higher than that of corticosteronc-treated ADX animals. It is concluded that circulating adrenal corticosteronc in ADX rats is involved in the expression of the behavioral and cardiac effect of central CRH. 0 I9Y2 Awdrmic I’rcs. Inc.
The corticotropin-releasing hormone (CRH) is considered to be a common mediator of stress responses via behavioral, autonomic, and neuroendocrine mechanisms (Fisher, 1989). High-affinity binding sites for CRH have been identified in several brain regions including areas of the limbic system and regions involved in the regulation of the autonomic nervous system (De Souza, Insel, Perrin. Rivier, Vale, and Kuhar, 1985; De Souza, 1988; Sawchenko, and Swanson, 1985; Wynn, Harwood, Catt, and Aguilera, 1985). The behavioral profile of intracerebroventricularly (icv) administered CRH in aversive situations suggests that it increases emotionality (Koob and Bloom, 1985). CRH is able to elicit a number of responses normally regarded as characteristic of anxiety or stress (Dunn, and Berridge, 1990). Furthermore, CKH plays an important role in the activation of the hypothalamic-pituitary-adrenal axis, reflected in an in’ To whom correspondence and reprint requests should be addressed
376
KORTEETAL.
crease in plasma corticosterone levels (Rivier, Brownstein, Spiess, and Vale, 1982). The secretion of adrenal cortical and also medullary hormones may be an integrated ultimate output of the CRH-mediated stress response. The stress hormone of the adrenal cortex corticosterone has long been considered an important modulator of adaptive behaviors in stressful conditions in the rat (Bohus, De KIoet, and Veldhuis, 1982; De Kloet, 1991; McEwen, De Kloet, and Rostene, 1986). In this species, the expression and extinction of fear-motivated immobility behavior is attenuated by short-term ADX and restored by corticosterone (Bohus, and De Kloet, 1981; Bohus, 1987). Adrenomedullary epinephrine also affects memory processes and thereby behavior (Borrell, De Kloet, Versteeg, and Bohus, 1983; Introini-Collison, and McGaugh, 1991), but its role in the behavioral stress reaction is not clear. However, its involvement in cardiac reaction is rather tenable. In the present study the question of whether CRH-induced stress responses, i.e., be.havioral activation and increase in heart rate, are dependent on the adrenal function is addressed. Accordingly, heart rate and behavior were measured before, during, and after the icv infusion of CRH in habituated rats in their home cages. The effects of removal of the source of adrenal hormones by ADX and ADX in combination with corticosterone replacement were studied. MATERIALS AND METHODS Animals and Housing
Male Wistar rats 3 months of age, weighing 290-340 g at the beginning of the experiments, were used. They were housed six to a cage. After surgery they were placed in individual clear Plexiglas cages (25 x 25 x 30 cm) on a 12-hr light-dark regime (07.30-19.30 hr light on) at a room temperature of 21 ? 2°C. All animals had free access to standard food (Hope Farms rat chow) and tap water. Following adrenalectomy the rats received saline (0.9% NaCl in water) to drink. The experiments were run between 9 AM and 14.00 PM. Surgery
All surgery was performed under ether anesthesia. To record the electrocardiogram (ECG) transcutaneous steel electrodes made of standard paperclips were implanted as described previously (Bohus, 1974). The cannula system for icv infusion was made of stainless steel (ID 0.1; OD 0.29 mm) (Scheurink, Steffens, and Gaykema, 1990) and was placed unilaterally in a lateral cerebral ventricle. Four days later adrenalectomy (ADX) was performed through a lumbar dorsal midline incision. The rats were given 10 days to recover from surgery.
CRH AND STRESS RESPONSES
377
Drug Treatment and Experimental Procedure Thirty minutes before the start of an experiment the infusion tubes were filled with corticotropin releasing hormone (CRH human, rat, Peninsula Laboratories, USA) or vehicle (aCSF; sterile artificial ccrebrospinal fluid containing (in mM) 127.64 NaCl, 2.55 KC!, 1.26 CaC!,, and 0.93 MgC!*. CRH (10, 30, 100, or 300 ng) were infused at a rate of 0.5 ~1 aCSF/min for 20 min. Heart rate was measured during l-min periods before, during, and after CRH infusion (at t = -10, -5, 5, 10, 15, 20, 30, 40, and 55 min) in freely moving rats in their home cages. To study the effects of CRH administration following adrenalectomy and corticosterone replacement, a dose of 300 ng CRH was selected. Pilot studies showed no differences in the behavioral and cardiac effects of infusion or a single injection of this particular dose. Therefore, the study was continued using bolus injections of CRH. The ADX rats received a subcutaneous injection of 100 pg/lOO g body wt of corticosteronc (Organon Int., Oss. The Netherlands) 1 hr before the CRH injection. The steroid was prepared shortly before treatment by dissolving it in ethanol and dilution with 0.9% saline to yield the required dose in a constant volume injection of 0.5 ml containing 8% ethanol. The control rats were preinjetted with this ethanol/saline vehicle. Behavioral Measurements The following behavioral elements were distinguished: exploring-investigation of any part of the home cage; grooming-wiping the fur with forepaws and tongue; burying-pushing the bedding material with rapid movements of the snout or forepaws; eating-chewing chow or feces. These behavioral elements were scored via the “point” sample technique; four samples every minute with 15-WCintervals. The number of sample periods in which grooming, burying, eating, and exploring occurred per total number of sample periods was expressed as percentage behavioral activity. ECG Recording and Analysis The ECG of freely moving rats was monitored te!emetrical!y (Bohus, 1974; Korte, and Bohus, 1990) by means of a miniature FM Transmitter (E.D.B., Harcn, The Netherlands). The transmitter was connected to the transcutaneous electrodes and secured around the chest of the rat by means of a strap. The strap alone had been fitted four times before, for 60 min, to habituate the animals to the testing circumstances. The transmitted signal was stored on tape for off line computer analysis (Olivetti M24). Prerecorded ECG samples of 1 min were played back via a cardiotachometcr pulse generator which generated a square wave pulse at each R wave. The time between the onset of two consecutive pulses, the interbeat interval (IBI). was measured within the range of 100 to 220 msec. The mean of IBI’s during 1 min was recalculated in bcats/min.
378
KORTE ET AL.
TABLE 1 The Effects of Corticotropin-Releasing Hormone (CRH) Infusion into Lateral Cerebral Ventricle on the Behavior and Cardiac Rate of Well Habituated Rats Behavioral activity (percentage time)
ng CRH 0 (n 30 (n 100 (n 300 (n
= = = =
7) 4) 4) 5)
0.1 20.0 17.0 70.4
Heart rate (beats/min) -__ 3% + 8 369 + 21 398 -c- 19 428 + 18*
d 0.1 2 20.0 r 13.9 -r- 3.7%
Nore. The data are presented as means -C SEM of four to seven observations per dose. The doses refer to the total amount per 20 min. The measurements were taken 20 min after the termination of the infusion period at I = 40 min. *P < 0.01 (Mann-Whitney U test and f test, respectively).
Statistics
Statistical analysis of the heart rate data was carried out using (multivariate) analysis of variance ((M)ANOVA of SPSS\ PC + ). The ANOVA’s were followed by t tests. Behavioral data were analyzed with Kruskal-Wallis ANOVA and followed by the Mann-Whitney U test. A probability level of P 4 0.05 was taken as statistical significance for all tests. RESULTS Behavioral and Cardiac Effects of icv CRH Infusion: Relations
Dose-Response
Table 1 shows the behavioral and cardiac effects of a 20-min infusion of CRH (0, 30, 100, or 300 ng) into the lateral cerebral ventricle of the well habituated rat. The infusion of CRH resulted in a dose-response relationship for both behavioral activation and tachycardiac response. These responses persisted after the termination of the infusion and were maximal at t = 40 min. Therefore, only data at t = 40 min are shown. Analysis of variance of the mean heart rate at t = 40 min revealed a significant treatment effect (F(3, 15) = 4.97, P = 0.014), with a significant increment in heart rate for the highest dose of CRH (P < 0.01). Behavioral activity was increased by CRH infusion (P = 0.01; Kruskal-Wallis test), particularly by the dose 300 ng (P < 0.01). The Effects of Adrenalectomy and Corticosterone Administration CRH-Induced Behavioral and Cardiac Activation
on the
Figure 1 shows that administration of CRH (300 ng) caused a significant increase in behavioral activity in all animals before surgery (P = 0.002, Mann-Whitney U test). The preinjection behavioral activity was not af-
CRH AND STRESS RESPONSES
intact
379
ADX
:I!j---y
10 0
t= -5 40 -5 40 t= -5 40 -5 40 t= -5 40 vehicle CRH CRH vehicle CRH FIG. 1. tkhavioral activity (%) as measured before injection at t = -5 min and after icv injection of CRH (300 ng) at t = 40 min in intact, adrenalectomized (ADX). and ADX animals with corticosterone (CORT) replaccmcnt. Means + SEM from five to six rats per group.
fected by ADX and corticosterone, whereas ADX totally abolished the behavioral response to CRH. Replacement with corticosterone in the ADX animals restored the behavioral activity to values similar to those found in the intact animals (P = 0.07, Mann-Whitney U test). Figure 2 shows the increase in heart rate after injection of 300 ng CRH in the intact animals. This response persisted during the next 40 min. MANOVA of the heart rate data of CRH relative to vehicle-treated animals revealed a significant main effect of the peptide treatment (F( 1, 15) = 6.00, P = 0.027), but not of the period. CRH injection led to a significant increase in heart rate at t = 10, 15, 20, and 30 min (P Q 0.05). After ADX, injection of CRH failed to produce significant effects relative to vehicle-treated animals, whereas basal heart rate in ADX compared to intact animals showed a tendency to be elevated (41, 13) = 3.55, P = 0.08). Corticostcronc replacement in these ADX animals restored basal heart rate to a pre-ADX level. Furthermore, CRH administration led to an increase in heart rate which was almost comparable to the response seen in the pre-ADX condition. MANOVA revealed a significant effect of heart rate data of ADX animals with corticosterone replacement relative to vehicle treatment (F(1, 10) = 5.30, P = 0.044). Basal heart rate of ADX rats with corticosterone replacement was lower than without corticosterone treatment at t = - 10 min (P = 0.06) and t = - 5 min (P = 0.02). After injection of CRH in the corticosterone-pretreated ADX animals, heart rate remained lower relative to vehicle-treated ADX rats at t = 5, 10, and 20 min (P 6 0.05).
380
KORTE
1
intact
ET AL.
ADX
46or-----l
2420E 3i z 4002 B 2 380r P = 360-
-20
0
20 40 60 -20 0 20 40 6C time (min) time (mid FIG. 2. The effect of icv injection CRH (300 ng) on heart rate (beats/min) in intact rats, adrenalectomized (ADX), and ADX animals with CORT replacement. Means k SEM from five to six animals per group.
DISCUSSION The present study shows that behavioral activation after icv CRH treatment was associated with an increase in heart rate. CRH treatment produced neither an increase in heart rate nor behavioral activation in ADX animals. Corticosterone replacement in a dose known to restore basal circulating hormone levels and occupation of specific corticoid receptor sites in the hippocampus of the rat (De Kloet, 1991; Bohus and De Kloet, 1981) restored the CRH-induced behavioral response to preoperative levels, whereas the CRH-induced tachycardia was partially restored. The behavioral finding in the rats’ home cage is consistent with observations that icv administered CRH produces a dose-dependent increase in activity in a familiar situation (Koob and Bloom, 1985; Sutton, Koob, Le Moal, Rivier, and Vale, 1982). A remarkable property of the peptide is to cause a long-lasting behavioral activation. Sutton et al. (1982) found an activating effect of a single injection even after 3 hr. The neurobiological background of this long-lasting behavioral activation is not yet known. The present study shows that this behavioral activation was absent in ADX rats and could be restored by corticosterone replacement thereby suggesting that it is corticosterone dependent. ADX leads to an absence of feedback signal of corticosterone resulting in an enhancement of CRH immunostaining in hypothalamic paraven-
CRH AND STRESS RESPONSES
381
tricular nucleus (PVN), in the bed nucleus of the stria terminalis and in the central nucleus of the amygdala (Merchenthalcr, Vigh, Petrusz, and Schally, 1983). Furthermore, ADX leads to an increase in hybridizable CRH mRNA level in the neurons in the hypothalamic paraventricular nucleus (Kovacs and Makara, 1988). These results suggest an increased synthesis of the pcptide. Increased CRH levels do not result in a general down-regulation and desensitization of all CRH receptors. Therefore, the effects of ADX cannot be simply explained by changes in CRH receptors in the brain. CRH receptors in the anterior pituitary, but not in the intermediate lobe, hippocampus, amygdala, or lateral septum undergo down-regulation in conditions such as ADX and glucocorticoid administration (Wynn et al., 1985; Wynn, Hauger, Holmes, Millan, Catt, and Aguilera 1984; Hauger, Millan, Catt, and Aguilera, 1987; Hauger, Millan, Lorang, Harwood, and Aguilera, 1988). An alternative explanation for the higher heart rate in ADX animals may be the fact that pituitary adrenocorticotropic hormone (ACTH) synthesis and release is substantially increased 10 days after ADX (Buckingham and Hodges, 1974; Dallman, Demanincor, and Shinsako, 1974). It is known that the treatment with the ACTH-like peptide (ACTH4-,J leads to an increase in heart rate (Bohus, 1975). It is also plausible that corticoid receptors in both the periphery and the brain are involved. This is not necessarily in contradiction to the findings of Britton (1989), who suggested that the activation of the pituitary-adrenal axis is neither sufficient nor necessary for the expression of the behavioral effects of CRH in intact animals. This statement does not exclude the possibility that saturation of the mineralocorticoid receptors (MRs) and/or partly occupied glucocorticoid receptors (GRs) with corticosterone (Reul and DC Kloet, 1985) in ADX rats is a prerequisite for the complete behavioral and autonomic activation after CRH administration. Removal of endogenous glucocorticoids by ADX induces a brain-spccific increase in the binding capacity of the type MRs and GRs, an increase which can be blocked by corticosteronc administration (Chao, Choo, and McEwen, 1989). The observed corticosteronc dependency of CRH-induced responses may therefore be explained by an altered stress state in ADX animals due to changes in binding capacity of corticoid receptors. It cannot be excluded that CRH failed to increase heart rate in ADX animals because of the ceiling or near-ceiling effect of the high baseline heart rate in these animals. The elevated heart rate observed in the ADX group compared to intact controls is consistent with previous observations in ADX rats (Yagil, Koreen, and Krakoff, 1986). Our study suggests that the presence of corticosterone is a prerequisite for a normal heart rate. The mechanisms underlying higher baseline heart rate in ADX animals remain to be determined. The fact that replacement with dexamethasonc,
382
KORTE ET AL.
but not with aldosterone, normalizes heart rate in ADX rats suggests that GRs are involved (Yagil et al., 1986). Lower brain areas involved in the control of heart rate (e.g., nucleus tractus solitarius) are known to contain intracellular GRs (De Kloet, 1991). Also a direct peripheral action of glucocorticoid hormones on the heart cannot be excluded because glucocorticoid insufficiency induced by adrenalectomy has been found to impair cardiac performance (Bhaskar, Stith, Brackett, Wilson, Lerner, and Reddy, 1989). CRH induced elevations of heart rate are suggested to be mediated by enhanced cardiac sympathetic outflow and decreased baroreceptor-induced activation of cardiac vagal motor neurons (Fisher, 1989b; Overton, Davis-Gorman, and Fisher, 1990). The present study suggests that the cardiovascular action of CRH parallels the behavioral activity. It remains to be determined to what extent the tachycardia can be explained by a somatic-autonomic coupling or by a primary sympathetic activation by CRH. The substantial differences in the dose of CRH used in the present study (300 ng) and by others (10 pg) (Fisher, 1989b; Fisher, Rivier, Spiess, Vale, and Brown, 1982) do not allow rejection of the possibility that both mechanisms are operating but at different doses of CRH. In conclusion, icv CRH administration causes behavioral activation in association with elevations in heart rate. The question whether the heart response is at least in part related to the increase in behavioral activity will need further investigation. It is suggested that corticosterone is involved in CRH-induced cardiac as well as behavioral responses in ADX rats, In addition, a normal baseline heart rate was corticosterone dependent. Whether these are peripheral or central effects of corticosterone remains to be elucidated. Further investigations are needed to specify whether GRs or MRs are involved. ACKNOWLEDGMENTS This study was supported in part by the Foundation for Medical and Health Research (NWO/MEDIGON) (Grant 900-551-044).We thank Organon International, Oss, The Netherlands, for the gift of corticosterone.
REFERENCES Bhaskar, M., Stith, R. D., Brackett, D. J., Wilson, M. F., Lerner, M. R., and Reddy, Y. S. (1989). Changes in myocardial contractile protein ATPases in chronically adrenalectomized rats with and without glucocorticoid replacement. Biochem. Med. Mer. Bio/. 42, 118-124. Bohus. B. (1974). Telemetered heart rate responses of the rat during free and learned behavior. Biorelemefry 1, 193-201. Bohus, B. (1987). Limbic-midbrain mechanisms and behavioral physiology interactions with CRF. ACTH and adrenal hormones. In Hellhamm, D., Florin, I., and Weiner, H. (Eds.), Neurobiological approaches to human disease. pp. 267-285. Huber, Toronto. Bohus, B., and De Kloet. E. R. (1981). Adrenal steroids and extinction behavior antagonism
CRH AND STRESS RESPONSES
383
by progesterone, deoxycorticosterone, and dexamethasone of specific effect of corticosterone. Life Sci. 28, 433-440. Bohus, B., De Kloet, E. R., and Veldhuis, H. D. (1982). Adrenal steroids and behavior adaptation: relationship to brain corticoid receptors. In Ganten, D., and Pfaff, D. W. (Eds.), Current topics in neuroendocrinology. pp. 107-142. Springer Verlag, Berlin. Borreli, J., De Kioet, E. R., Versteeg, D. H. G., and Bohus, B. (1983). Inhibitory avoidance deficit following short-term adrenaiectomy in the rat: The role of adrenal catecholamines. Behav. Neural Biol. 39, 241-258. Britton, D. R. (1989). Stress-related behavioral effect of corticotropin-releasing factor. In Tacht, Y., Morley, J. E., and Brown, M. R. (Eds.), Neuropepfides and stress. pp. 3948. Springer-Verlag, New York. Buckingham, J. C., and Hodges, J. R. (1974). Interrelationships of pituitary and piasma corticosterone during adrenocorticai regeneration in rat. J. Endocrinol. 67, 411-417. Chao, H. M., Choo, P. H., and McEwen, B. S. (1989). Giucocorticoid and mineraiocorticoid 50, 365-371. receptor mRNA expression in rat brain. Neuroendocrinology Dallman, M. F., Demanincor, D., and Shinsako, J. (1974). Diminishing corticotrope capacity to release ACTH during sustained stimulation: The twenty-four hours after bilateral adrenaiectomy in the rat. Endocrinology 95, 65-73. De Kloet, E. R. (1991). Brain corticosteroid receptor balance and homeostatic control. Frontiers Neuroendocrinol. U(2), 95-164. De Souza, E. B. (198%). Localization and modulation of brain and pituitary receptors involved in stress responses. Psychopharmacol. Bull. 24(3), 360-364. De Souza, E. B., Insei, T. R., Perrin, M. H., Rivier. J., Vale, W. W., and Kuhar, M. J. (1985). Corticotropin-releasing factor receptors are widely distributed within the rat central nervous system: an autoradiographic study. J. Neurosci. 5, 3189-3203. Dunn, A. J., and Berridge, G. W. (1990). Physiological and behavioral responses to corticotropin-releasing factor administration: is CRF a mediator of anxiety or stress responses? Brain Res. 15, 71-100. Fisher, L. A. (1989a). Central autonomic modulation of cardiac baroreflex by corticotropinreleasing factor. Am. J. Physiol. 256 (Hearf Circ. Physiol. 25), H949-H955. Fisher, L. A. (1989b). Corticotropin-releasing factor: Endocrine and autonomic integration of responses to stress. Trends Pharmacol. Sci. 10, 189-193. Fisher, L. A., Rivier, J., Rivier, C., Spiess, J., Vale, W., and Brown, M. R. (1982). Corticotropin-releasing factor (CRF): Central effects on mean arterial pressure and heart rate in rats. Endocrinology 110, 2222-2224. Hauger, R. L., Miiian, M. A., Catt, K. J., and Aguilera, G. (1987). Differential regulation of brain and pituitary corticotropin-releasing factor receptors by corticosterone. Endocrinology 120, 1527-1533. Hauger, R. L., Miilan, M. A., Lorang, M., Harwood, J. P.. and Aguiiera, G. (1988). Corticotropin-reieasing factor receptors and pituitary adrenal responses during immobilization stress. Endocrinology 123, 396-405. Introini-Coilison, I. B., and McGaugh, J. L. (1991). Interaction of hormones and neurotransmitter systems in the modulation of memory storage. In Frederickson, R. C, A., McGaugh, J. L., and Feiten, D. L. (Eds.), Peripheral signaling of he brain: Role in neural-immune interactions, learning and memory, pp. 275-301. Hogrefe Rr Huber, Toronto. Koob. G. F., and Bloom, F. E. (1985). Corticotropin-releasing factor and behavior. Fed. Proc. 44, 259-263.
Korte, S. M., and Bohus, B. (1990). The effect of ipsapirone on behavioural and cardiac responses in the shock-probe/defensive burying test in male rats. Eur. J. Pharmacoi. 181, 307-310.
384
KORTE ET AL.
Kovacs, K. J., and Makara, G. B. (1988). Corticosterone and dexamethasone act at different brain sites to inhibit adrenalectomy-induced adrenocorticotropin hypersecretion. Brain Res. 474, 205-210. McEwen, B. S., De Kloet, E. R., and Rostene, W. (1986). Adrenal steroid receptors and actions in the nervous system. Physiol. Rev. 66, 1121-1188. Merchenthaler, I., Vigh, S., Petrusz, P., and Schally, A. V. (1983). The paraventriculoinfundibular corticotropin releasing factor pathway as revealed by immunocytochemistry in long-term hypophysectomized or adrenalectomized rats. Reg. Peptides 5, 295-305. Overton, J. M., Davis-German, G., and Fisher, L. A. (1990). Central nervous system cardiovascular actions of CRF in sinoaortic-denervated rats. Am. J. Physiol. 258 (Reg. Integrative Comp. Physiol. 27), R596-R601. Reul, J. M. H. M., and De Kloet, E. R. (1985). Two receptor systems for corticosterone in raf brain: Microdistribution and differential occupation. Endocrinology 117, 117123. Rivier, C., Brownstein, M., Spiess, J.. Rivier, J., and Vale, W. (1982). In vivo corticotropinreleasing factor-induced secretion of adrenocorticotropin, beta-endorphin, and corticosterone. Etzdocrinology 110, 272-278. Sawchenko, P. E., and Swanson, L. W. (1985). Localization, colocalization, and plasticity of corticotropin-releasing factor immunoreactivity in rat brain. Fed. Proc. U(l), 221227. Scheurink, A. J. W., St&fens, A. B., and Gaykema, R. P. A. (1990). Paraventricular hypothalamic adrenoceptors and energy metabolism in exercising rats. Am. J. Physiol. 259, R478-R484. Sutton, R. E., Koob, G. F., Le Meal, M., Rivier, J., and Vale, W. (1982). Corticotropin releasing factor produces behavioural activation in rats. Nature 297, 331-333. Wynn, P. C., Hauger, R. L., Holmes, M. C., Millan, M. A., Catt, K. J., and Aguilera, G. (1984). Brain and pituitary receptors for corticotropin releasing factor: Localization and differential regulation after adrenalectomy. Peptides 5, 1077-1084. Wynn, P. C., Harwood, J. R., Catt, D. J., and Aguilera, G. (1985). Regulation of corticotropin-releasing factor (CRF) receptors in the rat pituitary gland: Effects of adrenalectomy on CRF receptors and corticotroph responses. Endocrinology 116, 1653-1659. Yagil, Y., Koreen, R., and Krakoff, L. R. (1986). Role of mineralocorticoids and glucocorticoids in blood pressure regulation in normotensive rats. Am. J. Physiol. Z51(Hearr Circ. Physiol. 20), H1354-H1360.
375-384 (1992)
Behavioral and Cardiac Responses after lntracerebroventricular Corticotropin-Releasing Hormone (CRH) Administration: Role of Adrenal Cortical Hormones S. M. KORTE,’ W. EISINGA, W. TIMMERMAN, C. NYAKAS, AND B. BOHUS Department of Animul Physialogy,
University of Groningen, The Netherlands
I’. 0. Box 14, 9750 AA Haren,
lntracerebroventricularly (icv) administered corticotropin-releasing hormone (CRH) produces a dose-dependent increase in heart rate in association with hehavioral activation. The present study was designed to investigate whether these CRH-induced responses are dependent on adrenal function. The effects of adrenalectomy (ADX) and subsequent corticosterone replacement were studied. Administration icv of 300 ng of CRH failed to produce behavioral activation and tachycardia in ADX rats. Corticosterone replacement restored the CRH-induced behavioral response to preoperative levels, whereas the CRH-induced tachycardia was partially restored. This latter result may be related to the fact that the baseline heart rate of ADX animals appeared to be significantly higher than that of corticosteronc-treated ADX animals. It is concluded that circulating adrenal corticosteronc in ADX rats is involved in the expression of the behavioral and cardiac effect of central CRH. 0 I9Y2 Awdrmic I’rcs. Inc.
The corticotropin-releasing hormone (CRH) is considered to be a common mediator of stress responses via behavioral, autonomic, and neuroendocrine mechanisms (Fisher, 1989). High-affinity binding sites for CRH have been identified in several brain regions including areas of the limbic system and regions involved in the regulation of the autonomic nervous system (De Souza, Insel, Perrin. Rivier, Vale, and Kuhar, 1985; De Souza, 1988; Sawchenko, and Swanson, 1985; Wynn, Harwood, Catt, and Aguilera, 1985). The behavioral profile of intracerebroventricularly (icv) administered CRH in aversive situations suggests that it increases emotionality (Koob and Bloom, 1985). CRH is able to elicit a number of responses normally regarded as characteristic of anxiety or stress (Dunn, and Berridge, 1990). Furthermore, CKH plays an important role in the activation of the hypothalamic-pituitary-adrenal axis, reflected in an in’ To whom correspondence and reprint requests should be addressed
376
KORTEETAL.
crease in plasma corticosterone levels (Rivier, Brownstein, Spiess, and Vale, 1982). The secretion of adrenal cortical and also medullary hormones may be an integrated ultimate output of the CRH-mediated stress response. The stress hormone of the adrenal cortex corticosterone has long been considered an important modulator of adaptive behaviors in stressful conditions in the rat (Bohus, De KIoet, and Veldhuis, 1982; De Kloet, 1991; McEwen, De Kloet, and Rostene, 1986). In this species, the expression and extinction of fear-motivated immobility behavior is attenuated by short-term ADX and restored by corticosterone (Bohus, and De Kloet, 1981; Bohus, 1987). Adrenomedullary epinephrine also affects memory processes and thereby behavior (Borrell, De Kloet, Versteeg, and Bohus, 1983; Introini-Collison, and McGaugh, 1991), but its role in the behavioral stress reaction is not clear. However, its involvement in cardiac reaction is rather tenable. In the present study the question of whether CRH-induced stress responses, i.e., be.havioral activation and increase in heart rate, are dependent on the adrenal function is addressed. Accordingly, heart rate and behavior were measured before, during, and after the icv infusion of CRH in habituated rats in their home cages. The effects of removal of the source of adrenal hormones by ADX and ADX in combination with corticosterone replacement were studied. MATERIALS AND METHODS Animals and Housing
Male Wistar rats 3 months of age, weighing 290-340 g at the beginning of the experiments, were used. They were housed six to a cage. After surgery they were placed in individual clear Plexiglas cages (25 x 25 x 30 cm) on a 12-hr light-dark regime (07.30-19.30 hr light on) at a room temperature of 21 ? 2°C. All animals had free access to standard food (Hope Farms rat chow) and tap water. Following adrenalectomy the rats received saline (0.9% NaCl in water) to drink. The experiments were run between 9 AM and 14.00 PM. Surgery
All surgery was performed under ether anesthesia. To record the electrocardiogram (ECG) transcutaneous steel electrodes made of standard paperclips were implanted as described previously (Bohus, 1974). The cannula system for icv infusion was made of stainless steel (ID 0.1; OD 0.29 mm) (Scheurink, Steffens, and Gaykema, 1990) and was placed unilaterally in a lateral cerebral ventricle. Four days later adrenalectomy (ADX) was performed through a lumbar dorsal midline incision. The rats were given 10 days to recover from surgery.
CRH AND STRESS RESPONSES
377
Drug Treatment and Experimental Procedure Thirty minutes before the start of an experiment the infusion tubes were filled with corticotropin releasing hormone (CRH human, rat, Peninsula Laboratories, USA) or vehicle (aCSF; sterile artificial ccrebrospinal fluid containing (in mM) 127.64 NaCl, 2.55 KC!, 1.26 CaC!,, and 0.93 MgC!*. CRH (10, 30, 100, or 300 ng) were infused at a rate of 0.5 ~1 aCSF/min for 20 min. Heart rate was measured during l-min periods before, during, and after CRH infusion (at t = -10, -5, 5, 10, 15, 20, 30, 40, and 55 min) in freely moving rats in their home cages. To study the effects of CRH administration following adrenalectomy and corticosterone replacement, a dose of 300 ng CRH was selected. Pilot studies showed no differences in the behavioral and cardiac effects of infusion or a single injection of this particular dose. Therefore, the study was continued using bolus injections of CRH. The ADX rats received a subcutaneous injection of 100 pg/lOO g body wt of corticosteronc (Organon Int., Oss. The Netherlands) 1 hr before the CRH injection. The steroid was prepared shortly before treatment by dissolving it in ethanol and dilution with 0.9% saline to yield the required dose in a constant volume injection of 0.5 ml containing 8% ethanol. The control rats were preinjetted with this ethanol/saline vehicle. Behavioral Measurements The following behavioral elements were distinguished: exploring-investigation of any part of the home cage; grooming-wiping the fur with forepaws and tongue; burying-pushing the bedding material with rapid movements of the snout or forepaws; eating-chewing chow or feces. These behavioral elements were scored via the “point” sample technique; four samples every minute with 15-WCintervals. The number of sample periods in which grooming, burying, eating, and exploring occurred per total number of sample periods was expressed as percentage behavioral activity. ECG Recording and Analysis The ECG of freely moving rats was monitored te!emetrical!y (Bohus, 1974; Korte, and Bohus, 1990) by means of a miniature FM Transmitter (E.D.B., Harcn, The Netherlands). The transmitter was connected to the transcutaneous electrodes and secured around the chest of the rat by means of a strap. The strap alone had been fitted four times before, for 60 min, to habituate the animals to the testing circumstances. The transmitted signal was stored on tape for off line computer analysis (Olivetti M24). Prerecorded ECG samples of 1 min were played back via a cardiotachometcr pulse generator which generated a square wave pulse at each R wave. The time between the onset of two consecutive pulses, the interbeat interval (IBI). was measured within the range of 100 to 220 msec. The mean of IBI’s during 1 min was recalculated in bcats/min.
378
KORTE ET AL.
TABLE 1 The Effects of Corticotropin-Releasing Hormone (CRH) Infusion into Lateral Cerebral Ventricle on the Behavior and Cardiac Rate of Well Habituated Rats Behavioral activity (percentage time)
ng CRH 0 (n 30 (n 100 (n 300 (n
= = = =
7) 4) 4) 5)
0.1 20.0 17.0 70.4
Heart rate (beats/min) -__ 3% + 8 369 + 21 398 -c- 19 428 + 18*
d 0.1 2 20.0 r 13.9 -r- 3.7%
Nore. The data are presented as means -C SEM of four to seven observations per dose. The doses refer to the total amount per 20 min. The measurements were taken 20 min after the termination of the infusion period at I = 40 min. *P < 0.01 (Mann-Whitney U test and f test, respectively).
Statistics
Statistical analysis of the heart rate data was carried out using (multivariate) analysis of variance ((M)ANOVA of SPSS\ PC + ). The ANOVA’s were followed by t tests. Behavioral data were analyzed with Kruskal-Wallis ANOVA and followed by the Mann-Whitney U test. A probability level of P 4 0.05 was taken as statistical significance for all tests. RESULTS Behavioral and Cardiac Effects of icv CRH Infusion: Relations
Dose-Response
Table 1 shows the behavioral and cardiac effects of a 20-min infusion of CRH (0, 30, 100, or 300 ng) into the lateral cerebral ventricle of the well habituated rat. The infusion of CRH resulted in a dose-response relationship for both behavioral activation and tachycardiac response. These responses persisted after the termination of the infusion and were maximal at t = 40 min. Therefore, only data at t = 40 min are shown. Analysis of variance of the mean heart rate at t = 40 min revealed a significant treatment effect (F(3, 15) = 4.97, P = 0.014), with a significant increment in heart rate for the highest dose of CRH (P < 0.01). Behavioral activity was increased by CRH infusion (P = 0.01; Kruskal-Wallis test), particularly by the dose 300 ng (P < 0.01). The Effects of Adrenalectomy and Corticosterone Administration CRH-Induced Behavioral and Cardiac Activation
on the
Figure 1 shows that administration of CRH (300 ng) caused a significant increase in behavioral activity in all animals before surgery (P = 0.002, Mann-Whitney U test). The preinjection behavioral activity was not af-
CRH AND STRESS RESPONSES
intact
379
ADX
:I!j---y
10 0
t= -5 40 -5 40 t= -5 40 -5 40 t= -5 40 vehicle CRH CRH vehicle CRH FIG. 1. tkhavioral activity (%) as measured before injection at t = -5 min and after icv injection of CRH (300 ng) at t = 40 min in intact, adrenalectomized (ADX). and ADX animals with corticosterone (CORT) replaccmcnt. Means + SEM from five to six rats per group.
fected by ADX and corticosterone, whereas ADX totally abolished the behavioral response to CRH. Replacement with corticosterone in the ADX animals restored the behavioral activity to values similar to those found in the intact animals (P = 0.07, Mann-Whitney U test). Figure 2 shows the increase in heart rate after injection of 300 ng CRH in the intact animals. This response persisted during the next 40 min. MANOVA of the heart rate data of CRH relative to vehicle-treated animals revealed a significant main effect of the peptide treatment (F( 1, 15) = 6.00, P = 0.027), but not of the period. CRH injection led to a significant increase in heart rate at t = 10, 15, 20, and 30 min (P Q 0.05). After ADX, injection of CRH failed to produce significant effects relative to vehicle-treated animals, whereas basal heart rate in ADX compared to intact animals showed a tendency to be elevated (41, 13) = 3.55, P = 0.08). Corticostcronc replacement in these ADX animals restored basal heart rate to a pre-ADX level. Furthermore, CRH administration led to an increase in heart rate which was almost comparable to the response seen in the pre-ADX condition. MANOVA revealed a significant effect of heart rate data of ADX animals with corticosterone replacement relative to vehicle treatment (F(1, 10) = 5.30, P = 0.044). Basal heart rate of ADX rats with corticosterone replacement was lower than without corticosterone treatment at t = - 10 min (P = 0.06) and t = - 5 min (P = 0.02). After injection of CRH in the corticosterone-pretreated ADX animals, heart rate remained lower relative to vehicle-treated ADX rats at t = 5, 10, and 20 min (P 6 0.05).
380
KORTE
1
intact
ET AL.
ADX
46or-----l
2420E 3i z 4002 B 2 380r P = 360-
-20
0
20 40 60 -20 0 20 40 6C time (min) time (mid FIG. 2. The effect of icv injection CRH (300 ng) on heart rate (beats/min) in intact rats, adrenalectomized (ADX), and ADX animals with CORT replacement. Means k SEM from five to six animals per group.
DISCUSSION The present study shows that behavioral activation after icv CRH treatment was associated with an increase in heart rate. CRH treatment produced neither an increase in heart rate nor behavioral activation in ADX animals. Corticosterone replacement in a dose known to restore basal circulating hormone levels and occupation of specific corticoid receptor sites in the hippocampus of the rat (De Kloet, 1991; Bohus and De Kloet, 1981) restored the CRH-induced behavioral response to preoperative levels, whereas the CRH-induced tachycardia was partially restored. The behavioral finding in the rats’ home cage is consistent with observations that icv administered CRH produces a dose-dependent increase in activity in a familiar situation (Koob and Bloom, 1985; Sutton, Koob, Le Moal, Rivier, and Vale, 1982). A remarkable property of the peptide is to cause a long-lasting behavioral activation. Sutton et al. (1982) found an activating effect of a single injection even after 3 hr. The neurobiological background of this long-lasting behavioral activation is not yet known. The present study shows that this behavioral activation was absent in ADX rats and could be restored by corticosterone replacement thereby suggesting that it is corticosterone dependent. ADX leads to an absence of feedback signal of corticosterone resulting in an enhancement of CRH immunostaining in hypothalamic paraven-
CRH AND STRESS RESPONSES
381
tricular nucleus (PVN), in the bed nucleus of the stria terminalis and in the central nucleus of the amygdala (Merchenthalcr, Vigh, Petrusz, and Schally, 1983). Furthermore, ADX leads to an increase in hybridizable CRH mRNA level in the neurons in the hypothalamic paraventricular nucleus (Kovacs and Makara, 1988). These results suggest an increased synthesis of the pcptide. Increased CRH levels do not result in a general down-regulation and desensitization of all CRH receptors. Therefore, the effects of ADX cannot be simply explained by changes in CRH receptors in the brain. CRH receptors in the anterior pituitary, but not in the intermediate lobe, hippocampus, amygdala, or lateral septum undergo down-regulation in conditions such as ADX and glucocorticoid administration (Wynn et al., 1985; Wynn, Hauger, Holmes, Millan, Catt, and Aguilera 1984; Hauger, Millan, Catt, and Aguilera, 1987; Hauger, Millan, Lorang, Harwood, and Aguilera, 1988). An alternative explanation for the higher heart rate in ADX animals may be the fact that pituitary adrenocorticotropic hormone (ACTH) synthesis and release is substantially increased 10 days after ADX (Buckingham and Hodges, 1974; Dallman, Demanincor, and Shinsako, 1974). It is known that the treatment with the ACTH-like peptide (ACTH4-,J leads to an increase in heart rate (Bohus, 1975). It is also plausible that corticoid receptors in both the periphery and the brain are involved. This is not necessarily in contradiction to the findings of Britton (1989), who suggested that the activation of the pituitary-adrenal axis is neither sufficient nor necessary for the expression of the behavioral effects of CRH in intact animals. This statement does not exclude the possibility that saturation of the mineralocorticoid receptors (MRs) and/or partly occupied glucocorticoid receptors (GRs) with corticosterone (Reul and DC Kloet, 1985) in ADX rats is a prerequisite for the complete behavioral and autonomic activation after CRH administration. Removal of endogenous glucocorticoids by ADX induces a brain-spccific increase in the binding capacity of the type MRs and GRs, an increase which can be blocked by corticosteronc administration (Chao, Choo, and McEwen, 1989). The observed corticosteronc dependency of CRH-induced responses may therefore be explained by an altered stress state in ADX animals due to changes in binding capacity of corticoid receptors. It cannot be excluded that CRH failed to increase heart rate in ADX animals because of the ceiling or near-ceiling effect of the high baseline heart rate in these animals. The elevated heart rate observed in the ADX group compared to intact controls is consistent with previous observations in ADX rats (Yagil, Koreen, and Krakoff, 1986). Our study suggests that the presence of corticosterone is a prerequisite for a normal heart rate. The mechanisms underlying higher baseline heart rate in ADX animals remain to be determined. The fact that replacement with dexamethasonc,
382
KORTE ET AL.
but not with aldosterone, normalizes heart rate in ADX rats suggests that GRs are involved (Yagil et al., 1986). Lower brain areas involved in the control of heart rate (e.g., nucleus tractus solitarius) are known to contain intracellular GRs (De Kloet, 1991). Also a direct peripheral action of glucocorticoid hormones on the heart cannot be excluded because glucocorticoid insufficiency induced by adrenalectomy has been found to impair cardiac performance (Bhaskar, Stith, Brackett, Wilson, Lerner, and Reddy, 1989). CRH induced elevations of heart rate are suggested to be mediated by enhanced cardiac sympathetic outflow and decreased baroreceptor-induced activation of cardiac vagal motor neurons (Fisher, 1989b; Overton, Davis-Gorman, and Fisher, 1990). The present study suggests that the cardiovascular action of CRH parallels the behavioral activity. It remains to be determined to what extent the tachycardia can be explained by a somatic-autonomic coupling or by a primary sympathetic activation by CRH. The substantial differences in the dose of CRH used in the present study (300 ng) and by others (10 pg) (Fisher, 1989b; Fisher, Rivier, Spiess, Vale, and Brown, 1982) do not allow rejection of the possibility that both mechanisms are operating but at different doses of CRH. In conclusion, icv CRH administration causes behavioral activation in association with elevations in heart rate. The question whether the heart response is at least in part related to the increase in behavioral activity will need further investigation. It is suggested that corticosterone is involved in CRH-induced cardiac as well as behavioral responses in ADX rats, In addition, a normal baseline heart rate was corticosterone dependent. Whether these are peripheral or central effects of corticosterone remains to be elucidated. Further investigations are needed to specify whether GRs or MRs are involved. ACKNOWLEDGMENTS This study was supported in part by the Foundation for Medical and Health Research (NWO/MEDIGON) (Grant 900-551-044).We thank Organon International, Oss, The Netherlands, for the gift of corticosterone.
REFERENCES Bhaskar, M., Stith, R. D., Brackett, D. J., Wilson, M. F., Lerner, M. R., and Reddy, Y. S. (1989). Changes in myocardial contractile protein ATPases in chronically adrenalectomized rats with and without glucocorticoid replacement. Biochem. Med. Mer. Bio/. 42, 118-124. Bohus. B. (1974). Telemetered heart rate responses of the rat during free and learned behavior. Biorelemefry 1, 193-201. Bohus, B. (1987). Limbic-midbrain mechanisms and behavioral physiology interactions with CRF. ACTH and adrenal hormones. In Hellhamm, D., Florin, I., and Weiner, H. (Eds.), Neurobiological approaches to human disease. pp. 267-285. Huber, Toronto. Bohus, B., and De Kloet. E. R. (1981). Adrenal steroids and extinction behavior antagonism
CRH AND STRESS RESPONSES
383
by progesterone, deoxycorticosterone, and dexamethasone of specific effect of corticosterone. Life Sci. 28, 433-440. Bohus, B., De Kloet, E. R., and Veldhuis, H. D. (1982). Adrenal steroids and behavior adaptation: relationship to brain corticoid receptors. In Ganten, D., and Pfaff, D. W. (Eds.), Current topics in neuroendocrinology. pp. 107-142. Springer Verlag, Berlin. Borreli, J., De Kioet, E. R., Versteeg, D. H. G., and Bohus, B. (1983). Inhibitory avoidance deficit following short-term adrenaiectomy in the rat: The role of adrenal catecholamines. Behav. Neural Biol. 39, 241-258. Britton, D. R. (1989). Stress-related behavioral effect of corticotropin-releasing factor. In Tacht, Y., Morley, J. E., and Brown, M. R. (Eds.), Neuropepfides and stress. pp. 3948. Springer-Verlag, New York. Buckingham, J. C., and Hodges, J. R. (1974). Interrelationships of pituitary and piasma corticosterone during adrenocorticai regeneration in rat. J. Endocrinol. 67, 411-417. Chao, H. M., Choo, P. H., and McEwen, B. S. (1989). Giucocorticoid and mineraiocorticoid 50, 365-371. receptor mRNA expression in rat brain. Neuroendocrinology Dallman, M. F., Demanincor, D., and Shinsako, J. (1974). Diminishing corticotrope capacity to release ACTH during sustained stimulation: The twenty-four hours after bilateral adrenaiectomy in the rat. Endocrinology 95, 65-73. De Kloet, E. R. (1991). Brain corticosteroid receptor balance and homeostatic control. Frontiers Neuroendocrinol. U(2), 95-164. De Souza, E. B. (198%). Localization and modulation of brain and pituitary receptors involved in stress responses. Psychopharmacol. Bull. 24(3), 360-364. De Souza, E. B., Insei, T. R., Perrin, M. H., Rivier. J., Vale, W. W., and Kuhar, M. J. (1985). Corticotropin-releasing factor receptors are widely distributed within the rat central nervous system: an autoradiographic study. J. Neurosci. 5, 3189-3203. Dunn, A. J., and Berridge, G. W. (1990). Physiological and behavioral responses to corticotropin-releasing factor administration: is CRF a mediator of anxiety or stress responses? Brain Res. 15, 71-100. Fisher, L. A. (1989a). Central autonomic modulation of cardiac baroreflex by corticotropinreleasing factor. Am. J. Physiol. 256 (Hearf Circ. Physiol. 25), H949-H955. Fisher, L. A. (1989b). Corticotropin-releasing factor: Endocrine and autonomic integration of responses to stress. Trends Pharmacol. Sci. 10, 189-193. Fisher, L. A., Rivier, J., Rivier, C., Spiess, J., Vale, W., and Brown, M. R. (1982). Corticotropin-releasing factor (CRF): Central effects on mean arterial pressure and heart rate in rats. Endocrinology 110, 2222-2224. Hauger, R. L., Miiian, M. A., Catt, K. J., and Aguilera, G. (1987). Differential regulation of brain and pituitary corticotropin-releasing factor receptors by corticosterone. Endocrinology 120, 1527-1533. Hauger, R. L., Miilan, M. A., Lorang, M., Harwood, J. P.. and Aguiiera, G. (1988). Corticotropin-reieasing factor receptors and pituitary adrenal responses during immobilization stress. Endocrinology 123, 396-405. Introini-Coilison, I. B., and McGaugh, J. L. (1991). Interaction of hormones and neurotransmitter systems in the modulation of memory storage. In Frederickson, R. C, A., McGaugh, J. L., and Feiten, D. L. (Eds.), Peripheral signaling of he brain: Role in neural-immune interactions, learning and memory, pp. 275-301. Hogrefe Rr Huber, Toronto. Koob. G. F., and Bloom, F. E. (1985). Corticotropin-releasing factor and behavior. Fed. Proc. 44, 259-263.
Korte, S. M., and Bohus, B. (1990). The effect of ipsapirone on behavioural and cardiac responses in the shock-probe/defensive burying test in male rats. Eur. J. Pharmacoi. 181, 307-310.
384
KORTE ET AL.
Kovacs, K. J., and Makara, G. B. (1988). Corticosterone and dexamethasone act at different brain sites to inhibit adrenalectomy-induced adrenocorticotropin hypersecretion. Brain Res. 474, 205-210. McEwen, B. S., De Kloet, E. R., and Rostene, W. (1986). Adrenal steroid receptors and actions in the nervous system. Physiol. Rev. 66, 1121-1188. Merchenthaler, I., Vigh, S., Petrusz, P., and Schally, A. V. (1983). The paraventriculoinfundibular corticotropin releasing factor pathway as revealed by immunocytochemistry in long-term hypophysectomized or adrenalectomized rats. Reg. Peptides 5, 295-305. Overton, J. M., Davis-German, G., and Fisher, L. A. (1990). Central nervous system cardiovascular actions of CRF in sinoaortic-denervated rats. Am. J. Physiol. 258 (Reg. Integrative Comp. Physiol. 27), R596-R601. Reul, J. M. H. M., and De Kloet, E. R. (1985). Two receptor systems for corticosterone in raf brain: Microdistribution and differential occupation. Endocrinology 117, 117123. Rivier, C., Brownstein, M., Spiess, J.. Rivier, J., and Vale, W. (1982). In vivo corticotropinreleasing factor-induced secretion of adrenocorticotropin, beta-endorphin, and corticosterone. Etzdocrinology 110, 272-278. Sawchenko, P. E., and Swanson, L. W. (1985). Localization, colocalization, and plasticity of corticotropin-releasing factor immunoreactivity in rat brain. Fed. Proc. U(l), 221227. Scheurink, A. J. W., St&fens, A. B., and Gaykema, R. P. A. (1990). Paraventricular hypothalamic adrenoceptors and energy metabolism in exercising rats. Am. J. Physiol. 259, R478-R484. Sutton, R. E., Koob, G. F., Le Meal, M., Rivier, J., and Vale, W. (1982). Corticotropin releasing factor produces behavioural activation in rats. Nature 297, 331-333. Wynn, P. C., Hauger, R. L., Holmes, M. C., Millan, M. A., Catt, K. J., and Aguilera, G. (1984). Brain and pituitary receptors for corticotropin releasing factor: Localization and differential regulation after adrenalectomy. Peptides 5, 1077-1084. Wynn, P. C., Harwood, J. R., Catt, D. J., and Aguilera, G. (1985). Regulation of corticotropin-releasing factor (CRF) receptors in the rat pituitary gland: Effects of adrenalectomy on CRF receptors and corticotroph responses. Endocrinology 116, 1653-1659. Yagil, Y., Koreen, R., and Krakoff, L. R. (1986). Role of mineralocorticoids and glucocorticoids in blood pressure regulation in normotensive rats. Am. J. Physiol. Z51(Hearr Circ. Physiol. 20), H1354-H1360.