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Pulsatile growth hormone secretion: inhibitory role of medial preoptic area
240
Brain Research, 148 (1978) 240 -244 (~3Elsevier/North-Holland Biomedical Press
Pulsatile growth hormone secretion: inhibitory role of medial preoptic area
JOHN O. WILLOUGHBY* and JOSEPH B. MARTIN Division of Neurology, Montreal General Hospital and Montreal Neurological Institute, Montreal, Quebec (Canada)
(Accepted December 15th, 1977)
Many neural structures have been implicated in regulating growth hormone (GH) secretion 1°. The ventromedial nucleus (VMH) and arcuate (ARC) region of the hypothalamus have been shown to be facilitatory to G H secretion in both electrolytic lesion6,1°,12 and stimulation studies 7A°,11. Several groups have confirmed the importance of VMH and ARC using the Halasz deafferentation technique 1°,13,14,19. Animals with complete deafferentation maintain or have augmented G H secretion, suggesting that the medial basal hypothalamus can generate G H secretion, probably through an intrinsic G H releasing factor (GRF) systemlL There is more recent evidence that the medial preoptic area (MPOA) is inhibitory to G H secretion. Thus (a) electrical stimulation of the MPOA in anaesthetized rats reduces r G H plasma concentrations 1°, (b) somatostatin has been identified immunohistologically in the MPOA and the anterior periventricular area of several species 1,9, and (c) ablation of the preoptic area results in stress augmentation of rGH levels, rather than suppression 1~. Finally earlier studies by MitchelP 3,~4 and co-workers, in which selective anterior hypothalamic deafferentation produced increased growth in rats, also raised the possibility that an anterior structure could inhibit rGH secretion. In undisturbed male rats, r G H secretion is characterized by rhythmic secretory bursts of r G H every 3.3 h, separated by intervals when r G H is unmeasurable ls,tg. Troughs between r G H bursts are thought to result from somatostatin secretion s (and Terry and Martin, unpublished), and the MPOA, which is the source of somatostatin in the medial eminence 5, might therefore be implicated in r G H regulation in the unstressed animal. The following study examined this possibility. Male albino Sprague-Dawley rats (290-310 g) were pentobarbitone anaesthetized and single mid-line 40-60 mC anodal current lesions were placed in the MPOAsuprachiasmatic region, using an insulated platinum electrode, with 0.5 mm exposed at the tip. De Groot co-ordinators of the lesions were anterior --7.40, lateral 0.00, and depth --2.50. Control animals were subjected to electrode insertion but no current was passed. * Present address and address for reprints: Department of Medicine (Neurology), The Flinders University of South Australia, Bedford Park, South Australia 5042.
241 400
~
MPOA 12-1 300 200 100 0
"1-
'
300 200' 100 O~ ;;; 1000 1100 1200 1300
1400 1500
1600
TIME-hr Fig. 1. Six-hour rGH profiles from two control animals. Normal, characteristic rGH secretory patterns
are evident: prominent surges of rGH secretion occur approximately every 3.3 h, and commence followingintervals when rGH is unmeasurable. Surviving animals were prepared with chronic indwelling right atrial cannulae after the method of Brown and Hedge 2, and were housed in isolation in blood sampling boxes, arranged so that the cannula opening on the rats' head could be connected to tubing leading to the outside of the box. This system permitted withdrawal of blood samples without disturbing the animals 12. After recovery from the cannulation procedure, blood samples (0.4 ml) were removed every 15 min for 6 h, and the plasma and red cells separated by centrifugation. The plasma (0.2 ml) was frozen for subsequent assay, and the red cells were resuspended in 0.2 ml physiological saline and reinjected into the rat after removal of the next sample. Plasma rGH concentrations were assayed using materials provided by NIAMDD, using a double antibody technique. Six hour rGH profiles were constructed for lesioned and control rats and compared. All rats were subjected to formalin perfusion under anaesthesia; the brains were removed, fixed and subsequently sectioned at 40 #m, to determine accuracy of placement of lesions. Sections were stained with thionin and cresyl violet. Results. rGH profiles were altered in rats with MPOA lesions (n ----6) compared to control animals (n ----9). Defining interpeak intervals arbitrarily as the average time between successive rGH bursts that exceeded 200 ng/ml, lesioned animals had more frequent bursts of rGH secretion than controls, every 2.11 ± 0.74 vs. 3.63 ± 0.40 h, respectively (P < 0.001), and during troughs rGH values also tended to remain elevated. Fig. 1 illustrates a typical rGH secretory profile from control animals, and Fig. 2 shows profiles from 6 rats with MPOA lesions, together with a diagrammatic illustration of each lesion. Histological recontruction of lesions showed that most of the MPOA was destroyed in all animals, together with the suprachiasmatic nuclei and part of the anterior hypothalamic area in some (see Fig. 2).
242 Growth Hormone Profiles in Rats with MPOA Lesions 400"
MPOA
~
A Z4 AC
'°°1 MPOA
7`8
V SCN I00. I0
O.
I'~11 MPOAh 3-2
0 I
A 7`4
!
2
400
Mo.
,,.,
200
100,
-
A7.6
MPOA
3O0
300
2Oo
200
I00
100
~oo .oo i:,oo
i ~ o ~40o ~soo ~6oo TIME - hr
ct
Iooo
A 7,4
i10o ~2b0 13oo ~4bo isbo
~,bo
TIME - hr
Fig. 2. Six-hour rGH profiles from 6 animals with MPOA lesions. The location and maximal crosssectional area of the lesions are illustrated with each profile. More frequent surges of rGH secretion are evident, with a tendency for rGH values to remain elevated in trough periods. A, anterior coordinate of de Groot; AC, anterior commissure; V, ventricle: SCN, suprachiasmatic nucleus; OC, optic chiasm. Thus, changes produced in the pattern of pulsatile r G H secretion by MPOA lesions suggest that the MPOA is inhibitory to r G H secretion in the intact non-stressed animal and, specifically, that it is implicated in producing troughs between consecutive r G H secretory episodes. Surgical isolation of the MBH has previously been shown to be capable of generating augmented pulsatile r G H secretion, similar in pattern to r G H profiles in rats with MPOA lesions 19. It could be postulated, therefore, that the MPOA rhythmically inhibits a spontaneously discharging G R F system in the MBH, and so produces the characteristic 3.3-hourly pulsatile r G H rhythm (Fig. 1). In view of studies showing the importance of somatostatin in regulating physiological r G H secretion, it is more likely that the MPOA rhythmically secretes somatostatin into the pituitary portal circulation to inhibit r G H release. Both direct MPOA-median eminence and MPOA-VMH projections have been shown electrophysiologically4,1s and by autoradiography ~, so either route, or both, might mediate rhythmic inhibition of r G H secretion by MPOA. Because MPOA-regulated gonadotropin secretory patterns specific for males and females are the consequence of differences in early MPOA maturation, it is possible that sexual differences in r G H rhythms 17 also result from differences in MPOA development. Whether the MPOA is the only determinant of periodicity of the r G H rhythm, or is in turn influenced by other neural structures, is unresolved. An inhibitory
243 noradrenergic projection, from b r a i n stem to a somatostatinergic n e u r o n a l system in M P O A , has recently been postulated to be stimulatory for r G H rhythmic secretion 2°. Ms. Judy A u d e t provided c o m p e t e n t technical assistance a n d Mrs Jane G i a c o b b e typed the manuscript. D r A l b e r t Parlow kindly provided materials for r a d i o - i m m u n o assay of r G H . J o h n O. W i l l o u g h b y was a Fellow of the Medical Research C o u n c i l of C a n a d a , a n d Joseph B. M a r t i n is a Medical Research Council Associate. The study was supported by grants from the Medical Research Council of C a n a d a .
1 Alpert, L. C., Brawer, J. R., Patel, Y. C. and Reichlin, S., Somatostatinergic neurons in the anterior hypothalamus: immunohistochemicallocalization, Endocrinology, 98 (1976) 255-258. 2 Brown, M. R. and Hedge, G. A., Thyroid secretion in the anaesthetised, stress-free rat and its suppression by pentobarbital, Neuroendocrinology, 9 (1972) 158-174. 3 Conrad, L. C. A. and Pfaff, D. W., Efferents from medial basal forebrain and hypothalamus in the rat. II. An autoradiographic study of the anterior hypothalamus, J. comp. Neurol., 169 (1976) 221-161. 4 Dyer, R. G. and Cross, B. A., Antidromic identification of units in the preoptic and anterior hypothalamic areas projecting directly to the ventromedial and arcuate nuclei, Brain Research, 43 (1972) 254-258. 5 Epelbaum, J., Willoughby, J. O., Brazeau, P. and Martin, J. B., Effects of brain lesions and hypothalamic deafferentation on somatostatin distribution in the rat brain, Endocrinology, 101 (1977) 1495-1502. 6 Frohman, L. A. and Bernardis, L. L., Growth hormone and insulin levels in weanling rats with ventromedial hypothalamic lesions, Endocrinology, 82 (1968) 1125-1132. 7 Frohman, L. A., Bernardis, L. L. and Kant, K., Hypothalamic stimulation of growth hormone secretion, Science, 162 (1968) 580-582. 8 Ferland, L., Labrie, F., Jobin, M., Arimura, A. and Schally, A. V., Physiologicalrole ofsomatostatin in the control of growth hormone and thyrotropin secretion, Biochem. biophys. Res. Commun., 68 (1976) 149-156. 9 H~ikfeit, T., Efendic, E. S., HellmstrOm, C., Johansson, O., Luft, R. and Arimura, A., Cellular localization of somatostatin in endocrine-likecells and neurons of the rat with special references to the A cells of the pancreatic islets and to the hypothalamus, Acta endocr. (Kbh.), 80, Suppl. 200 (1975) 1-41. 10 Martin,J.B.,Brainregulationofgrowthhormonesecretion.InL. MartiniandW. F. Ganong(Eds.), Frontiers in Neuroendocrinology, Vol. 4, Raven Press, New York, 1976, pp. 129-168. 11 Martin, J. B., Plasma growth hormone response to hypothalamic or extra hypothalamic electrical stimulation, Endocrinology, 91 (1972) 107-115. 12 Martin, J. B., Renaud, L. P. and Brazeau, P., Pulsatile growth hormone secretion: suppression by hypothalamic ventromedial lesions and by long acting somatostatin, Science, 186 (1974) 538-540. 13 Mitchell, J. A., Hutchins, M., Schindler, W. J. and Critchlow, V., Increases in plasma growth hormone concentration and naso-anal length in rats following isolation of the medial basal hypothalamus, Neuroendocrinology, 12 (1973) 161-173. 14 Mitchell, J. A., Smyrl, R., Hutchins, M., Schindler, W. J. and Critchlow, V., Plasma growth hormone levels in rats with increased naso-anal lengths due to hypothalamic surgery, Neuroendocrinology, 10 (1972) 31-45. 15 Renaud, L. P., Influence of medial preoptic-anterior hypothalamic area stimulation on the excitability of mediobasal hypothalamic neurones in the rat, J. Physiol. (Lond.), 264 (1977) 541-564. 16 Rice, R. W. and Critchlow, V., Extrahypothalamic control of stress-induced inhibition of growth hormone secretion in the rat, Endocrinology, 99 (1976) 970-976. 17 Terry, L. C., Saunders, A., Audet, J., Willoughby, J. O., Brazeau, P. and Martin, J. B., Physiologic secretion of growth hormone and prolactin in male and female rats, Clin. Endocrinol., 6 (1977) 19s-28s. 18 Tannenbaum, G. S. and Martin, J. B., Evidence for an endogenous ultradian rhythm governing growth hormone secretion in the rat, Endocrinology, 98 (1976) 562-570.
244 19 Willoughby, J. O., Terry, L. C., Brazeau, P. and Martin, J. B., Pulsatile growth hormone, prolactin and thyrotropin secretion in rats with hypothalamic deafferentation, Brain Research, 127 (1977) 137-152. 20 Willoughby, J. O. and Martin, J. B., Neural structures and neurotransmitters regulating growth hormone and prolactin secretion. In K. Lederis and R. Couper (Eds.), Recent Studies in Hypothalamic Function, Karger, Basle, 1977, in press.
Brain Research, 148 (1978) 240 -244 (~3Elsevier/North-Holland Biomedical Press
Pulsatile growth hormone secretion: inhibitory role of medial preoptic area
JOHN O. WILLOUGHBY* and JOSEPH B. MARTIN Division of Neurology, Montreal General Hospital and Montreal Neurological Institute, Montreal, Quebec (Canada)
(Accepted December 15th, 1977)
Many neural structures have been implicated in regulating growth hormone (GH) secretion 1°. The ventromedial nucleus (VMH) and arcuate (ARC) region of the hypothalamus have been shown to be facilitatory to G H secretion in both electrolytic lesion6,1°,12 and stimulation studies 7A°,11. Several groups have confirmed the importance of VMH and ARC using the Halasz deafferentation technique 1°,13,14,19. Animals with complete deafferentation maintain or have augmented G H secretion, suggesting that the medial basal hypothalamus can generate G H secretion, probably through an intrinsic G H releasing factor (GRF) systemlL There is more recent evidence that the medial preoptic area (MPOA) is inhibitory to G H secretion. Thus (a) electrical stimulation of the MPOA in anaesthetized rats reduces r G H plasma concentrations 1°, (b) somatostatin has been identified immunohistologically in the MPOA and the anterior periventricular area of several species 1,9, and (c) ablation of the preoptic area results in stress augmentation of rGH levels, rather than suppression 1~. Finally earlier studies by MitchelP 3,~4 and co-workers, in which selective anterior hypothalamic deafferentation produced increased growth in rats, also raised the possibility that an anterior structure could inhibit rGH secretion. In undisturbed male rats, r G H secretion is characterized by rhythmic secretory bursts of r G H every 3.3 h, separated by intervals when r G H is unmeasurable ls,tg. Troughs between r G H bursts are thought to result from somatostatin secretion s (and Terry and Martin, unpublished), and the MPOA, which is the source of somatostatin in the medial eminence 5, might therefore be implicated in r G H regulation in the unstressed animal. The following study examined this possibility. Male albino Sprague-Dawley rats (290-310 g) were pentobarbitone anaesthetized and single mid-line 40-60 mC anodal current lesions were placed in the MPOAsuprachiasmatic region, using an insulated platinum electrode, with 0.5 mm exposed at the tip. De Groot co-ordinators of the lesions were anterior --7.40, lateral 0.00, and depth --2.50. Control animals were subjected to electrode insertion but no current was passed. * Present address and address for reprints: Department of Medicine (Neurology), The Flinders University of South Australia, Bedford Park, South Australia 5042.
241 400
~
MPOA 12-1 300 200 100 0
"1-
'
300 200' 100 O~ ;;; 1000 1100 1200 1300
1400 1500
1600
TIME-hr Fig. 1. Six-hour rGH profiles from two control animals. Normal, characteristic rGH secretory patterns
are evident: prominent surges of rGH secretion occur approximately every 3.3 h, and commence followingintervals when rGH is unmeasurable. Surviving animals were prepared with chronic indwelling right atrial cannulae after the method of Brown and Hedge 2, and were housed in isolation in blood sampling boxes, arranged so that the cannula opening on the rats' head could be connected to tubing leading to the outside of the box. This system permitted withdrawal of blood samples without disturbing the animals 12. After recovery from the cannulation procedure, blood samples (0.4 ml) were removed every 15 min for 6 h, and the plasma and red cells separated by centrifugation. The plasma (0.2 ml) was frozen for subsequent assay, and the red cells were resuspended in 0.2 ml physiological saline and reinjected into the rat after removal of the next sample. Plasma rGH concentrations were assayed using materials provided by NIAMDD, using a double antibody technique. Six hour rGH profiles were constructed for lesioned and control rats and compared. All rats were subjected to formalin perfusion under anaesthesia; the brains were removed, fixed and subsequently sectioned at 40 #m, to determine accuracy of placement of lesions. Sections were stained with thionin and cresyl violet. Results. rGH profiles were altered in rats with MPOA lesions (n ----6) compared to control animals (n ----9). Defining interpeak intervals arbitrarily as the average time between successive rGH bursts that exceeded 200 ng/ml, lesioned animals had more frequent bursts of rGH secretion than controls, every 2.11 ± 0.74 vs. 3.63 ± 0.40 h, respectively (P < 0.001), and during troughs rGH values also tended to remain elevated. Fig. 1 illustrates a typical rGH secretory profile from control animals, and Fig. 2 shows profiles from 6 rats with MPOA lesions, together with a diagrammatic illustration of each lesion. Histological recontruction of lesions showed that most of the MPOA was destroyed in all animals, together with the suprachiasmatic nuclei and part of the anterior hypothalamic area in some (see Fig. 2).
242 Growth Hormone Profiles in Rats with MPOA Lesions 400"
MPOA
~
A Z4 AC
'°°1 MPOA
7`8
V SCN I00. I0
O.
I'~11 MPOAh 3-2
0 I
A 7`4
!
2
400
Mo.
,,.,
200
100,
-
A7.6
MPOA
3O0
300
2Oo
200
I00
100
~oo .oo i:,oo
i ~ o ~40o ~soo ~6oo TIME - hr
ct
Iooo
A 7,4
i10o ~2b0 13oo ~4bo isbo
~,bo
TIME - hr
Fig. 2. Six-hour rGH profiles from 6 animals with MPOA lesions. The location and maximal crosssectional area of the lesions are illustrated with each profile. More frequent surges of rGH secretion are evident, with a tendency for rGH values to remain elevated in trough periods. A, anterior coordinate of de Groot; AC, anterior commissure; V, ventricle: SCN, suprachiasmatic nucleus; OC, optic chiasm. Thus, changes produced in the pattern of pulsatile r G H secretion by MPOA lesions suggest that the MPOA is inhibitory to r G H secretion in the intact non-stressed animal and, specifically, that it is implicated in producing troughs between consecutive r G H secretory episodes. Surgical isolation of the MBH has previously been shown to be capable of generating augmented pulsatile r G H secretion, similar in pattern to r G H profiles in rats with MPOA lesions 19. It could be postulated, therefore, that the MPOA rhythmically inhibits a spontaneously discharging G R F system in the MBH, and so produces the characteristic 3.3-hourly pulsatile r G H rhythm (Fig. 1). In view of studies showing the importance of somatostatin in regulating physiological r G H secretion, it is more likely that the MPOA rhythmically secretes somatostatin into the pituitary portal circulation to inhibit r G H release. Both direct MPOA-median eminence and MPOA-VMH projections have been shown electrophysiologically4,1s and by autoradiography ~, so either route, or both, might mediate rhythmic inhibition of r G H secretion by MPOA. Because MPOA-regulated gonadotropin secretory patterns specific for males and females are the consequence of differences in early MPOA maturation, it is possible that sexual differences in r G H rhythms 17 also result from differences in MPOA development. Whether the MPOA is the only determinant of periodicity of the r G H rhythm, or is in turn influenced by other neural structures, is unresolved. An inhibitory
243 noradrenergic projection, from b r a i n stem to a somatostatinergic n e u r o n a l system in M P O A , has recently been postulated to be stimulatory for r G H rhythmic secretion 2°. Ms. Judy A u d e t provided c o m p e t e n t technical assistance a n d Mrs Jane G i a c o b b e typed the manuscript. D r A l b e r t Parlow kindly provided materials for r a d i o - i m m u n o assay of r G H . J o h n O. W i l l o u g h b y was a Fellow of the Medical Research C o u n c i l of C a n a d a , a n d Joseph B. M a r t i n is a Medical Research Council Associate. The study was supported by grants from the Medical Research Council of C a n a d a .
1 Alpert, L. C., Brawer, J. R., Patel, Y. C. and Reichlin, S., Somatostatinergic neurons in the anterior hypothalamus: immunohistochemicallocalization, Endocrinology, 98 (1976) 255-258. 2 Brown, M. R. and Hedge, G. A., Thyroid secretion in the anaesthetised, stress-free rat and its suppression by pentobarbital, Neuroendocrinology, 9 (1972) 158-174. 3 Conrad, L. C. A. and Pfaff, D. W., Efferents from medial basal forebrain and hypothalamus in the rat. II. An autoradiographic study of the anterior hypothalamus, J. comp. Neurol., 169 (1976) 221-161. 4 Dyer, R. G. and Cross, B. A., Antidromic identification of units in the preoptic and anterior hypothalamic areas projecting directly to the ventromedial and arcuate nuclei, Brain Research, 43 (1972) 254-258. 5 Epelbaum, J., Willoughby, J. O., Brazeau, P. and Martin, J. B., Effects of brain lesions and hypothalamic deafferentation on somatostatin distribution in the rat brain, Endocrinology, 101 (1977) 1495-1502. 6 Frohman, L. A. and Bernardis, L. L., Growth hormone and insulin levels in weanling rats with ventromedial hypothalamic lesions, Endocrinology, 82 (1968) 1125-1132. 7 Frohman, L. A., Bernardis, L. L. and Kant, K., Hypothalamic stimulation of growth hormone secretion, Science, 162 (1968) 580-582. 8 Ferland, L., Labrie, F., Jobin, M., Arimura, A. and Schally, A. V., Physiologicalrole ofsomatostatin in the control of growth hormone and thyrotropin secretion, Biochem. biophys. Res. Commun., 68 (1976) 149-156. 9 H~ikfeit, T., Efendic, E. S., HellmstrOm, C., Johansson, O., Luft, R. and Arimura, A., Cellular localization of somatostatin in endocrine-likecells and neurons of the rat with special references to the A cells of the pancreatic islets and to the hypothalamus, Acta endocr. (Kbh.), 80, Suppl. 200 (1975) 1-41. 10 Martin,J.B.,Brainregulationofgrowthhormonesecretion.InL. MartiniandW. F. Ganong(Eds.), Frontiers in Neuroendocrinology, Vol. 4, Raven Press, New York, 1976, pp. 129-168. 11 Martin, J. B., Plasma growth hormone response to hypothalamic or extra hypothalamic electrical stimulation, Endocrinology, 91 (1972) 107-115. 12 Martin, J. B., Renaud, L. P. and Brazeau, P., Pulsatile growth hormone secretion: suppression by hypothalamic ventromedial lesions and by long acting somatostatin, Science, 186 (1974) 538-540. 13 Mitchell, J. A., Hutchins, M., Schindler, W. J. and Critchlow, V., Increases in plasma growth hormone concentration and naso-anal length in rats following isolation of the medial basal hypothalamus, Neuroendocrinology, 12 (1973) 161-173. 14 Mitchell, J. A., Smyrl, R., Hutchins, M., Schindler, W. J. and Critchlow, V., Plasma growth hormone levels in rats with increased naso-anal lengths due to hypothalamic surgery, Neuroendocrinology, 10 (1972) 31-45. 15 Renaud, L. P., Influence of medial preoptic-anterior hypothalamic area stimulation on the excitability of mediobasal hypothalamic neurones in the rat, J. Physiol. (Lond.), 264 (1977) 541-564. 16 Rice, R. W. and Critchlow, V., Extrahypothalamic control of stress-induced inhibition of growth hormone secretion in the rat, Endocrinology, 99 (1976) 970-976. 17 Terry, L. C., Saunders, A., Audet, J., Willoughby, J. O., Brazeau, P. and Martin, J. B., Physiologic secretion of growth hormone and prolactin in male and female rats, Clin. Endocrinol., 6 (1977) 19s-28s. 18 Tannenbaum, G. S. and Martin, J. B., Evidence for an endogenous ultradian rhythm governing growth hormone secretion in the rat, Endocrinology, 98 (1976) 562-570.
244 19 Willoughby, J. O., Terry, L. C., Brazeau, P. and Martin, J. B., Pulsatile growth hormone, prolactin and thyrotropin secretion in rats with hypothalamic deafferentation, Brain Research, 127 (1977) 137-152. 20 Willoughby, J. O. and Martin, J. B., Neural structures and neurotransmitters regulating growth hormone and prolactin secretion. In K. Lederis and R. Couper (Eds.), Recent Studies in Hypothalamic Function, Karger, Basle, 1977, in press.