Corticosterone, Prolactin, and Growth Hormone Responses to Handling and New Environment in the Rat*

Transcription

1 Corticosterone, Prolactin, and Growth Hormone Responses to Handling and New Environment in the Rat* GREGORY M. BROWN, MD, PHD, FRCP(C) AND JOSEPH B. MARTIN, MD, PHD, FRCP(C) Temporal characteristics of hormonal responses to handling and novel environment were studied in the rat in order to determine whether the same factors which modify corticosterone responses, i.e., time of day and type of stimulus, also modify prolactin and GH responses. Resting corticosterone showed the expected difference between crest and trough of the diurnal cycle. In contrast, GH and prolactin showed no difference at these times. Prolactin elevation occurred in response to the same stimuli which produced adrenal activation. Prolactin responses, however, differed from adrenal responses in being more rapid at the trough of the adrenal cycle and slower at the crest. In contrast to prolactin and corticosterone, GH showed a drop (nonsignificant) following stimulation. It is concluded that prolactin and corticosterone both respond to identical stimuli but that the prolactin response shows different characteristics from the adrenal response suggesting that different regulatory mechanisms are involved. INTRODUCTION Adrenal activation in the rat is known to occur in response to psychological influences. The corticosterone response to acute stimulation such as handling or exposure to novel environment is rapid in onset and short in duration. The pattern of the response, i.e., the magnitude of the response, the rate of increase in plasma levels, and the duration of the elevation, are each affected in a characteristic fashion by factors such as the time of the diurnal cycle, intensity and type of stimulus, and the history of exposure of the organism to stimulation (1-4). From the Department of Psychiatry, University of Toronto and the Neuroendocrinology Research Section, Clarke Institute of Psychiatry, Toronto, Canada, and the Department of Neurology, McGill University, and the Division of Neurology, Department of Medicine, Montreal General Hospital, Montreal, Canada. Presented in part at the annual meeting of the American Psychosomatic Society, Denver, April 6, Address for reprint requests: Dr. Gregory M. Brown, Head, Neuroendocrinology Research Section, Clarke Institute of Psychiatry, 250 College Street, Toronto, Ontario M5T 1R8. Received for publication September 7,1973; revision received December 10, Following the development of radioimmunoassays capable of measuring plasma levels of prolactin and growth hormone (GH), it was demonstrated that these hormones, which are not directly concerned in adrenal regulation, are also markedly responsive to physical or chemical stimulation in the rat. Thus plasma prolactin, which was formerly thought to be solely related to milk production and gonadal regulation, rises rapidly after ether administration (5-7), while plasma growth hormone, which was formerly thought to be only involved in growth, shows a precipitous fall following ether, hypertonic glucose, insulin-induced hypoglycemia, epinephrine, and cold exposure (8-11). Psychophysiologic influences such as noise and vibration, exposure to novel environment and auditory stress have also been shown to cause a drop in GH (10,12, 13), and novel environment produces a rise in prolactin (12). The present study was done to compare the temporal characteristics of the responses of these hormones to handling and novel environment in order to determine whether some of the same factors which modify corticosterone responses, Psychosomatic Medicine Vol. 36, No. 3 (May-June 1974) 241 Copyright 1974 by the American Psychosomatic Society, Inc. Published by American Elsevier Publishing Company, Inc.

2 GREGORY M. BROWN, MD, AND JOSEPH B. MARTIN, MD i.e., time of day and type of stimulus also affect prolactin and GH responses. MATERIALS AND METHODS Male wistar rats (250 gm) obtained from High Oak Ranch were housed singly in 9X7X13 in. cages at 25±1 C on a 12 hr/12 hr lighting cycle with rat chow and water available ad libitum for a minimum of three weeks prior to experimentation. During that time, one-half of the animals were in a room with the lights on from 11 p.m. to 11 a.m. and were subsequently stimulated at 11:30 a.m. while the rest were in a room with the lights on from 9 a.m. to 9 p.m. and were subsequently stimulated at 9:30 a.m. Stimuli used were either 5-sec handling ("reaction to handling") or 3-min exposure to a novel environment (placement in a 65-cm dia cardboard drum with sides 65-cm high and with an open top) (1,12). Groups of animals were sacrificed before stimulation and 1, 5, 10, and 15 min after stimulation. Previous detailed (4 hourly) studies of the diurnal corticosterone rhythm in our laboratory in rats, from the same supplier, have demonstrated that minimal corticosterone levels are found between 2 hr before and 2 hr after the lights are switched on while maximum levels occur 12 hr later (Seggie J, Shaw B, and Brown GM, unpublished observations). A maximum of eight animals were sacrificed in one room on one day in order to preclude a sequential sacrificing effect (1). No one was permitted to enter the experimental room during the 16 hr preceding stimulation. Animals were transported to a separate room and sacrificed by decapitation within 20 sec of removal of the cage from the home rack. Blood was collected in a heparinized centrifuge tube, centrifuged, and frozen until assay. Assay of corticosterone was done by the CBG method as described previously (14). Assay of prolactin and GH was by radioimmunoassay using the NIAMD kits and are reported in terms of NIAMD standards. Each experimental group (i.e., each data point in the curves) consisted of either seven or eight animals and corticosterone and prolactin data were examined by Student's t test. Because of the great variability of GH levels, this data was studied by the median test (15). RESULTS Resting corticosterone (Fig. 1) showed the expected diurnal differences between e 8 * /ml. 15 H ng/ml. S!jj 40 c 75 < 5.0 PLAS I' Fig L + 30 D + 30 L + 30 D + 30 L + 30 D + 30 In rats sacrificed 30 min after lights on (L + 30), corticosterone is significantly lower than in animals sacrificed 30 min after lights off (D + 30). In contrast, there is no significant difference in prolactin or GH. GH levels, however, are highly variable. In each bar, the mean value is represented by the horizontal bar. 242 Psychosomatic Medicine Vol. 36, No. 3 (May-June 1974)

3 HORMONAL RESPONSES IN THE RAT crest and trough (t=6.4, d/=14, p<0.01). In contrast, prolactin and GH levels showed no diurnal variation at the time studied (t=2.07), d/=13, p>0.05 and p>0.05, respectively). GH, unlike corticosterone and prolactin, was highly variable under the conditions of this experiment. Corticosterone responses to stimulation differed at crest and trough with the maximum response (increment from resting levels) occurring more rapidly at crest than at trough (Fig. 2). Three minutes of new environment produced a larger adrenal response than 5 sec of handling at crest of the adrenal cycle (t=2.5, d/=14, p<0.05). Responses to the two levels of stimulation were equivalent at trough (t=0.2,d/=14, NS). At crest of the adrenal cycle, 3 min of new environment produced a larger prolactin response than 5 sec of handling (t = 2.8, df- 12, p < 0.02), and the levels were still increasing at 15 min (Fig. 3). At trough, maximum prolactin responses occurred at 5 min, and there was no difference in the magnitude of response to the two levels of stimulation (t = 0.1, df = 12, NS). The magnitude of response to 3 min of new environment (highest level of prolactin reached) appears to be greater at crest than at trough, but this effect is not significant (t=1.57, d/= 11), although crest animals have not necessarily reached their maximum by 15 min. GH appeared to drop dramatically following stimulation (Fig. 4) but because of the high variability this apparent drop was not significant. Responses of all three hormones to 3 min of new environment are shown in Fig. 5. The adrenal and GH responses appeared to go in opposite directions and seem very similar in time course at both crest and trough of the adrenal cycle with GH drop somewhat preceding the corticosterone rise but as noted above, GH changes were not signifi- 5 Sec. Handling Y///A 3 Min. New Environment,f Crest \ T Fig. 2. The patterns of plasma corticosterone responses differ at crest and trough of the adrenal cycle and with 5 sec vs 3 min of stimulation. In this and subsequent figures, vertical lines indicate standard error and the hatched insert represents the 3 min of exposure to a new environment. Psychosomatic Medicine Vol. 36, No. 3 (May-June 1974) 243

4 GREGORY M. BROWN, MD, AND JOSEPH B. MARTIN, MD 5 Sec. Handling Fig. 3. Patterns of prolactin responses differ at crest and trough of the adrenal cycle, although there is no difference in the initial prestimulation values. 5 Sec Handling XI//A '///* 3Min - New Environment " Fig. 4. Plasma GH appears to drop after stimulation but the apparent drop is not significant as the GH levels are highly variable. DISCUSSION In confirmation of previous observa- tions (6), there is no significant difference in resting prolactin levels at crest and cant. Prolactin responses clearly differ in time course from adrenal responses. At crest, prolactin responses are slower than adrenal responses while at trough the opposite relationship is found. 244 Psychosomatic Medicine Vol. 36, No. 3 (May-June 1974)

5 HORMONAL RESPONSES IN THE RAT 5 Min. New Environment Y////A J CREST TROUCH i I * 1 20 I I Fig. 5. At crest of the adrenal cycle, the corticosterone response precedes the prolactin response; while at trough, the prolactin response precedes corticosterone. trough of the adrenal cycle, indicating that any diurnal rhythm in prolactin must differ from the adrenal rhythm. The high variability in GH, which only disappears when levels drop following stimulation, has also been reported previously (8,9). The present study confirms previous observations with respect to adrenal responses. The maximum level is reached more rapidly at crest than at trough of the adrenal cycle, while there is a greater magnitude of response at trough than at crest (1,4). Finally, there is a greater magnitude of response with 3 min of stimulation than with 5-sec stimulation at crest but not at trough, suggesting that the animals are less sensitive to stimulation at crest as they require a longer stimulus to produce a major response (1). Patterns in the present study are not absolutely identical to previous studies but this could be expected as many differences exist between these studies, e.g., our animals come from a different supplier, were housed singly and were sampled at slightly different times in the diurnal cycle. Like corticosterone, prolactin rises following stimulation. Prolactin, like corticosterone, shows a greater sensitivity to stimulation at trough of the adrenal cycle (1) in that 5-sec stimulation produced a response equivalent to that of 3-min stimulation, whereas at crest only longer stimulation produced a major response. It is of some interest that differential response of prolactin is not related to the initial prestimulation levels as unlike corticosterone these do not differ. Despite these similarities to corticosterone, prolactin responses show a clear-cut difference in time to corticosterone suggesting that prolactin and adrenal time courses are controlled independently. These differences between the adrenal and prolactin responses suggest that there are different regulating mechanisms acting via differ- Psychosomatic Medicine Vol. 36, No. 3 (May-June 1974) 245

6 ent releasing factors. Dunn and coworkers have reported a greater magnitude of prolactin responses to ether anesthesia at crest of the adrenal cycle (6). In the present study, there was a similar tendency following 3 min of new environment but it was not significant. It should be noted that a phase difference between prolactin and corticosterone cycles has been reported (6). A more complete understanding of the regulatory mechanisms will require a comparison of prolactin responses at crest and trough of the prolactin cycle. GH levels in this study are highly variable, so variable in fact, that the apparent drop in GH is not significant. This extreme variability has been observed in previous studies and appears to be characteristic of GH in the rat (8-10). The tendency (nonsignificant) of GH to drop following stimulation in this study is in keeping with previous reports showing an acute lowering of GH following physical or chemical stimulation (8-10). In conclusion, comparison of corticosterone and prolactin responses to handling and to novel environment at different times of the diurnal cycle demonstrate that (a) each of these hormones responds dramatically to both of these stimuli and (b) prolactin responses show different characteristics than adrenal responses suggesting that different regulatory mechanisms control prolactin and corticoster- SUMMARY It has recently been shown that circulating hormones not directly concerned in adrenal regulation also show acute changes in response to psychological stimulation in the rat. Thus prolactin, which was formerly thought to be involved only GREGORY M. BROWN, MD, AND JOSEPH B. MARTIN, MD in lactation and gonadal regulation, rapidly rises in response to stimulation, while growth hormone, formerly thought to be involved solely in growth, shows a precipitous fall. The present study was done to compare the temporal characteristics of the responses of these hormones to stimulation. Two different types of stimuli, 5 sec of handling and 3 min of new environment, were used and responses were examined at both peak and trough of the corticosterone rhythm. These conditions were chosen because the type of stimulus and the time in the diurnal cycle have each previously been shown to have differential effects on corticosterone responses. Singly housed animals were sacrificed by rapid decapitation. Corticosterone was assayed by the competitive proteinbinding method, and growth hormone and prolactin were measured by radioimmunoassay. Resting corticosterone showed the expected difference between peak and trough. In contrast, prolactin and GH levels showed no diurnal variation at the times studied. In confirmation of previous reports, corticosterone responses to stimulation differed at peak and trough with a more rapid response at peak (1-5 min) and slower response (15 min) of greater magnitude at trough. Three min of new environment produced a larger adrenal response than 5 sec of handling. Prolactin levels rose in response to the same stimuli causing adrenal activation. Prolactin responses were more rapid than adrenal responses at trough but were slower than adrenal responses at peak. The differences between the prolactin and adrenal responses were highly significant. In contrast to prolactin, GH showed a drop (nonsignificant) following stimulation. GH responses in each instance appeared to be the mirror image of the corresponding adrenal response, al- 246 Psychosomatic Medicine Vol. 36, No. 3 (May-June 1974)

7 HORMONAL RESPONSES IN THE RAT though the GH response consistently pre- The authors wish to acknowledge the ceded the corticosterone change. assistance of Mrs. /. Chambers, Mrs. S. In conclusion, these studies demon- Kirpalani, and Mr. R. Rodman. This work strate that prolactin and corticosterone re- was supported in part by MRC grant MA spond to the same stimuli but that prolac and MRC MA Materials for tin responses differ in magnitude and tern- GH and prolactin radioimmunoassay poral characteristics from corticosterone were kindly provided by the NIAMD. responses indicating that separate mechanisms control the secretion of these hormones. REFERENCES 1. Ader R, Freidman SB: Plasma corticosterone response to environmental stimulation: effects of duration of stimulation and the 24-hour adrenocortical rhythm. Neuroendocrinology 3: , Friedman SB, Ader R: Adrenocortical response to novelty and noxious stimulation. Neuroendocrinology 2: , Zimmerman E, Critchlow V: Effects of diurnal variation in plasma corticosterone levels on adrenocortical response to stress. Proc Soc Exp Biol Med 125: , Dunn, J, Scheving L, Millet P: Circadian variation in stress-evoked increases in plasma corticosterone. Amer J Physiol 223: , Neill JP: Effect of stress on serum prolactin and luteinizing hormone levels during the estrous cycle of the rat. Endocrinology 87: , Dunn JP, Arimura A, Scheving LE: Effect of stress on circadian periodicity in serum LH and prolactin concentration. Endocrinology 90:29-33, Ajika K, Kalra SP, Fawcett CP, Krulich L, McCann SM: The effects of stress and nembutal on plasma levels of gonadotropins and prolactin in ovariectomized rats. Endocrinology 90: , Schalch DS, Reichlin S: Plasma growth hormone concentration in the rat determined by radioimmunossay: influence of sex, pregnancy, lactation, anesthesia, hypophysectomy and extrasellar pituitary transplants. Endocrinology 79: , Takahashi K, Daughaday WH, Kipnis DM: Regulation of immunoreactive growth hormone secretion in male rats. Endocrinology 88: , Kokka N, Garcia JF, George R, Elliott, HW: Growth hormone and ACTH secretion: evidence for an inverse relationship in rats. Endocrinology 90: , Brown GM, Reichlin S: Psychologic and neural regulation of growth hormone secretion. Psychosom Med 34:45-61, Brown GM, Uhlir IV, Seggie J, Schally AV, Kastin AJ: Effect of septal lesions on plasma levels of MSH, corticosterone, GH and prolactin before and after exposure to novel environment: role of MSH in the septal syndrome. Endocrinology 94: , Collu R, Jequier, JC, Letarte J, Leboeuf G, Ducharme JR: Effect of stress and hypothalamic deafferentation on the secretion of growth hormone in the rat. Neuroendocrinology 11: , Seggie J, Brown GM: Septal lesions and resting adrenal function. A possible explanation of conflicting findings. Neuroendocrinology 8: , Siegel S: Nonparametric Statistics for the Behavioral Sciences. New York, McGraw-Hill, 1956, pp Psychosomatic Medicine Vol. 36, No. 3 (May-June 1974) 247