PRL and ACTH secretion following acute heat exposure, in intact and in hypothalamic deafferentated male rats

Brain Research, 178 (1979) 459-466 © Elsevier/North-Holland Biomedical Press

459

PRL AND ACTH SECRETION FOLLOWING ACUTE HEAT EXPOSURE, IN INTACT AND IN HYPOTHALAMIC DEAFFERENTATED MALE RATS

R. A. SIEGEL, S. FELDMAN, N. CONFORTI, M. BEN-DAVID and I. CHOWERS

Laboratories of Experimental Endocrinology and Experimental Neurology, Departments of Neurology and Pharmacology, Hadassah University Hospital and Hebrew University-Hadassah Medical School, Jerusalem (Israel) (Accepted March 22nd, 1979)

Key words: PRL - - ACTH - - heat stress

SUMMARY

Adult male rats, intact or bearing complete, anterior or posterior hypothalamic deafferentiations (CHD, AHD and PHD, respectively) were acutely exposed to environmental temperature of 36 °C, and serum PRL and ACTH concentrations were determined by RIA. In intact animals, heat exposure resulted in elevated serum PRL and ACTH levels. None of the deafferentations affected basal serum PRL concentrations, whereas those of ACTH were elevated in both CHD and AHD, but not in PHD groups, as compared to intact controls. The PRL heat response was completely absent in CHD, attenuated in AHD, and delayed in PHD animals, and the ACTH heat response was absent in all three groups. These results demonstrate (1) that acute exposure to elevated environmental temperature stimulates secretion of PRL and of ACTH; (2) that this stimulation is carried out by diverse neural pathways; and, (3) that hypothalamic modulation of the secretion of PRL and ACTH is effected by independent mechanisms.

INTRODUCTION

While the lactogenic properties of prolactin (PRL) are well established, it has recently become apparent that this hormone may possess additional effects. Its responsiveness to a wide spectrum of sensory stimuli suggests that it may comprise an integral part of the general stress response, along with the hypothalamo-hypophysealadrenal (HHA) axisl,Z2,z4. Furthermore, demonstrations of the sensitivity of PRL to central osmotic stimuli3, and of its renotrophic properties 18, raise the possibility of a functiofial interrelationship between it and the thermoregulatory system. The HHA axis is involved in thermoregulation. The efficiency with which body temperature is maintained during exposure to severe environmental conditions depends

460 upon circulating glucocorticoids4,6. Corticosterone secretion is stimulated during heat exposure 5 and also alterations in the temperature of the temperature-regulating centers (TRCs) of the CNS 7. While these changes in glucocorticoid secretion have been assumed to be mediated by adenohypophyseal corticotrophin (ACTH), this has not been proven. Extra-hypothalamic modulation of both basal and heat-induced secretion of these two hormones is incompletely understood. Basal serum PRL concentrations seem to be maintained in the absence of neural input to the medial basal hypothalamus (MBH)Z°, 29, whereas in the hypothalamic deafferentated animal the HHA axis may be hyperactive~z. During exposure to elevated environmetal temperature, thermosensitive neurons located in the preoptic area (POA) and in the midbrain are activated14,15, and afferents from these to the hypothalamus permit the various thermoregulatory responses to be elicited 11. The anatomical and functional connections between these TRCs and the prolactin-inhibiting-factor (PIF), prolactin-releasingfactor (PRF), and cortico-trophin-releasing-factor (CRF) neurons of the MBH are unknown, however. The aims of the present experiments were (1) to describe the PRL and ACTH responses to acute exposure to elevated environmental temperature; (2) to determine which afferent neural pathways to the MBH are responsible for maintaining basal PRL and ACTH secretion, and for their responses to heat exposure. To this end we have estimated serum PRL and ACTH concentrations, in intact and in variously hypothalamic deafferentated male rats, in thermoneutral conditions and following acute exposure to environmental temperature of 36 °C. MATERIALS AND METHODS The experiments were carried out on male rats of the Hebrew University strain, weighing 220-260 g. They were housed in the animal room of our laboratory in groups of 5-6 per cage, under artificial illumination between 06.00 and 18.00 h, and were given Purina Chow and water ad libitum. The temperature of the animal room was 24 °C, and relative humidity (RH) was 55 ~. Hypothalamic deafferentation was performed according to the method of Halasz and Pupp 12, with minor modifications. The dimensions of the knife were: radius, 1.4 mm; height, 2.4 ram. Three types of deafferentation were performed. (i) Anterior hypothalamic deafferentation (AHD): a semicircular cut is made around the rostral hypothalamus, at the level of the posterior border of the optic chiasm, such that the POA, and the suprachiasmatic and paraventricular nuclei are functionally separated from the MBH. (2) Posterior hypothalamic deafferentation (PHD): in this preparation a semicircular cut is made around the caudal extreme of the hypothalamus. The cut is positioned just posterior to the mammillary nuclei. (3) Complete hypothalamic deafferentation (CHD): in this preparation the MBH is completely isolated from the remainder of the CNS. This is achieved by joining the anterior and posterior deafferentations with two cuts in the rostral-caudal direction. The location of these longitudinal cuts is 1.4 mm lateral to the midline. Functional integrity of the MBH was verified one week postoperatively by

461 examining the plasma corticosterone response to ether stress t°. Only animals which responded adequately were used in the subsequent experiment. One day prior to their being used in an experiment the rats were transferred to individual cages. All experiments were performed between 09.00 and 11.00 h. Heat exposure consisted of placing the animals, for periods of either 10 min or 30 min duration, in a climatic chamber (Hotpack) maintained at 36 °C and 37-42 ~ RH. Immediately upon termination of a given exposure period, the rats were decapitated. Trunk blood was collected for RIA, and brains were stored for histological verification of the accuracy and completeness of the deafferentation. Data obtained from animals in which the operation was deemed unsatisfactory were rejected. Control animals were simply removed from their cages and sacrificed in an identical manner; we have found that placing the animals in the climatic chamber at 24 °C has no significant effect on either serum PRL or ACTH levels. Serum PRL was determined using RIA materials supplied by NIH. Serum ACTH concentrations were determined using a RIA kit purchased from CIS (France). The data were analyzed using a one-way analysis of variance and, where significant effects were observed, a more precise inspection was carried out by means of the Scheffe multiple range test. Logarithmic (In) transformations of the raw data were tested, in order to achieve homogeneity of variance. RESULTS (1) Prolactin. Serum PRL concentrations (ng/ml) in the various experimental groups are presented in Table I (A) and the analysis of variance and Scheffe test on this data are shown in Table I (B). Basal serum PRL concentrations in the PHD group tended to be elevated, but the difference between this value and those determined in N, CHD and AHD animals did not acheive significance (0.05 < P < 0.10). In the N group, heat exposure led to elevated serum PRL levels (P < 0.001). The Scheffe test demonstrated that both the 10 min and the 30 min groups differed significantly from the C group, but were not different one from the other. In CHD animals, no PRL response whatsoever to heat exposure was observed. In the AHD group, exposure to elevated environmental temperature significantly affected serum PRL concentrations (P < 0.001). The Scheffe test demonstrated no significant difference in the effects of 10 min or 30 min of exposure in these animals, but did show that the effects of both of these exposures were significantly less marked in AHD than in N rats. Heat exposure also caused a marked elevation in serum PRL levels in PHD animals (P < 0.001), and the Scheffe test demonstrated significant differences between the effects of 10 min and 30 min of exposure. Furthermore, serum PRL concentrations in PHD rats following 10 min of exposure were intermediate between those found in N animals and in CHD animals, but at 30 min were not significantly different from those found in the N group.

462 TABLE IA Serum P R L concentrations (ng/ml) at 24 °C, andJbllowing 10 min and 30 rain o f exposure to 36 ~C, in intact rats and in rats with hypothalamic dea~rerentations

Results are expressed as arithmetic means ± standard errors. N, intact animals; CHD, complete hypothalamic deafferentation; AHD, anterior hypothalamic deafferentation; PHD, posterior hypothalamic deafferentation. Numbers in brackets refer to the number of animals tested. 36 °C; 37-42 % RH

C (controls) 24 C

N CHD AHD PHD

31 j_ 3(31) 34i5(8) 29:~2(17) 44 F 4 (8)

10 rain exposure

30 rain exposure

161 33 75 100

197 ± 13(36) 3 0 ± 2 (15) 82±8 (19) 148 :L 4 (7)

~: 10(18) ± 2 (6) & 10(10) ± 17 (7)

TABLE [B Analysis o f variance and Scheffe multiple range test on logarithmic (In) transJbrmations Of serum PRL concentrations presented in Table 1,4

N, intact animals; CHD, complete hypothalamic deafferentation; AHD, anterior hypothalamic deafferentation; PHD, posterior hypothalamic deafferentation. C, controls - - 24 c'C; 10 rain, 36 °C --10 rain exposure; 30 min, 36 °C - - 30 rain exposure. Analysis o f variance

N CHD AHD PHD C 10 min 30 min

Scheffe

source

df

F

P ,~

P ~< 0.05

Heat exposure Heat exposure Heat exposure Heat exposure Operation Operation Operation

2 2 2 2 3 3 3

89 0.3 19 25 3.0 24 41

0.001 NS 0.001 0.001 NS 0.001 0.001

C vs (10 min and 30 min) NS C vs (10 min and 30 min) C vs 10 rain vs 30 rain NS N vs CHD vs (AHD and PHD) (N and PHD) vs C H D vs A H D

(2) A CTH. Serum ACTH concentrations (pg/ml), and their analyses of variance and Scheffe comparisons, are presented in Tables II (A) and II (B) respectively. Basal serum ACTH levels were significantly affected by the various hypothalamic deafferentations (P < 0.001). The Scheffe test demonstrated that both complete and anterior deafferentations resulted in resting serum ACTH concentrations which were higher than those found in both the N and the P H D groups. In the N animals, exposure to elevated environmental temperature led to elevated serum ACTH concentrations (P -< 0.001), such that values obtained at both 10 min and 30 min were significantly different from those of controls (Scheffe). In neither C H D nor A H D animals were control serum levels of this hormone altered upon exposure to 36 °C. There was a tendency towards a decrease in this parameter in C H D rats; however this difference did not acheive significance. Similarly, in PHD animals the H H A response to thermal stress was blocked, such that no significant change in serum ACTH concentrations occurred upon heat exposure.

463 TABLE IIA Serum A C T H concentrations (pg/ml) at 24 °C, and following 10 min and 30 min o f exposure to 36 °C in intact rats and in rats with hypothalamic deafferentations

Abbreviations as in Table IA. C (controls) 24 °C

N CHD AHD PttD

101 194 187 115

d: I i 4-

36 °C; 37-42% R H

11 (44) 24 (36) 29 (16) 18 (15)

10 min exposure

30 min exposure

181 4- 24(15) --99 -4- 14 (8)

246 134 183 148

:t: 27 (23) ± 38 (9) i 42 (9) 4- 25 (8)

TABLE IIB Analysis o f variance and Scheffe multiple range test on logarithmic (In) transformations o f serum A C T H concentrations presented in Table IIA

Abbreviations as in Table IB. Analysis o f variance

N CHD AHD PHI) C 10 min 30 min

Scheffe

source

df

F

P <

P < 0.05

Heat exposure Heat exposure Heat exposure Heat exposure Operation Operation Operation

2 1 1 2 3 1 3

19 2.0 0.4 0.9 5.9 6.1 2.8

0.001 NS NS NS 0.001 0.02 0.05

C vs (10 min and 30 min) NS NS NS (N and PHD) vs (CHD and AHD) N vs PHD NS

DISCUSSION I n t a c t rats r e s p o n d e d to acute e x p o s u r e to elevated e n v i r o n m e n t a l t e m p e r a t u r e with a m a r k e d elevation in P R L secretion, as witnessed b y the greater t h a n 6-fold rise in s e r u m c o n c e n t r a t i o n s o f this h o r m o n e . T h e response was r a p i d , e s t i m a t i o n s at 10 rain a l m o s t as great as those at 30 min, a n d the r a p i d i t y is t h u s similar to r e p o r t e d P R L responses to v a r i o u s o t h e r stresses21, 30. I n t a c t a n i m a l s also d e m o n s t r a t e d elevated s e r u m A C T H c o n c e n t r a t i o n s following h e a t e x p o s u r e ; this is a characteristic A C T H stress response 27,3t. T h e p r e s e n t d a t a c o r r e l a t e well with serum c o r t i c o s t e r o n e levels d u r i n g similar exposures 5, p r o v i d i n g evidence for a causal r e l a t i o n s h i p between the secretion o f these two h o r m o n e s , u p o n h e a t exposure. I n C H D a n d A H D animals, b a s a l s e r u m P R L c o n c e n t r a t i o n s were n o r m a l , a n d the elevation in this p a r a m e t e r f o u n d in P H D rats d i d n o t achieve significance. These d a t a s u p p o r t previous reportsZ0, 29, a n d suggest t h a t the P I F - - a n d P R F - - secreting n e u r o n s o f the M B H r e m a i n f u n c t i o n a l l y intact in the absence o f n e u r a l i n p u t f r o m

464 extra-hypothalamic sites. Since in both C H D and A H D preparations MBH DA content is normal, whereas that of noradrenaline (NA) is reduced 2, the present results support the hypothesis of a dominant role for DA in the tonic control of PRL release. In contrast to their lack of effect upon basal PRL secretion, both complete and anterior hypothalamic deafferentations resulted in elevated serum A C T H concentrations in resting animals. These data support previous reports in which adenohypophyseal A C T H 1~ and plasma corticosterone 29 were the dependant variables. The present results demonstrate the existence of neural fibers, entering the MBH from the rostral direction, which tonically dampen the activity of the H H A axis. In view of the fact that these fibers are largely noradrenergic 2, our data support the hypothesis of tonic noradrenergic inhibition of the axis, at the level of the MBH zs. In PHD animals, in contrast thereto, no change in basal ACTH secretion was observed. Since MBH serotoninergic activity is interfered with in this preparation 25, these findings underline the lack of a role for hypothalamic 5-HT in the tonic regulation of the H H A axis. Complete hypothalamic deafferentation resulted in a total inhibition of the normal PRL heat response, and this is compatible with a previous report regarding a different sensory stimulus '~0.Our data underline the completeness of the neural isolation of the MBH in this preparation; they also demonstrate that, in contrast to findings regarding the hypothalamo.hypophyseal-thyroid axis, extra-hypothalamic modulation of PRL secretion is not effected by the transfer of bioactive substances to the MBH via the CSF. In animals with A H D only, the PRL response to exposure to 36 °C was attenuated, and in P H D animals a delay was apparent. These data demonstrate that this reponse is elicited by diverse nervous pathways entering the MBH from both the rostral and the caudal directions. Both the POA and the midbrain contain elements which are sensitive to environmental temperatureg, 14. Those of the midbrain are to a large extent serotoninergicS, 32 and they therefore impinge upon the MBH from the caudal direction2L The TRC of the POA, on the other hand, is directly linked with the MBH via nerve fibers which arrive at the latter tissue from the rostral direction 26. Our results therefore suggest that thermosensitive neurons of both the POA and the midbrain are responsible for stimulating PRL secretion during acute heat exposure. In addition to this neural drive, the aforesaid PRL response may be partially due to nonspecific stress stimulation, transmitted to the PIF and/or PRF neurons of the MBH via the medial forebrain bundle. The total blockade of the normal A C T H response to 30 min of exposure to elevated environmental temperature which we found in C H D animals supports a previous report in which the estimated parameter was plasma corticosterone concentrations 5. Both anterior and posterior hypothalamic deafferentations resulted in complete abolition of the A C T H heat response, demonstrating the participation of neural pathways impinging upon the MBH from both the rostral and the caudal directions. It is logical to consider that the extra-hypothalamic modulation of the H H A axis, during heat exposure, is similar to that described above vis-a-vis PRL. A distinct difference between the manner in which this modulation affects the CRF neurons and that in which it influences the PIF-PRF neurons emerges, however. This

465 is attested to by the observations that partial interruption (by either anterior or posterior deafferentation) of the neural input to the C R F neurons completely blocked the normal response of these cells, whereas the same partial interruption led to a partial, and not a total, inhibition of the P R L response. The neurophysiological mechanism underlying this interesting distinction remains to be investigated. One plausible explanation involves the existence of both a releasing and an inhibiting neurohormone mediating PRL secretion, whereas hypothalamic control of A C T H dynamics is carried out by a releasing hormone only. In view of the numerous instances of parellel changes in the secretion of PRL and ACTH, it has been suggested that the hypothalamic control of their secretion from the adenohypophysis may be effected by common mechanisms 16. Our present data, which demonstrate a number of instances in which changes in the secretion of these two polypeptides are not parallel, repudiate this hypothesis. Basal secretion rates of P R L and of ACTH, in both C H D and A H D animals, as compared to intact, illustrate this point. Furthermore, the separation of effect of both anterior and posterior deafferentation on their release following acute exposure to elevated environmental temperature underlines the independence of the hypothalamic systems modulating PRL and A C T H release, during stress. This paper has described the changes in PRL and A C T H secretion taking place upon exposure to elevated environmental temperature, and has demonstrated the anatomical location of neural pathways effecting these responses. Furthermore, the independence of the hypothalamic mechanisms modulating their secretion has been illustrated. ACKNOWLEDGEMENTS The technical assistance of Ms. N. Bergman and A. Izik is appreciated, as is the generous supply of RIA materials by Dr. A. F. Parlow of N I A M D D . Supported by Agreement no. 5 from the United States-Israel Binational Science Foundation.

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