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The role of norepinephrine in the neuroendocrine regulation of luteinizing hormone release in the rat Bergen, Hugo Theodore 1988

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THE ROLE OF NOREPINEPHRINE IN THE NEUROENDOCRINE REGULATION OF LUTEINIZING HORMONE RELEASE IN THE RAT by HUGO THEODORE BERGEN B.Sc. (Hons.), The University of Manitoba, I960 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Physiology) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA JULY 1988 (E) Hugo Theodore Bergen, 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada DE-6 (2/88) Abstract An excitatory r o l e for norepinephrine (NE) i n the regulation of l u t e i n i z i n g hormone (LH) release was f i r s t suggested when i t was demonstrated that noradrenergic receptor antagonists were able to block ovulation. More recently i t has been proposed that NE has both an excitatory r o l e and an i n h i b i t o r y r o l e i n the neuroendocrine regulation of LH release. The excitatory e f f e c t s may be mediated by alpha-adrenergic receptors and the i n h i b i t o r y e f f e c t s may be mediated v i a beta-adrenergic receptors. These experiments were performed to better understand the ro l e of NE, the receptor type through which NE exerts i t s eff e c t s , and the ro l e of the two major NE pathways i n the brain, on LH secretion* i n the rat. To further understand the r o l e of NE i n p u l s a t i l e LH release, NE or one of i t s agonists was infused into the t h i r d v e n t r i c l e of ovariectomlzed rat s pretreated with an adrenergic antagonist. In the second set of experiments ascending noradrenergic pathways were e l e c t r i c a l l y stimulated to determine t h e i r e f f e c t on p u l s a t i l e LH release. These experiments demonstrated that the i n h i b i t o r y e f f e c t of NE on p u l s a t i l e LH release i s blocked when a l p h a - 1 - or a l p h a - 2 - receptors are blocked but not when beta-receptors are blocked. E l e c t r i c a l stimulation experiments i n unprimed ovariectomlzed r a t s demonstrated that a c t i v a t i o n of the dorsal noradrenergic t r a c t (DNT) but not the ventral noradrenergic t r a c t (VNT) i n h i b i t e d p u l s a t i l e LH release. Another s e r i e s of experiments were performed to determine the r o l e NE i n the regulation of LH release i n the steroid-primed ovariectomlzed rat. These experiments demonstrated that a c t i v a t i o n of alpha- or beta-adrenergic receptors i n h i b i t e d the LH surge when adrenergic agonists are infused during the r i s i n g phase of the surge. In a s i m i l a r manner e l e c t r i c a l stimulation of ei t h e r the DNT or VNT in h i b i t e d LH release i f stimulation occured during the r i s i n g phase of the surge. The i n h i b i t o r y e f f e c t s of the DNT appear to be v i a ac t i v a t i o n of alpha-adrenergic receptors since i n h i b i t i o n was prevented by an alpha-adrenergic antagonist. Under a variety of s t e r o i d a l conditions and stimulation parameters, a c t i v a t i o n of the DNT or VNT did not enhance LH release. The lone exception to t h i s was stimulation of the VNT i n anaesthetized, steroid-primed ovariectomized r a t s pretreated with an alpha-adrenergic antagonist. In t h i s case stimulation of the VNT did enhance LH release over non-stimulated and e l e c t r i c a l l y stimulated, s a l i n e - t r e a t e d controls. These r e s u l t s suggest that LH release i s enhanced by stimulation of the VNT only when alpha-adrenergic receptors are blocked. In conclusion, i t i s evident from these studies that a c t i v a t i o n of alpha-adrenergic receptors e i t h e r by i n t r a -v e n t r i c u l a r infusion of NE or alpha-agonists, as well as e l e c t r i c a l stimulation of noradrenergic t r a c t s i n h i b i t s LH secretion. This suggests that the i n h i b i t o r y e f f e c t s of NE may be more of a factor i n the regulation of LH release than has been previously proposed. In conclusion, NE, i n addition to i t s well established excitatory role, may also have an important i n h i b i t o r y r o l e i n the regulation of LH release. It appears that both i n h i b i t o r y and excitatory e f f e c t s of NE on LH release may be mediated by both alpha- and beta-receptors. i v Table of Contents Abstract L i s t of Figures L i s t of Abbreviations Acknowledgements General Introduction I. Introduction I I . LHRH Synthesis I I I . LHRH Neuroanatomy IV. NE Neuroanatomy V. The LHRH Pulse Generator VI. The Neural Regulation of LH Release: Role of NE A. Introduction B. Stimulatory action of NE on LH release C. Inhibitory action of NE on LH release D. Role of brain NE pathways i n the re g u l a t i o n of LH Release E. Summary VII. Summary Page i i v i i x i i x i i i 1 1 7 8 13 16 20 20 26 33 38 43 45 V Norepinephrine I n h i b i t i o n of P u l s a t i l e LH Release  i n Ovariectomized Rats: Receptor S p e c i f i c i t y I. Introduction 48 I I . Materials and Methods 50 I I I . Results 55 IV. Discussion 67 E l e c t r i c a l Stimulation of Ventral versus Dorsal Mesencephalic  Tegmental Areas i n the Conscious Ovariectomized Rats: E f f e c t s on P u l s a t i l e LH Release I. Introduction 86 I I . Materials and Methods 87 I I I . Results 90 IV. Discussion 99 Suppression of the Progesterone-induced Gonadotrophin  Surge by Adrenergic Agonists i n Estrogen-Primed Ovariectomized Rats I. Introduction 109 I I . Materials and Methods 110 I I I . Results 112 IV. Discussion 122 v i E l e c t r i c a l Stimulation of Ascending Noradrenergic Tracts  i n the Midbrain: E f f e c t s on LH Release i n  Steroid-Primed Ovariectomized Rats I. Introduction 135 I I . Materials and Methods 137 I I I . Results 143 IV. Discussion 165 General Summary 178 References 182 v i i L i s t of Figures Page F i g . 1. Schematic s a g i t t a l section depicting the 15 noradrenergic neuronal system i n the r a t brain (from Ungerstedt, 1971). F i g . 2. Representative examples of l u t e i n i z i n g hormone 57 (LH) release i n ovariectomized (ovx) rats injected with s a l i n e before i n t r a v e n t r i c u l a r (ivt) infusion of norepinephrine (NE). F i g . 3. E f f e c t of i v t infusion of NE on LH release i n 58 mature ovx rats pretreated with s a l i n e (n = 9). F i g . 4. Representative examples of LH release i n ovx 59 r a t s i n j e c t e d with phenoxybenzamine before i v t i n f u s i o n of NE. F i g . 5. E f f e c t of i v t infusion of NE on LH release i n 60 ovx r a t s pretreated with phenoxybenzamine either i . v . (n = 4) or i . p . (n = 6). F i g . 6. Representative examples of LH release i n ovx 63 ra t s i n j e c t e d with propranolol 70 minutes before i v t i n f u s i o n of NE. F i g . 7. E f f e c t of i v t infusion of NE on LH release i n 64 ovx r a t s pretreated with propranolol e i t h e r i . v . (n = 4) or i. p . (n = 8) . F i g . 8. Representative examples of LH release i n ovx 65 ra t s i n j e c t e d with propranolol 15 minutes before i v t i n f u s i o n of NE. F i g . 9. Representative examples of LH release i n ovx 68 r a t s i n j e c t e d with prazosin before i v t infusion of NE. F i g . 10. E f f e c t of i v t infusion of NE on LH release i n 69 ovx r a t s pretreated with prazosin (n = 7). F i g . 11. Representative examples of LH release i n ovx 70 r a t s i n j e c t e d with piperoxan. F i g . 12. E f f e c t of i v t infusion of NE on LH release i n 71 ovx r a t s pretreated with piperoxan (n = 7). v i i i F i g . 13. Location of electrode t i p s f o r e l e c t r i c a l 91 stimulation (ES) of the dorsal, and ven t r a l noradrenergic t r a c t s (DNT and VNT, r e s p e c t i v e l y ) , and medial forebrain bundle (MFB) i n ovx r a t s . The inset i s a schematic s a g i t t a l section depicting the ascending noradrenergic system, with the s i t e s of ES indicated by the A (MFB), B (VNT), and C (DNT). In the f r o n t a l brain sections the s t i p p l e d area on the right-hand side depicts the MFB (A), VNT (B), and DNT (C), r e s p e c t i v e l y . The s i t e s of ES are depicted on the left-hand side of each f r o n t a l section as being e i t h e r i n s i d e ( f i l l e d c i r c l e s ) or outside (empty c i r c l e s ) the t r a c t s . Arc=arcuate nucleus; CG=central grey; CP=cerebral peduncle; DBC=decussation of the brachium conjunctivum; DR=dorsal raphe; f=fornix; IP=interpeduncular nucleus; mfb=MFB; ml=medial lemniscus; PH=posterior hypothalamus; SN=substantia nigra. Adapted from the a t l a s of Paxinos and Watson (1982). F i g . 14. Photomicrographs of representative f r o n t a l 92 sections i n the r a t midbrain depicting the s i t e s of electrode implantation i n the VNT (A) and the DNT (B). Fi g . 15. E f f e c t of ES of the VNT on p u l s a t i l e LH release 94 i n 3 ovx r a t s . The period of ES i s indicated by the black box. F i g . 16. E f f e c t s of mesencephalic ES on mean LH l e v e l s 95 i n ovx r a t s . Electrode t i p s are located i n s i d e or outside the VNT or DNT as depicted i n F i g . 12. Fi g . 17. E f f e c t of ES of the DNT on p u l s a t i l e LH release 96 i n 3 ovx r a t s . The period of ES i s indicated by the black box. F i g . 18. Representative examples on the e f f e c t of ES of 97 the zona i n c e r t a (A. ZI) and MFB (B.) on p u l s a t i l e LH release i n ovx r a t s . F i g . 19. E f f e c t s of i v t infusion of s a l i n e (n = 5) and 113 phenylephrine (n = 6) on the progesterone-induced LH surge i n EB-primed ovx r a t s . The s o l i d l i n e and broken l i n e s represent the rats given s a l i n e or phenylephrine, r e s p e c t i v e l y . F i g . 20. E f f e c t s of i v t infusion of s a l i n e and 114 isoproterenol on the progesterone-induced LH surge i n EB-primed ovx r a t s . The s o l i d l i n e and broken l i n e s represent the ra t s given s a l i n e or isoproterenol, r e s p e c t i v e l y . i x F i g . 21. Representative examples of LH release i n 115 progesterone and EB-primed ovx r a t s given an i v t i n f u s i o n of e i t h e r saline, phenylephrine, or isoproterenol. Arrows indicate time of i n f u s i o n . F i g . 22. E f f e c t s of i v t i n f u s i o n of s a l i n e (n = 5) and 117 phenylephrine (n = 6) on the progesterone-induced FSH surge i n EB-primed ovx r a t s . The s o l i d l i n e and broken l i n e s represent the rats given s a l i n e or phenylephrine, re s p e c t i v e l y . F i g . 23. Representative examples of FSH release i n 118 progesterone and EB-primed ovx rats given an i v t i n f u s i o n of e i t h e r saline, phenylephrine, or isoproterenol. Arrows indicate time of i n f u s i o n . F i g . 24. E f f e c t s of i v t i n f u s i o n of s a l i n e (n = 7) and 120 NE (n = 11) on the progesterone-induced LH surge i n EB-primed ovx r a t s . The s o l i d l i n e and broken l i n e s represent the r a t s given s a l i n e or NE, r e s p e c t i v e l y . Arrow indicates time of i n f u s i o n . F i g . 25. E f f e c t s of i v t i n f u s i o n of s a l i n e (n = 9) and 121 methoxamine (n = 7) on the progesterone-induced LH surge i n EB-primed ovx r a t s . The s o l i d l i n e and broken l i n e s represent the rats given s a l i n e or methoxamine, res p e c t i v e l y . Arrow indicates time of infusion. F i g . 26. Location of electrode t i p s f o r e l e c t r i c a l 144 stimulation (ES) of the dorsal, and v e n t r a l noradrenergic t r a c t s (DNT and VNT, r e s p e c t i v e l y ) , i n steroid-primed ovx rats. The inset i s a schematic s a g i t t a l section depicting the ascending noradrenergic system, with the s i t e s of ES indicated by the A (VNT), and B (DNT). In the f r o n t a l brain sections the s t i p p l e d area on the right-hand side depicts the VNT (A), and DNT (B), r e s p e c t i v e l y . The s i t e s of ES are depicted on the left-hand side of each f r o n t a l section as being e i t h e r i n s i d e ( f i l l e d c i r c l e s ) or outside (empty c i r c l e s ) the t r a c t s . See F i g . 12 f o r abbreviations. Adapted from the a t l a s of Paxinos and Watson (1982). F i g . 27. Representative examples of LH release i n 4 146 i n d i v i d u a l steroid-primed rat s from 4 d i f f e r e n t groups. The period of ES i n t h i s and the proceeding figures i s indicated by the black box. F i g . 28. Mean serum LH l e v e l s i n DNT-stimulated and non- 147 stimulated control rats (number of r a t s i s i n brackets). E f f e c t of ES of DNT on the progesterone-induced LH surge. X F i g . 29. Percent change i n mean LH l e v e l s , as compared 148 to values at 16.00 h i n DNT-, VNT-, or non-stimulated control r a t s (number of r a t s i s i n brackets). E f f e c t s of ES of the DNT and VNT on the progesterone-induced LH surge. F i g . 30. Location of electrode t i p s i n rats which were 150 stimulated twice (from 16.00 h to 17.00 h, and 19.00 h to 20.00 h) i n e i t h e r the VNT (A) or DNT (B). See F i g . 24 f o r abbreviations. F i g . 31. Representative examples of LH release i n 4 152 i n d i v i d u a l steroid-primed r a t s from 4 d i f f e r e n t groups. E f f e c t s of two periods of ES on the progesterone-induced LH surge. F i g . 32. Mean serum LH l e v e l s i n DNT-stimulated and non- 153 stimulated controls. E f f e c t of two periods of ES of DNT on the progesterone-induced LH surge. F i g . 33. Percent change i n mean LH l e v e l s , as compared 154 to values at 16.00 h i n twice DNT-, VNT-, or non-stimulated control r a t s . E f f e c t s of two periods of ES of the DNT and VNT on the progesterone-induced LH surge. F i g . 34. Representative examples of LH release i n 156 steroid-primed r a t s which, p r i o r to the s t a r t of ES of the DNT, were in j e c t e d (iv) with e i t h e r pimozide (Pirn.) or propranolol (Prop.). Arrow indicates the time of receptor antagonist i n j e c t i o n . F i g . 35. Mean serum LH l e v e l s i n DNT-stimulated r a t s 157 which also received e i t h e r pimozide or propranolol p r i o r to the onset of ES. The X and the black box at 18.00 h represent the LH l e v e l s i n the non-stimulated and the DNT-stimulated r a t s , r e s p e c t i v e l y . F i g . 36. Representative examples of LH release i n 158 steroid-primed r a t s which, p r i o r to the s t a r t of ES of the DNT, were in j e c t e d (iv) with phenoxybenzamine. F i g . 37. Mean serum LH l e v e l s i n phenoxybenzamine 159 treated DNT-stimulated versus s a l i n e treated non-stimulated r a t s . F i g . 38. E f f e c t of b i l a t e r a l electrochemical stimulation 163 of the DNT or VNT on the steroid-induced LH surge as compared to non-stimulated cont r o l s . F i g . 39. E f f e c t of b i l a t e r a l electrochemical stimulation 164 of the DNT or VNT on the steroid-induced PRL surge as compared to non-stimulated cont r o l s . x i F i g . 40. E f f e c t of e l e c t r i c a l stimulation of the VNT and 166 adrenergic antagonists, on LH release i n anaesthetized steroid-primed r a t s . F i g . 41. Proposed mechanism f o r the dual action of NE on 179 the regulation of hypothalamic LHRH release i n the r a t . Stimulatory e f f e c t s of NE are exerted p r i m a r i l y v i a alpha-receptors on LHRH neurons. Inhibitory e f f e c t s of NE could be mediated by alpha- and beta-receptors on putative i n h i b i t o r y interneurons. x i i L i s t of Abbreviations CNS ce n t r a l nervous system DA dopamine DNT dorsal noradrenergic t r a c t E 2 e s t r a d i o l EB e s t r a d i o l benzoate FSH f o l l i c l e stimulating hormone GABA gamma-amino b u t y r i c a c i d IP i n t r a p e r i t o n e a l i v intravenous LC locus coeruleus LH l u t e i n i z i n g hormone LHRH l u t e i n i z i n g hormone r e l e a s i n g hormone MBH medial basal hypothalamus ME median eminence MFB medial forebrain bundle MPOA medial preoptic area NE norepinephrine ovx ovariectomized P 4 progesterone SE standard error of the mean 6-OH-DA 6-hydroxy-dopamine VNT ve n t r a l noradrenergic t r a c t ZI zona i n c e r t a x i i i Acknowledgements I would l i k e to g r a t e f u l l y acknowledge my supervisor, Dr. Peter Leung, f o r providing me the opportunity to pursue these studies and fo r his.continuous support and encouragement throughout the time spent i n h i s lab. I also wish to acknowledge my collegues, comrade J . Wang and M. Rodway, who made my time i n the lab a more f e l i c i t o u s experience, and were p a r t i c u l a r l y patient with me during the w r i t i n g of t h i s t h e s i s . A l l of a sudden I would also l i k e to thank P. Rose (who wishes to remain anonymous) f o r her assistance with the fig u r e s , and J . Robinson for her enthusiastic help i n the use and care of a personal computer. Last, but not l e a s t , I thank T. S u l l i v a n f o r her understanding and encouragement during the f i n a l stages of t h i s work. 1 General Introduction I. Introduction A r o l e f o r catecholamines i n the regulation of ovulation was f i r s t implicated when blockade of alpha-adrenergic receptors prevented ovulation i n rabbits and i n f u s i o n of norepinephrine (NE) into the t h i r d v e n t r i c l e was able to induce ovulation (Sawyer et a l . , 1947; Sawyer, 1952). I t was also previously demonstrated that e l e c t r i c a l stimulation of the hypothalamus, but not the p i t u i t a r y , induced ovulation i n the r a b b i t (Markee et a l . , 1946). These experiments le d to the concept that the p i t u i t a r y i s stimulated to release i t s ovulating hormone by a humoral factor released from the hypothalamus, rather than v i a a d i r e c t neural l i n k from the hypothalamus to the p i t u i t a r y (Markee et a l . , 1952). I t was postulated that the humoral agent i s released i n t o the hypothalamo-hypophyseal p o r t a l vessels and subsequently stimulates the p i t u i t a r y to release l u t e i n i z i n g hormone (LH). S i m i l a r l y i n the r a t , administration of alpha-adrenergic antagonists also blocked ovulation. Experiments i n rats demonstrated that adrenergic antagonists administered to proestrous rats before 14.00 h, blocked ovulation. I f these drugs were given a f t e r 16.00 h, ovulation was not blocked (Everett et a l . , 1950). The early studies of Everett, Sawyer, and others, gave r i s e to the concept that on the afternoon of proestrus a c r i t i c a l period (14.00 - 16.00 h) e x i s t s during 2 which time the hypothalamus becomes activated to secrete a gonadotrophin-releasing factor known as LH-releasing hormone (LHRH) or gonadotrophin-releasing hormone. The fa c t o r i s released at the l e v e l of the basal hypothalamus in t o the hypophyseal p o r t a l veins, and stimulates the p i t u i t a r y . The p i t u i t a r y then releases LH which stimulates the ovary and r e s u l t s i n ovulation of the mature f o l l i c l e ( s ) . In t h i s way the c e n t r a l nervous system (CNS) exercises influence on the p i t u i t a r y . These authors also reported that estrogen exerts i t s influence on LH release at the l e v e l of the CNS and that the neural elements responsible f o r the stimulation of LH have a 24 hour p e r i o d i c i t y (Everett and Sawyer, 1950). These studies stressed the r o l e of the CNS i n the regulation of LH release. Since then numerous studies have investigated the r o l e of various agents, present i n the CNS, i n the regulation of LH release. The l i s t of agents which have been implicated i n the control of LH secretion includes neurotransmitters, putative neurotransmitters or neuromodulators, st e r o i d s , and more recently peptides (Weiner and Ganong, 1978; Barraclough and Wise, 1982; Kalra and Kalra, 1983; McCann et a l . , 1984; Ramirez et a l . , 1984; Coen, 1987). This introduction w i l l focus on the r o l e of the catecholamines, p a r t i c u l a r l y norepinephrine (NE), i n the regulation of LH release. The f i r s t section w i l l b r i e f l y o u t l i n e the c h a r a c t e r i s t i c s of LH and LHRH secretion i n the r a t . LH, a glycoprotein hormone made up of an alpha-subunit (common to f o l l i c l e stimulating hormone (FSH) and thy r o i d 3 stimulating hormone) and a beta-subunit, i s secreted from p i t u i t a r y gonadotrophs. The gonadotrophs may contain LH alone, po s s i b l y FSH alone, or both LH and FSH (Childs et a l . , 1983). The proportion of gonadotrophs which contain FSH alone, i f any, i s not resolved (Childs et a l . , 1983; Dada et a l . , 1983). In any case, i t has been shown that the r e l a t i v e proportions of monohormonal and multihormonal c e l l types changes during the estrous cycle and monohormonal c e l l types can be stimulated to become multihormonal c e l l types (Childs et a l . , 1987). The gonadotrophs are stimulated to release LH following the binding of LHRH to s p e c i f i c receptors f o r LHRH on the c e l l surface. The reponse of the gonadotroph to a given amount of LHRH i s under complex regulation and can vary considerably under d i f f e r e n t conditions. The modulation of the gonadotroph•s response to LHRH w i l l not be discussed here. In the r a t , under normal laboratory conditions, the estrous cycle consists of 4 days. Throughout most of the cycle LH l e v e l s are r e l a t i v e l y low, except during the afternoon of proestrus when a massive surge of LH i s secreted by the p i t u i t a r y (Butcher et a l . , 1974; Smith et a l . , 1975). LH release i s p u l s a t i l e on a l l days of the cycle when l e v e l s are low, as well as during the LH surge which i s made up of large amplitude pulses i n rapid succession ( Gallo, 1981b; 1981c; Fox and Smith, 1985). This surge of LH r e s u l t s i n ovulation on the following day. The negative feedback of the ovarian hormones, e s t r a d i o l (E 2) and progesterone ( P 4 ) , on LH release during each day of the cycle has recently been outlined ( Leipheimer et 4 a l . , 1985, 1986; Gallo, 1987). On most days of the cycle, E 2 and P 4 act e i t h e r separately, or together, to i n h i b i t the amplitude and/or frequency of the LH pulses. On the afternoon of proestrus the E 2 l e v e l s are at t h e i r highest (Butcher et a l . , 1974; Leipheimer et a l . , 1986), and i t i s the sustained increase i n E 2 which eventually t r i g g e r s the LH surge (Legan et a l . , 1975; Legan and Karsch, 1975; Fink, 1979). Therefore, E 2 can exert both negative and p o s i t i v e feedback e f f e c t s on LH release. Removal of ovarian feedback, i n p a r t i c u l a r ovariectomy, r e s u l t s i n LH release characterized by high amplitude pulses occurring at regular i n t e r v a l s . The ascending limb of the LH pulse i s r e l a t i v e l y steep and b r i e f , and the descending limb i s longer and the decline more gradual (Gay and Sheth, 1972; Weick, 1981). During t h i s l a t t e r period, the LH l e v e l s i n the blood decrease at a rate s i m i l a r to the h a l f - l i f e of the hormone which suggests that LH release during t h i s period i s s l i g h t (Dierschke et a l . , 1970; Gay and Sheth, 1972). P u l s a t i l e LH release was f i r s t described i n the ovariectomized monkey, and s h o r t l y thereafter i n the r a t (Dierschke et a l . , 1970; Gay and Sheth, 1972). Si m i l a r p u l s a t i l e LH release i s also present i n the gonadectomized male. Since then i t has been found that LH release i s p u l s a t i l e i n adult castrates i n a l l species i n which LH l e v e l s have been measured. I t i s now generally accepted that the p u l s a t i l e release of LH from the p i t u i t a r y i s due to the p u l s a t i l e release of LHRH i n t o the p i t u i t a r y p o r t a l veins. A close c o r r e l a t i o n has 5 been demonstrated to occur between LH pulses i n the jugular v e i n and LHRH pulses i n the p i t u i t a r y p o r t a l v e i n i n the ovariectomlzed ewe (Clarke and Cummins, 1982; Levine et a l . , 1982). S i m i l a r r e s u l t s demonstrating p u l s a t i l e LHRH release i n ovariectomized monkeys and ovariectomized rat s have also been reported (Carmel et a l . , 1976; Levine and Ramirez, 1982; Levine et a l . , 1985). During a preovulatory LH surge the increase i n LH release i s at l e a s t p a r t i a l l y a r e s u l t of an increase i n LHRH release, i n addition to an increase i n the s e n s i t i v i t y of the gonadotrophs to LHRH (Sarkar et a l . , 1976). In o v a r i -ectomized r a t s , monkeys, and sheep, E 2 can induce an LH surge which i s also associated with an increase i n LHRH release into the p o r t a l veins (Clarke and Cummins, 1985a; Levine et a l . , 1985; Levine and Ramirez, 1982). Therefore changes i n LH release i n the i n t a c t animal r e f l e c t changes, at lea s t p a r t i a l l y , i n LHRH release (Clarke et a l . , 1984). Experiments i n sheep, monkeys, and humans have c l e a r l y demonstrated that the p u l s a t i l e nature of the LHRH signa l to the p i t u i t a r y i s important i n the maintenance of LH release. In the absence of LHRH, LH release from the p i t u i t a r y i s very low while continuous i n f u s i o n of LHRH r e s u l t s i n d e s e n s i t i z a t i o n of the p i t u i t a r y to LHRH and a subsequent decline i n LH l e v e l s (Belchetz et a l . , 1978). When endogenous LHRH release i s eliminated experimentally, a p u l s a t i l e i nfusion of LHRH i s required i n order to maintain LH l e v e l s i n the blood (Knobil, 1980; Clarke et a l . , 1984). The LHRH pulse frequency i s also an important f a c t o r i n determining the amplitude of the 6 LH pulses. An increase i n LHRH pulse frequency r e s u l t s i n a decrease i n LH pulse amplitude and a decrease i n pulse frequency r e s u l t s i n an increase i n pulse amplitude (Clarke et a l . , 1984, 1985b; Petrie et a l . , 1988). In addition to stimulating LH release, p u l s a t i l e LHRH release i s also important i n regulating the number of receptors f o r LHRH and stimulating the synthesis of LH (Katt et a l , 1985; Clarke and Cummins, 1985b, 1987; Papavasilou et a l , 1986; Hamernik and Nett, 1988) . I t i s therefore evident that the mode of LHRH secretion i s important i n the reproductive physiology of the animal since changes i n the p u l s a t i l e parameters of LHRH release have s i g n i f i c a n t e f f e c t s on the number of receptors for LHRH, LH pulse parameters, and LH synthesis. While the p u l s a t i l e release of LHRH i s important i n the regulation of LH release, the p u l s a t i l e pattern of LH release i s important i n the reproductive physiology of the animal. During the r a t or sheep estrous cycle, LH release has a c h a r a c t e r i s t i c pattern on each day depending on the l e v e l of ovarian feedback present at the time (Karsch et a l . , 1980; Goodman et a l . , 1981; Gallo, 1987). S i m i l a r l y , changes i n the p u l s a t i l e pattern of LH release are also observed at d i f f e r e n t stages of the menstrual cycle of humans and monkeys (Reame et a l . , 1984; Norman et a l . , 1984). The c h a r a c t e r i s t i c pattern of LH release at any p a r t i c u l a r point i n time i s a function of the l e v e l of ovarian feedback and the interplay between the ovary and the hypothalamo-pituitary u n i t . I t i s t h i s complex in t e r p l a y which i s e s s e n t i a l i n maintaining the c y c l i c a l 7 pattern of E 2 , P 4, LH, and FSH release observed i n females which have regular estrous or menstrous cycles. I t i s i n t e r e s t i n g that changes i n the reproductive physiology or status of an animal are associated with changes i n p u l s a t i l e LH release. This i s p a r t i c u l a r l y evident i n 1. the onset of puberty, 2. the onset of reproductive senescence, and 3. the reproductive changes that occur i n seasonal breeders as they enter and depart breeding seasons. Therefore the pattern of LHRH release and subsequent LH release i s a very important f a c t o r i n the reproductive physiology of the animal (Knobil, 1980; Lincoln and Short, 1980; Karpas et a l . , 1983; Ojeda et a l . , 1984). I I . LHRH Synthesis LHRH i s a decapeptide which was f i r s t i s o l a t e d and synthesized i n 1971 (Matsuo et a l , 1971a; Matsuo et a l . , 1971b). Similar to other peptide hormones, such as vasopressin and adrenocorticotropin, LHRH i s i n i t i a l l y synthesized as part of a larger precursor molecule. The nucleotide sequence f o r human placental cDNA encoding the precursor protein f o r LHRH has been characterized. The precursor (proLHRH) consists of 92 amino acids and LHRH i s preceded by a 23 amino ac i d s i g n a l peptide (Seeburg and Adelman, 1984). I t was l a t e r reported that the DNA sequence f o r hypothalamic proLHRH i s i d e n t i c a l to that i s o l a t e d from the placenta (Adelman et a l . , 1986). They also reported that a 56 amino ac i d peptide which i s part of the precursor f o r LHRH i n h i b i t e d p r o l a c t i n release i n p i t u i t a r y 8 c e l l c ultures (Nikolics et a l . , 1985). This i s of in t e r e s t since under some conditions the l e v e l s of LH and p r o l a c t i n are inv e r s e l y r e l a t e d . A d d i t i o n a l l y , i t has been demonstrated that another peptide, which i s also part of the precursor f o r LHRH, stimulated LH and FSH release i n v i t r o independent of the LHRH receptor ( M i l l a r et a l . , 1986). The p h y s i o l o g i c a l s i g n i f i c a n c e of these peptides, i n the regulation of gonadotrophin release, i s not known. I I I . LHRH Neuroanatomy LHRH, synthesized i n nerve c e l l bodies, i s released from v e s i c l e s i n nerve terminals i n the median eminence (ME) into the p i t u i t a r y p o r t a l veins. The LHRH molecules then t r a v e l down the veins to the p i t u i t a r y where they bind to s p e c i f i c receptors and stimulate the release of LH. The LHRH-containing neurons can be divided into two populations, one population which has i t s nerve terminals i n the ME and one population which has terminals i n regions which are outside the hypothalamus. The population of LHRH-releasing c e l l s that innervate the ME w i l l be discussed here since i t i s these c e l l s which are d i r e c t l y involved i n the stimulation of LH release from the p i t u i t a r y . I t has been suggested that only one h a l f of the LHRH-containing c e l l s i n the an t e r i o r hypothalamic areas innervate the ME (Silverman et a l . , 1987). The possible function of the LHRH neurons which are located outside the hypothalamus i s not known, although they may also be involved i n reproduction. I t i s thought that i n the extrahypothalamic 9 projections, LHRH may be acting as a neurotransmitter. I t may be mentioned that the d i s t r i b u t i o n s of these two populations of LHRH-synthesizing c e l l bodies do not appear to be anatomically or c y t o l o g i c a l l y d i s t i n c t from each other (Jennes and Stumpf, 1986; Silverman et a l . , 1987). There have been a number of immunocytochemical studies which have examined the d i s t r i b u t i o n of LHRH-containing c e l l bodies, axons, and terminals i n the r a t brain. The general concensus i s that the majority of the c e l l bodies containing LHRH-immunoreactive material are located i n the preoptic, anterior hypothalamic, and septa l areas including the diagonal band of Broca (Bennett-Clark and Joseph, 1982; Hoffman and Gibbs, 1982; King et a l . , 1982; Witkin et a l . , 1982; Merchenthaler et a l . , 1984). The majority of LHRH c e l l s involved i n the control of gonadotrophin release ( i n the rat) are located i n the medial preoptic area (MPOA). The LHRH c e l l s which have terminals i n the ME are not i n a d i s t i n c t group but are d i s t r i b u t e d d i f f u s e l y i n a n t e r i o r hypothalamic areas (Silverman et a l . , 1987). This d i f f u s e d i s t r i b u t i o n of LHRH c e l l s has also recently been confirmed by i n s i t u h y b r i d i z a t i o n techniques which demonstrated that c e l l s i n these areas do synthesize LHRH mRNA (Shivers et a l . , 1986). The question of whether LHRH c e l l bodies are present i n the arcuate nucleus of the r a t i s somewhat c o n t r o v e r s i a l . A few studies have reported LHRH c e l l bodies i n the arcuate nucleus area of the r a t (Naik, 1976; Kawano and Daikoku, 1981; K e l l y et a l . , 1982; ), as observed i n a number of other species (e.g., dog, cat, rabbit, 10 monkey, human) (Barry et a l . , 1985). However, most of the immunocytochemical studies have not observed c e l l s i n the arcuate nucleus and t h e i r presence i n t h i s region i s doubtful (Rethelyi et a l . , 1981; Kozlowski and Les Dees, 1984; Merchenthaler et a l . , 1984; Barry et a l . , 1985). L o c a l i z a t i o n of LHRH mRNA by i n s i t u h y b r i d i z a t i o n has also f a i l e d to detect LHRH c e l l s i n the arcuate nucleus (Shivers et a l . , 1986; Rothfeld et a l . , 1987). LHRH-containing neurons send t h e i r axons p o s t e r i o r l y from a d i f f u s e group of c e l l s to the ME i n a d i f f u s e septo-preoptico-infundibular t r a c t (Barry et a l . , 1985). The o r i g i n of t h i s t r a c t i s well known (see above) but determination of the precise paths that the d i f f e r e n t components of t h i s t r a c t take i s d i f f i c u l t . The d i f f i c u l t y a r i s e s due to the d i f f u s e nature of the LHRH c e l l u l a r f i e l d and the diffuseness of the t r a c t i t s e l f . Studies have described two (King et a l . , 1982), three (Bennett-Clarke and Joseph, 1982; Merchenthaler et a l . , 1980, 1984) or four pathways from the septo/preoptic region to the ME (Hoffman and Gibbs, 1982). King et a l . (1982) described two d i s t i n c t but d i f f u s e pathways, a p e r i v e n t r i c u l a r pathway and a l a t e r a l pathway, which are separated f o r most of the hypothalmus by medial hypothalamic n u c l e i . As they pass caudally they converge to terminate i n the ME. Merchenthaler and co-workers (1980) used systematic knife-cuts around the hypothalamus followed by LHRH immunocytochemistry, and reported a median, a medial, and a l a t e r a l path. They also found that each t r a c t has terminals a l l along the length of the ME. A 11 s i m i l a r d i s t r i b u t i o n of f i b r e s was found by others (Bennet-Clark and Joseph, 1982; Hoffman and Gibbs, 1982). Hoffman and Gibbs (1982) also reported an ad d i t i o n a l sub-chiasmatic pathway which by i t s e l f was able to sustain gonadotrophic function. Disruption of the f i b r e s from the septum and diagonal band of Broca decreased LHRH l e v e l s i n the ME by 30 %, but ovulation occurred normally (Koves and Molnar, 1986). Ovulation was disrupted only when k n i f e cuts of LHRH pathways r e s u l t e d i n LHRH l e v e l s i n the ME below 40 % of controls (Koves and Molnar, 1986) . The precise r o l e that each of these pathways plays i n the regulation of gonadotrophin release i s not known, although some pathways appear to be more important than others i n t h i s regard (Hoffman and Gibbs, 1982; Koves and Molnar, 1986). Whether these d i f f e r e n t LHRH pathways are d i f f e r e n t i a l l y innervated by other neuronal systems i s not known. I t i s possibl e that these d i f f e r e n t t r a c t s provide a means by which other neuronal systems might d i f f e r e n t i a l l y e f f e c t changes i n LHRH and subsequent gonadotrophin release. A number of recent studies have reported on the anatomical r e l a t i o n s h i p s of the c l a s s i c a l transmitters to LHRH-containing neurons i n the hypothalamus. The o r i g i n a l studies demonstrated a close overlap (at the l i g h t microscope level) between neurons containing dopamine (DA) and LHRH-containing neurons i n the ME using monoamine histofluorescence and LHRH immunocytochemistry on the same, or adjacent, h i s t o l o g i c a l sections (McNeill and Sladek, 1978; Ajika, 1979; Ibata et a l . , 1979; McNeill et a l . , 1980). A j i k a (1979), employing a 12 technique which allowed v i s u a l i z a t i o n at the electron microscopic l e v e l , also reported axo-axonic contacts between dopaminergic and LHRH c e l l s i n the ME near the p o r t a l vessels. This suggested a means by which catecholamines could a l t e r the release of LHRH into the p o r t a l veins. Hoffman et a l . (1982) also demonstrated close contact between LHRH neurons and catecholaminergic neurons (those that synthesize tyrosine hydroxylase). They reported that an apparent contact of LHRH c e l l s with catecholaminergic c e l l s d i d not depend on the density of the catecholaminergic neurons and that contacts were apparently made i n regions of heavy as well as l i g h t catecholaminergic f i b r e density. Close contacts of catecho-laminergic f i b r e s (which they suggest are noradrenergic) to LHRH c e l l s were seen i n the MPOA, and LHRH axon swellings contacted dopaminergic c e l l s i n the arcuate nucleus (Hoffmann et a l . , 1982; Hoffman, 1985). In addition, the c e l l s which do not p r o j e c t to the ME apparently receive l e s s catecholaminergic input than c e l l s which project to the ME. Jennes et a l . (1982, 1983), employing immunohistochemical double s t a i n i n g techniques, also reported on the anatomical r e l a t i o n s h i p of catecholamines to the LHRH neuronal system. They reported that dopaminergic and NE-containing neurons both contacted LHRH-containing c e l l bodies i n the MPOA, however, i n the ME very l i t t l e overlap of NE- and LHRH-containing neurons occurred. They therefore suggested that at the l e v e l of the ME, e f f e c t s of NE on LHRH-containing neurons may be mediated by DA. However, Palkovits et a l . (1982) have provided evidence that NE-containing terminals do make d i r e c t connections to LHRH neurons. They found that following transection of the ventral noradrenergic t r a c t (VNT) degenerating nerve terminals were seen i n contact with LHRH processes i n the arcuate nucleus. These studies together suggest that the catecholamines do make synaptic contacts with LHRH neurons although more studies, p a r t i c u l a r l y at the electron microscope l e v e l , are required to i d e n t i f y the p a r t i c u l a r connections of inputs to the LHRH-containing c e l l bodies, axons, and terminals. I t would be of p a r t i c u l a r i n t e r e s t to determine whether the d i f f e r e n t LHRH-containing f i b r e t r a c t s , from the MPOA to the ME are d i f f e r e n t i a l l y innervated by the d i f f e r e n t catecholaminergic f i b r e pathways. IV. NE Neuroanatomy In the hypothalamus, NE i s r e s t r i c t e d to nerve terminals and axons whose c e l l bodies are located i n the NE c e l l groups of the brainstem (Moore and Bloom, 1979; L i n d v a l l and Bjorklund, 1983) . The NE-containing c e l l groups can be divided in t o three major systems: 1. the locus coeruleus-subcoeruleus c e l l group (LC) (A6 and A4 c e l l groups o r i g i n a l l y designated by Dahlstrom and Fuxe, 1964), 2. the l a t e r a l tegmental group (Al, A3, A5, and A7), and 3. the dorsal medullary group (A2). While most of the NE-containing c e l l groups do have some terminals i n the hypothalamus, i t i s presently thought that the A5 and A7 c e l l groups have very few or no terminals at a l l i n the hypothalamus (Li n d v a l l and Bjorklund, 1983). The LC supplies 14 only a minor po r t i o n of the NE innervation of the hypothalamus as compared to the l a t e r a l tegmental and dorsal medullary groups. In p a r t i c u l a r , the major portion of the NE-containg terminals i n the hypothalamus a r i s e from the A l and A2 groups (Palkovits et a l . , 1980; L i n d v a l l and Bjorklund, 1983). Ascending noradrenergic f i b r e s from the brainstem pass through the midbrain v i a two major pathways, the dorsal noradrenergic t r a c t (DNT) and the ventral noradrenergic t r a c t (VNT) (see F i g . 1) ( L i n d v a l l and Bjorklund, 1978; Ungerstedt, 1971; Palkovits, 1981). Fibres o r i g i n a t i n g from the LC ascend to the hypothalamus i n the DNT, and innervate the arcuate, p e r i v e n t r i c u l a r , paraventricular, dorsomedial n u c l e i of the hypothalamus and also the a n t e r i o r hypothalamic area. (Palkovits et a l , 1980; L i n d v a l l and Bjorklund, 1983; Moore and Card, 1984). Retrograde t r a c e r studies reveal some projections from the LC to the MPOA, and the l a t e r a l and p o s t e r i o r hypothalamic areas (Berk and F i n k e l s t e i n , 1981) . Lesions of the LC r e s u l t s i n degenerating terminals i n the ME which suggests of a d i r e c t connection between these regions (Palkovits et a l . , 1977). I t i s evident that the DNT, carrying f i b r e s from the LC, innervates a number of areas i n the hypothalamus. NE-containing f i b r e s ascending from the A l and A2 c e l l groups t r a v e l i n the VNT to provide the major po r t i o n of the NE innervation of the hypothalamus (Ungerstedt, 1971; Palkovits, 1981; L i n d v a l l and Bjorklund, 1983). These two c e l l groups provide almost a l l of the NE-containing f i b r e s innervating the 15 Fig. 1. Schematic s a g i t t a l section depicting the noradrenergic neuronal system i n the rat brain (from Ungerstedt, 1971). MPOA, paraventricular nucleus and supraoptic nucleus (Day et a l . , 1980; Berk and F i n k e l s t e i n , 1981; Sawchenko and Swanson, 1982) . Fibres from the A l and A2 c e l l groups also p r o j e c t to the anterior, l a t e r a l , and po s t e r i o r hypothalamic areas (Sakumoto et a l . , 1978; Ricardo and Koh, 1978). Lesioning of these two c e l l groups or b i l a t e r a l transections of the VNT resulted i n a decrease of over 60% of NE l e v e l s i n the arcuate nucleus and ME (O'Donohue et a l . , 1979; Palkovits et a l . , 1980). Lesioning of the VNT also resulted i n a d r a s t i c decline i n noradrenergic terminals i n the arcuate nucleus, ventromedial nucleus, and the ME (Kizer et a l . , 1976). More re c e n t l y i t was demonstrated that transection of the VNT r e s u l t e d i n degenerating nerve terminals on LHRH-containing p r o j e c t i o n s i n the arcuate nucleus (Palkovits et a l . , 1982). I t i s evident that f i b r e s of both pathways innervate the hypothalamus with the MPOA and ME ( s i t e s of LHRH-containing c e l l bodies and terminals, respectively) r e c e i v i n g innervation l a r g e l y from the Al and A2 c e l l groups v i a the VNT. The possible r o l e s of these c e l l groups, v i a t h e i r respective f i b r e pathways, i n regulating LH release i s not completely understood. V. The LHRH Pulse Generator The release of LH from the p i t u i t a r y appears to be pri m a r i l y a function of release of LHRH from nerve terminals i n the ME, into the p i t u i t a r y p o r t a l veins. LH release i s p u l s a t i l e throughout the estrous cycle as well as i n the ovariectomized r a t (Gay and Sheth, 1972; Gallo, 1981b, 1981c). 17 This p u l s a t i l e release i s probably due to p u l s a t i l e LHRH release from the ME (Clarke and Cummmins, 1982; Levine et a l . , 1982). The release of LHRH from nerve terminals i s a r e s u l t of LHRH neuronal a c t i v i t y , i . e . , action p o t e n t i a l s i n the nerve terminal t r i g g e r release of the synaptic v e s i c l e s into the p o r t a l c i r c u l a t i o n . The LHRH-containing nerve terminals i n the ME are situated on fenestrated c a p i l l a r i e s i n t o which the v e s i c l e s of LHRH are emptied. The blood i n these c a p i l l a r i e s t r a v e l s down the p o r t a l vessels to the p i t u i t a r y . In order f o r a pulse of LHRH to reach the p i t u i t a r y i t i s necessary that i n d i v i d u a l neurons release "LHRH-packets" i n pulses, at approximately the same time as other LHRH-containing neurons. The mechanism of coordination of a c t i v i t y between LHRH neurons i s not known but LHRH-containing neurons do have synaptic connections with other LHRH neurons i n the MPOA (Leranth et a l . , 1985; P e l l e t i e r , 1987). In addition, l o c a l c i r c u i t s of interneurons might also coordinate the a c t i v i t y of the LHRH-containing neurons. To account f o r p u l s a t i l e release of LHRH, i t has been proposed that LHRH release i s under the control of a c e n t r a l pattern (or pulse) generator (Lincoln et a l . , 1985). Presumably t h i s c e n t r al pulse generator determines the frequency of LHRH neuronal a c t i v i t y and thereby determines the frequency of LHRH p u l s a t i l e release. The nature(s) of the neurons which make up t h i s pulse generator i s (are) not known. I t i s also not known whether the p u l s a t i l i t y of LHRH neuronal a c t i v i t y i s i n t r i n s i c to the LHRH-releasing neurons. In the Rhesus monkey, i n contrast to rats, the mediobasal 18 hypothalamus (MBH) appears to be the s i t e of the majority of LHRH-containing neurons. The MBH also appears to be the s i t e of the pulse generator c o n t r o l l i n g LHRH secretion since with s u r g i c a l i s o l a t i o n of the MBH, p u l s a t i l e LH release i s maintained and les i o n s i n the MBH disrupt LH release (Krey et a l . , 1975; Plant et a l . , 1978). The implantation of recording electrodes i n the MBH showed that each LH pulse i s preceded by a r a p i d increase i n neuronal multiunit a c t i v i t y (Wilson et a l . , 1984). Increases i n the multiunit a c t i v i t y of the MBH were al s o reported to occur, p r i o r to an LH pulse, i n ovariectomized r a t s (Kawakami et a l . , 1982). Presumably the increase i n mul t i u n i t a c t i v i t y r e f l e c t s a synchronous increase i n the a c t i v i t y of LHRH-containing neurons and/or the neural network which drives t h e i r a c t i v i t y , i . e . , the pulse generator, or an increase i n the a c t i v i t y of inputs to the pulse generator. Undoubtedly the pulse generator responsible for LHRH release receives numerous inputs which a f f e c t the frequency at which the pulse generator operates. In support of t h i s , a l p h a ^ adrenergic antagonists, and a dopaminergic antagonist, which both i n h i b i t e d p u l s a t i l e LH release, s i m i l a r l y i n h i b i t the p u l s a t i l i t y of the multiunit a c t i v i t y i n the MBH (Kaufman et a l . , 1985). The a c t i v i t y of the LHRH pulse generator has also been monitored by measuring LHRH release both i n v i t r o and i n vivo. I t has been reported that b r a i n fragments containing the MBH-MPOA-suprachiasmatic nucleus, release LHRH i n a p u l s a t i l e mode during i n v i t r o superfusion (Kim and Ramirez, 1986; Meyer, 19 1987). LHRH release was much more e r r a t i c i f the fragments excluded the MPOA and suprachiasmatic nucleus (Meyer, 1987). This suggests that components of the pulse generator, or inputs to the pulse generator, reside i n the MPOA and/or suprachiasmatic region. Push-pull perfusion of the MBH or ant e r i o r p i t u i t a r y i n unanaesthetized animals i s another means of monitoring the LHRH pulse generator (Ramirez and Dluzen, 1987). Dluzen and Ramirez (1985, 1986b) found that push-p u l l perfusion of the MBH i n castrated r a t s (male and female) did not r e s u l t i n a dramatic increase i n LHRH as one would expect from the elevated l e v e l s of LH i n the serum. However, push-pull perfusion of the anterior p i t u i t a r y i n gonadectomized males revealed elevated LHRH l e v e l s at the l e v e l of the p i t u i t a r y compared to LHRH l e v e l s i n i n t a c t animals. (Dluzen and Ramirez, 1987). Ramirez and Dluzen (1987) have hypothesized that the large LH pulses c h a r a c t e r i s t i c of gonadectomized r a t s may be more a function of increased synchrony of release by the LHRH neurons rather than an o v e r a l l increase i n the amount of LHRH released (Dluzen and Ramirez, 1987; Ramirez and Dluzen, 1987). I n t e r e s t i n g l y , a s i m i l a r hypothesis has been proposed from studies that employ neural recording techniques (Summerlee, 1986). In these studies the o s c i l l a t o r y pattern of a c t i v i t y of a si n g l e putative LHRH neuron, i n the MPOA, i s co r r e l a t e d with the surrounding multiunit a c t i v i t y . From these studies i t has been proposed that LHRH neurons are inherently o s c i l l a t o r y i n a c t i v i t y i . e . , they e x h i b i t bursts of a c t i v i t y between periods of no a c t i v i t y , and with each burst of a c t i v i t y LHRH i s released i n t o the p o r t a l vessels. The random bursting of neurons r e s u l t s i n a baseline of LHRH release. A pulse of LHRH release occurs when the bursting a c t i v i t y of a population of LHRH c e l l s occurs at approximately the same time, and t h i s r e s u l t s i n a coordinated release of LHRH into the p o r t a l vein which w i l l subsequently r e s u l t i n a pulse of LH i n the peripheral c i c u l a t i o n (Summerlee, 1986). This increase i n synchrony would presumably be maximal when LH release i s maximal i . e . , during a gonadotrophin surge. This hypothesis may account f o r the fac t that o v e r a l l LHRH neuronal a c t i v i t y i s not markedly enhanced i n gonadectomized animals whereas the release of LH i s grea t l y enhanced. How the synchrony of LHRH neurons might be achieved i s not known. Presumably i n such a system a pulse generator would be involved, i n coordinating the various inputs to the LHRH neurons, to bring about the "p h y s i o l o g i c a l l y appropriate", coordinated release of LHRH. Although the existence of a neural network responsible f o r episodic LHRH release seems quite c e r t a i n , the d e t a i l s concerning which neuronal systems are involved and how they i n t e r a c t to produce coordinated pulsations i n LHRH neuronal a c t i v i t y remain to be elucidated. VI. The Neural Regulation of LH Release: Role of NE  A. Introduction The neural regulation of LHRH release into the p i t u i t a r y p o r t a l veins i s under the complex control of a v a r i e t y of 21 neurotransmitters and/or neuromodulators. The l i s t of agents p o t e n t i a l l y involved i n the p h y s i o l o g i c a l release of LHRH has grown s t e a d i l y over the years. The monoamines , serotonin, DA, NE, and epinephrine, have a l l been implicated i n the control of LHRH release (Ramirez et a l . , 1984; Coen, 1987). In recent years the r o l e of peptides i n the regulation of anterior p i t u i t a r y hormone secretion has received increased attention (McCann et a l . , 1984). This has p a r t i a l l y come about as a r e s u l t of immunohistochemical and biochemical mapping studies demonstrating the presence of numerous peptides i n the brain (Krieger, 1983; Palkovits, 1984). The l i s t of neuropeptides i n the b r a i n has now grown to include more than 40, and a number of these have been implicated i n the control of gonadotrophin release (McCann et a l . , 1984). Many of these peptides are thought to act as neurotransmitters or neuromodulators i n the CNS (Krieger, 1983). The l i s t of peptides that may be involved i n the regulation of LHRH includes some of the other hypothalamic neurohormones such as c o r t i c o t r o p i n r e l e a s i n g factor, oxytocin, and LHRH i t s e l f (Gambacciani et a l . , 1986a, 1986b; Valenca et a l . , 1987; DePaulo et a l . , 1987). The endogenous opioid family of peptides (beta-endorphin, enkephalins, and dynorphins) are also thought to be involved i n the regulation of LH release. In p a r t i c u l a r , they may have a r o l e i n mediating the negative feedback e f f e c t s of gonadal ste r o i d s (Kalra and Kalra, 1984). Vasoactive i n t e s t i n a l polypeptide, pancreatic polypeptide, neuropeptide Y, cholecystokinin, and substance P are members of another family of peptides, f i r s t discovered i n the g a s t r o - i n t e s t i n a l t r a c t , that have also been reported to influence LH release (Vijayan and McCann, 1979; Vijayan et a l . , 1979a, 1979b; Kimura et a l . , 1983; Kalra and Crowley, 1984a, 1984b; Alexander et a l . , 1985; MacDonald et a l . , 1985a, 1985b). Neurotensin, angiotensin I I , and most rec e n t l y a t r i a l n a t r i u r e t i c peptide, have also been implicated i n the regulation of LHRH/LH release (Vijayan and McCann, 1979; F e r r i s et a l . , 1984; Steele and Ganong, 1986; Samson et a l . , 1988). In addition to a l t e r i n g LH release when infused into the t h i r d v e n t r i c l e , some of these peptides can a l t e r neuronal e x c i t a b i l i t y and t h e i r concentrations i n the br a i n do change over the course of the estrous cycle (Frankfurt et a l . , 1986; Pan et a l . , 1986). Immunocytochemical studies have also demonstrated that l e a s t two of these peptides (neurotensin and substance P) are present i n neurons which are c l o s e l y associated with LHRH-containing neurons i n the MPOA, thus providing an anatomical substrate f o r the demonstrated e f f e c t s of these peptides on LH release. These r e s u l t s suggest that several of the neuropeptide systems may be involved i n the regulation of LH release. Amino acids acting as neurotransmitters have also been associated with changes i n LH release. In p a r t i c u l a r gamma-aminobutyric acid (GABA) appears to have an i n h i b i t o r y r o l e i n the regulation of LH release (Donoso et a l . , 1986). The i n h i b i t o r y feedback e f f e c t s of E 2 have been postulated to occur v i a a c t i v a t i o n of GABA-ergic neurons which i n h i b i t the pre-synaptic release of NE. NE i s generally considered stimulatory 23 to LHRH release, therefore a decrease i n presynaptic NE release would be expected to decrease LHRH, and subsequent LH release (Wuttke et a l . , 1985). Histamine, which i s thought to act as a neurotransmitter i n the brain, i s able to stimulate LHRH release i n v i t r o v i a a s p e c i f i c histamine receptor (Miyake et a l . , 1987). I t has also recently been demonstrated that several metabolites of arachidonic a c i d (prostaglandin E 2 and leukotrienes C 4 and C 5) are present i n the hypothalamus and are potent stimulators of LHRH release (Ojeda et a l . , 1979; Heaulme and Dray, 1984; Gerozissis et a l . , 1985, 1987). The p h y s i o l o g i c a l s i g n i f i c a n c e of these various neuroactive agents i n the reg u l a t i o n of LHRH/LH release i s not known. However i t i s apparent that the neural regulation of LHRH i s complex, and a wide assortment of agents may be involved, i n various ways, employing d i f f e r e n t mechanisms, and under d i f f e r e n t hormonal conditions, i n the p h y s i o l o g i c a l l y appropriate release of LHRH. The most widely studied neurotransmitter that has been implicated i n LHRH/LH i s NE. There i s l i t t l e doubt that NE plays a r o l e i n the regulation of LH release from the p i t u i t a r y . I t i s also generally considered that the influence of NE on LH release i s p r i m a r i l y v i a i t s e f f e c t s on LHRH release from the ME. NE could p o t e n t i a l l y e f f e c t changes i n LHRH release by acting d i r e c t l y on LHRH-containing neurons at the l e v e l of the c e l l bodies, axons, and/or terminals. A d d i t i o n a l l y , NE released from nerve terminals could act i n d i r e c t l y to e f f e c t changes i n LHRH release. This could be achieved by enhancing or i n h i b i t i n g the a c t i v i t y of interneurons (excitatory and/or inhibitory) which are capable of i n f l u e n c i n g the a c t i v i t y of LHRH-containing neurons. A further degree of complexity a r i s e s because NE could also act v i a d i f f e r e n t receptors to e f f e c t d i f f e r e n t changes (excitatory or i n h i b i t o r y ) i n the post-synaptic c e l l . Employing various adrenergic agonists and antagonists, and under d i f f e r e n t s t e r o i d a l conditions, numerous reports have provided evidence on the e f f e c t s of adrenergic receptor a c t i v a t i o n on LHRH release, both i n v i t r o and i n vivo. Since the i n i t i a l proposal that NE has an excitatory r o l e i n the regulation of LHRH/LH release (see above) a great deal of evidence has accumulated im p l i c a t i n g the noradrenergic neuronal system as being f a c i l i t a t o r y to LHRH release (Barraclough and Wise, 1982; Kalra and Kalra, 1983; Ramirez et a l . , 1984; Coen, 1987). More recently i t has become evident that i n addition to i t s exc i t a t o r y r o l e , NE may also have an i n h i b i t o r y r o l e i n the regulat i o n of LH release (Taleisnik and Sawyer, 1986). The e f f e c t s of NE on LHRH release have been reported to occur v i a several pharmacologically d i s t i n c t receptors. Adrenergic receptors have been c l a s s i f i e d into two main groups, alpha and beta. The alpha-receptors and beta-receptors have each been further divided into 2 subtypes, alpha^ and alpha 2, and b e t a 1 and b e t a 2 , respectively (Cooper et a l . , 1982). In the p e r i p h e r a l nervous system, the al p h a ^ r e c e p t o r s were o r i g i n a l l y proposed as being located postsynaptically and the alpha 2-receptors were proposed as being presynaptic and associated with the regulation of synaptic NE release (Langer 25 et a l . , 1974). However, i n the CNS the alpha 2 receptors are probably located postsynaptically as well as presynaptically (Aghajanian, 1984; U'Prichard et a l . , 1980). A c t i v a t i o n of alpha 1~receptors i n hypothalamic neurons has been reported to r e s u l t i n d e p o l a r i z a t i o n and an increase i n the f i r i n g rate of the neurons (Ogata and Matsuo, 1986; Kow and P f a f f , 1987). Aghajanian (1984) has proposed that alpha 1~receptors are important i n regulating the e x c i t a b i l i t y of the postsynaptic c e l l and act by lowering the threshold of the c e l l to other e x c i t a t o r y inputs, rather than by transmitting the signals themselves. There i s also evidence which suggests that alpha 2~ receptor a c t i v a t i o n r e s u l t s i n hyperpolarization of the postsynaptic membrane and a consequent i n h i b i t i o n of neuronal a c t i v i t y (Aghajanian, 1984; Kow and P f a f f , 1987). On the other hand, beta-receptors have been reported to e i t h e r i n h i b i t neuronal a c t i v i t y (Ogata and Matsuo, 1986) or have a mixed i n h i b i t o r y and excitatory e f f e c t on neuronal a c t i v i t y (Kow and P f a f f , 1987). In the pyramidal c e l l s of the hippocampus beta-receptors enhance neuronal a c t i v i t y by blocking the accomodation of the neuron to continous excitatory input (Madison and N i c o l l , 1982; Haas and Konnerth, 1983). I n t e r e s t i n g l y , NE has been observed to exert both stimulatory and i n h i b i t o r y e f f e c t s i n the supraoptic nucleus, ventromedial nucleus, arcuate nucleus, o l f a c t o r y cortex, cerebellum, and a number of other b r a i n regions (Szabadi, 1979; C o l l i n s et a l . , 1984; B a s i l e and Dunwiddie, 1984; Ogata and Matsuo, 1986; Kow and P f a f f , 1985, 1987; Pan et a l , 1986). I t i s evident that NE i s able to produce a range of e f f e c t s on the postsynaptic c e l l . The c e l l may become depolarized or hyperpolarized depending on the c e l l and/or the receptor mediating the response as well as the concentration of the NE at the synapse. Since the d i f f e r e n t receptors may a l l be present on the same c e l l , the reponse of the c e l l to NE could be modulated by changes i n the r e l a t i v e porportions of the receptors on the postsynaptic c e l l . I t has been demonstrated that the r e l a t i v e proportions as well as the absolute number of putative transmitter receptors i s modulated by the s t e r o i d a l environment (Agnati et a l . , 1981; McEwen et a l . , 1981, Wilkinson et a l . , 1981). In addition to a l t e r i n g adrenergic recptors, gonadal steroids also a f f e c t a v a r i e t y of other neuronal systems (peptidergic) which may i n turn a l t e r the e f f e c t s of NE on the postsynaptic c e l l (Stumpf and Jennes, 1984). These various factors should be kept i n mind when attempting to i n t e r p r e t the e f f e c t s of NE on LH release. B. Stimulatory Action of NE on LH Release As mentioned previously, an excitatory r o l e f o r NE i n the con t r o l of gonadotrophin release was f i r s t suggested by Sawyer and co-workers (1947). I t was l a t e r demonstrated that NE, when infused into the t h i r d v e n t r i c l e , could induce ovulation when the endogenous proestrous LH surge was blocked by pentobarbital (Rubinstein and Sawyer, 1970). I n t r a v e n t r i c u l a r i n f u s i o n of NE also induced ovulation i n r a t s made anovulatory by anter i o r hypothalamic lesions or continuous i l l u m i n a t i o n (Tima and 27 Flerko, 1974) . Simultaneous i n j e c t i o n of E 2 and P 4 into ovariectomized r a t s s e n s i t i z e s the gonadotroph to LHRH 3 days l a t e r (Ramirez and McCann, 1963). In ovariectomized rats primed with E 2 and P^, NE stimulated LH release (Krieg and Sawyer, 1976; Gallo and Drouva, 1979) by increasing the amount of LHRH released i n t o the p o r t a l blood (Ching and Krieg, 1986). This e f f e c t was v i a alpha-receptors since i n t r a v e n t r i c u l a r i n f u s i o n of alpha-agonists, but not beta-agonists, increased LH release (Leung et a l . , 1982b). Push-pull perfusion of the MBH with NE i n i n t a c t or E 2~primed ovariectomized rabbits also increased LHRH release i n t h i s area (Ramirez et a l . , 1986; Pau and Spies, 1986) . An increase i n NE release i n the MPOA was also detected, using a push-pull cannula, p r i o r to the onset of the E 2~induced LH surge i n the ovariectomized r a t (Demling et a l . , 1985). These r e s u l t s demonstrate that NE can enhance LHRH release and that there i s an increase i n NE release before an increase i n LHRH release. Drugs which deplete b r a i n monoamines or i n h i b i t catecholamine synthesis also i n h i b i t LH release on proestrus and block ovulation (Barraclough and Sawyer, 1957; Kalra and McCann, 1974) . Transection of the DNT i n i n t a c t female r a t s also i n i t i a l l y disrupted the estrous cycle. In spi t e of the f a c t that t h i s transection r e s u l t e d i n an 80% depletion of NE l e v e l s i n the hypothalamus, estrous cycles were present 2 3 days a f t e r the transections were performed ( C l i f t o n and Sawyer, 1979). In contrast to t h i s , Kawakami and A r i t a (1980) reported that transection of the DNT on the morning of proestrus d i d not a f f e c t the LH surge or ovulation. The reason 28 f o r t h i s discrepancy i s not cl e a r . In any case, depletion of hypothalamic NE l e v e l s with 6-hydroxy-dopamine (6-OH-DA), a neurotoxin of catecholaminergic neurons, suppressed preovulatory,and steroid-induced, LH surge release (Hancke and Wuttke, 1979; Simpkins et a l . , 1979). Sarkar and Fink (1981) also reported that i n h i b i t o r s of catecholamine synthesis reduced the magnitude of the LHRH and LH surge. The stimulatory e f f e c t of NE on LHRH release i s probably mediated v i a alpha-adrenergic receptors since phenoxybenzamine (an alpha-adrenergic antagonist) reduced both the LHRH and LH surge whereas propranolol and yohimbine (a beta- and an alpha 2~ adrenergic antagonist, respectively) had no e f f e c t on the estrogen-induced LH surge (Sarkar and Fink, 1981; Drouva et a l . , 1982). A few recent reports have suggested that excitatory e f f e c t s of NE may also be mediated v i a a c t i v a t i o n of beta-receptors. In pentobarbital-blocked proestrous rats i n t r a -v e n t r i c u l a r i n f u s i o n of a s e l e c t i v e beta 2~agonist, but not a beta 1~agonist, stimulated ovulation and also enhanced LH release i n E 2-primed ovariectomized rat s (Al-Hammood et a l . , 1985). These authors have also reported that the post-c a s t r a t i o n increase i n LH release i s p a r t i a l l y i n h i b i t e d by a beta 2-antagonist (Al-Hamood et a l . , 1987). Their r e s u l t s implicate beta-receptors as p a r t i a l l y mediating the excitatory e f f e c t s of NE on LH release. A stimulatory r o l e f o r NE on LHRH release (mediated by alpha-adrenergic receptors) may also be i n f e r r e d from the 29 r e s u l t s of several i n v i t r o studies. I t has been shown that NE-induced LHRH release i n ME fragments i n a dose-dependent manner and these e f f e c t s were blocked by alpha-, but not beta-, adrenergic antagonists (Negro-Vilar et a l . , 1979; Ojeda et a l . , 1982) . Using a s i m i l a r ME incubation system, Heaulme and Dray (1984) demonstrated that phenylephrine (an alp h a 1 adrenergic agonist) stimulated LHRH release whereas clonidine (an alpha 2~ adrenergic agonist) and isoproterenol (a beta-adrenergic agonist) had no e f f e c t on basal LHRH release. They also found that an alpha-adrenergic antagonist, but neither an a l p h a 2 - or a beta-adrenergic antagonist, blocked the stimulatory e f f e c t s of NE on LHRH release. NE also stimulated LHRH release from superfused hypothalamic fragments (Nowak and Swerdloff, 1985). Similar to the r e s u l t s of experiments on fragments of the ME, the e f f e c t s of NE on hypothalamic fragments were dose dependent and blocked by an alpha-adrenergic antagonist. An excitatory r o l e f o r NE i n LH release i s also supported by studies which have examined catecholamine turnover rates i n s p e c i f i c hypothalamic regions. The rate of catecholamine turnover i s measured by f i r s t blocking catecholamine synthesis and then measuring catecholamine l e v e l s at regular i n t e r v a l s a f t e r the administration of the catecholamine synthesis i n h i b i t o r . Presumably the amount of catecholamine depleted a f t e r catecholamine synthesis has been halted, r e f l e c t s the amount of catecholamine which has been released from the nerve terminals. Changes i n turnover rates have been correlated with a l t e r a t i o n s i n plasma LH l e v e l s i n i n t a c t female rats, ovariectomized rats, ovariectomized plus estrogen, and ovariectomized plus estrogen- and P 4~primed rats (Honma and Wuttke, 1980; Ranee et a l . , 1981a, 1981b; Wise et a l . , 1981a; Crowley, 1982). In proestrous r a t s , the NE turnover rate increased i n i t i a l l y i n the ME p r i o r to i n i t i a t i o n of the LH surge; once the surge has sta r t e d NE turnover rates are also increased i n the MPOA, suprachiasmatic nucleus and the arcuate nucleus (Ranee et a l . , 1981b). Honma and Wuttke (1980) also reported an increase i n rate of NE turnover on the afternoon of proestrous i n the MPOA and MBH; the increase i n NE turnover i n the ME and MBH correlated w ell with increased LH l e v e l s i n ovariectomized rats primed e i t h e r with estrogen alone, or with estrogen and P 4 < Ranee et a l . (1981a) found that i n ovariectomized rats primed with estrogen, increases i n NE turnover occurred i n the ME, MPOA, and suprachiasmatic n u c l e i at the time of an increase i n LH secretion. An increase i n NE turnover rate i n the ME also occurred i n proestrous rat s at the time of the LH surge and t h i s increase was asssociated with a decline i n LHRH l e v e l s i n the ME (Ranee et a l . , 1981b; Wise et a l . , 1981b). This decrease i n LHRH concentration at the l e v e l of the ME i s thought to be a r e s u l t of an increase i n the amount of LHRH released into the p o r t a l veins which i n turn increases LH release. In r a t s treated with estrogen and P 4, these increases i n NE turnover are advanced i n time as i s the LH surge (Wise et a l . , 1981). Taken together, these studies suggest that an increase i n the a c t i v i t y of the noradrenergic neurons i s involved i n t r i g g e r i n g and maintaining the LH surge, 31 and the feedback e f f e c t s of estrogen and P 4 on LH release may be mediated by t h e i r e f f e c t s on noradrenergic neurons i n the hypothalamus. These noradrenergic neurons i n the hypothalamus presumably act to enhance the release of LHRH i n t o the p o r t a l veins. As well as being implicated i n the onset of the proestrous surge and steroid-induced LH surge, NE i s also thought to be involved i n maintaining the p u l s a t i l e release of LH observed i n ovariectomized r a t s . Systemic i n j e c t i o n of phentolamine or phenoxybenzamine (alpha-adrenergic antagonists) into ovariectomized rat s resulted i n marked suppression of p u l s a t i l e LH release (Gnodde and Schuiling, 1976; Weick, 1978). In ovariectomized monkeys, alpha-adrenergic antagonists decreased the frequency of p u l s a t i l e LH release and i n h i b i t e d multiunit a c t i v i t y i n the MBH which i s associated with p u l s a t i l e LH release (Kaufman, et a l . , 1985). This suggests that these drugs have a c e n t r a l s i t e of action. I n h i b i t i o n of NE synthesis i n ovariectomized r a t s , which resulted i n decreased hypothalamic NE concentration, also resulted i n marked suppression of p u l s a t i l e LH release (Gnodde and Schuiling, 1976; Drouva and Gallo, 1976; Negro-Vilar et a l . , 1982) . The i n i t i a l increase i n LH release observed i n gonadectomized r a t s has been at t r i b u t e d to increased a c t i v a t i o n of both alpha- and beta-adrenergic receptors (Ojeda and McCann, 1973; Al-Hamood et a l . , 1987). ojeda and McCann (1979) reported that s e l e c t i v e blockade of NE synthesis i n h i b i t e d the post-castration r i s e i n LH, as d i d the administration of alpha-32 adrenergic antagonists. More recently i t has been suggested that the increase i n LH release following c a s t r a t i o n may be p a r t i a l l y a r e s u l t of an increase i n the a c t i v a t i o n of beta 2~, and a decrease i n the a c t i v a t i o n of beta^-adrenergic receptors (Al-Hamood et a l . , 1987). These authors reported that b e t a 2 ~ agonists and beta^antagonists increased while b e t a 2 ~ antagonists and be t a ^ a g o n i s t s decreased the post-castration r i s e i n LH release. However, i n long-term ovariectomized rats, the p u l s a t i l e release of LH appears to be maintained by NE v i a a c t i v a t i o n of alpha-receptors since alpha-antagonists i n h i b i t e d LH release while beta-receptor antagonists had l i t t l e or no e f f e c t on LH release (Ojeda and McCann, 1973; Weick, 1978). Gallo and Kalra (1983) found that, as i n ovariectomized rats, i n h i b i t i o n of NE synthesis i n i n t a c t r a t s i n diestrous 1 also blocked p u l s a t i l e LH release. Similar to ovariectomized rats, alpha-adrenergic but not beta-adrenergic antagonists reduced mean blood LH l e v e l s by decreasing both LH pulse amplitude and pulse frequency i n i n t a c t rats on diestrous 1. Taken together, these r e s u l t s suggest that NE acting p r i m a r i l y v i a alpha-adrenergic receptors i s important i n maintaining the p u l s a t i l e pattern of LH release i n ovariectomized and i n t a c t r a t s . I t has been proposed that the ro l e of NE i s to provide a permissive environment i n which the regular discharge of the LHRH neurons can occur (Kalra and Kalra, 1983) and that the p u l s a t i l e release of LHRH i s not a r e s u l t of p u l s a t i l e NE input to the LHRH pulse generator. Depletion of NE l e v e l s i n ovariectomized rats eliminates p u l s a t i l e LH release but pulses can be restored, at l e a s t p a r t i a l l y , by a single i n j e c t i o n of clonidine (alpha 2~agonist) (Estes et a l . , 1982). The r e s u l t s demonstrating i n h i b i t i o n by alpha-antagonists, of multiunit a c t i v i t y i n the MBH of ovariectomized monkeys also argues against p u l s a t i l e NE input (Kaufman et a l . , 1985). I t was suggested that since blockade of alpha-receptors i n h i b i t e d the frequency of the pulse generator a c t i v i t y i t does not seem l i k e l y that the antagonists could change the frequency of NE input. I t seems more probable that blockade of the alpha-adrenergic receptors decreased the frequency at which the pulse generator drives the LHRH neural network. C. Inhi b i t o r y Action of NE on LH Release I t i s only rather recently that an i n h i b i t o r y action of NE on LH release has been described. Gallo and Drouva (1979) f i r s t suggested the p o s s i b i l i t y that a c t i v a t i o n of noradrenergic receptors could r e s u l t i n i n h i b i t i o n of LH release when i t was demonstrated that NE in f u s i o n into the t h i r d v e n t r i c l e i n h i b i t e d p u l s a t i l e LH release i n o v a r i -ectomized r a t s . This i n h i b i t o r y e f f e c t i s probably v i a alpha-adrenergic receptors since alpha-adrenergic agonists such as clonidine and phenylephrine are more e f f e c t i v e than isoproterenol, a beta-adrenergic agonist, i n suppressing LH release (Leung et a l . , 1982a). Continuous i n t r a v e n t r i c u l a r i n f u s i o n of NE i n ovariectomized rats resulted i n decreased LH pulse frequency while pulse amplitude remained unchanged (Gallo, 1984) . This suggests that i t i s the frequency of the LHRH pulse generator that i s affec t e d and not the amount of LHRH released per pulse into the p o r t a l veins. (Gallo, 1984). Leipheimer and Gallo (1985) found that the MPOA i s one s i t e where NE could act to suppress the putative LHRH pulse generator. Using a push-pull perfusion cannula they found that perfusion of the MPOA with NE resul t e d i n suppression of LH pulse frequency while pulse amplitude remained unchanged, s i m i l a r to continuous i n f u s i o n of NE into the t h i r d v e n t r i c l e (Leipheimer and Gallo, 1985). I n t r a v e n t r i c u l a r i n f u s i o n of NE i n ovariectomized r a t s also i n h i b i t e d multiunit a c t i v i t y i n the MPOA, diagonal band of Broca and anter i o r hypothalamic area (regions r i c h i n LHRH c e l l bodies) which was corre l a t e d with a decrease i n p u l s a t i l e LH release (Leung et a l . , 1981b, 1982c). This decrease i n multiunit a c t i v i t y may r e f l e c t a decrease i n the a c t i v i t y of the central pulse generator. Int e r e s t i n g l y , NE inf u s i o n increased multiunit a c t i v i t y i n E 2 and P 4 primed rat s when i t also increased LH l e v e l s i n the blood (Leung et a l . , 1981b, 1982c) . I t has also been reported that i n anaesthetized r a t s , e l e c t r i c a l stimulation of ascending noradrenergic f i b r e s at the l e v e l of the mesencephalon resulted i n i n h i b i t i o n of p u l s a t i l e LH release s i m i l a r to i n t r a -v e n t r i c u l a r infusion of NE (Leung et a l . , 1981a). I t i s not known where the e f f e c t of a c t i v a t i o n of these f i b r e s i s mediated but one p o s s i b i l i t y i s the MPOA since the suppression of LH release i s s i m i l a r to that seen with perfusion of t h i s area, i . e . a decrease i n pulse frequency (Leipheimer and Gallo, 1985). Whether these i n h i b i t o r y e f f e c t s are mediated by an 35 i n h i b i t o r y interneuron between the ascending NE f i b r e s and the LHRH neuron i s not known. The mechanism and p h y s i o l o g i c a l s i g n i f i c a n c e of NE i n h i b i t i o n of p u l s a t i l e LH release i n the ovariectomized r a t i s not known. One p o s s i b i l i t y f o r the mechanism of i n h i b i t i o n i s that the LHRH neurons may become desensitized to a d d i t i o n a l exogenous noradrenergic input, r e s u l t i n g i n reduced LHRH output and subsequently lower LH l e v e l s i n the blood. In t h i s context, i t i s of i n t e r e s t to note that the increased release of LH induced by electrochemical stimulation of the MPOA could also be p a r t i a l l y suppressed by perfusion of the t h i r d v e n t r i c l e with high concentrations of NE and epinephrine (Cramer and Barraclough, 1978). Gallo (1982) showed that continuous i n t r a v e n t r i c u l a r i n f u s i o n of NE i n ovariectomized steroid-primed rats i n i t i a l l y increased LH release but LH l e v e l s returned to p r e i n f u s i o n l e v e l s within ninety minutes thus demonstrating that the LHRH neurons can become desensitized to continuous e x c i t a t o r y NE input. These r e s u l t s suggest that d e s e n s i t i z a t i o n of the excitatory NE receptors does occur r e a d i l y . I n t e r e s t i n g l y , while d e s e n s i t i z a t i o n to the stimulatory e f f e c t s of NE occurred within ninety minutes, the receptors activated by NE that are i n h i b i t o r y to p u l s a t i l e release, maintained t h e i r responsiveness to continuous NE i n f u s i o n even a f t e r twenty hours and therefore are not e a s i l y desensitized (Gallo, 1982). An i n h i b i t o r y r o l e f o r NE i n regulating LH release has also been proposed by T a l e i s n i k and co-workers (Caceres and 36 T a l e i s n i k , 1980b, 1982; Dott i and Ta l e i s n i k , 1982, 1984). I t was demonstrated that electrochemical stimulation of the fr o n t a l lobe of the cerebral cortex on the day of proestrous, blocked the preovulatory LH surge and ovulation (Caceres and Ta l e i s n i k , 1980a). Stimulation of the ant e r i o r cingulate cortex also blocked the steroid-induced surge i n ovariectomized r a t s and LH release induced by e l e c t r i c a l stimulation of the MPOA. This i n h i b i t i o n was found to be mediated v i a beta-adrenergic receptors since propranolol, a beta-adrenergic antagonist, prevented the i n h i b i t o r y e f f e c t s of c o r t i c a l stimulation (Caceres and Ta l e i s n i k , 1980b). I t was also shown that i n t r a - v e n t r i c u l a r i n f u s i o n of isoproterenol (a beta-adrenergic agonist), i n h i b i t e d LH release induced by e l e c t r i c a l stimulation of the MPOA (Caceres and Ta l e i s n i k , 1980b; 1982). In t e r e s t i n g l y , a c t i v a t i o n of NE c e l l groups i n the brainstem (LC, A5, A l , c e l l groups) also i n h i b i t e d ovulation and LH release (Dotti and T a l e i s n i k , 1982). The i n h i b i t o r y noradrenergic f i b r e s from the LC ascend through the mescencephalon i n the DNT and eventually end i n the premammillary nucleus, where NE activates beta-adrenergic receptors located on interneurons which e i t h e r d i r e c t l y or i n d i r e c t l y i n h i b i t LHRH release (Dotti and T a l e i s n i k , 1982; 1984) . Beltramino and T a l e i s n i k (1984) found that premammillary cuts and lesions of the pos t e r i o r hypothalamus which presumably interrupted these ascending i n h i b i t o r y f i b r e s , r e s u l t e d i n an e a r l i e r proestrous LH surge while stimulation of the premammillary or pos t e r i o r hypothalamic n u c l e i resulted i n blockade of the LH surge and ovulation. I t was suggested that these i n h i b i t o r y inputs acting v i a beta-adrenergic receptors may be involved i n s e t t i n g the timing and height of the LH surge. The divergent e f f e c t s of alpha- and beta-agonists led to the proposal that alpha-receptors mediate the excitatory e f f e c t s and beta-receptors mediate the i n h i b i t o r y e f f e c t s of NE on LH release ( T a l e i s n i k and Sawyer, 1986). A pos s i b l e r o l e f o r beta-adrenergic receptors i n mediating the i n h i b i t o r y e f f e c t s of NE on LH release was also proposed when i t was demonstrated that i n t r a v e n t r i c u l a r i n f u s i o n of a beta-adrenergic agonist resulted i n suppression of a steroid-induced LH surge (Leung et a l . , 1982b). Caceres and T a l e i s n i k (1980b) found that i n t r a v e n t r i c u l a r i n f u s i o n of NE had no e f f e c t on LH release induced by e l e c t r i c a l stimulation of the MPOA but NE was more than two and a h a l f times more e f f e c t i v e i n releasing LH when beta-adrenergic receptors were blocked. Presumably the beta-receptors are i n h i b i t o r y and when they are blocked the alpha-receptors can exert t h e i r e x c i t a t o r y e f f e c t s v i a excitatory alpha-receptors, unopposed by the simultaneous a c t i v a t i o n of i n h i b i t o r y beta-receptors. I t has also been suggested that neural elements i n h i b i t o r y to LH release, i n t h i s case NE, may have an important p h y s i o l o g i c a l r o l e to play i n the regul a t i o n of the LH surge. One p o s s i b i l i t y i s that a f t e r a LH surge has been triggered, i n h i b i t o r y components could become acti v a t e d to suppress the amplitude and duration of the LH surge. I f the 38 changes i n LH release are seen as a l t e r a t i o n s i n the tone of exc i t a t o r y and i n h i b i t o r y inputs, as has recently been suggested by Beltramino and T a l e i s n i k (1984), i n i t i a t i o n of the LH surge could r e s u l t from a r e l a t i v e increase i n excitatory tone whereas an increase i n i n h i b i t o r y tone could act to r e s t r a i n the height and/or duration of the surge. This r e l a t i v e increase i n i n h i b i t o r y tone may p a r t i a l l y be a r e s u l t of the r e l a t i v e d e s e n s i t i z a t i o n properties of the two types of receptors (Gallo, 1982, 1984). I t has been demonstrated that some i n h i b i t o r y tone i s present even during a LH surge and the amount of LH released can be enhanced with elimination of t h i s i n h i b i t o r y tone. Blockade of op i o i d receptors with naloxone enhanced LH release i n proestrous rat s and the E 2~induced LH surge i n ovariectomized rats ( I e i r i et a l . , 1980; Sylvester et a l . , 1980; Kubo et a l . , 1983). The proestrous LH surge was also enhanced by transections of f i b r e s i n the v e n t r o l a t e r a l region of the midbrain (Kawakami and A r i t a , 1980). The biochemical nature of the f i b r e s , involved i n t h i s e f f e c t , i s not known. Again, these f i b r e s would presumably be involved i n the timing and amplitude of the surge. D. Role of Brain NE Pathways i n the Regulation of LH Release Noradrenergic nerve terminals i n the hypothalamus have t h e i r c e l l bodies i n the brainstem and the axons ascend through the midbrain v i a the DNT and VNT (L i n d v a l l and Bjorklund, 1978; Moore and Bloom, 1979; see above). The r e l a t i v e r o l e s of these two noradrenergic pathways i n the regulation of gonadotrophin 39 release are not f u l l y understood. I t i s thought that impulses t r a v e l l i n g v i a these pathways are able to modulate the frequency and/or the amplitude of the LHRH pulses, thus a f f e c t i n g LH secretion. One approach to better understanding the r o l e of NE (and the possible d i f f e r e n t roles of these two pathways) i n the regulation of LH release, has involved l e s i o n i n g or stimulating the ascending noradrenergic pathways and monitoring ovulation and/or LH release. The r o l e of the DNT i n the regulation of gonadotrophin release i s not completely c l e a r . Attempting to understand the r o l e of the DNT i s p a r t i c u l a r l y d i f f i c u l t when i n t e r p r e t i n g and comparing data from acute and chronic studies i n i n t a c t , o v a r i -ectomized, or steroid-primed ovariectomized r a t s . I t appears that, s i m i l a r to NE (see above), the DNT can have an excitatory and an i n h i b i t o r y e f f e c t on LH release. I t has been proposed that the DNT c a r r i e s f i b r e s which can f a c i l i t a t e LH release. Carrer and T a l e i s n i k (1970) reported that electrochemical stimulation of the DNT i n proestrus r a t s had no e f f e c t on ovulation. However they d i d f i n d that stimulation of the DNT induced ovulation i n rats made anovulatory by constant i l l u m i n a t i o n , and increased serum LH l e v e l s i n estrogen-primed ovariectomized r a t s . However, the f i b r e s which f a c i l i t a t e LHRH release do not appear to be r e a d i l y activated since e l e c t r i c a l stimulation of the DNT i n ovariectomized, steroid-primed r a t s had no e f f e c t on LH release (Leung et a l . , 1981a). I t should be noted that i n t r a -v e n t r i c u l a r infusion of NE re s u l t e d i n an increase i n LH 40 release i n s i m i l a r l y steroid-primed r a t s . However, i f f i b r e s e x c i t a t o r y to LH release are present they are not e s s e n t i a l since normal estrous cycles resume a f t e r a period of time following DNT transection ( C l i f t o n and Sawyer, 1979). S i m i l a r l y , chronic hypothalamic depletion of NE also did not prevent the r i s e i n blood LH l e v e l s nor the p u l s a t i l e release of LH observed i n long term ovariectomized r a t s ( C l i f t o n and Sawyer, 1980a, 1980b; C l i f t o n and Steiner, 1985). The i n h i b i t o r y e f f e c t s of NE on p u l s a t i l e LH release i n the ovariectomized rat are probably mediated, at l e a s t i n part, by NE release from nerve f i b r e s ascending v i a the DNT. E l e c t r i c a l stimulation of the DNT i n anaesthetized o v a r i -ectomized r a t s i n h i b i t e d p u l s a t i l e LH release (Leung et a l . , 1981a). A NE-mediated mechanism was suggested since the e f f e c t was blocked by a catecholamine, but not a serotonin, synthesis i n h i b i t o r . Further evidence f o r a alpha-adrenergic mechanism i s provided by i n t r a v e n t r i c u l a r i n f u s i o n experiments, i n that the i n h i b i t i o n produced by e l e c t r i c a l stimulation i s s i m i l a r to that observed with i n t r a v e n t r i c u l a r i n f u s i o n of alpha-adrenergic agonists (Leung et a l . , 1982a). The e f f e c t of LC a c t i v a t i o n i n suppressing ovulation, and LH release, also appears to be mediated by f i b r e s i n the DNT since i n h i b i t i o n i s blocked by lesions of the DNT but not of the VNT (Dotti and T a l e i s n i k , 1982). This i n h i b i t i o n i s apparently mediated by a c t i v a t i o n of a beta-adrenergic receptor s i t u a t e d i n the premammilary nucleus (Caceres and T a l e i s n i k , 1982; Dotti and T a l e i s n i k , 1984). Dotti and T a l e i s n i k (1984) suggested that 41 beta-adrenergic receptors i n h i b i t o r y to LH release are not situated a n t e r i o r to the premammillary nucleus. From these reports i t appears that the DNT c a r r i e s f i b r e s which can act at alpha-receptors or beta-receptors to suppress LH release. These may be separate f i b r e s acting at d i f f e r e n t receptors or the same f i b r e s with separate branches projecting and acting on d i f f e r e n t receptors. I t i s known that noradrenergic c e l l bodies i n the LC send out numerous axon c o l l a t e r a l s to a v a r i e t y of s i t e s i n the b r a i n (Moore and Bloom, 1979) . A l t e r n a t i v e l y , the s t e r o i d treatment (E 2) may also have played a r o l e i n a l t e r i n g the r e l a t i v e populations of the d i f f e r e n t receptors. Wilkinson et a l . , (1979, 1981) have reported that gonadal s t e r o i d s can influence the number of adrenergic receptors present i n the hypothalamus. S p e c i f i c a l l y , E 2 decreased alpha-receptor density and increased beta-receptor density i n the hypothalamus (Wilkinson et a l . , 1979). I t i s possible that t h i s e f f e c t of E 2 may have had a r o l e i n changing the i n h i b i t o r y e f f e c t from an alpha-receptor to a beta-receptor. Taken together, these r e s u l t s suggest that the DNT contains f i b r e s which are p r i m a r i l y , although not n e c e s s a r i l y ex c l u s i v e l y , i n h i b i t o r y to LH release and that the i n h i b i t i o n can occur v i a a c t i v a t i o n of e i t h e r alpha- or beta-adrenergic receptors. The r o l e of the VNT i n regulating LH (LHRH) release i s not well understood. The VNT has been assigned an i n h i b i t o r y r o l e by some in v e s t i g a t o r s , and an excitatory r o l e by others. 42 Carrer and T a l e i s n i k (1970) reported that electrochemical stimulation of the VNT i n h i b i t e d ovulation i n proestrous r a t s . I t has also been suggested that the f i b r e s i n t h i s t r a c t are pr i m a r i l y e x c i t a t o r y to LH release (Ramirez et a l . , 1984). Martinovic and McCann (1977) reported that chemical lesions of the VNT produced by 6-OH-DA, blocked the proestrous LH and FSH surges i n i n t a c t r a t s and the steroid-induced surges i n ovariectomized r a t s . Hancke and Wuttke (1979) reported that a f t e r 6-OH-DA was inj e c t e d into the MPOA, or the VNT, there was a t r a n s i e n t d i s r u p t i o n of the estrous cycle, with resumption of regular estrous cycles a f t e r 1-3 days. However, on the second proestrus a f t e r 6-OH-DA administration, surge l e v e l s of LH were suppressed compared to saline-treated controls. I t was suggested that NE may not be mandatory fo r ovulation but the VNT i s involved i n f a c i l i t a t i n g LH release during a surge. In contrast to the study of Martinovic and McCann (1977), Nicholson et a l . (1978) reported that l e s i o n i n g of the VNT with 6-OH-DA, reduced the NE content of the hypothalamus and preoptic area, but had l i t t l e or no e f f e c t on the occurence of estrous c y c l e s . These r e s u l t s also demonstate that the VNT may not be necessary f o r a LH surge to occur. Nicholson et a l . (1978) suggested that the acute i n h i b i t o r y e f f e c t s of 6-OH-DA on the proestrous surge that Martinovic and McCann (1977) reported, may be due to nonspecific e f f e c t s of the neurotoxin and the degeneration of the NE f i b r e s takes 7-14 days before degeneration of the f i b r e s i s complete. While these r e s u l t s are not i n complete agreement, i t appears that acute depletion 43 of NE l e v e l s may i n h i b i t LH release but chronic depletion has l i t t l e or no e f f e c t on estrous cycles. Kawakami and A r i t a (1980) reported that transections of the VNT on proestrous blocked the LH surge and ovulation. They also demonstrated that i f NE was infused into the t h i r d v e n t r i c l e at the time of transection, ovulation occurred (Kawakami and Ando, 1981). I t i s not known whether normal estrous cycles were able to resume a f t e r VNT transections. E l e c t r i c a l stimulation of the VNT can bring about a s l i g h t increase i n LH release i n E 2~primed ovariectomized r a t s which suggests an excitatory r o l e f o r the VNT (Dyer et a l . , 1985). Taken together i t appears that the VNT i s probably f a c i l i t a t o r y to LHRH release, but i t i s not completely c l e a r whether i t i s required f o r normal LH release since ovulation and regular estrous cycles occur despite chemical l e s i o n i n g of the t r a c t . There are no reports examining a possible r o l e f o r the VNT i n the regulation of p u l s a t i l e LH release i n gonadectomized animals. I t seems probable that i f the VNT does play an ex c i t a t o r y r o l e i n the regulation of LH release i t i s v i a alpha-receptors since the bulk of the evidence points to alpha-receptors as mediating the excitatory e f f e c t s of NE on LH release (Ramirez et a l . , 1984; T a l e i s n i k and Sawyer, 1986; also see above). E. Summary The primary si g n a l regulating the release of LH from the p i t u i t a r y i s LHRH. The release of LHRH into the p i t u i t a r y p o r t a l system i s the f i n a l step i n the neuroendocrine regul a t i o n of LH secretion. The regulation of LHRH secretion appears to be under the complex con t r o l of a wide v a r i e t y of fa c t o r s . Catecholamines, serotonin, amino acids, arachidonic a c i d metabolites, and numerous neuropeptides, have a l l been i d e n t i f i e d as having a pharmacological e f f e c t on the secretion of LHRH and/or LH release. A d d i t i o n a l l y , ovarian s t e r o i d feedback at the l e v e l of the brain plays a major r o l e i n the regulat i o n of LHRH release. How these various factors act together, presumably i n a coordinated manner, i n the neuro-endocrine regulation of LH release i s not known. The r o l e of NE i n the regulation of LH release has received the most attention of a l l the neurotransmitters. There i s abundant evidence demonstrating an excitatory r o l e for NE i n the regulation of LH release. The excitatory e f f e c t s may involve, but are not necessarily l i m i t e d to, NE acting d i r e c t l y on the LHRH neurons. The alpha-adrenergic receptors are most often implicated as mediating the excitatory e f f e c t s of NE on LHRH, but a few reports have also suggested that beta-receptors under c e r t a i n conditions might also mediate excitatory e f f e c t s of NE on LH release. The s i t e of these receptors has not been determined conclusively although catecholaminergic nerve terminals have been closely associated with LHRH-containing neurons at the l e v e l of the MPOA and ME. This would suggest that at l e a s t some of the e f f e c t s of NE are as a d i r e c t r e s u l t of t h e i r i n t e r a c t i o n with LHRH-containing neurons. I t i s becoming evident that the NE has an i n h i b i t o r y r o l e as well as 45 an excitatory r o l e i n the regulation of LH release. The i n h i b i t o r y e f f e c t s of NE can be demonstrated i n ovariectomized as well as steroid-primed r a t s . The i n h i b i t o r y e f f e c t s i n these two cases are mediated by an alpha- and a beta-receptor, r e s p e c t i v e l y . However, i t does appear that under c e r t a i n conditions the excitatory e f f e c t s may be mediated by both alpha- and beta-adrenergic receptors and the i n h i b i t o r y e f f e c t s of NE can also be mediated by both alpha- and beta-receptors. The r o l e s of the d i f f e r e n t NE pathways i n LH release are not well understood. There are reports which suggest that the DNT i s i n h i b i t o r y and the VNT i s excitatory to LH release. There are however r e l a t i v e l y few studies which have examined the possible r o l e s of these pathways i n LH release under d i f f e r e n t s t e r o i d a l conditions. VII. Summary A r o l e f o r catecholamines i n the regulation of LH release was f i r s t proposed 40 years ago. Since that time a great number of experiments have examined, and attempted to shed l i g h t on, the neuroendocrine regulation of LH release. Much of t h i s work has focussed on the r o l e of NE i n the r e g u l a t i o n of LHRH release. Neuroanatomical studies have attempted to determine the anatomical r e l a t i o n s h i p between the LHRH and NE neuronal systems. While some studies have provided evidence f o r the neuroanatomical substrate f o r the e f f e c t s of NE on LHRH release, the precise nature and d i s t r i b u t i o n of noradrenergic inputs to LHRH-containing neurons i s not known. I t i s also not 46 yet known the types of receptors located on the LHRH neurons through which NE might exert i t s e f f e c t . In any case i t i s known that NE does influence the release of LHRH e i t h e r d i r e c t l y or i n d i r e c t l y (via interneurons) and that i t can influence the a c t i v i t y of the LHRH pulse generator. Since NE can be both excitatory and i n h i b i t o r y to LH release i t seems u n l i k e l y that the same receptor population would mediate both e f f e c t s . This has prompted the proposal that NE exerts i t s d i f f e r e n t i a l e f f e c t s v i a separate and anatomically d i s t i n c t receptor populations (Leung et a l . , 1982a; Bergen and Leung, 1987a). One population of receptors (alpha-receptors) presumably located d i r e c t l y on LHRH neurons are stimulatory to LHRH release and t o n i c a c t i v a t i o n of these neurons would maintain p u l s a t i l e LH release. Increased a c t i v a t i o n of these f i b r e s might also mediate f a c i l i t a t i o n of the LH surge. Other populations of receptors (alpha and beta) i n h i b i t o r y to LHRH release may be located on the LHRH neurons or may influence interneurons to i n h i b i t the a c t i v i t y of the LHRH neurons. There i s probably a dynamic i n t e r p l a y of excitatory versus i n h i b i t o r y influences on LHRH neuronal a c t i v i t y and i t i s the r e s u l t of t h i s i n t e r p l a y which determines the release of LHRH at any given point i n time. Although NE does appear to have a r o l e i n the regulation of LH release there are doubtlessly other neuronal systems involved, and feedback from the gonads i s also c r u c i a l i n regulating the r e l a t i v e importance of each of the various neuronal systems (Stumpf and Jennes, 1984). The experiments which are reported herein were undertaken to examine further the r o l e of NE i n the neuroendocrine regulation of LH release i n ovariectomized, and steroid-primed ovariectomized r a t s . The following two chapters report on the r o l e of NE i n the regulation of p u l s a t i l e LH release i n the ovariectomized r a t when gonadal s t e r o i d l e v e l s are low. In p a r t i c u l a r , the e f f e c t s of s p e c i f i c adrenergic receptor antagonists on p u l s a t i l e LH release and the e f f e c t of those antagonists on the NE-induced i n h i b i t i o n of LH release were determined. I t was then examined whether the two major NE t r a c t s i n the b r a i n are able to i n h i b i t p u l s a t i l e LH release, s i m i l a r to exogenous i n f u s i o n of NE into the t h i r d v e n t r i c l e . The l a s t two chapters report on the e f f e c t s of NE receptor a c t i v a t i o n , and stimulation of the NE t r a c t s i n the r e g u l a t i o n of LH release i n d i f f e r e n t , well-established, experimental models where LH release i s a f f e c t e d by the administration of ovarian s t e r o i d s . In p a r t i c u l a r i t was of i n t e r e s t to determine the e f f e c t s of d i f f e r e n t receptor agonists on the steroid-induced LH surge. I t was also of i n t e r e s t to determine the e f f e c t s of stimulation of the two major t r a c t s on LH release under a number of d i f f e r e n t conditions i . e . , at times of low LH release and at times of high LH release. These experiments were performed to understand, more completely, the r o l e of the d i f f e r e n t adrenergic receptor subtypes and d i f f e r e n t NE t r a c t s i n the r e g u l a t i o n of LH release. In each chapter i s a more d e t a i l e d account of the objectives of the experiments described i n that chapter. 48 Norepinephrine I n h i b i t i o n of P u l s a t i l e LH Release i n Ovariectomized Rats: Receptor S p e c i f i c i t y I. Introduction In v i r t u a l l y a l l mammalian species, as well as i n other vertebrates which have been studied, LH release i s p u l s a t i l e i n both the male and the female. I t i s also well documented that changes i n the p u l s a t i l e release pattern of LH i . e . , the amplitude and frequency of the pulses, are associated with changes i n the reproductive status of the animal (see General Introduction pp.3-7, re: p u l s a t i l e LH re l e a s e ) . An almost uni v e r s a l c h a r a c t e r i s t i c of LH release i n mammals i s that following gonadectomy, i n males and females, LH pulse amplitude and frequency increase dramatically, and the LH release pattern becomes r e l a t i v e l y constant. In the r a t LH release remains c o n s i s t e n t l y p u l s a t i l e throughout a 24 hour time period. Evidence suggests that following removal of the gonads, feedback e f f e c t s are removed at the l e v e l of the p i t u i t a r y as well as the ce n t r a l nervous system. Since p u l s a t i l e LH release i s a widespread phenomenon i t i s of i n t e r e s t to understand how the frequency and amplitude of LH release, and presumably LHRH release i s regulated. Due to the extremely low le v e l s of LHRH i n the peripheral c i r c u l a t i o n and the t e c h n i c a l d i f f i c u l t y i n obtaining blood samples from the p i t u i t a r y p o r t a l vessels i n order to measure LHRH release, i t becomes necessary to measure LH release and assume (with 49 some caution) that changes i n LH release r e f l e c t changes i n LHRH release. Presumably changes i n the frequency of LH release r e f l e c t changes i n the frequency of LHRH release at the l e v e l of the hypothalamus, p a r t i c u l a r l y when p i t u i t a r y s e n s i t i v i t y to LHRH remains constant. S i m i l a r l y , i f p i t u i t a r y s e n s i t i v i t y i s unchanged, changes i n the amplitude of the LH pulses measured i n the peripheral c i r c u l a t i o n presumably r e f l e c t changes i n the amount of LHRH released per pulse into the p i t u i t a r y p o r t a l veins. Numerous studies have examined the r o l e of (putative) neurotransmitters and neuromodulators i n the regul a t i o n of LH (LHRH) . In p a r t i c u l a r , attention has focussed on the r o l e of monoamines (norepinephrine, epinephrine, dopamine, and serotonin) i n the regulation of LH release i n both i n t a c t , gonadectomized, and gonadectomized steroid-primed r a t s with a wide v a r i e t y of experimental techniques being employed (see General Introduction, pp.20-43). Gallo and Drouva (1979) demonstrated that i n t r a v e n t r i c u l a r i n f u s i o n of NE and DA, but not epinephrine, to ovariectomized rat s resulted i n suppression of p u l s a t i l e LH release which suggested that NE could i n h i b i t LHRH release. I t has also been demonstrated that the i n t r a -v e n t r i c u l a r i n f u s i o n of alpha-adrenergic agonists are more e f f e c t i v e than beta-adrenergic agonists i n i n h i b i t i n g p u l s a t i l e LH release i n the ovariectomized r a t (Leung et a l . , 1982a). In contrast, i t has also been demonstrated that systemic i n j e c t i o n of phenoxybenzamine or other alpha-adrenergic antagonists r e s u l t s i n marked suppression of p u l s a t i l e LH release, which suggests that alpha-adrenergic receptors are important i n maintaining p u l s a t i l e discharges of LH i n the ovariectomized r a t (Weick, 1978). In view of these paradoxical findings, t h i s study was conducted to examine 1) the e f f e c t of systemic i n j e c t i o n of alpha- or beta- antagonists on p u l s a t i l e LH release i n long term ovariectomized rats and 2) t h e i r possible modulation of the e f f e c t s of a subsequent i n t r a v e n t r i c u l a r i n f u s i o n of NE on LH release. I I . Materials and Methods  General Adult female Sprague-Dawley rats were maintained i n a temperature- (23 ± 1°C) and l i g h t - c o n t r o l l e d (14-h l i g h t , 10-h dark) room. Rat chow and water were av a i l a b l e ad li b i t u m . One or two days a f t e r the rats a r r i v e d i n the animal holding f a c i l i t i e s , they were anaesthetized with sodium methohexital ( B r i e t a l , 50 mg/kg of body weight) and ovariectomized. Surgical cannulation of the t h i r d v e n t r i c l e was performed 14-16 days a f t e r ovariectomy as follows. The r a t was anaesthetized with sodium pentobarbital (Somnotol, 45 mg/kg of body weight) and then placed i n a stereotaxic instrument with i t s head i n the f l a t - s k u l l p o s i t i o n as described by Paxinos and Watson (1982). The s k u l l was exposed and four holes were d r i l l e d , into which anchoring screws were secured. A small hole, the centre of which was 6.4 mm anterior to the i n t e r a u r a l l i n e , was then d r i l l e d m i d s a g i t a l l y i n the s k u l l . The dura mater was then exposed and a 22 gauge s t a i n l e s s s t e e l cannula 51 was lowered 8.0 mm into the br a i n . Placement of the cannula at t h i s p o s i t i o n r e s u l t e d i n the t i p of the cannula i n the t h i r d v e n t r i c l e . Dental cement (Hygenic Cold Cure) was placed around the cannula and the s k u l l screws, to secure the cannula to the s k u l l . To prevent leakage of cerebrospinal f l u i d , a 28 gauge s t a i n l e s s s t e e l s t y l e t t e exactly the same length as the cannula was inserted i n the cannula and i t s handle was cemented i n place u n t i l the day of the experiment. A f t e r v e n t r i c u l a r cannulation the r a t s were placed i n ind i v i d u a l cages and were given at l e a s t one week to recover from the surgery. On the day before the c o l l e c t i o n of blood samples the rats were anaesthetized b r i e f l y with B r i e t a l ($45 min.) and a catheter made of s i l a s t i c tubing was inserted i n t o the r i g h t jugular v e i n using the method described by Harms and Ojeda (1974). B r i e f l y , the r i g h t jugular vein was exposed and a curved 22-gauge needle was used to place the s i l a s t i c catheter into the vein. The catheter was s l i d into the vei n and made to enter or approach the r i g h t atrium. The catheter was then anchored onto the muscle under which the jugular v e i n passes. The exposed end of the catheter was then passed underneath the skin and e x t e r i o r i z e d at the back of the neck and t i e d shut. The r a t s were then returned to t h e i r cages u n t i l the next day. On the day of the experiment the jugular catheter was connected to polyethylene tubing (PE-50) which had been f i l l e d with heparinized s a l i n e . Using t h i s catheter, blood samples were withdrawn with minimal disturbance to the r a t . Before blood sampling was started, 200 ±1 heparin (1000 U/ml) was 52 i n j e c t e d i n t o the rats (250-3 00 g) v i a the i n t r a - a t r i a l catheter to prevent c l o t t i n g of the blood i n the tubing. The rat s (conscious and unrestrained) were then given e i t h e r an intravenous (iv) or int r a p e r i t o n e a l (ip) i n j e c t i o n with one of several adrenergic antagonists before the period of blood c o l l e c t i o n was started. A f t e r the pre-infusion period of blood c o l l e c t i o n the rats were given an i n t r a v e n t r i c u l a r infusion of NE (L-norepinephrine b i t a r t r a t e from Sigma Chemicals; 0.06 tmol i n 2 11 over 2 minutes, pH 5.5). Following heparin and antagonist i n j e c t i o n , blood sampling was started. In one experiment the antagonist was injected during the period of blood sampling i . e . 15 minutes before infusion of NE (see Expt. #1) . At 7- or 10-minute i n t e r v a l s , 50 t l of whole blood was withdrawn from the rats v i a the i n t r a - a t r i a l catheter. The whole blood samples were placed into tubes (on ice) containing 450 t l of phosphate buffered s a l i n e with 0.1% g e l a t i n and a f t e r the experiment the samples were kept frozen (-20"C) u n t i l LH radioimmunoassay (RIA). In a l l cases, 5 min. before the s t a r t of i n f u s i o n of NE, a 28 gauge infusion s t y l e t t e was placed i n s i d e the implanted 22 gauge cannula such that the t i p of the i n f u s i o n s t y l e t t e was i n the t h i r d v e n t r i c l e . In many cases cerebrospinal f l u i d was seen flowing out of the cannula. This was taken as evidence that the cannula was i n the t h i r d v e n t r i c l e . The infusion s t y l e t t e had been previously connected to a Hamilton m i c r o l i t r e syringe i n a syringe pump with PE-20 tubing f i l l e d with the NE sol u t i o n . The infusion volume was 2 t l infused over 2 minutes. 53 At the end of the bleeding session, the rats were anesthetized with pentobarbital and then perfused i n t r a -c a r d i a l l y with s a l i n e followed by 10% formaldehyde solu t i o n i n s a l i n e . The brains were removed from the s k u l l and l a t e r sectioned to v e r i f y the loc a t i o n of the t h i r d v e n t r i c l e cannula. The r a t s that had the cannula outside the v e n t r i c l e were not used i n the analysis. The blood samples were analyzed f o r LH with a LH RIA k i t provided by the National I n s t i t u t e of A r t h r i t i s , Diabetes, and Digestive and Kidney Diseases, National Hormone and P i t u i t a r y Program. The assay was a s l i g h t modification of the double antibody RIA described by Niswender et a l . (1968). The reference preparation was NIADDK-rat-LH-RP-2. The mean between-pulse i n t e r v a l ( i . e . , the time between LH pulse peaks) was determined for pre-infusion period a f t e r pulses were detected using the c o e f f i c i e n t of v a r i a t i o n method of Gallo (1981b). B r i e f l y , the mean LH l e v e l and c o e f f i c i e n t of v a r i a t i o n of a l l the LH values of a prospective pulse, i . e . , the values comprising the ascending limb as well as the descending limb, were calculated. A pulse was defined when the c o e f f i c i e n t of v a r i a t i o n was 1.5 times the intra-assay c o e f f i c i e n t of v a r i a t i o n . Also, as described previously, (Leung et a l . , 1982a) the post-infusion between-pulse i n t e r v a l was taken as the time between the l a s t LH peak before infusion and the f i r s t LH peak a f t e r infusion of NE, i . e . , i t r e f l e c t s the i n t e r v a l immediately a f t e r NE administration, during which no apparent LH pulse was present. In addition, the mean blood 54 LH l e v e l s were determined f o r the pre-infusion period and the 60-minute period immediately a f t e r i n f u s i o n of NE. For a l l s a l i n e and drug treatments, the i n t r a p e r i t o n e a l and intravenous groups were combined fo r data a n a l y s i s since preliminary r e s u l t s showed that the route of drug or s a l i n e administration used ( i . e . , i n t r a p e r i t o n e a l vs. intravenous) did not s i g n i f i c a n t l y a f f e c t the r e s u l t s . The dosages chosen were previously shown to be e f f e c t i v e i n blocking adrenergic receptors (Weick, 1978; Caceres and T a l e i s n i k , 1980b, Coen and Coombs, 1983). S t a t i s t i c a l differences of the data (between pulse i n t e r v a l s and mean LH le v e l s ) were determined with one way analysis of variance followed by Scheffe's t e s t . Values i n the text represent the means ± SE. Experiment #1 In the f i r s t experiment the r a t s were i n j e c t e d with eithe r i s o t o n i c s a l i n e , propranolol hydrochloride (beta-adrenergic antagonist obtained from Sigma Chemical) at a dose of 6.0 mg/kg body weight, or phenoxybenzamine (alpha-adrenergic antagonist, dibenzyline obtained as a g i f t from Smith, Kline and French, Canada) at a dose of 15.0 mg/kg body weight, immediately p r i o r to the beginning of the blood sampling period. In a number of animals, s a l i n e or propranolol was injected a f t e r the blood sampling had started, i . e . 15 minutes before i n t r a v e n t r i c u l a r i n f u s i o n of NE. In a l l r a t s , blood samples were c o l l e c t e d at 10 minute i n t e r v a l s f o r 70 minutes before NE i n f u s i o n (time 0) and 120 minutes a f t e r NE i n f u s i o n . Experiment #2 55 In t h i s experiment the rats were inj e c t e d with e i t h e r s a l i n e , prazosin hydrochloride (alpha-l-adrenergic antagonist obtained as a g i f t from P f i z e r Canada) at a dose of 2 mg/kg body weight, or yohimbine (alpha-2-adrenergic antagonist obtained from Sigma Chemical) at a dose of 2 mg/kg body weight immediately p r i o r to the s t a r t of the blood sampling period. In t h i s experiment blood samples were c o l l e c t e d at 7 minute i n t e r v a l s to more accurately determine LH p u l s a t i l e release. The pre-infusion period was lenghthened to 98 minutes ( i . e . 15 samples p r i o r to NE infusion) to better determine the pre-infu s i o n pattern of LH release, and the pos t - i n f u s i o n period was shortened to 70 minutes ( i . e . 10 samples following NE infusion) since the e f f e c t s of NE were previously seen to generally l a s t only 45-50 minutes. Experiment #3 In t h i s experiment the ra t s were inj e c t e d with e i t h e r prazosin hydrochloride at a dose of 5 mg/kg body weight, or piperoxan (alpha-2-adrenergic antagonist obtained from Rhone-Poulenc) at a dose of 50 mg/kg body weight p r i o r to the period of blood sampling. Blood sampling and i n f u s i o n of NE was performed s i m i l a r to that o u t l i n e d i n experiment #2. I I I . Results  Experiment #1 In the control r a t s , i n j e c t e d e i t h e r i n t r a p e r i t o n e a l l y or intravenously with s a l i n e at £s -70 minutes, LH release was p u l s a t i l e before i n t r a v e n t r i c u l a r i n f u s i o n of NE at time 0. During t h i s pre-NE in f u s i o n period, the between-pulse i n t e r v a l w a s 26 ± 2 minutes and the mean blood LH l e v e l was 3.5 ± 0.2 ng/ml (n = 9). Three representative examples are shown i n F i g . 2. A f t e r i n t r a v e n t r i c u l a r i n f u s i o n of NE (0.06 imol) at time 0 the between-pulse i n t e r v a l lengthened s i g n i f i c a n t l y to 50 ± 4 minutes (P<0.01). As indicated i n F i g . 3 for the pooled data f o r the control r a t s , the mean LH l e v e l showed a s i g n i f i c a n t d e c l i ne to 2.5 ± 0.2 ng/ml (P<0.05) when compared with pre-NE i n f u s i o n l e v e l s . The e f f e c t s of NE l a s t e d J45-50 minutes, at which time p u l s a t i l e LH release resumed. As reported previously, (Leung et a l . , 1982a), i n f u s i o n of a c i d i f i e d s a l i n e i n t o the t h i r d v e n t r i c l e of ovariectomized rats f a i l e d to a f f e c t the p u l s a t i l e pattern of LH release (data not shown). Administration of the alpha-adrenergic antagonist, phenoxybenzamine (iv or ip) markedly suppressed the p u l s a t i l e pattern of LH release i n most of the r a t s . As shown i n the representative examples i n F i g . 4, i n the majority of the rats i n the phenoxybenzamine-treated group, there was e i t h e r only one LH pulse present during the e n t i r e 70 minute period before i n f u s i o n (top panel) or p u l s a t i l e LH release was not evident at a l l (bottom panel). In a few animals, however , treatment with phenoxybenzamine was not e f f e c t i v e i n a l t e r i n g p u l s a t i l e LH release (Fig. 4, middle panel). Because of t h i s v a r i a b i l i t y , the between-pulse i n t e r v a l s could not be calculated f o r the phenoxybenzamine-treated group. Nevertheless, when a l l the phenoxybenzamine treated r a t s were analyzed together (n = 10), the mean blood LH l e v e l during the 70 minute period a f t e r 5 7 LH(ng/mL) l 1 1 I - 6 0 0 6 0 120 Time (min) Fig. 2. Representative examples of l u t e i n i z i n g hormone (LH) release i n ovariectomized (ovx) r a t s i n j e c t e d v i t h s a l i n e before i n t r a v e n t r i c u l a r ( i v t ) i n f u s i o n of norepinephrine (NE). 58 LH(ng/mL) Time (min) Fig. 3. E f f e c t of i v t in f u s i o n of NE on LH release i n mature ovx r a t s pretreated with s a l i n e (n = 9). LH (ng/mL) Time (min) Fig. 4. Representative examples of LH release i n ovx r a t s i n j e c t e d with phenoxybenzamine before i v t in f u s i o n of NE. 60 LH(ng/mL) Phenoxybenzamine (i.v.) 5r I 3- \ Phenoxybenzamine (i.p.) 0 •H- / V V +H Vent NE - 6 0 0 60 120 Time (min) Fig. 5. Eff e c t of i v t i n f u s i o n of NE on LH release i n ovx r a t s pretreated with phenoxybenzamine ei t h e r i . v . <n = 4) or i.p. (n = 6). 61 phenoxybenzamine administration was 2.8 ± 0.3 ng/ml, which was s i g n i f i c a n t l y lower (P<0.05) than that observed during the same 70-minute period i n the s a l i n e - t r e a t e d controls (3.5 ± 0.2 ng/ml, n = 9) . A f t e r i n t r a v e n t r i c u l a r i n f u s i o n of NE to the phenoxy-benzamine-treated r a t s at time 0 the pattern of LH release was again more v a r i a b l e than i n the control animals. For several r a t s i n t h i s group p u l s a t i l e LH release had apparently returned at t h i s time and was not further affected by i n t r a v e n t r i c u l a r i n f u s i o n of NE at time 0 (Fig. 4, top panel). Importantly i n a l l of the animals i n the e n t i r e phenoxybenzamine-treated group NE was i n e f f e c t i v e i n suppressing LH release as observed i n the control animals. In f a c t , a few r a t s i n t h i s group appeared to respond to subsequent i n t r a v e n t r i c u l a r infusion of NE with an increase i n LH release (Fig. 4, bottom panel). Thus as seen i n F i g . 5, when a l l the phenoxybenzamine-treated rats were analyzed together (n = 10) , the mean LH l e v e l s during the 60 minute period a f t e r NE administration was s l i g h t l y higher than that observed during the pre-infusion period i n the same animals (3.5 ± 0.3 ng/ml, P<0.05). In ra t s i n j e c t e d with the beta-adrenergic antagonist propranolol (ip or i v ) , the p u l s a t i l e LH release pattern before i n f u s i o n of NE was s i m i l a r to that seen i n the s a l i n e - t r e a t e d ra t s (Fig. 6) . During t h i s period the between-pulse i n t e r v a l i n the propranolol-treated r a t s was 29 ± 2 minutes and the mean LH l e v e l was 3.2 ± 0.2 ng/ml (n = 12). A f t e r NE infusion, LH release i n these r a t s was suppressed i n a manner s i m i l a r to 62 that observed i n saline-treated controls (Fig. 6 and 7) . The between-pulse i n t e r v a l lengthened to 62 ± 5 minutes which was s i g n i f i c a n t l y longer (P<0.01) than the pre-infusion between-pulse i n t e r v a l and the mean LH l e v e l decreased s i g n i f i c a n t l y to 2.3 ± 0.1 ng/ml (P<0.05). The e f f e c t s of NE on LH release i n the propranolol-treated rats also lasted f o r approximately 45 to 50 minutes. To eliminate the p o s s i b i l i t y that the e f f e c t of beta-receptor blockade by propranolol may have already cleared by the time of NE inf u s i o n and thus was i n e f f e c t i v e i n a l t e r i n g the action of NE at time 0. propranolol was in j e c t e d into another group of rats (n = 6) at time -15 minutes. As seen i n F i g . 8 f o r three representative examples, t h i s treatment also f a i l e d to a l t e r the pattern of the LH response to NE, i . e . , i n t r a v e n t r i c u l a r i n f u s i o n of NE at time 0 markedly lengthened the between-pulse i n t e r v a l (24 ± 3 vs. 53 ± 8 minutes, P<0.01) as well as suppressed the mean blood LH l e v e l s when compared with the 70 minute pre-infusion period i n the same animals (P<0.05). Experiment #2 As observed i n experiment #1, LH release was p u l s a t i l e i n sal i n e - t r e a t e d r a t s before i n t r a v e n t r i c u l a r i n f u s i o n of NE. During t h i s pre-NE in f u s i o n period, the mean blood LH l e v e l was 3.16 ± 0.14 ng/ml with a between-pulse i n t e r v a l of 20.7 ± 1.6 minutes (n = 6). Following infusion of NE, p u l s a t i l e LH release was i n h i b i t e d and mean LH l e v e l s declined s i g n i f i c a n t l y (p<0.001) to 1.68 ± 0.22 ng/ml. P u l s a t i l e release resumed towards the end of the bleeding session 40-50 minutes a f t e r 63 LH (ng/mL) NE Time (min) Fig. 6. Representative examples of LH release i n ovx rats i n j e c t e d with propranolol 70 minutes before i v t i n f u s i o n of NE. 64 LH (ng/mL) 5j- Propranolol (i.v.) t \ i 6r Propranolol (i.p.) - i II Vent NE /I 0 -60 0 60 Time (min) 120 Fig. 7. E f f e c t of i v t i n f u s i o n of NE on LH release i n ovx r a t s pretreated with propranolol e i t h e r i . v . (n = 4) or i.p. (n = 8). 65 j i l l j I I 1 i i i i -60 0 60 120 Time (min.) Fig. 8. Representative examples of LH release i n ovx r a t s injected with propranolol 15 minutes before i v t i n f u s i o n of NE. 66 infusion of NE. To determine through which type of adrenergic receptor ( i . e . alpha-1 or alpha-2) the e f f e c t s of i n f u s i o n of NE are mediated s p e c i f i c alpha-1- and alpha-2-adrenergic antagonists were injected into the rats p r i o r to infusion of NE. Prazosin (alpha-1 antagonist) i n j e c t i o n at a dose of 2 mg/kg (n * 7) resulted i n higher LH l e v e l s as compared to s a l i n e - t r e a t e d controls (3.84 ± 0.28 vs. 3.16 ± 0.14 ng/ml) and a f t e r infusion of NE, LH l e v e l s were again s i g n i f i c a n t l y lower (p<0.05) than pre-infusion l e v e l s (3.84 ± 0.28 vs. 2.48 ± 0.32) (data not shown). Yohimbine (alpha-2-antagonist) was a l s o tested to determine whether i t had any e f f e c t on p u l s a t i l e IS. release or NE-induced i n h i b i t i o n of LH release. Mean LH l e v e l s during the pre-infusion period i n yohimbine-treated rat s (n = 7, 2.70 ± 0.16 ng/ml) were not s i g n i f i c a n t l y d i f f e r e n t than the same period i n the s a l i n e - t r e a t e d controls. Following i n f u s i o n of NE, LH l e v e l s declined and post-infusion l e v e l s were s i g n i f i c a n t l y lower than pre-infusion l e v e l s (2.18 ± 0.18 vs. 2.70 ± 0.16 ng/ml, respectively) (data not shown). Experiment #3 Since infusion of NE i n the prazosin and yohimbine treated rats of experiment #2 did not appear to be as e f f e c t i v e i n lowering LH l e v e l s as i n the s a l i n e - t r e a t e d controls, another experiment was conducted. In t h i s experiment the objective was to determine whether a higher dose of prazosin (5 mg/kg) might be more e f f e c t i v e i n blocking the NE-induced suppression of LH release and whether piperoxan (another alpha-67 2 antagonist, 50 mg/kg) might have any e f f e c t on the NE-induced suppression of LH release. In both groups of r a t s , the drug treatment d i d not appear to have as dramatic e f f e c t s on p u l s a t i l e release as the phenoxybenzamine-treated rat s of experiment #1. Although the pulses d i d not seem to be as dramatic as seen previously i t i s d i f f i c u l t to be c e r t a i n of any consistent e f f e c t of the drug on p u l s a t i l i t y since LH release was not monitored before the i n j e c t i o n of the adrenergic antagonist. This i s p a r i c u l a r l y the case since the pattern of LH release while remaining r e l a t i v e l y constant i n each animal, can and does vary between animals (Gay and Sheth, 1972). In the prazosin-treated r a t s (n = 7), the post-infusion l e v e l s were not s i g n i f i c a n t l y d i f f e r e n t than the mean LH l e v e l s during the pre-infusion period (2.95 ± 0.14 vs. 2.87 ± 0.08 ng/ml, respectively) (Fig. 9 and 10). S i m i l a r l y i n the piperoxan-treated rats (n =7) the mean post-infusion LH l e v e l s were not s i g n i f i c a n t l y d i f f e r e n t than the pre-infusion l e v e l s (2.34 ± 0.11 vs. 2.52 ± 0.08 ng/ml, respectively) (Fig. 11 and 12) . IV. Discussion The r e s u l t s of the present study provide further evidence that NE i s involved i n the regulation of p u l s a t i l e LH release i n ovariectomized r a t s . In the sa l i n e - t r e a t e d control r a t s LH release i s p u l s a t i l e p r i o r to i n t r a v e n t r i c u l a r i n f u s i o n of NE. S i m i l a r l y , rats injected with propranolol also had p u l s a t i l e LH release p r i o r to infusion of NE. In contrast, the rats 68 H 0 -L-1 1 1 ~ 1 1 1 1 r --150 -120 -90 -60 -30 0 30 60 Time (rnin ) F i g . 9. Representative examples of LH release i n ovx r a t s i n j e c t e d with prazosin before i v t infusion of NE. Fig. 10. E f f e c t of i v t infusion of NE on LH release i n ovx r a t s pretreated prazosin (n = 7). Fig. 11. Representative examples of LH release i n ovx r a t s i n j e c t e d with piperoxan. Fig. 12. E f f e c t of i v t infusion of NE on LH release i n ovx r a t s pretreated plperoxan (n = 7 ) . 72 injected with phenoxybenzamine had a LH release p r o f i l e i n which LH pulses were e i t h e r sporadic or absent (the majority of animals) . In these r a t s the mean blood LH l e v e l s were s l i g h t l y , but s i g n i f i c a n t l y suppressed during the 70 minute period p r i o r to infusion of NE (Fig. 3 and 5) . This agrees well with other reports demonstrating that blockade of alpha-but not beta-adrenergic receptors interrupts p u l s a t i l e LH release i n rat s and monkeys (Gnodde and Schuiling, 1976; Weick, 1978; Gallo and Kalra, 1983; Bhattacharya et a l . , 1972). Weick (1978) reported that p u l s a t i l e discharges of LH were blocked i n ovariectomized rats (majority of cases) for periods of 1-2 hours, by adrenergic antagonists phentolamine and phenoxy-benzamine, but not propranolol. More recently, Gallo and Kalra (1983) reported that i n i n t a c t r a t s on diestrus 1, blockade of alpha- but not beta-adrenergic receptors decreased blood LH l e v e l s and suppressed p u l s a t i l e LH secretion, decreasing both LH pulse frequency and amplitude. Huang and McCann (1983) have also reported that i n t r a v e n t r i c u l a r propranolol had no e f f e c t on LH release i n ovariectomized r a t s . Taken together these r e s u l t s suggest that alpha-receptors are more important i n the regulation of p u l s a t i l e release than beta-receptors. In t h i s study administration of the s p e c i f i c a l p h a 2 ~ adrenergic antagonist, yohimbine, had l i t t l e or no e f f e c t on the release of LH when compared to the s a l i n e - t r e a t e d controls. However, the alpha-adrenergic antagonist prazosin at a dose of 2 mg/kg increased mean LH l e v e l s over controls during the pre-in f u s i o n period with an increase i n pulse amplitude, compared to controls. I n t e r e s t i n g l y , neither a s p e c i f i c a l p h a 1 ~ nor a s p e c i f i c alpha 2-adrenergic antagonist alone, even at r e l a t i v e l y high doses, had an e f f e c t comparable to the e f f e c t of phenoxy-benzamine on p u l s a t i l e LH release ( i . e . , i n h i b i t i o n of LH relea s e ) . Although phenoxybenzamine and acts p r i m a r i l y at alpha-adrenergic receptors, at high doses i t might also act at alpha 2~adrenergic receptors. These r e s u l t s suggest that since neither an a l p h a 1 ~ nor an alpha 2-adrenergic antagonist alone i s able to i n h i b i t p u l s a t i l e LH release, the i n h i b i t o r y e f f e c t s of phenoxybenzamine (and phentolamine i n other studies) on p u l s a t i l e LH release reported here and elsewhere may be a r e s u l t of the drug acting at both alpha^- and alpha 2-adrenergic receptors. I t i s possible that the i n h i b i t i o n of p u l s a t i l e LH release produced by administration of adrenergic antagonists only occurs when both a l p h a 1 - and alpha 2~adrenergic receptors are blocked. In addition phenoxybenzamine may have a more dramatic e f f e c t on p u l s a t i l e LH release because of other physico-chemical properties that allow i t easier access to a greater number of receptors involved i n the regulation of p u l s a t i l e LH release. As an example i t may be that following i n j e c t i o n phenoxybenzamine might reach the alpha-receptors more r e a d i l y than prazosin or piperoxan due a more rap i d rate of d i f f u s i o n from the general c i r c u l a t i o n to the receptors. In any case, presumably blockade of these alpha-adrenergic receptors suppressed p u l s a t i l e LH release by i n h i b i t i n g both the frequency of the putative LHRH generator and the amount of LHRH released per pulse (Gallo and Kalra, 1983; Kalra and Kalra, 74 1983) . In agreement with previous studies (Gallo, 1984; Gallo and Drouva, 1979; Leung et a l . , 1982a), these r e s u l t s demonstrate that i n t r a v e n t r i c u l a r infusion of NE i n h i b i t s the p u l s a t i l e release of LH normally seen i n ovariectomized r a t s . Following i n t r a v e n t r i c u l a r i n f u s i o n of NE i n sal i n e - t r e a t e d rats mean LH l e v e l s are s i g n i f i c a n t l y suppressed as compared to pre-infusion l e v e l s . Leung et a l . (1982a ) reported that t h i s i n h i b i t o r y e f f e c t could be e l i c i t e d by eithe r alpha- or beta-adrenergic agonists, with alpha-agonists more potent than beta-agonists i n suppressing LH release. In the present study, i t was of i n t e r e s t to determine the e f f e c t of s p e c i f i c adrenergic antagonists on the NE-induced i n h i b i t i o n of LH release i n ovariectomized r a t s . The r e s u l t s presented here demonstrate that i n the phenoxybenzamine-treated r a t s , subsequent i n f u s i o n of NE into the t h i r d v e n t r i c l e ( i . e . , 70 minutes a f t e r phenoxybenzamine injection) d i d not r e s u l t i n lower mean LH l e v e l s , as i n the sal i n e - t r e a t e d c o n t r o l r a t s . In none of the phenoxybenzamine-treated r a t s was a suppression of p u l s a t i l e discharges of LH observed a f t e r i n t r a v e n t r i c u l a r NE at time 0. Since phenoxy-benzamine, at the dosage used, e f f e c t i v e l y blocked central alpha-adrenergic receptors i n the majority of animals i n t h i s group (see above), the subsequent administration of NE would have acted on beta-adrenergic receptors. The f a i l u r e of NE to further i n h i b i t LH release i s consistent with a previous report that beta-adrenergic agonists are less potent than alpha-75 adrenergic agonists i n suppressing p u l s a t i l e LH release (Leung et a l . , 1982a) and therefore beta-receptors are probably not involved i n mediating NE-induced i n h i b i t i o n of LH release. The increase i n LH release i n some of the phenoxybenzamine-treated ra t s , i n response to NE, may be a r e s u l t of NE acting on beta-receptors which are excitatory to LH release. As mentioned previously, there may be a population of beta-receptors that are able to enhance LH release (Al-Hamood et a l . , 1985, 1987). In addition to having no observable e f f e c t on p u l s a t i l e LH release, propranolol also had no e f f e c t on the NE-induced suppression of LH release when i t was administered e i t h e r 70 or 15 minutes before infusion of NE. Suppression of LH release was s i m i l a r to that seen i n the s a l i n e - t r e a t e d controls. Presumably propranolol, at the dosage given, blocked central beta-adrenergic actions since a s i m i l a r dosage has been previously shown to be e f f e c t i v e i n blocking beta-receptors (Caceres and T a l e i s n i k , 1980b; 1982; Dotti and T a l e i s n i k , 1982). The suppression of LH release by i n t r a v e n t r i c u l a r NE i n the propranolol-treated rat s i s add i t i o n a l evidence of an i n h i b i t o r y alpha-adrenergic component i n the c o n t r o l of LH release i n the ovariectomized r a t , and a lack of an i n h i b i t o r y beta-adrenergic component. I f NE did act to i n h i b i t LH release v i a beta-receptors, blockade of the receptors would have blocked the i n h i b i t o r y e f f e c t of NE infusion on LH release. This study also demonstrates that more s p e c i f i c alpha-receptor antagonists are able to block the e f f e c t s of NE i n f u s i o n . While low doses of prazosin (alpha.) (2 mg/kg) and 76 yohimbine (alpha 2) (2 mg/kg) were r e l a t i v e l y i n e f f e c t i v e i n blocking the e f f e c t of NE on LH release, a higher dose of prazosin (5 mg/kg) and piperoxan (alpha 2) (50 mg/kg) were both e f f e c t i v e i n blocking NE-induced suppression of LH release. This provides further evidence, together with the r e s u l t s obtained with phenoxybenzamine, that the i n h i b i t o r y e f f e c t s of NE on LH release are mediated by alpha-receptors. A number of previous studies employing pharmacological manipulations have implicated catecholamines i n the regulation of p u l s a t i l e LH release i n r a t s . These studies have suggested that NE and possibly epinephrine are important i n maintaining regular p u l s a t i l e LH release. I n h i b i t i o n of dopamine-beta-hydroxylase a c t i v i t y , which res u l t e d i n decreased NE synthesis, suppressed p u l s a t i l e release i n ovariectomized and i n t a c t r a t s (Gnodde and Schuiling, 1976; Drouva and Gallo,1976; Gallo and Kalra, 1983). S i m i l a r l y alpha-receptor antagonists are e f f e c t i v e i n i n h i b i t i n g the regular pulsations of LH i n rats and monkeys (see above). The r e s u l t s reported here, on the e f f e c t of adrenergic blockers on p u l s a t i l e release, also support the current view that alpha-adrenergic receptors are important i n the maintenance of p u l s a t i l e discharges of LH. In contrast to the ro l e proposed f o r NE i n maintaining p u l s a t i l e LH release a number of studies have also demonstrated that catecholamines can i n h i b i t LH release when infused into the t h i r d v e n t r i c l e (Gallo and Drouva, 1979; Gallo, 1981d; 1984; Leung et a l . , 1982a). A c t i v a t i o n of DA-ergic receptors with e i t h e r DA or i t s agonists i n h i b i t s the p u l s a t i l e release 77 of LH i n the ovariectomized r a t (Drouva and Gallo, 1976; 1977; Gallo, 1981d; Gallo and Drouva, 1979). S i m i l a r l y , a c t i v a t i o n of adrenergic receptors also i n h i b i t s LH release i n ovariectomized r a t s (Gallo and Drouva, 1979; Gallo, 1984; Leung et a l . , 1982a). Evidence suggests that NE may have two roles i n the regulation of p u l s a t i l e LH release. I t may play an important r o l e i n maintaining p u l s a t i l e release and may also play an i n h i b i t o r y r o l e i n the regulation of LH release. I n t e r e s t i n g l y , the high amplitude p u l s a t i l e LH release observed under conditions when gonadal s t e r o i d l e v e l s are low, appears to be p a r t i c u l a r l y s e n s i t i v e to i n h i b i t o r y inputs. In p a r t i c u l a r a number of peptides, including angiotensin I I , neuropeptide Y, pancreatic polypeptides, cholecystokinin, and vasoactive i n t e s t i n a l polypeptide also i n h i b i t LH release when infused i n t o the t h i r d v e n t r i c l e of ovariectomized rats (Vijayan et a l . , 1979a, 1979b; Kalra and Crowley, 1984a, 1984b; Alexander et a l . , 1985; Steele et a l . , 1985; McDonald et a l . , 1985a, 1985b). The reason that LH release i s susceptible to i n h i b i t i o n i s not known. I t may be that the c e n t r a l pattern generator i s d r i v i n g the LHRH neurons at a near maximal frequency and a disturbance such as the introduction of a neuroactive agent w i l l tend to disrupt the pulse generator. The mechanism by which NE could be both i n h i b i t o r y and necessary i n the maintenance of p u l s a t i l e LH release i s not known. I t has been proposed that i t i s u n l i k e l y the same post-synaptic receptor (alpha 1) i s responsible for mediating these opposite e f f e c t s of NE (Leung, 1985). Rather, there are 78 separate ( i . e . , anatomically d i s t i n c t ) populations of adrenergic receptors (at l e a s t two) through which these d i f f e r e n t e f f e c t s are mediated. Anatomical studies using immunocytochemical and histofluorescence techniques have demonstrated that catecholaminergic f i b r e s p r e f e r e n t i a l l y contact the LHRH containing c e l l s i n the MPOA which innervate the ME as opposed to the LHRH c e l l s which send terminals to the organum vasculosum lamina terminalis and extrahypothalamic regions (Hoffman, 1985). At the l e v e l of the caudal ME, catecholaminergic f i b r e s overlap extensively with LHRH containing v a r i c o s i t i e s (McNeill, Scott, and Sladek, 1980), while i n the r o s t r a l ME, there i s l i t t l e or no overlap of the catecholaminergic and LHRH containing f i b r e s . Jennes et a l . (1982) also demonstrated that i n the MPOA, NE f i b r e s are cl o s e l y associated with LHRH c e l l s , but i n the ME, NE acts on LHRH containing neurons v i a interneurons, s p e c i f i c a l l y DA-ergic , to e f f e c t changes i n LHRH secretion. In any case, i t i s apparent that NE could act d i r e c t l y at the l e v e l of the LHRH containing-, c e l l bodies i n the MPOA (Hoffman et a l . , 1982; Hoffman, 1985; Jennes et a l . , 1982), c e l l processes (axons?) i n the arcuate (Palkovits et a l . , 1982), and/or nerve terminals i n the ME (McNeill et a l . , 1980). NE could also act i n d i r e c t l y v i a interneurons at a l l three l e v e l s ( c e l l body, axon, terminal), but evidence suggests that i n d i r e c t e f f e c t s occur at the l e v e l of the MPOA and ME (Jennes et a l . , 1982; Sawyer and C l i f t o n , 1980). The chemical nature or function of the afferents to the LHRH containing neurons i s unknown. S i m i l a r l y 79 the nature of the s p e c i f i c receptors situated on the LHRH neurons i s also unknown. It has been postulated that one group of alpha 1~ adrenergic receptors, possibly located d i r e c t l y on LHRH neurons i s stimulatory to LH release and i t s a c t i v a t i o n i s important i n the maintenance of p u l s a t i l e LH release i n ovariectomized rats and for the f a c i l i t a t i o n of the LH surge i n proestrus and steroid-primed ovariectomized r a t s . I t has also been postulated that another population of alpha-adrenergic receptors may be located on i n h i b i t o r y interneurons. In t e r e s t i n g l y , P a r v i z i and E l l e n d o r f f (1982) have demonstrated that not only i s the e f f e c t of NE on LH release dependent on the hormonal status of the animal (as mentioned previously) but i t i s also s i t e and dose dependent. M i c r o i n j e c t i o n of NE into the dorsal hypothalamus resulted i n an increase i n LH release while s i m i l a r i n j e c t i o n into the dorsomedial nucleus of the hypothalamus i n h i b i t e d LH release. Presumably a c t i v a t i o n of the alpha-receptors located on the putative i n h i b i t o r y interneurons would r e s u l t i n suppression of p u l s a t i l e discharges of LH v i a an increase i n a c t i v i t y of these i n h i b i t o r y interneurons impinging, d i r e c t l y or i n d i r e c t l y , on LHRH neurons. Blockade of c e n t r a l alpha-adrenergic receptors by phenoxybenzamine, would presumably block the excitatory alpha-receptors and thus lower the mean blood LH l e v e l s and LH pulse frequency. At the same time, phenoxybenzamine would also be expected to block the alpha-receptors that i n h i b i t LH release, and therefore NE inf u s i o n would not r e s u l t i n suppression of p u l s a t i l e LH 80 release. A possible candidate for the putative interneuron that i s i n h i b i t o r y to LH release i s the tuberoinfundibular dopaminergic neuron i n the MBH (Fuxe et a l . , 1984). Serotoninergic neurons which have t h e i r c e l l bodies i n the raphe n u c l e i may also be involved i n the i n h i b i t i o n of p u l s a t i l e LH release (Gallo and Moberg, 1977; Arendash and Gallo, 1978). However, Gallo (1984) has reported that the suppressive e f f e c t of NE on p u l s a t i l e release LH i n ovariectomized r a t s , was not prevented by p r i o r blockade of e i t h e r dopamine or serotonin receptors. Thus the precise nature of the putative i n h i b i t o r y interneuron, through which the i n h i b i t o r y e f f e c t of NE on LHRH (LH) release i n ovariectomized rats i s mediated, remains to be determined. I t i s of i n t e r e s t to note that a recent study reported that hypothalamic multiunit a c t i v i t y increased i n some areas and decreased i n other areas, p r i o r to an LH pulse (Martin and Thiery, 1987). When these areas were e l e c t r i c a l l y stimulated they e i t h e r increased or decreased LH secretion, r e s p e c t i v e l y . This would suggest that p u l s a t i l e LH release i s a r e s u l t of an increase i n the a c t i v i t y of LHRH containing neurons and a decrease i n the a c t i v i t y of neurons i n h i b i t o r y to LHRH release. Another candidate f o r mediating the i n h i b i t o r y action of NE on p u l s a t i l e LH release i s the alpha 2-receptor. I t was previously been shown that i n t r a v e n t r i c u l a r administration of c l o n i d i n e (an alpha 2~agonist) markedly suppressed LH release i n ovariectomized r a t s and that i t was more e f f e c t i v e than phenylephrine (an alpha..-agonist) i n t h i s regard (Leung et a l . , 81 1982a). The present study provides further evidence that NE induced i n h i b i t i o n may, at l e a s t i n part, be mediated by alpha 2-receptors. Although yohimbine d i d not block the i n h i b i t o r y e f f e c t of NE, piperoxan was e f f e c t i v e i n t h i s regard. Also, as mentioned previously, the e f f e c t s of phenoxybenzamine may also be mediated by i t s antagonistic action at the alpha 2-receptor. The present study suggests that since both prazosin and piperoxan (alpha 1~ and alpha 2-receptor antagonists, respectively) were both e f f e c t i v e i n blocking the i n h i b i t o r y action of NE, both receptors may be involved i n mediating t h i s e f f e c t . Kow and P f a f f (1987) have shown that a c t i v a t i o n of alpha 2~receptors i n h i b i t s neuronal e x c i t a b i l i t y i n the ventromedial hypothalamus. Also, NE can e l i c i t e x c i t a t o r y and i n h i b i t o r y e f f e c t s but the i n h i b i t o r y responses occur at a lower dose, suggesting that the i n h i b i t o r y e f f e c t s may be more r e a d i l y induced. Whether these same neurons i n the ventromedial hypothalamus are involved i n the regulation of LH release i s not known j u s t as the l o c a t i o n of these putative receptors which are i n h i b i t o r y to p u l s a t i l e LH release i s not known. The alpha 1~receptor may be on the LHRH neuron or i t may be located on interneurons, e i t h e r increasing the a c t i v i t y of i n h i b i t o r y interneurons or decreasing the a c t i v i t y of e x c i t a t o r y interneuronal a c t i v i t y . In a s i m i l a r manner the alpha 2-receptor may act to a l t e r the a c t i v i t y of i n h i b i t o r y or e x c i t a t o r y interneurons. This i s of course speculative, and how a c t i v a t i o n of these receptors influences LHRH release and t h e i r respective mechanisms i s a t o p i c of future studies. 82 Another candidate f o r mediating the NE-induced i n h i b i t i o n of LH release i s the beta-adrenergic receptor. Caceres and T a l e i s n i k (1980b) were the f i r s t to propose that NE can exert an i n h i b i t o r y e f f e c t on LH secretion through beta-receptor a c t i v a t i o n . In the ovariectomized, estrogen-primed r a t , LH release induced by i n t r a v e n t r i c u l a r i n f u s i o n of NE was markedly enhanced i n rats which were pretreated with propranolol (beta-antagonist) 1 hour before NE administration. Propranolol had no e f f e c t on LH release i n the absence of NE. They suggested that i t i s possible the excitatory action of NE on LH release may conceal an i n h i b i t o r y influence, which can be revealed by i t s suppression with a beta-adrenergic antagonist, thereby r e l e a s i n g an unopposed stimulatory action v i a alpha-receptor. They also found that LH release induced by e l e c t r i c a l stimulation of the MPOA was i n h i b i t e d by i n t r a v e n t r i c u l a r i n f u s i o n of isoprenaline (a beta-agonist). This e f f e c t was also blocked by propranolol, providing further evidence f o r an i n h i b i t o r y beta-adrenergic receptor which may be involved i n the regulation of LHRH release. T a l e i s n i k et a l . (Caceres and Ta l e i s n i k , 1982; Dotti and T a l e i s n i k , 1984; T a l e i s n i k and Sawyer, 1986) have suggested that noradrenergic f i b r e s , which are i n h i b i t o r y to LHRH release, o r i g i n a t e i n the LC and act v i a a c t i v a t i o n of beta-receptors on interneurons located i n the premammillary nuclei since l e s i o n s of these n u c l e i , or i n j e c t i o n of propranolol into the n u c l e i , blocks the LC-induced i n h i b i t i o n of LH release. An i n h i b i t o r y beta-receptor was also suggested by Leung 83 et a l (1982b), when they reported that i n t r a v e n t r i c u l a r i n f u s i o n of isoproterenol, a beta-adrenergic agonist, markedly suppressed the r i s e i n serum LH l e v e l induced by progesterone i n ovariectomized estrogen-primed rats (Leung et a l . 1982a). In t r a v e n t r i c u l a r isoproterenol also suppressed p u l s a t i l e LH release i n ovariectomized r a t s , a l b e i t to a l e s s e r extent than alpha-adrenergic agonists and NE (Leung et al.,1982a), thereby further supporting an i n h i b i t o r y r o l e f o r the beta-receptor. However, the present r e s u l t s with adrenergic antagonists are consistent with the notion that the putative i n h i b i t o r y alpha-adrenergic receptors are more dominant than beta-receptors i n ovariectomized unprimed rats i n mediating the NE-induced suppression of p u l s a t i l e release. I t i s p o s s i b l e that the i n h i b i t o r y e f f e c t s mediated by beta-receptors are more prevalent a f t e r exposure to E 2 since the i n h i b i t o r y e f f e c t of beta-receptors occurred a f t e r administration of E 2 to ovariectomized r a t s . Wilkinson et a l . (1979) have reported that the number of beta-receptors i n the hypothalamus increased i n ovariectomized r a t s a f t e r an i n j e c t i o n of E 2« Whether t h i s e f f e c t plays a r o l e i n changing the r e l a t i v e influence of beta-receptors on LHRH release i s unknown. In conclusion, the following hypothesis i s proposed f o r the adrenergic regulation of p u l s a t i l e LH release i n ovariectomized r a t s . From the work of many in v e s t i g a t o r s , i t seems c l e a r that adrenergic neurotransmitters, s p e c i f i c a l l y alpha-adrenergic receptors, are important i n maintaining the regular p u l s a t i l e pattern of LH release, probably by a f f e c t i n g 84 both the frequency of the putative LHRH pulse generator and the amount of LHRH released per pulse (Barraclough and Wise, 1982; Kalra and Kalra, 1983; Ramirez et a l . , 1984). On the other hand, i t has been c l e a r l y shown that i n t r a v e n t r i c u l a r i n f u s i o n of NE can suppress the p u l s a t i l e LH release pattern i n ovariectomized rats (Gallo, 1984; Gallo and Drouva, 1979; Leung et a l . , 1982a). Therefore, NE appears to play an apparently paradoxic and opposite r o l e i n ovariectomized r a t s . Perhaps a basal l e v e l of adrenergic neurotransmission i s required to ac t i v a t e an excitatory alpha-adrenergic receptor population on the LHRH neuron f o r the maintenance of the normal p u l s a t i l e pattern of LH release. On the other hand, a r e l a t i v e l y large increase i n NE, such as v i a i n t r a v e n t r i c u l a r administration (Gallo, 1984; Gallo and Drouva,1979; Leung et a l . , 1982a) or e l e c t r i c a l stimulation of b r a i n NE pathway (Leung et a l . , 1981a), may r e s u l t i n an abrupt inter r u p t i o n of the same p u l s a t i l e LH release pattern as a r e s u l t of a c t i v a t i o n of i n h i b i t o r y receptors. These receptors may be e i t h e r alpha- or beta since both alpha- and beta-adrenergic agonists can mimic the i n h i b i t o r y action of NE on LH release (Leung et a l . , 1982a). However, our present r e s u l t s suggest that the NE induced suppression of LH release i s coupled more t i g h t l y to i n h i b i t o r y alpha-adrenergic receptors, at l e a s t i n the ovariectomized unprimed r a t . The l o c a t i o n of these receptors i s not known although the MPOA (Leipheimer and Gallo, 1985) and the dorsal medial hypothalamus ( P a r v i z i and E l l e n d o r f f , 1982) are two regions i n which NE i n j e c t i o n i n h i b i t s p u l s a t i l e LH 85 s e c r e t i o n . E l e c t r i c a l stimulation of the dorsomedial hypothalamus can also i n h i b i t p u l s a t i l e LH release (Gallo, 1981a). I t may also be that i n t r a v e n t r i c u l a r i n f u s i o n of NE i s e f f e c t i v e i n suppressing LH release as a r e s u l t of the anatomical d i s t r i b u t i o n of the two types of receptors i . e . , the i n h i b i t o r y receptors are situated c l o s e r to the t h i r d v e n t r i c l e and are therefore more r e a d i l y activated than the excitatory receptors. The r o l e of these receptors, which are i n h i b i t o r y to LHRH release, i n the regulation of LHRH release i s not known. One p o s s i b i l i t y i s that a pulse of LHRH may not only be a r e s u l t of increased excitatory inputs to LHRH containing neurons but also a r e s u l t of decreased i n h i b i t o r y inputs (Martin and Thiery, 1987). I t i s the task of future studies to unravel the nature of the various inputs to LHRH neurons, and attempt to understand how these d i f f e r e n t inputs i n t e r a c t with each other to determine the eventual frequency and amplitude of the LHRH s i g n a l , which i s of c r u c i a l importance i n the reproductive biology of the animal. 86 E l e c t r i c a l Stimulation of Ventral versus Dorsal Mesencephalic  Tegmental Areas i n the Conscious Ovariectomized Rat:  E f f e c t s on P u l s a t i l e LH Release I. Introduction NE i s thought to have both an exc i t a t o r y and an i n h i b i t o r y r o l e i n the control of gonadotrophin release (Kalra and Kalra, 1983; Ramirez et a l . , 1984; T a l e i s n i k and Sawyer, 1986; see General Introduction, pp. 20-37). Presumably NE may act d i r e c t l y on LHRH neurons, and/or i n d i r e c t l y v i a interneurons which then act d i r e c t l y and/or i n d i r e c t l y to enhance or i n h i b i t LHRH neuronal a c t i v i t y , and thereby modulate gonadotrophin release (Sawyer and C l i f t o n , 1980). The c i r c u i t r y of inputs to the LHRH neurons through which NE may may d i r e c t l y and/or i n d i r e c t l y i s not known. I t i s also not well understood what r o l e s the DNT and the VNT play i n the regulat i o n of LH secretion (see pp. 37-43). The present study was undertaken to examine the possible r o l e of d i f f e r e n t CNS f i b r e pathways i n the regulation of p u l s a t i l e LH release i n the long term ovariectomized r a t s . Long term ovariectomized r a t s were e l e c t r i c a l l y stimulated i n e i t h e r the DNT, VNT, medial forebrain bundle (MFB), or zona i n c e r t a (ZI). To avoid complications i n i n t e r p r e t a t i o n of the data, the experiments were performed i n nonanaesthetized, unrestrained rats rather than pentobarbital-anaesthetized rats as i n a previous report. 87 I I . Materials and Methods Adult female Sprague-Dawley rats were purchased from Charles River Canada Inc. (Montreal, Canada) and housed i n a c o n t r o l l e d environment (temperature: 22 ± 1 °C; 14h L: lOh D, l i g h t s on 5.00h-19.00 h). The r a t s were ovariectomized, while under sodium methohexital anaesthesia ( B r i e t a l , 50 mg/kg of body weight; weight range of 175-200g) one or two days a f t e r t h e i r a r r i v a l to the animal holding f a c i l i t i e s . Implantation of a b i p o l a r electrode into the r a t brain was performed approximately two weeks a f t e r ovariectomy, as described below. The rat was anaesthetized with sodium pentobarbital (Somnotol, 45 mg/kg of body weight) and then placed i n a stereotaxic instrument with the head i n the f l a t -s k u l l p o s i t i o n as described by Paxinos and Watson (1982). The s k u l l was exposed and four holes were d r i l l e d around the prospective s i t e of the electrode. Small screws were secured into the holes f o r the purpose of l a t e r anchoring the electrode to the s k u l l . A small hole was then d r i l l e d i n the s k u l l , into one of three previously determined s i t e s , through which the electrode was lowered. The four s i t e s and t h e i r respective coordinates (see Paxinos and Watson, 1982) are: 1. Dorsal tegmental area, the s i t e of the dorsal noradrenergic t r a c t (DNT) (ventral and l a t e r a l to the mesencephalic grey) ( i n t e r a u r a l +1.2 mm, l a t e r a l 1.3 mm, v e r t i c a l -6.4 mm), 2. Ventral tegmental area, the s i t e of the ventral noradrenergic t r a c t (VNT) (in t e r a u r a l +3.2 mm, l a t e r a l 1.0 mm, v e r t i c a l -7.5 mm), 3. Medial forebrain bundle (MFB) ( i n t e r a u r a l +5.2 mm, 88 l a t e r a l 1.8 mm, v e r t i c a l -8.5 mm), and 4. Zona in c e r t a (ZI) (in t e r a u r a l +6.2 mm, l a t e r a l 1.8 mm, v e r t i c a l -7.5 mm). The electrode was made of two lengths of insulated s t a i n l e s s s t e e l wire (0.005 inch diameter; C a l i f o r n i a Fine Wire Co.) which were twisted together and then completely insulated with epoxylite except at the t i p . A gold connecting pin (Amphenol # 22 0-P02) was crimped onto each of the two free ends of the electrode and the two gold pins were inserted into a small p l a s t i c connector (Amphenol #223-1505). One or two days before e l e c t r i c a l stimulation the rats were f i t t e d with an i n t r a - a t r i a l s i l a s t i c catheter, f o r withdrawal of blood, as described i n the previous chapter. E l e c t r i c a l stimulation experiments were begun no sooner than one week a f t e r completion of the brain surgery, at which time a l l animals appeared healthy and weighed 250-300g. On the day of e l e c t r i c a l stimulation, s a l i n e - f i l l e d polyethylene tubing (PE-50) was connected to the s i l a s t i c catheter, and the rat s were inj e c t e d with 200 IU of heparin p r i o r to the beginning of blood sampling to prevent c l o t t i n g of the blood i n the tubing. Blood samples of 50 i l (25 t l duplicates) were c o l l e c t e d at 7-min i n t e r v a l s throughout the bleeding session and put d i r e c t l y into assay tubes containing phosphate-buffered s a l i n e with 0.1% g e l a t i n . The samples were then frozen at -70 'C u n t i l the LH radioimmunoassay (RIA). The r a t s which had an electrode implanted i n the DNT or VNT were bled a t o t a l of 3.5 hours: 1.5 hours before e l e c t r i c a l stimulation, which lasted f or 1 hour, and 1 hour a f t e r 89 e l e c t r i c a l stimulation. The r a t s which had an electrode implanted i n the MFB or ZI were bled i n a separate experiment i n a s l i g h t l y d i f f e r e n t manner. The bleeding period lasted approximately 3.4 hours: 2.1 hours before the onset of e l e c t r i c a l stimulation, and the period of stimulation l a s t e d 77 minutes. Samples were not c o l l e c t e d a f t e r stimulation was stopped. The bleeding schedule was changed p r i m a r i l y f o r two reasons: 1. To obtain a broader representation of pre-stimulation p u l s a t i l e LH release and 2. I t was previously observed that the e f f e c t s ( i f any) of e l e c t r i c a l stimulation on LH release lasted only as long as the period of stimulation, a f t e r which regular p u l s a t i l e LH release was resumed. P r i o r to e l e c t r i c a l stimulation, the electrode implanted into the r a t was connected to a Grass S88 stimulator v i a two Grass PSIU6 phot o e l e c t r i c stimulus i s o l a t i o n u n i t s with constant current output. The stimulus consisted of biphasic pulses of 400 tsec each, separated by 50 tsec, occurring at 30 Hz. The stimulus was applied 10 seconds on, and 10 seconds o f f . The current was gradually increased up to 100 i A or u n t i l behavioral changes (general arousal, e.g., grooming, running) were j u s t noted and then decreased s l i g h t l y to avoid these e f f e c t s i n response to the stimulation. The current (50-100 iA) and waveform were monitored p e r i o d i c a l l y during the stimulation period using a Kikusui o s c i l l o s c o p e . E l e c t r i c a l stimulation under these parameters i n the medial preoptic area area (MPOA) has previously shown to be highly e f f e c t i v e i n e l i c i t i n g LH release (Jamieson and Fink, 1976) and the amount 90 + 3 of i r o n (Fe ) deposited at the electrode t i p i s r e l a t i v e l y low (Terasawa and Sawyer, 1969). At the conclusion of the experiment, the rats were anaesthetized with pentobarbital and t h e i r brains were perfused with s a l i n e followed by 10% formalin i n s a l i n e . The brains were l a t e r removed from the s k u l l s , sectioned at 50 im and stained with c r e s y l v i o l e t to determine the electrode t i p loc a t i o n s . The locations of the electrode t i p s were judged as being insi d e or outside the noradrenergic t r a c t s without regard to the LH records. The blood or serum values were assayed using RIA k i t s provided by the NIADDK National Hormone and P i t u i t a r y Program using a method previously reported. The values of blood LH were expressed i n terms of NIADDK-Rat-LH-RP-2. The whole blood samples were assayed for LH using a s i m i l a r procedure as reported previously, and the intra-assay c o e f f i c i e n t of v a r i a t i o n (CV) was 11.3%. The c o e f f i c i e n t of v a r i a t i o n (CV) method of Gall o (1981b) was used to define a pulse (as described i n previous chapter) . S t a t i s t i c a l s i g n i f i c a n c e of the data was determined by analysis of variance followed by the Newman-Keul t e s t to determine s i g n i f i c a n c e between means (Zar, 1984) . Values f o r LH i n the text represent the means ± SE. I I I . Results The locations of the electrode t i p s implanted into the midbrain are shown i n F i g . 13 and F i g . 14. The rats i n which the electrode was implanted i n the v e n t r a l tegmental area (n = A B C Fig. 13. Location of electrode t i p s for e l e c t r i c a l stimulation (ES) of the dorsal, and ventral noradrenergic t r a c t s (DNT and VNT, r e s p e c t i v e l y ) , and medial forebrain bundle (MFB) i n ovx rats. The inset i s a schematic s a g i t t a l section depicting the ascending noradrenergic system, with the s i t e s of ES indicated by the A (MFB), B (VNT), and C (DNT). In the f r o n t a l brain sections the stippled area on the right-hand side depicts the MFB (A), VNT (B), and DNT (C), respectively. The s i t e s of ES are depicted on the left-hand side of each f r o n t a l section as being either inside ( f i l l e d c i r c l e s ) or outside (empty c i r c l e s ) the tracts. Arc=arcuate nucleus; CG=central grey; CP=cerebral peduncle; DBC=decussation of the brachium conJunctivum; DR=dorsal raphe; f=fornix; IP=interpeduncular nucleus; mfb=MFB; ml=medial lemniscus; PH=posterior hypothalamus. Adapted from the a t l a s of Paxinos and Watson (1982). Fig. 14. Photomicrographs of representative f r o n t a l sections in the rat midbrain depicting the s i t e s of electrode implantation i n the VNT (A) and the DNT (B). The s i t e i n each case i s marked by a black c i r c l e . ii) 14; F i g . 13 B) were divided into two groups depending on whether the electrode t i p was inside (n = 9) or outside (n = 5) the VNT as ou t l i n e d by Ungerstedt (1971) . In both groups, LH release remained p u l s a t i l e throughout the en t i r e bleeding period, i . e . , e l e c t r i c a l stimulation of the ventral tegmental region whether insid e or outside the VNT had no e f f e c t on p u l s a t i l e LH release. Three representative examples of the VNT-stimulated group are shown i n F i g . 15. In the VNT-stimulated r a t s , the interpulse i n t e r v a l p r i o r to stimulation was 21 ± 2 min. which was not s i g n i f i c a n t l y d i f f e r e n t from that of 19 ± 2 min. observed during the period of e l e c t r i c a l s timulation (p>0.05). Likewise, the mean LH l e v e l s before, during, or a f t e r stimulation of the VNT were not s i g n i f i c a n t l y d i f f e r e n t from each other (p>0.05; F i g . 16). The r a t s which had an electrode implanted i n the dorsal tegmental area (n = 16) were also divided into two groups, depending on whether the electrode t i p was inside (n = 10) or outside (n = 6) the DNT as outlined by Swanson and Hartman (1975) (Fig. 13 C) . In r a t s which had the electrode t i p near, but outside the DNT, LH release remained r e l a t i v e l y p u l s a t i l e throughout the bleeding period (not shown) and the mean LH l e v e l s were not a l t e r e d s i g n i f i c a n t l y (p>0.05) during e l e c t r i c a l stimulation when compared with the LH l e v e l s observed before, or a f t e r e l e c t r i c a l stimulation (Fig. 16). In contrast, i n the rats i n which the electrode t i p was located i n s i d e the DNT region, mean LH l e v e l s were s i g n i f i c a n t l y lower during the period of e l e c t r i c a l stimulation when compared with 94 o -> 1 1 1 r - 1 0 1 2 Time (hours) Fig. 15. E f f e c t of ES of the VNT on p u l s a t i l e LH release i n 3 ovx rats. The period of ES i s indicated by the black box. 9 5 4 -3-Inside VNT JL T Outside VNT X X Pre-ES ES Post-ES Pre-ES ES Post-ES Inside DNT Pre-ES ES Post-ES Outside DNT Pre-ES ES Post-ES F i g . 16. E f f e c t s of mesencephalic ES on mean LH l e v e l s i n ovx rats. Electrode t i p s are located inside or outside the VNT or DNT as depicted i n Fi g . 12. 96 O-J 1 , , r - 1 0 1 2 Time (hours) Fig. 17. E f f e c t of ES of the DNT on p u l s a t i l e LH release In 3 ovx ra t s . The period of ES i s Indicated by the black box. 97 B j 0 zona * 98 l e v e l s e i t h e r before or a f t e r stimulation (p<0.01); F i g . 16). Three representative examples are shown i n F i g . 17. In the DNT-stimulated r a t s , the inter-pulse i n t e r v a l p r i o r to e l e c t r i c a l stimulation was 25 ± 2 minutes. However, during e l e c t r i c a l stimulation, LH pulses were completely absent i n 4 r a t s and only one pulse was present i n 5 of the remaining 6 r a t s . In these l a t t e r cases, the pulse occurred at 47 ± 2 minutes following the onset of e l e c t r i c a l stimulation. Taken together, the i n t e r v a l from the LH pulse j u s t p r i o r to the beginning of e l e c t r i c a l stimulation to the next LH pulse was 67 ± 5 minutes f o r the e n t i r e group (n = 10) of DNT-stimulated animals. E l e c t r i c a l stimulation i n r a t s which had the electrode i n the region of the ZI (n=6) had no e f f e c t on mean LH l e v e l s , pulse amplitude, or pulse frequency, when compared to pre-stimulation values. A representative example of LH release i n a Zl-stimulated r a t i s shown i n F i g . 18 A. Electrodes were implanted i n the region of the MFB i n 9 r a t s of which, 5 were determined to have the t i p of the electrode i n s i d e the MFB as depicted i n F i g . 13 A. E l e c t r i c a l stimulation, e i t h e r inside or outside the MFB, had no e f f e c t on mean LH l e v e l s , pulse amplitude, or pulse frequency, when compared to pre-stimulation values. A representative example of LH release i n a MFB-stimulated rat i s shown i n F i g . 18B. 99 IV. Discussion The present study demonstrates that e l e c t r i c a l stimulation of the DNT i n the dorsal tegmental region of the midbrain i n f r e e l y moving ovariectomized unprimedrats r e s u l t s i n a marked suppression of p u l s a t i l e LH release and a s i g n i f i c a n t decrease i n mean LH l e v e l s . This i n h i b i t o r y e f f e c t appears to be anatomically s i t e - s p e c i f i c ; e l e c t r i c a l stimulation i n the dorsal tegmentum near but outside the DNT region had no e f f e c t on p u l s a t i l e LH release i n the ovariectomized r a t s . This provides further evidence that a c t i v a t i o n of NE f i b r e s i n the midbrain can i n h i b i t p u l s a t i l e LH release. Although there i s no neurochemical v e r i f i c a t i o n that e l e c t r i c a l stimulation of the DNT area a c t u a l l y stimulated endogenous NE release, ascending NE f i b r e s do traverse t h i s region of the mesencephalon (Swanson and Hartman, 1975; L i n d v a l l and Bjorklund, 1978). Moreover, i t has already shown (a l b e i t i n anaesthetized rats) that the decrease i n LH release induced by e l e c t r i c a l stimulation of the DNT could be blocked by alpha-methyl-p-tyrosine, (alpha-MPT) but not p-chlorophenyl-alanine (Leung et a l . , 1981b), thereby supporting a catecholaminergic mechanism. Although alpha-MPT also i n h i b i t s synthesis of DA, dopaminergic f i b r e s are not known to t r a v e l through t h i s region. Therefore i t seems probable that the i n h i b i t i o n of p u l s a t i l e LH release by e l e c t r i c a l stimulation of the DNT i s mediated through increased NE release from the nerve terminals of the DNT. I t i s now well documented that i n t r a v e n t r i c u l a r infusion 100 of NE can i n h i b i t p u l s a t i l e LH release i n both male and female castrated r a t s (Gallo and Drouva, 1979; Leung et a l . , 1982b; Gallo, 1984; Condon et a l . , 1986). This i n h i b i t i o n i s probably mediated by alpha-adrenergic receptors i n the CNS since alpha-agonists are more e f f e c t i v e than beta-agonists i n i n h i b i t i n g p u l s a t i l e LH release (Leung et a l . , 1981a). I t was also previously reported that alpha-adrenergic antagonists such as phenoxybenzamine and prazosin prevented the NE-induced i n h i b i t i o n of p u l s a t i l e LH release i n ovariectomized rats (Bergen and Leung, 1987a, see previous chapter). Intra-v e n t r i c u l a r infusions of NE or perfusion of the hypothalamic preoptic area with NE r e s u l t s i n i n h i b i t i o n of p u l s a t i l e LH release, p r i m a r i l y as a r e s u l t of a decrease i n pulse frequency and not pulse amplitude (Gallo, 1984; Leipheimer and Gallo, 1985). The r e s u l t s reported here also demonstrate that during e l e c t r i c a l stimulation of the DNT, the decrease i n mean LH l e v e l s i s , at l e a s t i n part, a r e s u l t of a decrease i n LH pulse frequency. In the majority of the DNT-stimulated r a t s , no LH pulse was seen during the 60 minutes of e l e c t r i c a l stimulation, whereas p r i o r to e l e c t r i c a l stimulation LH pulses occurred every 25 ± 2 minutes. The e f f e c t of e l e c t r i c a l stimulation on pulse amplitude i s not c l e a r from these data since only a few i s o l a t e d pulses of LH were observed i n some of the DNT-stimulated r a t s during the period of e l e c t r i c a l stimulation. As i l l u s t r a t e d i n F i g . 17, i n some animals a pulse of LH did appear at the end of the period of DNT stimulation, sh o r t l y before the stimululator was turned o f f . I t i s not possible to 101 determine from t h i s data whether stimulation of the DNT has the i d e n t i c a l e f f e c t s on p u l s a t i l e LH release as i n t r a v e n t r i c u l a r i n f u s i o n of NE ( i . e . , i n h i b i t i o n of pulse frequency with no e f f e c t on pulse amplitude). S i m i l a r l y , i t i s not known whether the infrequent pulse i s an i n d i c a t i o n of d e s e n s i t i z a t i o n of the i n h i b i t o r y receptors to continuous a c t i v a t i o n of the DNT, or simply that pulses of LH, while l e s s frequent, are not completely abolished. I t i s probably the l a t t e r , i n view of the e a r l i e r report that d e s e n s i t i z a t i o n to the i n h i b i t o r y e f f e c t of i n t r a v e n t r i c u l a r i n f u s i o n of NE on p u l s a t i l e LH release does not r e a d i l y occur, even with continuous i n t r a -v e n t r i c u l a r infusion of NE f o r periods up to 20 hours (Gallo, 1984) . I t i s also possible that the i n h i b i t o r y e f f e c t s of DNT stimulation (or i n t r a v e n t r i c u l a r i n f u s i o n of NE) on LH release may be mediated by a mechanism other than a c t i v a t i o n of i n h i b i t o r y alpha-adrenergic receptors. Electrochemical stimulation of the a n t e r i o r cingulate cortex or LC i s able to i n h i b i t LH release induced by e l e c t r i c a l stimulation of the MPOA i n EB-primed rats (Caceres and T a l e i s n i k , 1980a; D o t t i and T a l e i s n i k , 1982). I t has been proposed that t h i s i n h i b i t i o n i s mediated v i a beta-adrenergic receptors since i n h i b i t i o n of LH release i n both cases was blocked by propranolol (beta-adrenergic antagonist) (Caceres and T a l e i s n i k , 1980b; D o t t i and T a l e i s n i k , 1984). These i n h i b i t o r y beta-adrenergic receptors are at the l e v e l of the premammillary nucleus since propranolol but not phenoxybenzamine (alpha-adrenergic antagonist) 102 i n j e c t i o n into the premammillary nucleus blocked the i n h i b i t i o n of LH release produced by stimulation of the LC (Dotti and Ta l e i s n i k , 1984). Lesioning the premammillary n u c l e i was as e f f e c t i v e as propranolol administration i n blocking LC-induced i n h i b i t i o n of LH release (Dotti and T a l e i s n i k , 1984). They proposed that f i b r e s o r i g i n a t i n g i n the LC which can i n h i b i t the release of LH, f i r s t synapse i n the premammillary nucleus, and NE released, activates beta-receptors on premammillary neurons. Presumably, these premammillary neurons then act eit h e r d i r e c t l y or i n d i r e c t l y on LHRH-containing neurons to i n h i b i t LHRH release. As mentioned above, the studies of i n t r a v e n t r i c u l a r NE inf u s i o n i n ovariectomized r a t s suggested that the i n h i b i t i o n of p u l s a t i l e LH release by NE i s mediated p r i m a r i l y by alpha-receptors. Whether or not there i s an ad d i t i o n a l i n h i b i t o r y beta-adrenergic mechanism i s unclear. NE f i b r e s i n the DNT ascend from the brain to innervate a broad spectrum of r o s t r a l areas. Therefore, i t i s not possible to determine the l o c a t i o n of the nerve terminals of the axons that are being stimulated. However, the MPOA i s a possible s i t e where NE release from nerve terminals could be involved i n i n h i b i t i n g LH release. Leipheimer and Gallo (1985), using a push-pull perfusion cannula, reported that perfusion of the MPOA with NE i n h i b i t e d LH release by decreasing pulse frequency. Leung et a l . (1981b, 1982c) reported that i n t r a v e n t r i c u l a r i n f u s i o n of NE, at doses which i n h i b i t e d LH release i n ovariectomized r a t s , also i n h i b i t e d the multiunit a c t i v i t y of neurons i n the MPOA, diagonal band of Broca, and 103 a n t e r i o r hypothalamic area. A l l three regions are r e l a t i v e l y r i c h i n LHRH-containing neurons and therefore p o t e n t i a l s i t e s f o r mediating the i n h i b i t o r y e f f e c t s of NE on LH release. Immunocytochemical studies have also demonstrated close apposition of catecholaminergic neurons to LHRH-containing neurons i n the MPOA (Hoffman et a l . , 1982; Jennes et a l . , 1982; Hoffman, 1985). Fibres from the A l and A2 noradrenergic c e l l groups have a l s o been shown to project to the MPOA (Day et a l . , 1980). Kim et a l . (1987) recently reported that e l e c t r i c a l stimulation of the A l noradrenergic c e l l group resulted i n changes i n the e x t r a c e l l u l a r a c t i v i t y of neurons i n the MPOA. They also found that the neurons which d i d respond to A l stimulation were more l i k e l y to respond to ME stimulation. The neurons act i v a t e d by ME stimulation may be LHRH-secreting neurons which might also receive inputs from A l noradrenergic c e l l s . These studies taken together provide evidence that NE release from NE-containing terminals can a l t e r the a c t i v i t y of neurons (possibly LHRH) i n the MPOA. There i s evidence to suggest that neurons which can i n h i b i t LH secretion (possibly noradrenergic?) are involved i n the regulation of p u l s a t i l e LH release (Martin and Thiery, 1987) . The a c t i v i t y of i n h i b i t o r y neurons decreases, and the a c t i v i t y of excitatory neurons, or possib l y LHRH-secreting neurons, increases p r i o r to the onset of an LH pulse. I t i s possible that i n h i b i t o r y noradrenergic f i b r e s could act d i r e c t l y , and/or i n d i r e c t l y (via interneurons), on LHRH-secreting neurons to a l t e r t h e i r release of LHRH into the p o r t a l vessels. 104 In sharp contrast to the NE-induced i n h i b i t i o n of p u l s a t i l e LH release i n ovariectomized r a t s , numerous studies have established NE as a neurotransmitter which f a c i l i t a t e s LH release i n the r a t (for reviews see Barraclough and Wise, 1982; Kalra and Kalra, 1983; Ramirez et a l , 1984; see pp. 26-33.). Drugs which i n h i b i t NE synthesis block the LH surge i n proestrous and steroid-primed rats (Kalra et a l . , 1972, 1974, 1978) . Increased LH release has also been c o r r e l a t e d with increased NE turnover rates i n d i s c r e t e hypothalamic areas (Ranee et a l . , 1981a, 1981b; Wise et a l . , 1981a). Intra-v e n t r i c u l a r i n f u s i o n of NE induces LH and/or LHRH release i n ovariectomized, estrogen and progesterone-primed r a t s (Krieg and Sawyer, 1976; Gallo and Drouva, 1979; Ching and Krieg, 1986) and anaestrous r a t s or rats i n which the preovulatory surge has been blocked by pentobarbital (Rubinstein and Sawyer, 1970; Tima and Flerko, 1974). Likewise, electrochemical stimulation of the DNT was able to induce ovulation i n constant estrus r a t s , as well as to induce a small r i s e i n LH l e v e l s i n ovariectomized, EB-primed rats (Carrer and T a l e i s n i k , 1970). I t i s evident that i n i n t a c t r a t s or ovariectomized rats primed with ovarian steroids NE has a f a c i l a t a t o r y r o l e i n the regulation of LH and/or LHRH release. However when ovarian feedback i s removed, i . e . , low ovarian s t e r o i d a l l e v e l s , NE can have an i n h i b i t o r y r o l e i n the regulation of LH (LHRH) release. I n t e r e s t i n g l y , not only NE acts i n t h i s way but also other neural systems. E l e c t r i c a l stimulation of e i t h e r , the arcuate nucleus, the peri-suprachiasmatic region, or the dorsal raphe 105 nucleus i n h i b i t e d LH release i n ovariectomized rat s but enhanced LH release i n estrogen-primed ovariectomized rat s (Arendash and Gallo, 1979b; Gallo and Osland, 1976; K i t t s and Johnson, 1986) . In a s i m i l a r manner to the e f f e c t s of NE, i n t r a v e n t r i c u l a r infusion of peptides, which are present i n the hypothalamus, i n h i b i t e d LH and/or LHRH release i n gonadectomized animals and stimulated LH (LHRH) release i n i n t a c t or ovarian steroid-primed ovariectomized animals. The peptides reported to act i n t h i s way are pancreatic polypeptide (Kalra and Crowley, 1984a), neuropeptide Y (Kalra and Crowley, 1984b; Khorram et a l . , 1987), angiotensin II (Steele et a l . , 1985) and endogenous opioid peptides (Piva et a l . , 1986). The mechanism of action of these peptides i s not completely understood. However, the stimulatory e f f e c t s of both neuropeptide Y and angiotensin II on LH release may act v i a ac t i v a t i o n of NE systems. These peptides both appear to act v i a a c t i v a t i o n of alpha 2-adrenergic receptors that are excitatory to LHRH release since t h e i r e f f e c t s are blocked by alpha 2-adrenergic antagonists (Steele and Ganong, 1986b; A l l e n et a l . , 1987). The mechanism involved i n the reversal of the e f f e c t , of NE and the peptides mentioned above on LH release, i s not known. Certainly the steroids are involved. One p o s s i b i l i t y i s that the steroids may a l t e r the number and type of adrenergic receptors present at d i f f e r e n t brain s i t e s involved i n regulation of LHRH release. I t has been reported that estrogen increases the number of beta-receptors i n the 106 hypothalamus (Wilkinson et al.,1979; Wilkinson and Herdon, 1982). In t h i s way ovarian steroids may influence whether an excitatory or i n h i b i t o r y e f f e c t w i l l be dominant. I t i s also possible that the f a c i l i t a t o r y or i n h i b i t o r y e f f e c t depends on d i f f e r e n t populations of NE neurons. The d i f f e r e n t e f f e c t s may be a r e s u l t of a c t i v a t i o n of receptors located i n anatomically d i s t i n c t areas and the s t e r o i d a l environment plays a r o l e i n determining which f i b r e s (excitatory or i n h i b i t o r y ) w i l l be activated at any given time. S i m i l a r l y , Lenahan et a l . , (1986) have also suggested multiple serotonergic influences on the regulation of LH release. They propose stimulatory and i n h i b i t o r y r o l e s for both 5HT 1 and 5HT2 receptors through which serotonin could act at d i f f e r e n t times and s i t e s to exert a wide range of e f f e c t s on LH release. While e l e c t r i c a l stimulation of the DNT i n h i b i t e d LH release, s i m i l a r stimulation of the VNT had no s i g n i f i c a n t e f f e c t on LH release. Some studies have implicated the VNT i n the regulation of LH (LHRH) release although i t s precise r o l e i s not well understood. Carrer and T a l e i s n i k (1970) reported that electrochemical stimulation of the VNT i n ovariectomized ra t s d i d not s i g n i f i c a n t l y a l t e r mean LH l e v e l s , which i s i n agreement with the r e s u l t s reported here. On the other hand, previous reports have suggested that the VNT may be f a c i l i t a t o r y to LH release i n steroid-primed r a t s . Acute l e s i o n or s u r g i c a l transection of the VNT i n h i b i t e d the occurence of the proestrous or steroid-induced LH surge i n these animals (Kawakami and A r i t a , 1980; Martinovic and McCann, 107 1977) . Estrogen priming may be necessary before e l e c t r i c a l stimulation of the VNT can exert an e f f e c t on LH release since other studies which have suggested a r o l e for the VNT i n the regulation of LH release, have reported e f f e c t s i n i n t a c t or steroid-primed r a t s (see next chapter on the r o l e of the VNT i n these cases). Similar to e l e c t r i c a l stimulation of the VNT, stimulation of the MFB or ZI also d i d not s i g n i f i c a n t l y a l t e r p u l s a t i l e LH release. The MFB contains ascending catecholaminergic f i b r e s from the brainstem, some of which may innervate the hypothalamus. The r o l e of the MFB i n LH release i s not known. From t h i s study i t does not appear that the f i b r e s i n the MFB play a major r o l e i n regulation of p u l s a t i l e LH release i n the ovariectomized r a t . The ZI contains dopaminergic c e l l s and axons of the incerto-hypothalamic t r a c t which p r o j e c t to the MPOA, anter i o r hypothalamus, and p e r i v e n t r i c u l a r hypothalamus (Moore and Bloom, 1978; L i n d v a l l and Bjorklund, 1983). DA has been reported to have an i n h i b i t o r y influence on p u l s a t i l e LH release i n ovariectomized r a t s (Drouva and Gallo, 1976, 1977; Gallo, 1981d), and i t i s thought that the dopaminergic tubero-infundibular neurons may have an i n h i b i t o r y r o l e i n LH release. An excitatory r o l e f o r DA i n LH release has also been proposed and the dopaminergic neurons of the incerto-hypothalamic t r a c t have been implicated i n t h i s e f f e c t . DA or DA agonist injected into the ZI induced ovulation i n rat s on the day of proestrus and stimulated LH release i n estrogen-primed ovariectomized rats (MacKenzie et a l . , 1984; James et a l . , 1987). The present 108 study suggests that the ZI has l i t t l e or no influence on p u l s a t i l e LH release, as e l e c t r i c a l stimulation of t h i s area had no observable e f f e c t on LH l e v e l s . A p o s s i b i l i t y may be that ovarian s t e r o i d s need to be present i n order for a stimulatory e f f e c t of the ZI on LH secretion can be demonstrated. In any case i t i s evident that the i n h i b i t i o n of p u l s a t i l e LH release induced by e l e c t r i c a l stimulation of the DNT i s not a non-specific e f f e c t as e l e c t r i c a l stimulation outside the region of the DNT has l i t t l e or no e f f e c t on p u l s a t i l e LH secretion. The p r e c i s e r o l e that i n h i b i t o r y f i b r e s have i n the regulation of LH secretion i s not known. One p o s s i b i l i t y i s that a pulse of LHRH i s a r e s u l t of an increase i n the a c t i v i t y of LHRH sec r e t i n g neurons (and a general increase i n excitatory inputs to these c e l l s ) as well as a decrease i n the a c t i v i t y of i n h i b i t o r y inputs to LHRH secreting c e l l s . In conclusion, our re s u l t s demonstrate that i n the ovariectomized r a t , i n h i b i t i o n of LH release i s induced by e l e c t r i c a l stimulation of the DNT region of the dorsal tegmentum. E l e c t r i c a l stimulation of the vent r a l tegmentum, the MFB, or the ZI, f a i l e d to e f f e c t changes i n the p u l s a t i l e pattern of LH release i n the ovariectomized r a t . This suggests that f i b r e s i n the DNT may play a r o l e i n the regulation of p u l s a t i l e LH release i n the r a t . 109 Suppression of the Progesterone-Induced Gonadotrophin Surge  bv Adrenergic Agonists i n Estrogen-Primed  Ovariectomized Rats I. Introduction In the control of gonadotrophin release, NE appears to have a dual r o l e as discussed previously (see General Intro-duction, pp. 20-44) . I t has been postulated that alpha-receptors mediate the stimulatory e f f e c t of NE on LH release and the i n h i b i t o r y component i s mediated v i a beta-receptors (Leung et a l . , 1982b; D o t t i and T a l e i s n i k , 1984) and that ovarian steroids are important i n determining whether the i n h i b i t o r y or the e x c i t a t o r y component w i l l be dominant at any given time (Taleisnik and Sawyer, 1986) . In support of an i n h i b i t o r y r o l e f o r beta-receptors, i t was reported that i n t r a v e n t r i c u l a r i n f u s i o n of isoproterenol (a beta-agonist) res u l t e d i n a suppression of the estrogen and progesterone-induced LH surge i n ovariectomized rats (Leung et a l . , 1982b). In non-primed ovariectomized rats, alpha-agonists are more e f f e c t i v e than beta-agonists i n suppressing p u l s a t i l e LH release. However, alpha-adrenergic receptor a c t i v a t i o n has been associated with f a c i l i t a t i o n of LH and LHRH release and i n i t i a t i o n of the LH surge. As mentioned above, i t has been postulated that alpha- and beta-receptors mediate opposite e f f e c t s on LH release. Since a beta-agonist can i n h i b i t a s t e r o i d induced LH surge i t was of i n t e r e s t to determine the 110 e f f e c t s of i n t r a v e n t r i c u l a r infusions of NE and alpha-adrenergic agonists on gonadotrophin release during a s t e r o i d induced surge. This chapter reports on the e f f e c t s of i n t r a -v e n t r i c u l a r infusion of NE and adrenergic agonists on gonadotrophin release during an ongoing steroid-induced surge. I I . Materials and Methods Adult female Sprague-Dawley rats were housed i n a temperature- (22 ± 1 °C) and l i g h t - (14 L: 10 D, l i g h t s on 07.00-21.00 h) controlled environment. The rats were ovariectomized and 2 weeks l a t e r a cannula was implanted into t h e i r t h i r d v e n t r i c l e . Cannulation of the t h i r d v e n t r i c l e was performed according to a procedure d e t a i l e d e a r l i e r (p. 50) . To prevent leakage of cerebrospinal f l u i d , a 28-gauge s t a i n l e s s s t e e l s t y l e t t e was inserted i n t o the 22-gauge guide tube and i t s handle cemented i n place u n t i l the day of the experiment. The r a t s were allowed at l e a s t 1 week of recovery before s t e r o i d i n j e c t i o n s were performed. The rats (200-250 g) were given a subcutaneous i n j e c t i o n of e s t r a d i o l benzoate (EB, 5 ig/100 g body weight) i n o i l at 10.00 h on 2 consecutive days and on the 3rd day were given a subcutaneous i n j e c t i o n of 2 mg progesterone at 08.00 h rat colony time i n accordance to a previously published model (Leung et a l . , 1982b). On the 2nd day of EB i n j e c t i o n they were f i t t e d with an i n t r a - a t r i a l catheter f o r withdrawal of blood (Harms and Ojeda, 1974; see p.51). Blood samples (250 t l ) were taken on the day of progesterone i n j e c t i o n at 1-hour i n t e r v a l s from 12.00 to 19.00 I l l h colony time and the blood was replaced by an equal volume of s a l i n e . The blood was centrifuged a f t e r i t was allowed to c l o t at 4 'C. Serum samples were stored at -20 "C u n t i l RIA. Af t e r the blood sample was taken at 15.00 or 16.00 h (as described i n r e s u l t s ) , the rats were given i n t r a v e n t r i c u l a r infusions of eith e r s a l i n e or one of the following agents dissolved i n i s o t o n i c s a l i n e and adjusted to pH 5.5: L-norepinephrine b i t a r t r a t e (NE), L-phenylephrine hydrochloride, DL-isoproterenol hydrochloride or methoxamine hydrochloride (Burroughs Wellcome). A l l solutions were infused into the t h i r d v e n t r i c l e of the r a t brain (0.3 imol i n 2 t l s a l i n e over 2 min) using an infusion pump and Hamilton syringe connected to an i n f u s i o n s t y l e t t e which had been placed i n s i d e the v e n t r i c u l a r cannula of the r a t p r i o r to the bleeding session. These dosages were consistent with those used i n our previous report on the d i f f e r e n t i a l e f f e c t s of c e n t r a l adrenergic agonists on LH release (Leung et a l . , 1982a). A f t e r the experiment, the rats were anaesthetized with pentobarbital and t h e i r brains perfused with 10 % formalin i n buffered s a l i n e . Brains were l a t e r removed from the s k u l l s and sectioned to v e r i f y the placement of the v e n t r i c u l a r cannula. The experiment was done i n three parts. In the f i r s t part the rats were infused at 15:00 h with e i t h e r s a l i n e (n=5), phenylephrine (n=6) or isoproterenol (n=5). In the second and t h i r d parts of the experiment the rats were infused with s a l i n e (n=7 and n=9) and e i t h e r NE (N=ll) or methoxamine (n=7) , resp e c t i v e l y , at 16.00 h. The time of infusion i n the second 112 and t h i r d parts was changed to 16.00 h since i t was seen i n the previous experiment that i n some i n d i v i d u a l r a t s the gonadotrophin l e v e l s had not r i s e n s i g n i f i c a n t l y by 15.00 h, compared to l e v e l s at 12.00 h. Therefore, i t was decided that in f u s i o n at 16.00 h would allow for better detection of i n h i b i t i o n of a gonadotrophin surge. Serum samples were assayed for LH and FSH with RIA k i t s provided by the NIADDK r a t p i t u i t a r y hormone d i s t r i b u t i o n program (see p. 53). The reference preparation was NIADDK rLH-RP-2 and NIADDK rFSH-RP-1, respectively. S t a t i s t i c a l s i g n i f i c a n c e of the data was determined by analysis of variance and Scheffes t e s t f o r differences between d i f f e r e n t groups i n the f i r s t part of the experiment (multiple comparisons). In the second and t h i r d parts of the experiment, s t a t i s t i c a l s i g n i f i c a n c e was determined between the 2 groups by the unpaired t - t e s t . Differences within groups at d i f f e r e n t times were determined by the paired t - t e s t . Values f o r LH and FSH i n the text represent the means ± SE. II I . Results In the f i r s t part of the study, the e f f e c t s of i n t r a v e n t r i c u l a r i n f u s i o n of an alpha-adrenergic agonist (phenylephrine) and a beta-agonist (isoproterenol) on a steroid-induced gonadotrophin surge were determined. As shown i n F i g . 19, LH l e v e l s i n the con t r o l , s a l i n e - t r e a t e d , r a t s were s i g n i f i c a n t l y higher (p<0.01) at the time of i n f u s i o n (15.00 h) than at the beginning of the bleeding session (12.00 h) and Fig. 19. E f f e c t s of i v t infusion of s a l i n e <n = 5) and phenylephrine (n = 6) on the progesterone-induced LH surge i n EB-primed ovx rats. The s o l i d l i n e and broken l i n e s represent the rat s given s a l i n e or phenylephrine, r e s p e c t i v e l y . 114 30 r 20 -c 10 12.00 14.00 16.00 18.00 C o l o n y Time Fig. 20. E f f e c t s of Ivt Infusion of s a l i n e and isoproterenol on the progesterone-induced LH surge i n EB-primed ovx rats. The s o l i d l i n e and broken l i n e s represent the rat s given s a l i n e or isoproterenol, respectively. 115 LH (ng/mL) Colony Time F i g . 21. Representative examples of LH release i n progesterone and EB-primed ovx rats given an i v t inf u s i o n of e i t h e r s a l i n e , phenylephrine, or isoproterenol. Arrows indicate time of Infusion. 116 peak l e v e l s were seen at 17.00 h. At 15.00 h, j u s t p r i o r to i n t r a v e n t r i c u l a r i n f u s i o n , the serum LH l e v e l s were not s i g n i f i c a n t l y d i f f e r e n t between the saline-treated r a t s and those infused with phenylephrine. However, 1 hour a f t e r i n t r a v e n t r i c u l a r i n f u s i o n of phenylephrine, LH l e v e l s had declined and were not s i g n i f i c a n t l y d i f f e r e n t (p>0.05) from l e v e l s seen at 12.00 h. The LH l e v e l s at 16.00 h i n phenylephrine-treated r a t s were s i g n i f i i c a n t l y lower (p<0.05) than those i n s a l i n e - t r e a t e d r a t s (4.6 ± 1.2 vs. 18.5 ± 5.5 ng/ml). LH l e v e l s i n phenylephrine-treated r a t s increased s l i g h t l y over the next 2 hours but remained lower than the LH l e v e l s i n s a l i n e - t r e a t e d c o n t r o l r a t s . At 19.00 h the LH l e v e l s i n phenylephrine-treated rat s were not s i g n i f i c a n t l y d i f f e r e n t from those of the sali n e - t r e a t e d group. S i m i l a r to r e s u l t s reported previously (Leung et a l . , 1982b), isoproterenol was also e f f e c t i v e i n suppressing LH release during an ongoing s t e r o i d induced LH surge (Fig. 20) . Representative examples of LH release i n the three groups are seen i n Fig . 21. The pattern of FSH release i n these three groups was also determined i n t h i s experiment. As shown i n F i g . 22, the s t e r o i d regimen that induced an LH surge also induced a surge of FSH i n the s a l i n e - t r e a t e d control r a t s . In the s a l i n e -treated rats FSH l e v e l s continued to r i s e 2-3 hours a f t e r i n t r a v e n t r i c u l a r i n f u s i o n (15.00 h) . In rats infused with phenylephrine, FSH values were also on the r i s e at 15.00 h. Int r a v e n t r i c u l a r i n f u s i o n of phenylephrine r e s u l t e d i n 117 FSH (ug/mL) 22 r Colony Time Fig. 22. E f f e c t s of i v t i n f u s i o n of s a l i n e (n = 5) and phenylephrine (n = 6) on the progesterone-induced FSH surge i n EB-primed ovx rats. The s o l i d l i n e and broken l i n e s represent the r a t s given s a l i n e or phenylephrine, respectively. 118 J I I I I I I I I f I I JL. 1 I 1 1 1 1 13.00 15.00 17.00 19.00 13.00 15.00 17.00 19.00 Colony Time Fig. 23. Representative examples of FSH release In progesterone and EB-prlmed ovx rats given an Ivt Infusion of eith e r s a l i n e , phenylephrine, or isoproterenol. Arrows indicate time of infusion. 119 s i g n i f i c a n t l y lower FSH values at 17.00 h when compared with co n t r o l s . The i n i t i a l i n h i b i t i o n (16.00 h) was not as marked as that seen i n LH release. FSH l e v e l s at 16.00 h tended to remain the same as the l e v e l s seen at 15.00 h but were s i g n i f i c a n t l y lower (p<0.05) when compared to controls at 17.00 h (1.3 ± 0.1 vs. 1.9 ± 0.1 ig/ml). FSH l e v e l s i n phenylephrine-treated rat s began to r i s e 3-4 hours a f t e r i n f u s i o n while FSH l e v e l s were d e c l i n i n g at t h i s time i n cont r o l s . Thus, at 19.00 h the FSH l e v e l s of phenylephrine-treated r a t s were not d i f f e r e n t from those observed i n s a l i n e -treated animals. Si m i l a r to the e f f e c t s of isoproterenol on LH release, the e f f e c t of isoproterenol on FSH release was to cause a s i g n i f i c a n t i n h i b i t i o n i n FSH l e v e l s as compared to sa l i n e - t r e a t e d controls (group data not shown). Representative examples of FSH release i n the three groups i s seen i n F i g . 23. The patterns of LH secretion following i n t r a v e n t r i c u l a r i n f u s i o n of e i t h e r s a l i n e (n = 7) or NE (n = 11) at 16.00 h can be seen i n Fig.24. In a l l r a t s progesterone treatment at 08.00 h caused a highly s i g n i f i c a n t increase i n plasma LH l e v e l s by 15.00 h when compared with those seen at 13.00 h (p<0.01). The LH l e v e l s at 16.00 h (time of infusion) were not s i g n i f i c a n t l y d i f f e r e n t between the two groups of ra t s (p>0.05). In the rat infused with s a l i n e the LH l e v e l s at 17.00, 18.00, and 19.00 h were elevated when compared with the LH values at 13.00 (p<0.01), but were not d i f f e r e n t from those seen at 16.00 i n the same animals (p>0.05). In contrast, i n t r a v e n t r i c u l a r i n f u s i o n of NE caused a dramatic suppression of LH l e v e l s at 120 (J J — , , 1 1 1 1 1 1 13.00 15.00 17.00 19.00 Colony Time Fig. 24. E f f e c t s of i v t i n f u s i o n of s a l i n e <n = 7) and NE (n = 11) on the progesterone-induced LH surge i n EB-primed ovx rats. The s o l i d l i n e and broken l i n e s represent the r a t s given s a l i n e or NE, respectively. Arrow i n d i c a t e s time of infusion. 121 Fig. 25. E f f e c t s of i v t i n f u s i o n of s a l i n e (n = 9) and methoxamine (n = 7) on the progesterone-induced LH surge i n EB-primed ovx rats. The s o l i d l i n e and broken l i n e s represent the ra t s given s a l i n e or methoxamine, respectively. Arrow indi c a t e s time of infusion. 122 18.00 when compared with LH values at 16.00 i n the same animals (p<0.01). Also, at 19.00 h while the plasma LH values of the s a l i n e - i n f u s e d group were s t i l l elevated, those of the NE-infused group at 19.00 were not s i g n i f i c a n t l y d i f f e r e n t from the baseline values seen at 13.00 i n the same animals (p>0.05). When compared with the surge i n the saline-infused group, the LH surge i n the NE-infused rat s was interrupted and markedly suppressed. Since phenylephrine, a predominantly alpha-adrenergic agonist, i s known also to have some weak beta-receptor agonist properties, the e f f e c t of a more s p e c i f i c a l p h a ^ r e c e p t o r agonist, methoxamine, was tested. As shown i n F i g . 25 i n t r a v e n t r i c u l a r i n f u s i o n of methoxamine (n = 7) at the same molar concentration as phenylephrine, also resulted a suppression of the s t e r o i d induced LH surge. At 16.00 h, j u s t p r i o r to i n t r a v e n t r i c u l a r i n f u s i o n , the serum LH l e v e l s were not d i f f e r e n t between the s a l i n e treated r a t s (n =9) and those subsequently infused with methoxamine. Following methoxamine inf u s i o n , LH l e v e l s at 17.00 h were about 30% of LH l e v e l s i n control r a t s (p<0.05). LH l e v e l s remained s i g n i f i c a n t l y suppressed compared to the s a l i n e treated rat s (p<0.01) during the remainder of the bleeding session. IV. Discussion The stimulatory r o l e of NE and epinephrine on LH release i n i n t a c t r a t s and ovariectomized steroid-primed rats has become w e l l documented (see General Introduction, pp. 26-33). 123 Evidence of an i n h i b i t o r y r o l e for NE, i n addition to i t s excitatory r o l e , i n the regulation of LH release i s also accumulating (see General Introduction, pp. 33-38). I t has been proposed that the excitatory e f f e c t s of NE are mediated v i a alpha-receptor a c t i v a t i o n and the i n h i b i t o r y e f f e c t s are mediated v i a beta-receptor a c t i v a t i o n (Taleisnik and Sawyer, 1986) . There i s also evidence to suggest that NE could exert an i n h i b i t o r y e f f e c t on LH release v i a a c t i v a t i o n of alpha-receptors as well as beta-receptors. I t has been reported that i n t r a v e n t r i c u l a r i n f u s i o n of NE i n h i b i t s p u l s a t i l e LH release i n ovariectomized rat s and alpha-adrenergic agonists are more e f f e c t i v e than beta-agonists i n t h i s regard (Gallo and Drouva, 1979; Leung et a l . , 1982a; Gallo, 1984). I have also reported that the i n h i b i t o r y e f f e c t of NE on p u l s a t i l e LH release, i n the ovariectomized rat, i s blocked by alpha-adrenergic antagonists but not beta-antagonists (Bergen and Leung, 1986a; see General Introduction, pp. 48-85). This suggests that, at l e a s t i n the non-primed ovariectomized r a t , NE can exert i n h i b i t o r y e f f e c t s on LH release v i a alpha-receptors. I t has also been reported that i n t r a v e n t r i c u l a r infusion of a beta-adrenergic agonist blocked the increase i n LH release produced by stimulation of the MPOA i n estrogen-primed ovariectomized r a t s (Caceres and T a l e i s n i k , 1980b). E l e c t r o -chemical stimulation of the LC on the day of proestrus also blocked the LH surge and ovulation (Dotti and T a l e i s n i k , 1982). These e f f e c t s are probably mediated by beta-receptors since 124 propranolol (a beta-antagonist) but not phenoxybenzamine (an alpha-antagonist) prevented the i n h i b i t i o n produced by stimulation of the LC (Dotti and T a l e i s n i k , 1984). The possible i n h i b i t o r y r o l e of NE mediated by beta-adrenergic receptors was further demonstrated by Leung et a l . (1982b). They reported that i n t r a v e n t r i c u l a r infusion of isoproterenol, a beta-agonist suppressed LH l e v e l s when infused during the r i s i n g phase of the LH surge i n steroid-primed ovariectomized r a t s . This study confirms that isoproterenol can suppress the steroid-induced gonadotrophin surge. More i n t e r e s t i n g l y , however, the r e s u l t s reported here also suggest that a c t i v a t i o n of alpha-adrenergic receptors during a steroid-induced gonadotrophin surge can r e s u l t i n i n h i b i t i o n of the surge. These r e s u l t s also demonstrate that s i m i l a r to i n t r a v e n t r i c u l a r i n f u s i o n of isoproterenol, i n f u s i o n of NE or alpha-adrenergic agonists during the r i s i n g phase of the LH/FSH surge i n steroid-primed ovariectomized r a t s , r e s u l t s i n s i g n i f i c a n t l y lower LH and FSH l e v e l s when compared with s a l i n e - t r e a t e d controls. The i n h i b i t i o n of gonadotrophin release a f t e r t h i r d v e n t r i c u l a r infusion of NE, phenylephrine or methoxamine i s somewhat unexpected since i t i s currently accepted that, i n r a t s , p a r t i c u l a r l y steroid-primed rats, NE has an excitatory r o l e i n the regulation of LH release v i a a c t i v a t i o n of excitatory a l p h a ^ r e c e p t o r s . An i n h i b i t o r y e f f e c t of NE or alpha-agonists was previously only demonstrated i n unprimed ovariectomized r a t s . I t has c l e a r l y been established that i n t r a v e n t r i c u l a r 125 i n f u s i o n of NE or epinephrine stimulates LH release when administered to animals i n which a gonadotrophin surge has been a r t i f i c i a l l y blocked, such as by pentobarbital (Rubinstein and Sawyer, 1970), continuous i l l u m i n a t i o n (Tima and Flerkd, 1974), or high doses of estrogen and progesterone (Krieg and Sawyer, 1976; Gallo and Drouva, 1979; Leung et a l . , 1982a; Ching and Krieg, 1986; see General Introduction). The present study employed an often used animal model i n which an endogenous gonadotrophin surge i s trig g e r e d i n ovariectomized r a t s by the sequential administration of estrogen followed by progesterone (Leung et a l . , 1982b). From t h i s study and a prevous report (Leung et a l . , 1982b) i t appears that once a gonadotrophin surge has been triggered (presumably v i a exc i t a t o r y alpha-adrenoceptors), the surge can be suppressed with exogenous administration of adrenergic agonists into the t h i r d v e n t r i c l e . One possible explanation f o r these unexpected r e s u l t s i s that a c t i v a t i o n of adrenergic receptors can acti v a t e d i f f e r e n t neural systems regulating LHRH release: one which i s excitatory, and one which i s i n h i b i t o r y to LHRH release. The neural elements which i n h i b i t LHRH release may act to provide a braking mechanism f o r the surge and prevent an unbridled release of LHRH and subsequent gonadotrophin release. In other words, NE i s involved i n the stimulation or i n i t i a t i o n of the LH/FSH surge i n the appropriate s t e r o i d a l environment. However, once the gonadotrophin surge i s i n i t i a t e d , alpha-and/or beta-receptors, which are i n h i b i t o r y to LHRH, gradually become increasingly active as the LH surge progresses u n t i l the 126 influence of these receptors equals the influence of excitatory receptors, presumably at the peak of gonadotrophin release. During the descending limb of the gonadotrophin surge the influence of the i n h i b i t o r y elements exceeds the influence of the excitatory elements on the release of LHRH into the p i t u i t a r y p o r t a l veins. During the ascending limb the receptors i n h i b i t o r y to LH release may become s e n s i t i z e d such that further alpha- and/or beta-adrenergic receptor a c t i v a t i o n ( i . e . , i n t r a v e n t r i c u l a r i n f u s i o n of alpha- or beta-agonists) r e s u l t s i n a decrease rather than an increase i n LHRH release. I t has been previously reported by others that i n h i b i t o r y elements may be active during a LH surge. Kawakami and A r i t a (1980) reported that while knife-cuts i n the ven t r a l medial midbrain i n h i b i t e d the proestrous surge of LH and FSH, s i m i l a r knife-cuts i n the l a t e r a l part of the midbrain resulted i n s i g n i f i c a n t l y enhanced surges of LH and FSH. This suggests that ascending f i b r e s i n the l a t e r a l part of the midbrain may have a suppressive e f f e c t on the proestrous surge of gonadotrophins. S i m i l a r l y , knife-cuts placed j u s t i n front of the mammillary bodies, or le s i o n s of the po s t e r i o r hypothalamic n u c l e i or ventral premammillary n u c l e i , also resulted i n an e a r l i e r onset of the proestrous surge, and enhanced LH release during the surge (Beltramino and T a l e i s n i k , 1984). In the l a t t e r groups the number of ova ovulated was also increased over c o n t r o l s . Conversely, electrochemical stimulation of the po s t e r i o r hypothalamic nucleus, or the dorsal, or ventral premammillary n u c l e i i n h i b i t e d the preovulatory LH surge and 127 ovulation (Beltramino and T a l e i s n i k , 1984). Beltramino and T a l e i s n i k (1984) suggested that inputs from n u c l e i i n the p o s t e r i o r hypothalamus may play a r o l e i n determing the timing and the duration of the proestrous surge of LH. I n t e r e s t i n g l y , i t has also been suggested that a change i n the r a t i o of stimulatory and i n h i b i t o r y catecholaminergic inputs to LHRH-rel e a s i n g neurons may be responsible f o r the increase i n LH secretion, at the time of puberty i n the r a t (Raum and Swerdloff, 1986), and may also be involved i n the t r a n s i t i o n between anaestrous and estrous states i n the ewe (Meyer and Goodman, 1985). A strong candidate f o r mediating, at l e a s t p a r t i a l l y , the NE-induced i n h i b i t i o n of the proestrous or steroid-induced gonadotrophin surge i s the beta-adrenergic receptor. Caceres and T a l e i s n i k (1980a) were the f i r s t to propose that NE can exert an i n h i b i t o r y e f f e c t on LH secretion through beta-receptor a c t i v a t i o n . In the estrogen-primed ovariectomized r a t , the surge l e v e l of LH attained following i n t r a v e n t r i c u l a r NE was markedly enhanced i f the animals were pretreated with propranolol (a beta-adrenergic antagonist) one hour p r i o r to NE administration. Propranolol had no e f f e c t on the l e v e l of LH i n the absence of NE (Caceres and T a l e i s n i k , 1980b) . I t i s p o s s i b l e that the excitatory action of NE on LH release may conceal an i n h i b i t o r y influence and that t h i s i n h i b i t o r y e f f e c t can be revealed by suppressing i t with a beta-antagonist, thereby r e l e a s i n g an unopposed stimulatory action. As mentioned previously, electrochemical stimulation of the LC on 128 proestrous blocked the LH surge v i a a c t i v a t i o n of beta-receptors (Dotti and T a l e i s n i k , 1984). These beta-receptors appear to be i n the premammillary nucleus since i n j e c t i o n of propranolol but not phenoxybenzamine into t h i s nucleus, suppressed the i n h i b i t o r y e f f e c t of LC stimulation on LH release. Knife-cuts i n front of the premammillary nucleus or le s i o n s of t h i s nucleus also blocked the i n h i b i t o r y e f f e c t of LC stimulation on LH release (Dotti and T a l e i s n i k , 1984). Premammillary cuts also enhanced NE-induced LH release s i m i l a r to the enhancement produced by p r i o r administration of propranolol (Caceres and Ta l e i s n i k , 1982). T a l e i s n i k and Sawyer (1986) have suggested that these beta-receptors are located on interneurons which l i e caudal to the cuts. These interneurons, which are innervated by noradrenergic f i b r e s of the LC send t h e i r axons a n t e r i o r l y to influence LHRH release. The present study confirmed a previous f i n d i n g that i n t r a v e n t r i c u l a r i n f u s i o n of isoproterenol, a beta-agonist, can markedly suppress the steroid-induced gonadotrophin surge i n ovariectomized r a t s (Leung et a l . , 1982b). This e f f e c t of isoproterenol may be v i a a c t i v a t i o n of the premammillary beta-receptor discussed above, or a c t i v a t i o n of beta-receptors on interneurons located elsewhere ( i . e . , outside the premammillary nucleus). A l t e r n a t i v e l y , isoproterenol may act d i r e c t l y on LHRH neurons to bring about a decrease i n LHRH release. Beta-adrenergic receptor a c t i v a t i o n i n h i b i t s neuronal e x c i t a b i l i t y i n the hippocampus, cerebellum, cerebral cortex, and supraoptic nucleus (Moore and Bloom, 1979; Szabadi, 1979, Aghajanian, 129 1984, see General Introduction, pp. 20-47). I t i s possible that post-synaptic beta-receptors, when activated by NE, induce a hyperpolarization of LHRH c e l l membranes r e s u l t i n g i n a subsequent suppression i n neuronal a c t i v i t y . S i m i l a r l y i f a c t i v a t i o n of beta-receptors does induce hyperpolarization of the postsynaptic membrane of the putative interneuron i n the premammillary nucleus these interneurons would presumably be f a c i l i t a t o r y to LHRH release. The present study also demonstrates that NE and alpha-agonists can i n h i b i t the steroid-induced surge i n o v a r i -ectomized r a t s . As mentioned previously numerous studies have demonstrated an excitatory r o l e f o r NE (via alpha-receptor ac t i v a t i o n ) i n the regulation of LH (LHRH) release. I t seems u n l i k e l y (see below) that the same population of postsynaptic alpha-adrenergic receptors could mediate both stimulatory and i n h i b i t o r y e f f e c t s on gonadotrophin (LHRH) release. I t seems more probable that there e x i s t s anatomically d i s t i n c t populations (at lea s t two) of alpha-adrenergic receptors which mediate the d i f f e r e n t e f f e c t s of alpha-agonists on LHRH release. One population of alpha^adrenoceptors, possibly located d i r e c t l y on LHRH neurons, i s primarily stimulatory to LH release and therefore activated by i n t r a v e n t r i c u l a r NE and alpha-adrenergic agonists to i n i t i a t e an LH/FSH surge. Another population (or populations) of alpha-adrenergic receptors may be located on interneurons which can i n h i b i t LHRH neuronal a c t i v i t y . A c t i v a t i o n of these putative interneurons with NE or alpha-adrenergic agonists, as i n the present study, could 130 mediate a supression of LH/FSH secretion once an endogenous gonadotrophin surge has been triggered. The nature and location(s) of these putative interneurons i s (are) not known. Two p o s s i b l e s i t e s are the dorsomedial hypothalamic nuclei and the MPOA since NE i n j e c t i o n i n these areas i n h i b i t e d LH release ( P a r v i z i and E l l e n d o r f f , 1982; Leipheimer and Gallo, 1985). A t h i r d p o s s i b l e s i t e (at l e a s t i n the ovariectomized ewe) i s the medial area of the median eminence-retrochiasmatic area (Martin and Thiery, 1987). E l e c t r i c a l stimulation of the dorsomedial hypothalamic nucleus and medial area of the retrochiasmatic area i n h i b i t s LH release (Gallo, 1981a; Martin and Thiery, 1987) . P a r v i z i and E l l e n d o r f f (1982) have demonstrated that the LH response, to NE i n j e c t i o n i n the brain, depended on the s i t e and dose of administration. Taken together, these reports suggest that NE may act at d i f f e r e n t s i t e s i n the brain, presumably v i a d i f f e r e n t types of receptors on d i f f e r e n t types of interneurons, to act i n d i f f e r e n t ways, and at d i f f e r e n t times, under d i f f e r e n t s t e r o i d a l conditions to e f f e c t changes i n LHRH (LH) release. In t h i s way i n t r a v e n t r i c u l a r infusion of alpha- or beta-agonists could bring about the same e f f e c t on LH release (e.g., suppression of a surge) and alpha-receptor a c t i v a t i o n could have d i f f e r e n t e f f e c t s under d i f f e r e n t s i t u a t i o n s (e.g., stimulatory to LH release during low l e v e l release and i n h i b i t o r y during a steroid-induced surge). Another fac t o r which may be involved i n the i n h i b i t o r y e f f e c t s of adrenergic agonists on the steroid-induced surge i s 131 the d i f f e r e n t i a l rates of d e s e n s i t i z a t i o n between the d i f f e r e n t receptors. I t i s possible that d e s e n s i t i z a t i o n to stimulation occurs more r e a d i l y i n receptors which f a c i l i t a t e LHRH release than those which i n h i b i t LHRH release. I t i s of i n t e r e s t to note that continuous i n t r a v e n t r i c u l a r i n f u s i o n of NE i n steroid-primed ovariectomized rat s increased LH l e v e l s f or the i n i t i a l 40-60 minutes of inf u s i o n followed by a return to baseline LH l e v e l s , despite the continuous presence of NE (Gallo, 1982). This d e s e n s i t i z a t i o n i s c e n t r a l l y mediated since the p i t u i t a r y remained s e n s i t i v e to LHRH. This d e s e n s i t i z a t i o n to NE d i d not occur i f NE was administered i n pulses (Gallo, 1982). This suggests that the receptors mediating the excitatory e f f e c t s on LHRH (LH) are rap i d l y desensitized to continuous in f u s i o n of NE. By contrast, under conditions where i n t r a v e n t r i c u l a r i n f u s i o n of NE i n h i b i t s LH release (i.e.,non-primed ovariectomized rat) continuous i n f u s i o n of NE i n h i b i t e d p u l s a t i l e LH release f o r periods as long as 20 hours (Gallo, 1984). Therfore, the central receptors activated by NE that are i n h i b i t o r y to p u l s a t i l e LH release are not r e a d i l y desensitized to continuous NE input. These experiments indicate a c l e a r d i f f e r e n c e i n the response of noradrenergic receptors i n h i b i t o r y to LH release compared to those excitatory to LH secretion (Gallo, 1984). This d i f f e r e n c e i n des e n s i t i z a t i o n could at l e a s t p a r t i a l l y account f o r the suppression of the gonadotrophin surge by i n t r a -v e n t r i c u l a r infusion of NE and alpha-agonists. Presumably at the time of infusion excitatory receptors are being activated 132 and further stimulation (due to inf u s i o n of alpha-agonists) may r e s u l t i n d e s e n i t i z a t i o n of the receptors. The i n h i b i t o r y adrenergic receptors w i l l a l s o be activated as a r e s u l t of infu s i o n and they are not r e a d i l y desensitized. I t can be postulated that i n i t i a l l y , the r o l e of NE (which possesses both alpha- and beta-agonist properties) would be to ac t i v a t e excitatory alpha 1~receptors on LHRH neurons to t r i g g e r a gonadotrophin surge i n steroid-primed r a t s . However, once a LH/FSH surge has been i n i t i a t e d and perhaps due to a s e l e c t i v e d e s e n s i t i z a t i o n of the ex c i t a t o r y alpha-adrenergic receptors, the action of NE may be to p r i m a r i l y activate i n h i b i t o r y beta-receptors on LHRH neurons and/or to activate alpha 1-receptors located on putative i n h i b i t o r y interneurons, both processes leading to a suppression of gonadotrophin release and thus damping the ongoing surge. I t i s i n t e r e s t i n g to note that there are no reports of f a c i l i t a t i o n of LH/FSH release during a surge by NE or i t s agonists. Continuous intravenous infusion of epinephrine (but not NE or DA) on the afternoon of proestrus has al s o been reported to block (via a c e n t r a l mechanism) the proestrous surge of LH and ovulation i n the r a t (Blake, 1976) . Perfusion of the t h i r d v e n t r i c l e with r e l a t i v e l y high concentrations of NE can also decrease the amount of LH released by electrochemical stimulation of the MPOA (Cramer and Barraclough, 1978), suggesting LHRH release may indeed be decreased by an extended period of NE stimulation. A further p o s s i b i l i t y to account f o r i n h i b i t i o n of the LH 133 surge i s the increased release of a fac t o r that i s i n h i b i t o r y to LH release. I t has been reported that a factor i n the hypothalamus can suppress basal and LHRH stimulated LH release from p i t u i t a r i e s i n v i t r o (Johansson et a l . , 1975). More recently, a p a r t i a l l y p u r i f i e d factor, extracted from the hypothalamus, can suppress LHRH-induced LH release from dispersed p i t u i t a r y c e l l s and LHRH-induced LH release i n proestrous rats (Hwan and Freeman, 1987a, 1987b). An antiserum to t h i s i n h i b i t o r y f a c t o r also enhanced the estrogen-induced LH surge i n ovariectomized r a t s (Hwan and Freeman, 1987b). I f an i n h i b i t o r y factor i n the hypothalamus i s involved i n the regulation of LH release i t i s possib l e that i n f u s i o n of NE, or one of i t s agonists, may have r e s u l t e d i n i t s enhanced release. This could account f o r the decrease i n LH release observed i n the present experiment. Further study i s required to determine the r o l e , i f any, of t h i s LH r e l e a s e - i n h i b i t i n g factor i n the regulation of LH release. Another factor which might account f o r the decrease i n LH and FSH release i s a vasopressor e f f e c t of NE and a l p h a 1 ~ adrenergic agonists. These agonists could conceivably decrease LH release by decreasing blood flow i n the hypothalamo-hypophyseal vessels which would r e s u l t i n a decrease i n the amount of LHRH deli v e r e d to the gonadotropes. However, s i m i l a r dosages of NE and adrenergic agonists are able to increase LH release i n other experimental models and a vasopressor e f f e c t does not appear to be a major f a c t o r (Krieg and Sawyer, 1976; Leung et a l . , 1982a). Caceres and T a l e i s n i k (1980b) found that 134 when beta-receptors are blocked, the a b i l i t y of NE to enhance LH release (presumably only alpha-receptors are being activated) increased by more than 2.5 times. Therefore, while i n f u s i o n of NE, methoxamine, or phenylephrine, may r e s u l t i n v a s o c o n s t r i c t i o n of the hypophyseal p o r t a l vessels, i t seems u n l i k e l y that t h i s would account f o r the d r a s t i c decline i n gonadotrophin l e v e l s which were observed. In conclusion, i t must be said that the precise mechanism whereby a gonadotrophin surge can be i n h i b i t e d by i n f u s i o n of NE or an alpha- or beta-agonist i s unknown. However the po s s i b l e mechanisms have been discussed and i t should be stressed that these mechanisms are not generally mutually exclusive of each other, but rather the i n h i b i t i o n observed i n the present study may be due to several factors working together. I t i s evident that the regulation of LHRH release i s very complex. The large body of work which has attempted to unravel i t s regulation a t t e s t s to that fa c t . The evidence presented here suggests that i n addition to the well established stimulatory r o l e of NE and epinephrine i n the gonadotrophin surge, both alpha- and beta-adrenergic receptors may exert i n h i b i t o r y e f f e c t s on LH/FSH release, once a surge has been tri g g e r e d . 135 E l e c t r i c a l Stimulation of Ascending Noradrenergic Tracts  i n the Midbrain: E f f e c t s on LH Release i n  Steroid-Primed Ovariectomized Rats I. Introduction There i s extensive evidence which suggests that the c e n t r a l noradrenergic system i s involved i n the regulation of LH s e c r e t i o n (Barraclough and Wise, 1982; Ramirez et a l . , 1984; see General Introduction). NE-containing f i b r e s t r a v e l r o s t r a l l y from the brainstem and pass through the mesencephalon v i a two major pathways, the DNT and the VNT (L i n d v a l l and BjOrklund, 1978). The f i b r e s of both t r a c t s have been implicated i n the control of LH release, although t h e i r precise r o l e s are not known. E l e c t r i c a l stimulation of the DNT has been shown to i n h i b i t LH release i n the ovariectomized r a t , whereas stimulation of the VNT has no e f f e c t on p u l s a t i l e LH release (Leung et a l . , 1981b; Bergen and Leung, 1987b; see pp. 86-108). The i n h i b i t i o n of p u l s a t i l e LH release caused by DNT stimulation i s s i m i l a r to the i n h i b i t i o n of p u l s a t i l e LH release observed with i n f u s i o n of NE into the t h i r d v e n t r i c l e of the ovariectomized r a t (Gallo and Drouva, 1979; Gallo, 1984). On the other hand, using an estrogen-primed ovariectomized r a t model, i t was shown that NE as well as alpha- and beta-adrenergic agonists e f f e c t i v e l y i n h i b i t LH release when administered during the r i s i n g phase of an ongoing P.-induced LH surge (Leung et a l . , 136 1982a; Bergen and Leung, 1986; see pp. 109-134). These rather unexpected r e s u l t s implicate an i n h i b i t o r y component i n the r o l e of the c e n t r a l noradrenergic neurons i n the maintenance of the gonadotrophin surge. To further investigate t h i s hypothesis, the present study (Experiment #1) was designed to determine the e f f e c t s of e l e c t r i c a l stimulation of the DNT, or VNT, on LH release during an ongoing steroid-induced LH surge. In addition the DNT was stimulated i n rats that were pretreated with a receptor antagonist i n an attempt to determine the receptor through which e f f e c t s were being mediated. In Experiment #2 e i t h e r the DNT, or VNT, was e l e c t r i c a l l y stimulated i n steroid-primed ovariectomized r a t s p r i o r to the onset of the steroid-induced LH surge. This was done to t e s t whether a c t i v a t i o n of NE pathways would i n i t i a t e a LH surge i n appropriately primed ovariectomized r a t s . In Experiment #3 e i t h e r the DNT, or VNT, was stimulated i n rats which were primed only with estradiol-benzoate (E 2) 3 days previously. E l e c t r i c a l stimulation was c a r r i e d out before the onset of the E 2~induced LH surge. In these rats stimulation was e i t h e r continuous or intermittent to determine whether intermittent ( p u l s a t i l e ) administration of the stimulus might more r e a d i l y enhance the release of LH. In Experiment #4 i t was decided to t e s t the e f f e c t s of electrochemical stimulation of the DNT and/or the VNT on the gonadotrophin surge. Electrochemical stimulation of other brain regions has been found to have a s i g n i f i c a n t e f f e c t on gonadotrophin release. Again, ovariectomized r a t s were 137 sequentially primed with E 2 and P 4 to induce a gonadotrophin surge. In the f i n a l experiment described i n t h i s study (Experiment #5) the steroid-primed rats were anaesthetized f o r the period of stimulation and i n these rats the e l e c t r i c a l current used to stimulate the ra t s was two and a h a l f , to f i v e times the previous l e v e l s ( i . e . , those used i n Experiments #1-#4) to stimulate either the DNT or the VNT. Higher currents were used since the low currents used i n previous experiments ( i . e . , #2 and #3) might have been inadequate to stimulate f i b r e s excitatory to LHRH release. Materials and Methods  General Adult female Sprague-Dawley rats were obtained from Charles Rivers (Montreal, Canada) and housed i n a c o n t r o l l e d environment (temperature, 22 ± 1 °C; 14 h of l i g h t , 10 h of darkness; l i g h t s on, 0500-1900 h) . The ra t s were ovariectomized and 10-14 days l a t e r e i t h e r a b i p o l a r (for e l e c t r i c a l stimulation) or a monopolar (for electrochemical stimulation) electrode was implanted under pentobarbital anaesthesia i n eithe r the dorsal tegmental area or v e n t r a l tegmental area, according to the a t l a s of Paxinos and Watson (1982). The bipol a r electrode was made of twisted s t a i n l e s s s t e e l wire (0.005-in. diameter; C a l i f o r n i a Fine Wire Co., Grover City,CA.) completely insulated except at the t i p . The monopolar electrode was made of a s t a i n l e s s s t e e l wire (0.010-138 i n . diameter) insulated except at the t i p . In r a t s implanted with a monopolar electrode a wire was also attached to a s k u l l screw to act as the ground. The rats were given at l e a s t 1 week to recover from the surgery at which time they appeared healthy and weighed 250-300 g. In a l l experiments blood samples were c o l l e c t e d v i a i n t r a a t r i a l S i l a s t i c catheters which were implanted into the r a t s one or two days p r i o r to the day of stimulation and blood c o l l e c t i o n . The catheters were implanted by a procedure described e a r l i e r (p. 51) . In a l l cases the blood samples (250-300 t l ) were allowed to c l o t at 4 °C and then centrifuged. Immediately following a blood sample an equal volume of s a l i n e was inj e c t e d into the r a t . Unless s p e c i f i e d the rats were conscious and unrestrained during the period of blood sampling. Serum was removed following c e n t r i f u g a t i o n and the samples were stored at -70 'C u n t i l RIA. The serum samples were assayed using RIA k i t s provided by the NIADDK National Hormone and P i t u i t a r y Program using a procedure described e a r l i e r (p. 53) . The values of LH are expressed i n terms of NIADDK r a t RP-2. A f t e r the experiment, the ra t s were anaesthetized with pentobarbital, and t h e i r brains were perfused with s a l i n e , followed by 10% formalin i n s a l i n e . The brains were then stored i n 10% formalin and l a t e r sectioned at 50 tm and stained with a N i s s l s t a i n . The l o c a t i o n of the electrode t i p (either i n s i d e or outside the tra c t ) was determined without regard to the LH records. The r a t s i n which the electrode t i p was judged to be outside the DNT or VNT were excluded from t h e i r 139 respective groups and t h e i r LH l e v e l s were not included i n the data analysis. S t a t i s t i c a l analysis included one-way analysis of variance. I f s i g n i f i c a n t F values were obtained (p<0.05), Scheffe's t e s t was used to determine differences between the groups. Experiment #1 The s t e r o i d treatment consisted of E 2 (Sigma Chemical Co., St. Louis, MO) i n sesame seed o i l at a dose of 10 tg/100 g BW. The r a t s were then given an i n j e c t i o n of 72 hours l a t e r at 11.00 h. A f t e r P 4 i n j e c t i o n , s a l i n e - f i l l e d polyethylene tubing was connected to the i n t r a a t r i a l S i l a s t i c catheter that had been implanted into the r a t on the previous day. Blood samples of 250 t l were taken from the r a t at 1 hour i n t e r v a l s beginning at 14.00 h and ending at 19.00, 20.00, or 22.00 h, and the blood was replaced by an equal volume of s a l i n e . Before e l e c t r i c a l stimulation, the b i p o l a r electrode implanted i n t o the r a t s was connected to a Grass S88 stimulator with two Grass PSIU6 ph o t o e l e c t r i c stimulus i s o l a t i o n units (Grass Instruments, Quincy, MA) with constant current output f o r biphasic pulses. The stimulus consisted of biphasic pulses of 400 tsec each, separated by 50 tsec, occurring at 30 Hz. The stimulus was applied 10 sec on and 10 sec o f f . The current was gradually increased up to 100 tA or u n t i l behavioral changes (general arousal, e.g. grooming and running) were j u s t noted and then decreased s l i g h t l y to avoid these e f f e c t s i n response to stimulation. The current (50-100 tA) and waveform 140 were monitored p e r i o d i c a l l y during the stimulation period with an o s c i l l o s c o p e . E l e c t r i c a l stimulation under these parameters i n the medial preoptic area has previously been shown to be highly e f f e c t i v e i n e l i c i t i n g LH release, and the amount of 3+ i r o n (Fe ) deposited at the electrode t i p i s r e l a t i v e l y low (Jamieson and Fink, 1976; Terasawa and Sawyer, 1969). On the day of the experiment, the DNT- or VNT-implanted ra t s were assigned to one of s i x groups. One group of rats with an electrode implanted into e i t h e r the DNT or VNT was not stimulated during the bleeding session (group 1) . Two groups of r a t s with electrodes implanted into e i t h e r the DNT (group 2) or VNT (group 3) were stimulated e l e c t r i c a l l y , beginning immediately a f t e r the blood sample at 16.00 h was taken and ending a f t e r the 17.00 h blood sample was taken. The f i n a l blood sample was taken at e i t h e r 19.00 or 20.00 h. In group 4 (electrode i n DNT) and group 5 (electrode i n VNT) , the rats were stimulated from 16.00-17.00 h as i n groups 2 and 3, but i n addi t i o n were stimulated for a second time (with the same parameters) from 19.00-20.00 h. The f i n a l sample i n these rats was taken at 22.00 h. Group 6 ra t s were stimulated e l e c t r i c a l l y i n the DNT, as i n group 2, except that they were also given at 16.00 h a s i n g l e i v i n j e c t i o n of propranolol 15 mg/kg), phenoxybenzamine (15 mg/kg), or pimozide (0.65 mg/kg). These drugs, at the dosage described, have been previously shown to be e f f e c t i v e i n antagonizing the beta-adrenergic, alpha-adrenergic, and dopaminergic receptor, r e s p e c t i v e l y . 141 Experiment #2 In t h i s experiment r a t s were again implanted with a b i p o l a r electrode u n i l a t e r a l l y , into e i t h e r the DNT or VNT. A few electrodes were also implanted into the the arcuate nucleus to t e s t the a b i l i t y of the electrodes, with the stimulation parameters described above, to induce LH release at a s i t e previously shown to e f f e c t i v e i n t h i s regard. The rats were primed with E 2 and P 4 as above. E l e c t r i c a l stimulation was started at 12.00 h and lasted for one hour ( i . e . , before the onset of the steroid-induced surge). Blood sampling was started immediately p r i o r to the s t a r t of e l e c t r i c a l stimulation and blood samples were c o l l e c t e d at regular 20 minute i n t e r v a l s during the period of stimulation and at hourly i n t e r v a l s thereafter u n t i l 18.00 h. Serum samples were assayed f o r LH and FSH. Experiment #3 In t h i s experiment rats were stimulated i n the DNT v i a a b i p o l a r electrode. The rats were primed with E 2 at the same dose as i n the previous experiment and were stimulated approximately 72 hours a f t e r s t e r o i d i n j e c t i o n . In a l l cases, stimulation occurred before 13.00 h which i s p r i o r to the onset of the LH surge induced by the s t e r o i d treatment. In t h i s experiment the rats were divided a r b i t r a r i l y i n t o 3 groups. One group of rats was stimulated f o r one hour with the stimulation parameters as set i n experiment 1. Blood samples were c o l l e c t e d at 15 minute i n t e r v a l s during the 1 hour stimulation period and f o r 45 minutes a f t e r the period of 142 stimulation. In the second group, the rats were stimulated 4 times with each period of stimulation l a s t i n g 5 minutes. The time between stimulations was 20 minutes. Blood samples were c o l l e c t e d at regular i n t e r v a l before the onset of stimulation and immediately before cessation of stimulation as well as 5 minutes a f t e r stimulation was stopped. In the t h i r d group, the ra t s d i d not receive any e l e c t r i c a l stimulation over the course of blood sampling. Serum samples were assayed f o r LH, FSH and p r o l a c t i n (PRL). Experiment #4 In t h i s experiment rats were implanted b i l a t e r a l l y with monopolar electrodes into e i t h e r the VNT, the DNT, or had one electrode i n the DNT and one electrode i n the c o n t r a l a t e r a l VNT. The rats were primed sequentially with E 2 and P 4 to induce a gonadotrophin surge. The rats were a r b i t r a r i l y d ivided into the d i f f e r e n t groups. Of the three groups ( i . e . , b i l a t e r a l DNT, b i l a t e r a l VNT, u n i l a t e r a l DNT and VNT), i n d i v i d u a l rats were selected at random to receive stimulation and a number of r a t s i n each group were not stimulated. Blood samples were c o l l e c t e d at hourly i n t e r v a l s from 12.00 to 18.00 h and two ad d i t i o n a l samples were taken at 13.30 and 14.30 h. The r a t s were stimulated at 13.00 h with an anodal current of 100 tA f o r 60 seconds. These parameters have been reported to be e f f e c t i v e i n other studies. The serum samples were assayed f o r LH and PRL. Experiment #5 In t h i s experiment the r a t s were a l l implanted with a 143 b i p o l a r electrode i n the VNT. The rats were primed with E 2 (50 ig) and P 4 (25 mg) each. Three days l a t e r the rats were anaesthetized with pentobarbital. Blood samples were taken at regular i n t e r v a l s before, during, and a f t e r e l e c t r i c a l stimulation. The parameters used were same as those i n experiment #1 except that the current was increased to 250 iA instead of 50-100 tA. A l l other stimulation parameters were the same. On the day of the experiment the rats were a r b i t r a r i l y assigned to one of 4 groups. Group #1 d i d not receive e l e c t r i c a l stimulation and received s a l i n e 5 minutes before i t otherwise would have been stimulated. Group #2 was stimulated fo r one hour and also was i n j e c t e d with s a l i n e , 5 minutes before the onset of stimulation. Groups #3 and #4 were also stimulated f o r one hour and i n addition received phenoxybenzamine (15 mg/kg) or propranolol (10 mg/kg), resp e c t i v e l y , 5 minutes before the onset of stimulation. Results  Experiment #1 E l e c t r i c a l Stimulation of the DNT The r a t s i n groups 1 and 2 were used for t h i s part of the study. Group 1 consisted of r a t s that were implanted with electrodes but not stimulated ( i . e . , non-stimulated c o n t r o l s ) . Group 2 consisted of rats that were implanted with an electrode i n the DNT, as determined h i s t o l o g i c a l l y ; i n these animals e l e c t r i c a l stimulation was applied during the bleeding sessions A B Fig. 26. Location of electrode t i p s for e l e c t r i c a l stimulation (ES) of the dorsal, and ventral noradrenergic t r a c t s (DNT and VNT, r e s p e c t i v e l y ) , i n steroid-primed ovx rats. The inset i s a schematic s a g i t t a l section depicting the ascending noradrenergic system, with the s i t e s of ES indicated by the A (VNT), and B (DNT). In the f r o n t a l brain sections the s t i p p l e d area on the right-hand side depicts the VNT (A), and DNT (BJ, respectively. The s i t e s of ES are depicted on the left-hand side of each f r o n t a l section as being e i t h e r inside ( f i l l e d c i r c l e s ) or outside (empty c i r c l e s ) the t r a c t s . See Fig. 12 for abbreviations. Adapted from the a t l a s of Paxinos and Watson (1982). 145 ( i . e . , 16.00 h). F i g . 26 shows the l o c a t i o n of the electrode t i p s , with the f i l l e d c i r c l e s representing the electrodes designated as being i n the ascending NE t r a c t s i n the mesencephalon. Also included i n Fig . 26 are data obtained from other r a t s that were implanted with an electrode and stimulated e l e c t r i c a l l y as i n group 2, but the t i p of the electrode was designated as being outside of the DNT or VNT and are represented with empty c i r c l e s . These rats are designated the stimulated c o n t r o l s . F i g . 27 shows LH release i n 4 r a t s , which are representative of each of the d i f f e r e n t groups. i n non-stimulated control rats (Fig. 27A), a c h a r a c t e r i s t i c surge of LH (induced by P 4) was present which reached peak l e v e l s at approximately 18.00 h. As i l l u s t r a t e d i n F i g . 28, which represents the mean LH values of 18 non-stimulated control animals. LH l e v e l s increased s i g n i f i c a n t l y at 17.00 h compared with those at 16.00 h (P<0.05). In sharp contrast, i n rats that were stimulated i n the DNT between 16.00 and 17.00 h (n = 10), LH l e v e l s decreased (P<0.01) a f t e r e l e c t r i c a l stimulation (Fig. 28). At 17.00 and 18.00 h, the LH l e v e l s i n the DNT-stimulated r a t s were s i g n i f i c a n t l y lower (P<0.05) than the corresponding values i n control animals. In r a t s that were e l e c t r i c a l l y stimulated, but i n which the electrode t i p s were determined to be outside of e i t h e r the DNT or VNT ( i . e . , the stimulated co n t r o l s ; n = 6), the mean LH l e v e l s were not s i g n i f i c a n t l y d i f f e r e n t from those i n the nonstimulated rat s at any time during the bleeding sessions. A representative of the T I M E ( h ) Fig. 27. Representative examples of LH release i n 4 i n d i v i d u a l steroid-pr rat s from 4 d i f f e r e n t groups. The period of ES i n t h i s and the proceeding figures i s Indicated by the black box. 147 K.00 16.00 18.00 20.00 TIME (h) F i g . 28. Mean serum LH l e v e l s i n DNT-stimulated and non-stimulated control r a t s (number of rats i s i n brackets). E f f e c t of ES of DNT on the progesterone-induced LH surge. 16.00 18.00 20.00 TIME (h) Fig. 29. Percent change i n mean LH levels, as compared to values at 16.00 h i n DNT-, VNT-, or non-stimulated control rats (number of r a t s i s i n brackets). E f f e c t s of ES of the DNT and VNT on the progesterone-induced LH surge. M *. Oo 149 stimulated controls i s shown i n F i g . 27B. Because of the s l i g h t v a r i a t i o n s i n the timing of the LH surge i n r a t s , we have also expressed these r e s u l t s as a percent change i n LH secretion i n reference to the LH values observed at 16.00 h, i . e . , the beginning of e l e c t r i c a l stimulation. As seen i n Fig. 29, LH l e v e l s i n c o n t r o l r a t s increased at 17.00 and 18.00 h as compared to values at 16.00 h. In the DNT-stimulated rats, e l e c t r i c a l stimulation c o n s i s t e n t l y resulted i n a decrease i n LH l e v e l s at 17.00 h, which were s i g n i f i c a n t l y lower than the LH l e v e l s i n the non-stimulated controls (P<0.001). After cessation of e l e c t r i c a l stimulation i n the DNT r a t s , LH l e v e l s rose again, and at 19.00 and 20.00 h, the r e l a t i v e LH l e v e l s were not s i g n i f i c a n t l y d i f f e r e n t i n the DNT and c o n t r o l groups. E l e c t r i c a l Stimulation of the VNT F i g . 29 also i l l u s t r a t e s the e f f e c t s of e l e c t r i c a l stimulation of the VNT on the steroid-induced LH surge. As i n the DNT-stimulated animals, e l e c t r i c a l stimulation applied to the VNT ( i . e . , group 3; n = 8) at 16.00 h caused a marked suppression of LH l e v e l s at 17.00 h (P<0.001). At 17.00 and 18.00 h, the LH l e v e l s i n group 3 rats were s i g n i f i c a n t l y lower (P<0.01) than those i n co n t r o l animals ( i . e . , group 1), but were not d i f f e r e n t from those i n DNT-stimulated animals (group 2) . For the animals i n group 3 the l a s t blood sample was c o l l e c t e d at 19.00 h. E f f e c t s of Two Stimulation Sessions The e f f e c t s of two e l e c t r i c a l stimulation sessions, 2 hours apart, on LH release i n the same animal were investigated Fig. 30. Location of electrode t i p s i n rats which were stimulated twice (from 16.00 h to 17.00 h, and 19.00 h to 20.00 h) i n ei t h e r the VNT (A) or DNT (B). See Fig. 24 f o r abbreviations. £j O 151 i n groups 4 and 5. As indicated i n F i g . 30, electrodes were again implanted i n the mesencephalon, e i t h e r inside or outside the ascending NE t r a c t s . F i g . 31 shows the LH values i n 4 rats representative of the four d i f f e r e n t groups ( i . e . , non-stimu-lat e d c ontrols and rats stimulated inside the DNT, inside the VNT, or outside the NE t r a c t s ) . The rats were f i r s t stimulated from 16.00-17.00 h and again from 19.00-20.00 h on the same day and with the same e l e c t r i c a l stimulation parameters. As expected, e l e c t r i c a l stimulation of the DNT between 16.00 and 17.00 h r e s u l t e d suppression of the LH surge. This i s i l l u s t r a t e d i n the mean LH l e v e l f o r a group of seven DNT-stimulated animals (Fig. 32) . The mean LH l e v e l s of the DNT-stimulated r a t s were on the r i s e between 17.00 and 19.00 h. Quite dramatically, e l e c t r i c a l l y stimulation from 19.00-20.00 h again suppressed LH release. This i s evident by comparing the LH p r o f i l e s i n F i g . 32 to those of the one-time DNT-stimulated rats i n F i g . 28. In DNT stimulated r a t s not stimulated between 19.00 and 20.00 h (Fig. 28), LH values continued to increase, so that the LH l e v e l s at 20.00 h were not s i g n i f i c a n t l y d i f f e r e n t from those i n non-stimulated control animals. By contrast, i n r a t s stimulated a second time between 19.00 and 20.00 h (Figs. 31 and 32), LH release was again suppressed a f t e r the second e l e c t r i c a l stimulation, so that the mean LH l e v e l s were s i g n i f i c a n t l y lower (P<0.05) than those i n non-stimulated control rats (n = 8) at 20.00 h. This second i n h i b i t i o n of LH release i s also evident by comparing the percent change i n LH values i n the two-time DNT-stimulated rats TIME (h) F i g . 31. R e p r e s e n t a t i v e examples of LH r e l e a s e i n 4 I n d i v i d u a l s t e r o i d - p r i m e d r a t s from 4 d i f f e r e n t groups. E f f e c t s of two p e r i o d s o f ES on the p r o g e s t e r o n e -induced LH surge. U.00 16.00 18.00 20.00 22.00 TIME (h) Fig. 32. Mean serum LH l e v e l s i n DNT-stimulated and non-stimulated controls. E f f e c t of two periods of ES of DNT on the progesterone-induced LH surge. 16.00 18.00 20.00 22.00 TIME (h) F i g . 33. Percent change i n mean LH l e v e l s , as compared t o v a l u e s at 16.00 h i n twice DNT-, VNT-, or non-stimulated c o n t r o l r a t s . E f f e c t s o f two p e r i o d s o f ES of the DNT and VNT on the progesterone-induced LH surge. £JJ 155 (Fig. 33) with those i n the one-time stimulated ra t s i n Fig. 29. At 20.00 h, the LH l e v e l s i n the two-time DNT-stimulated r a t s were s i g n i f i c a n t l y lower than those i n the one-time DNT-stimulated animals (P<0.01). Likewise, e l e c t r i c a l stimulation of the VNT 2 hours a f t e r the f i r s t stimulation session r e s u l t e d i n a resuppression of LH release (Figs. 31 and 33). At 20.00 h, the LH values of the two-time VNT-stimulated rats (n = 6) were not d i f f e r e n t from those i n the two-time DNT-stimulated group. Receptor S p e c i f i c i t y of DNT Stimulation In t h i s experiment the r a t s were implanted with an electrode i n the DNT and p r i o r to the s t a r t of e l e c t r i c a l stimulation, which lasted 2 hours, were injected with either s a l i n e , pimozide, propranolol, or phenoxybenzamine. A f i f t h group also received s a l i n e but d i d not receive e l e c t r i c a l stimulation. As expected from previous r e s u l t s , e l e c t r i c a l s timulation of the DNT for 2 hours (from 16.00 to 18.00 h) res u l t e d i n s i g n i f i c a n t l y lower LH l e v e l s as compared with non-stimulated controls. The values at 18.00 h for the two groups are shown i n Fig. 35. F i g . 34 shows the LH release p r o f i l e s of 4 i n d i v i d u a l rats which received e i t h e r pimozide or propranolol before the s t a r t of e l e c t r i c a l stimulation. I t i s evident that neither propranolol (n = 8) nor pimozide (n = 8) was able to a l t e r the i n h i b i t i o n of LH release induced by stimulation of the DNT and LH le v e l s were s i g n i f i c a n t l y (p<0.01) lower than i n the non-stimulated controls (Fig. 35). For comparison, i n F i g . 35 the LH values at 18.00 h i n the non-stimulated and the TIME (h) Fig. 3 4 . Representative examples of LH release i n steroid-primed r a t s which, p r i o r to the s t a r t of ES of the DNT, were injected (iv) with e i t h e r pimozide (Pirn.) or propranolol (Prop.). Arrow indicates the time of receptor antagonist i n j e c t i o n . ^ Ql I I I I I I I K . 0 0 16.00 18.00 20.00 TIME (h) Fig. 35. Mean serum LH l e v e l s i n DNT-stimulated rats which also received e i t h e r pimozide or propranolol p r i o r to the onset of ES. The X and the black box at 18.00 h represent the LH l e v e l s i n the non-stimulated and the DNT-stimulated rats, respectively. U.00 16.00 18.00 20.00 TIME (h) F i g . 36. Representative examples of LH release i n s t e r o i d -primed r a t s which, p r i o r to the s t a r t of ES of the DNT, were in j e c t e d (iv) with phenoxybenzamine. F i g . 3 7 . Mean serum LH l e v e l s i n phenoxybenzamine t r e a t e d DNT-stimulated s a l i n e t r e a t e d non-stimulated r a t s . 160 s a l i n e - t r e a t e d stimulated r a t s are designated by an X and s o l i d box, r e s p e c t i v e l y . In contrast to these r e s u l t s , i n h i b i t i o n of the LH surge produced by e l e c t r i c a l stimulation of the DNT was blocked by p r i o r administration of phenoxybenzamine (Figs. 3 6 and 37). During the period of e l e c t r i c a l stimulation and at the end of the stimulation period, LH l e v e l s i n the phenoxy-benzamine-treated rats (n = 9) were not s i g n i f i c a n t l y d i f f e r e n t (p>0.05) than l e v e l s i n the non-stimulated controls, whereas e l e c t r i c a l stimulation of the DNT saline-treated rats (n = 7) again i n h i b i t e d LH release as compared to the non-stimulated contro l s . Experiment #2 E l e c t r i c a l Stimulation of DNT or VNT  before Steroid-Induced Surge In t h i s experiment, sequentially primed (E 2 followed by P 4, 3 days la t e r ) ovariectomized rat s were e l e c t r i c a l l y stimulated i n e i t h e r the DNT or VNT p r i o r to the onset of the gonadotrophin surge to determine whether a c t i v a t i o n of these t r a c t s from 12.00 to 13.00 h could i n i t i a t e or induce an early occurence of the surge. In the non-stimulated controls there was no s i g n i f i c a n t difference (p>0.05) i n LH l e v e l s between 12.00 and 13.00 h. LH l e v e l s i n DNT-stimulated rats (n = 4) were also not s i g n i f i c a n t l y d i f f e r e n t (p>0.05) at 13.00 h ( a f t e r stimulation) than at the onset of the stimulation period at 12.00 h (3.32 ± .44 vs 2.98 ± .79 ng/ml, r e s p e c t i v e l y ) . S i m i l a r l y , LH l e v e l s i n VNT-stimulated rats (n = 4) at 13.00 h were not s i g n i f i c a n t l y d i f f e r e n t (p>0.05) than l e v e l s at 12.00 161 h (2.71 ± .28 vs. 2.98 ± .38 ng/ml, r e s p e c t i v e l y ) . In both rats which were stimulated i n the arcuate nucleus, LH l e v e l s increased over the stimulation period (from 5.74 to 12.97 ng/ml i n one, and 2.36 to 15.24 ng/ml i n the other). In a l l the rats that were stimulated i n the DNT or the VNT, a LH surge occured l a t e r that afternoon which was not s i g n i f i c a n t l y d i f f e r e n t than that seen i n non-stimulated controls (data not shown). S i m i l a r l y , e l e c t r i c a l stimulation of the DNT or VNT also did not s i g n i f i c a n t l y a f f e c t the release of FSH. Experiment #3 E l e c t r i c a l Stimulation of DNT or VNT i n E 2~Primed Rat:  E f f e c t of Continuous or P u l s a t i l e Stimulation. In t h i s experiment ovariectomized rats were primed only with E 2 and then stimulated 3 days l a t e r , several hours before the onset of the LH surge. In rat s which were stimulated for one hour (n = 8) , the LH, FSH and PRL serum l e v e l s d i d not change s i g n i f i c a n t l y (p>0.05) over the course of e l e c t r i c a l stimulation of the DNT. Hormone l e v e l s remained unchanged throughout the blood sampling period s i m i l a r to non-stimulated controls (n = 5) (Data not shown). The second group of rats (n = 8) were stimulated f o r 5 minute periods separated by 20 minute periods. There were no s i g n i f i c a n t changes i n LH, FSH, and PRL l e v e l s over the course of the blood sampling period. Also, no trends i n hormone release, as a r e s u l t of e l e c t r i c a l stimulation, were observed i n any of the hormones assayed (Data not shown). 162 Experiment #4 Electrochemical Stimulation of the DNT or VNT i n E„ and P. Primed Rats — 2 4 In t h i s experiment the r a t s were divided into 4 groups. Group 1 (n = 8) had electrodes implanted d i d not receive any stimulation ( c o n t r o l ) . In these non-stimulated r a t s , the s i t e of electrode implantation d i d not have any observable e f f e c t on hormone release and therefore the r e s u l t s obtained from these rats were grouped together f o r s t a t i s t i c a l purposes. The second group was stimulated b i l a t e r a l l y i n the DNT (n = 7), the t h i r d group was stimulated i n the VNT (n = 7) , and group 4 received stimulation i n the DNT and VNT (n=7). In the non-stimulated controls a LH surge was evident towards the end of the bleeding period at 18.00 h. In the b i l a t e r a l l y stimulated DNT r a t s , LH l e v e l s showed l i t t l e i f any change over the course of the blood sampling period and at 17.00 h were s i g n i f i c a n t l y lower (p<0.05) than the non-stimulated controls (1.18 ± .26 vs. 7.61 ± 2.58 ng/ml, respectively) (Fig. 38) . LH l e v e l s remained s i g n i f i c a n t l y lower (p<0.05) at 18.00 h i n the DNT stimulated r a t s as compared to the non-stimulated controls (1.66 ± .36 vs. 13.03 ± 4.48, r e s p e c t i v e l y ) . S i m i l a r l y , LH l e v e l s i n the VNT and DNT/VNT (data not shown) stimulated r a t s were both s i g n i f i c a n t l y lower (p<0.05) than LH l e v e l s i n the non-stimulated controls (Fig. 38). Electrochemical stimulation of the DNT, VNT, or DNT and VNT together d i d not have a s i g n i f i c a n t e f f e c t (p>0.05) on PRL 163 15 r 12.00 U.00 16.00 18.00 TIME (h) F i g . 38. E f f e c t of b i l a t e r a l electrochemical stimulation of the DNT or VNT on the steroid-Induced LH surge as compared to non-stimulated controls. 164 600 r TIME (h) Fig. 39. E f f e c t of b i l a t e r a l electrochemical stimulation of the DNT or VNT on the steroid-induced PRL surge as compared to non-stimulated controls. 165 l e v e l s at any time when compared to non-stimulated controls. There was a s l i g h t but not s t a t i s t i c a l l y s i g n i f i c a n t trend for stimulation of the VNT to r e s u l t i n a s l i g h t delay i n the onset of the PRL surge (Fig. 39). Experiment #5 E l e c t r i c a l Stimulation of the VNT In t h i s experiment the a l l rats were under pentobarbital anaesthesia during the bleeding period. The r a t s that were not stimulated and received s a l i n e (Group #1, n = 4), LH l e v e l s , as expected, did not change s i g n i f i c a n t l y over the course of the 105 minute bleeding period. S i m i l a r l y , i n rats stimulated i n the VNT and treated e i t h e r with s a l i n e (Group #2, n = 7) , or propranolol (Group #4, n = 8) , LH l e v e l s d i d not show any s i g n i f i c a n t change i n LH release over the bleeding period. In the phenoxybenzamine-treated r a t s (Group#3 (n = 5) , blood LH l e v e l s were s l i g h t l y but s i g n i f i c a n t l y (P<0.05; paired t-test) higher at the end of the stimulation period than at the beginning of the stimulation period (Fig. 40). However, there were no s i g n i f i c a n t d i f f e r e n c e s between the groups at the end of the stimulation period. IV. Discussion This study demonstrates that e l e c t r i c a l stimulation of e i t h e r the DNT or VNT during a steroid-induced LH surge r e s u l t s i n i n h i b i t i o n of LH release. This i n h i b i t i o n i s s i t e s p e c i f i c since stimulation near, but outside the f i b r e t r a c t s does not r e s u l t i n i n h i b i t i o n of the LH surge (Figs. 27 and 31). There 0 30 60 TIME (min) Fig. 40. E f f e c t of e l e c t r i c a l stimulation of the VNT and adrenergic antagonists, on LH release i n anaesthetized steroid-primed rats. 167 are a number of factors which suggest that stimulation of these f i b r e s , p a r t i c u l a r l y the DNT, a c t i v a t e s noradrenergic f i b r e s and that i t i s the increased release of NE which r e s u l t s i n i n h i b i t i o n of the LH surge. The axons of NE c e l l bodies i n the hindbrain ascend through the midbrain v i a dorsal and ventral pathways, i . e . , DNT and VNT, r e s p e c t i v e l y ( L i n d v a l l and Bjorklund, 1978; Moore and Card, 1984). I t was previously demonstrated that e l e c t r i c a l stimulation of the DNT i n h i b i t s p u l s a t i l e LH release i n a manner s i m i l a r to that of i n t r a v e n t r i c u l a r i n f u s i o n of NE, and that the e f f e c t s of e l e c t r i c a l stimulation of the DNT could be blocked by alpha-methyl -p-tyrosine but not by E-chlorophenylalanine (Leung et a l . , 1981b; Bergen and Leung, 1987b; see pp. 48-85). Thus although the s i t e s of e l e c t r i c a l stimulation i n the midbrain are close to serotoninergic neurons, the a c t i v a t i o n of which can also have i n h i b i t o r y e f f e c t s on the LH surge (Morello and T a l e i s n i k , 1985), the lack of e f f e c t of p-chlorophenylalanine should r u l e out a serotonergic mechanism, at l e a s t f o r the DNT. A noradrenergic mechanism of i n h i b i t i o n v i a alpha-receptors i s also suggested since phenoxybenzamine (an alpha-antagonist) e f f e c t i v e l y blocked the i n h i b i t o r y e f f e c t s of DNT stimulation on the LH surge. Although i t has been previously demonstrated that phenoxybenzamine administered before the onset of a steroid-induced LH surge i n h i b i t e d the surge, i t i s evident that phenoxybenzamine administered during the surge d i d not i n h i b i t the surge but rather blocked the i n h i b i t o r y e f f e c t s of e l e c t r i c a l stimulation of the DNT (Drouva et a l . , 1982). A 168 possibl e explanation for t h i s paradoxical e f f e c t of phenoxybenzamine on LH release i s that with administration of phenoxybenzamine before the surge the excitatory alpha-receptors are blocked as i s the surge. However during the r i s i n g phase of the surge there i s a r e l a t i v e decrease i n the a c t i v i t y of excitatory inputs and a r e l a t i v e increase i n the a c t i v i t y of i n h i b i t o r y inputs to the LHRH neurons and therefore phenoxybenzamine given during t h i s time w i l l tend to have more of an e f f e c t on i n h i b i t o r y receptors rather than excitatory receptors and w i l l also block the i n h i b i t o r y e f f e c t of DNT stimulation which also occurs v i a alpha-receptors. In contrast i n h i b i t i o n was s t i l l present a f t e r administration of either a beta-adrenergic or dopaminergic antagonist. A noradrenergic mechanism i s also supported by previous studies which demonstrated that s i m i l a r to e l e c t r i c a l stimulation of the DNT or VNT, i n t r a v e n t r i c u l a r i n f u s i o n of NE or adrenergic agonists (alpha- or beta-) resulted i n suppression of the s t e r o i d -induced LH surge (see pp. 109-134). Therefore i t appears that stimulation of ascending noradrenergic t r a c t s i s as e f f e c t i v e as i n t r a v e n t r i c u l a r infusion of NE (or one of i t s agonists) i n i n h i b i t i n g the steroid-induced LH surge. The r e s u l t s from the experiments with two periods of stimulation demonstrate that e l e c t r i c a l stimulation of either the DNT or VNT i s e f f e c t i v e throughout the period of r i s i n g LH l e v e l s i n suppressing LH release. This would suggest that the exci t a t o r y d r i v e to increase LH release remains s e n s i t i v e to i n h i b i t o r y inputs over the two periods of stimulation. 169 S i m i l a r l y , the neural elements stimulating LH release do not become r e a d i l y desensitized to the i n h i b i t o r y inputs, at l e a s t not over the span of two hours. This may not be p a r t i c u l a r l y s u r p r i s i n g since i n ovariectomized unprimed rats the i n h i b i t i o n of p u l s a t i l e LH release induced by NE can be sustained f o r up to 20 hours (Gallo, 1984). In experiment #2 e l e c t r i c a l stimulation of e i t h e r the DNT or the VNT had l i t t l e or no e f f e c t on LH release when applied before the onset of the LH surge. I t appeared from previous experiments (see experiment #1 t h i s chapter, and pp. 48-85) that during the ascending portion of a gonadotrophin surge, stimulation of NE pathways or infusion of NE agonists r e s u l t s i n suppression of the surge. This may be because neural elements, which are i n h i b i t o r y to LH release, become s e n s i t i z e d as the surge progresses and these i n h i b i t o r y elements provide a braking mechanism f o r the surge. I f both e x c i t a t o r y and i n h i b i t o r y f i b r e s are present i n the ascending noradrenergic pathways, stimulation during an ongoing surge may favour the expression of i n h i b i t i o n of the surge rather than stimulation. However, e l e c t r i c a l stimulation from 12.00 to 13.00 h, or electrochemical stimulation at 13.00 h of e i t h e r the DNT or VNT before the onset of the surge, did not enhance LH release, and i n f a c t the surge was i n h i b i t e d i n the electrochemically stimulated groups. Presumably before the onset of the surge the neural elements stimulatory to the LH release would be more s e n s i t i z e d than the i n h i b i t o r y elements to e l e c t r i c a l stimulation and stimulation at t h i s time would favor 170 stimulation rather than i n h i b i t i o n of LH release. Interestingly, e l e c t r i c a l stimulation of the arcuate nucleus resulted i n LH release. Therefore the stimulation parameters used i n t h i s experiment can e l i c i t LH release i n areas previously reported to be e f f e c t i v e i n stimulating LH release i n steroid-primed ovariectomized rat s (Gallo and Osland, 1976; Caceres and T a l e i s n i k , 1980). S i m i l a r l y electrochemical stimulation, with the present parameters, has a l s o been e f f e c t i v e i n stimulating f i b r e s which e f f e c t changes i n LH release. Therefore i t i s apparent that i f f i b r e s e x c i t a t o r y to LHRH release are present i n the DNT or VNT, stimulation of these bundles does not r e s u l t i n enhanced LH release, due to the simultaneous a c t i v a t i o n of i n h i b i t o r y f i b r e s which would cancel the e f f e c t s of excitatory f i b r e a c t i v a t i o n . Another possible reason why a c t i v a t i o n of these f i b r e s di d not enhance LH release i s that with stimulation periods l a s t i n g one hour, excitatory f i b r e s may r a p i d l y become re f r a c t o r y or desensitized to continuous stimulation. This p o s s i b i l i t y was tested i n experiment #3. The DNT was stimulated i n E 2-primed rats for ei t h e r one hour continuously or was stimulated f o r 5 minute periods followed by 20 minutes of no stimulation. Four periods of e l e c t r i c a l stimulation were applied over a 100 minute period. As reported i n E 2 + P 4 primed ovariectomized rat s e l e c t r i c a l stimulation had no e f f e c t on LH release. E l e c t r i c a l stimulation applied f o r 5 minutes at 20 minute i n t e r v a l s also had no consistent e f f e c t on LH release. Therefore i t appears that under the conditions 171 which would seem to maximize the a b i l i t y of excitatory f i b r e s to e l i c i t an e f f e c t on LH release, no e f f e c t was observed. I t seems that i f f i b r e s are present i n the DNT which can f a c i l i t a t e LHRH (LH) release, they are intermingled with i n h i b i t o r y f i b r e s such that i f and when the excitatory f i b r e s are activated, t h e i r e f f e c t s are cancelled by the simultaneous a c t i v a t i o n of i n h i b i t o r y f i b r e s . S i m i l a r r e s u l t s were also obtained with e l e c t r i c a l and electrochemical stimulation of the VNT. In experiment #5, i n order to t e s t whether an increase i n the amount of current used to stimulate the VNT might have a d i f f e r e n t e f f e c t on LH release, E 2-primed ovariectomized rats were anaesthetized and then stimulated. As i n the previous experiments which have attempted to induce stimulation of LH release, by e l e c t r i c a l a c t i v a t i o n of f i b r e pathways, there was l i t t l e or no change i n LH l e v e l s . The only s t a t i s t i c a l l y s i g n i f i c a n t change came with e l e c t r i c a l stimulation of the VNT i n r a t s previously treated with phenoxybenzamine. In these animals LH l e v e l s were s i g n i f i c a n t l y higher at the end of the stimulation period than at the s t a r t of stimulation. One p o s s i b i l i t y i s that with alpha-adrenergic receptor blockade by phenoxybenzamine, i n h i b i t o r y f i b r e s activated by e l e c t r i c a l stimulation may have had t h e i r e f f e c t s blocked and t h i s allowed the s l i g h t excitatory e f f e c t s (possibly v i a beta-receptors) to be expressed. I t must however be taken in t o account that the increase was r e l a t i v e l y s l i g h t compared to the decrease i n LH release which can be induced by stimulation of e i t h e r the DNT 172 or VNT. This suggests that e l e c t r i c a l stimulation of the VNT i s only s l i g h t l y e f f e c t i v e i n increasing LH release. Under most conditions employing d i f f e r e n t stimulation parameters and under d i f f e r e n t s t e r o i d a l conditions e l e c t i c a l stimulation of the VNT (or DNT) does not enhance LH release. In addition to the well established excitatory r o l e of NE i n LH release, there i s an increasing amount of evidence i m p l i c a t i n g a noradrenergic system i n h i b i t o r y to LH release ( T a l e i s n i k and Sawyer, 1986; Caceres and T a l e i s n i k , 1982). Electrochemical stimulation of the LC i n estrogen-primed ovariectomized rats i n h i b i t e d LH release induced by e l e c t r i c a l s t imulation of the MPOA (Caceres and T a l e i s n i k , 1982; Dotti and T a l e i s n i k , 1984). This i n h i b i t i o n i s probably mediated by NE, since the e f f e c t can be blocked by propranolol or i n h i b i t i o n of NE synthesis (Dotti and T a l e i s n i k , 1984). I t appears that t h i s i n h i b i t i o n i s v i a the DNT and not the VNT because transection of the DNT, but not the VNT, prevented the i n h i b i t i o n . I t has also been demonstrated that electrochemical stimulation of the f r o n t a l lobe cortex also i n h i b i t e d LH release induced by MPOA stimulation i n ovariectomized estrogen-primed r a t s (Caceres and T a l e i s n i k , 1980a, 1980b). This i n h i b i t i o n i s also blocked by l e s i o n i n g ascending pathways (Caceres and Tal e i s n i k , 1981) as well as in t r a v e n t i c u l a r i n f u s i o n of propranolol (Caceres and T a l e i s n i k , 1980b). An i n h i b i t o r y e f f e c t of NE on LH release has been demonstrated i n unprimed ovariectomized r a t s . Infusion of NE or adrenergic agonists i n the t h i r d v e n t r i c l e of ovariectomized 1 7 3 r a t s i n h i b i t e d p u l s a t i l e LH release (Gallo and Drouva, 1 9 7 9 ; Gallo, 1984). We have recently demonstrated that e l e c t r i c a l stimulation of the DNT, but not the VNT, i n h i b i t e d p u l s a t i l e LH release (Bergen and Leung, 1987b). In t h i s regard, a possible s i t e of ac t i o n of NE i s the MPOA, since perfusion of t h i s area with NE e f f e c t i v e l y suppressed p u l s a t i l e LH release i n ovariectomized r a t s (Leipheimer and Gallo, 1985). In the present study, the s i t e of i n h i b i t i o n responsible f o r suppression of the ongoing LH surge i s not known. Presumably, NE could act e i t h e r d i r e c t l y on the LHRH neurons or i n d i r e c t l y v i a i n h i b i t o r y interneurons (Barraclough and Wise, 1982; Ramirez et a l . , 1984; Leung, 1985; T a l e i s n i k and Sawyer, 1986) . Caceres and T a l e i s n i k (1982) demonstrated that premammary l e s i o n s suppressed the i n h i b i t o r y noradrenergic e f f e c t s , suggesting that the i n h i b i t o r y noradrenergic f i b r e s synapse before the premammillary bodies and send i n h i b i t o r y interneurons r o s t r a l l y to LHRH neurons i n the hypothalamus. Whether the i n h i b i t o r y f i b r e s i n the present study correspond to the i n h i b i t o r y f i b r e s described by T a l e i s n i k and co-workers i s not known. Together with our previous observation that e l e c t r i c a l stimulation of the DNT i n h i b i t s p u l s a t i l e LH release i n ovariectomized r a t s , the present r e s u l t s provide further evidence f o r an i n h i b i t o r y noradrenergic system involved i n the regulation of LH release. While e l e c t r i c a l stimulation of the DNT but not the VNT, i n h i b i t s p u l s a t i l e LH release i n the ovariectomized r a t (Bergen and Leung, 1987b), a c t i v a t i o n of 174 e i t h e r the DNT or VNT i s e f f e c t i v e i n i n h i b i t i n g an ongoing LH surge. The reason for t h i s d i f f e r e n c e i s not c e r t a i n . Perhaps during periods of r a p i d l y increasing LH release, as seen i n the present study, the LHRH/LH secretory system i s more s e n s i t i v e to i n h i b i t o r y influences. This i s suggested i n experiments i n which adrenergic agonists are infused into the t h i r d v e n t r i c l e during an ongoing LH surge (Leung et a l . , 1982b; Bergen and Leung, 1986b). V e n t r i c u l a r administration of NE, beta-agonist (isoproterenol), or alpha-agonists (phenylephrine and methoxamine) are equally e f f e c t i v e i n disrupting the P 4~induced gonadotrophin surge i n estrogen-primed ovariectomized rats (Bergen and Leung, 1986b; pp. 109-134). By contrast, i n unprimed ovariectomized r a t s , t h i r d v e n t r i c l e i n f u s i o n of alpha-adrenergic agonists i s more potent than i n f u s i o n of a beta-agonist i n suppressing p u l s a t i l e LH release (Leung et a l . , 1982b; Bergen and Leung, 1986a). As proposed previously (Leung, 1985; Bergen and Leung, 1987a; Leung et a l . , 1981b; Leung et a l . , 1982a), the s t e r o i d treatment might have alt e r e d the r a t i o of i n h i b i t o r y to excitatory receptors. I t has been demonstrated that ovarian steroids are involved i n the regulation of adrenergic receptors (Agnati, et a l . , 1981; McEwen, et a l . , 1981). A l t e r n a t i v e l y , as Gallo (1982) has already proposed, LHRH neurons (or interneurons mediating the NE signal) respond best to p u l s a t i l e NE stimulation and become desensitized to continuous NE input. I t would seem possible that e l e c t r i c a l stimulation of the DNT or VNT i n the present study, which presumably i s superimposed on an already elevated 175 NE tone, may cause a more continuous pattern of NE release and, thus, be i n h i b i t o r y to the LH surge. However, b r i e f intermittent periods of DNT stimulation had l i t t l e or no e f f e c t on LH secretion i n E 2-primed r a t s . Also, stimulation of the VNT (experiment #5) d i d not increase LH l e v e l s i n s a l i n e treated c o n t r o l s . Therefore one can say that stimulation of the DNT and VNT does a c t i v a t e f i b r e s which are i n h i b i t o r y to LH release rather than the i n h i b i t i o n being s o l e l y a r e s u l t of d e s e n s i t i z a t i o n of excitatory inputs. The notion that i n h i b i t o r y inputs to LHRH ( d i r e c t l y or i n d i r e c t l y ) are involved i n the timing of the proestrous LH surge has been suggested by Beltramino and T a l e i s n i k (1984). Before the proestrous LH surge, LH release may be suppressed v i a t h i s pathway, since transection of ascending a f f e r e n t s to the hypothalamus r e s u l t e d i n an e a r l i e r surge and electrochemical stimulation of the posterior hypothalamus blocked the proestrous surge and ovulation. I t has also been suggested that the P 4~induced LH surge may i n part be a r e s u l t of decreased i n h i b i t o r y o p i o i d tone on LHRH neurons (Gabriel et a l . , 1983). In the present study, i t i s not known whether the i n h i b i t i o n of LH release induced by a c t i v a t i o n of the DNT or VNT involves i n h i b i t o r y o p i o i d e r g i c neurons. Nevertheless, our r e s u l t s are i n accord with the notion that f i b r e s of the DNT and VNT are involved i n the proper timing of the LH surge. As w e l l , some of these i n h i b i t o r y f i b r e s might provide a turning-o f f mechanism whereby a LH surge could be terminated. The f i n a l question which must be answered i s why 176 e l e c t r i c a l stimulation of e i t h e r of the two NE pathways i s unable to stimulate LH release to any great extent, but under several conditions r e s u l t s i n i n h i b i t i o n of LH release. The c l e a r e s t example of enhanced LH release as a r e s u l t of stimulation of a NE pathway i s a report of e l e c t r i c a l stimulation of the VNT f a c i l i t a t i n g LH release i n the E 2-primed ovariectomized r a t (Dyer et a l . , 1985). The reason f o r the discrepancy between that report and the r e s u l t s reported here, i s not c l e a r . Although e l e c t r i c a l stimulation of the VNT did s i g n i f i c a n t l y increase LH release i n that report, the increase i s r e l a t i v e l y s l i g h t . The authors of that report suggest the reason f o r the r e l a t i v e l y small increase i s that anaesthetization i n t e r f e r e s with the stimulation of the LHRH neurons and they predict that l a r g e r increases would be observed i n non-anaesthetized r a t s (Dyer et a l . , 1985). The r e s u l t s obtained with stimulation of the VNT i n conscious (non-anaesthetized) rats suggests that t h i s i s not the case. The only case where LH release was enhanced, was a f t e r e l e c t r i c a l s timulation of the VNT, i n r a t s that were injected with phenoxybezamine p r i o r to the onset of e l e c t r i c a l stimulation. This increase may be a r e s u l t of a c t i v a t i o n of f i b r e s that a c t i v a t e beta-receptors that are excitatory to LH release (Al-Hamood, et a l . , 1985). A d d i t i o n a l l y , the increase i n LH that Dyer et a l . (1985) report following stimulation of the VNT may be a r e s u l t of the s l i g h t l y d i f f e r e n t steroid-priming regimen. Their regimen did not include P, and t h i s difference may be important i n determining whether excitatory f i b r e s can 177 be s u f f i c i e n t l y activated, over i n h i b i t o r y f i b r e s , to r e s u l t i n an increase i n LH secretion. In summary i t i s evident that enhancement of LH release by stimulation of ascending noradrenergic f i b r e s does not r e a d i l y occur. While stimulation of these pathways may be a c t i v a t i n g f i b r e s e x c i tatory to LHRH (LH) release, f i b r e s i n h i b i t o r y to LH release are dominant. This i s p a r t i c u l a r l y evident i n s i t u a t i o n s when LH l e v e l s are high, as i n the case of unprimed ovariectomized rats and i n r a t s which have elevated LH l e v e l s as a r e s u l t of ovarian s t e r o i d treatment ( i . e . , during the steroid-induced LH surge). Since elevated l e v e l s of LH i n the i n t a c t r a t are the exception (proestrous LH surge) rather than the r u l e and the e f f e c t of ovarian steroids i s pr i m a r i l y one of i n h i b i t i o n i t should not be s u r p r i s i n g that stimulation of f i b r e s which can influence LHRH (LH) release, do so i n a negative way rather than i n a p o s i t i v e fashion. The case may be made that i n h i b i t o r y f i b r e s play a more important r o l e i n the regulation of LH release than previously suspected. In other words, elevated LH l e v e l s may be as much a function of a decrease i n the i n h i b i t o r y tone impinging on LHRH neurons, as an increase i n the a c t i v i t y of excitatory inputs to the LHRH neurons. In ovariectomized ewes i t has recently been reported that a decrease i n the a c t i v i t y of i n h i b i t o r y neuronal a c t i v i t y preceded a pulse of LH (Martin and Thiery, 1987). Ovarian steroids undoubtedly play an important r o l e i n modulating the r e l a t i v e strengths of the i n h i b i t o r y and exc i t a t o r y f i b r e s to LHRH release. 178 General Summary The precise nature of noradrenergic inputs to LHRH neurons i s unknown nor i s t h e i r r o l e i n the regulation of LH completely understood. Since NE can be both excitatory and i n h i b i t o r y to LH release, i t seems u n l i k e l y that the same receptor population could mediate both of these e f f e c t s . This prompted the proposal that NE exerts i t s d i f f e r e n t i a l e f f e c t s v i a separate and anatomically d i s t i n c t receptor populations (Leung et a l . , 1982). As hypothesized i n F i g . 41, one group of NE receptors (A-receptors) presumably located d i r e c t l y on LHRH neurons are stimulatory to LH release and ton i c a c t i v a t i o n of these neurons would maintain p u l s a t i l e LH release. Increased a c t i v a t i o n of these receptors might also mediate f a c i l i t a t i o n of the LH surge. Other populations of A- and A-adrenergic receptors may be located on i n h i b i t o r y interneurons which would act to suppress LH release. The numbers and d i s t r i b u t i o n of excitatory and i n h i b i t o r y receptors may vary according to the hormonal m i l i e u . In p a r t i c u l a r E 2 may play an important r o l e i n e f f e c t i n g changes i n the adrenergic receptor populations. This may be one way i n which NE exerts i t s dual e f f e c t s on LH secretion. The r o l e of NE i n c o n t r o l l i n g LH release at any point i n time i s probably a function of ovarian s t e r o i d l e v e l s and previous LH l e v e l s . In s i t u a t i o n s of low st e r o i d l e v e l s , such as i n ovx or i n t a c t r a t s on diestrous, a low l e v e l of NE 179 DNT Fig. 41. Proposed mechanism f o r the dual action of NE on the regulation of hypothalamic LHRH release i n the rat. Stimulatory e f f e c t s of NE are exerted primarily v i a alpha-receptors on LHRH-containing neurons. Inhibitory e f f e c t s of NE could be mediated by alpha- and beta-receptors on putative i n h i b i t o r y interneurons. 180 release i s present which maintains LH pulses since i t i s well established that NE synthesis i n h i b i t o r s or A-adrenergic antagonists can suppress p u l s a t i l e release. However, a further increase i n NE release, or i n t r a v e n t r i c u l a r i n f u s i o n of NE, would i n h i b i t LH release, presumably v i a i n h i b i t o r y interneurons. Under conditions of high s t e r o i d a l l e v e l s such as i n steroid-primed ovx r a t s or on proestrous, there i s increased a c t i v a t i o n of adrenergic receptors which r e s u l t s i n the i n i t i a t i o n of the LH surge. Nevertheless, the data presented here suggests that continued elevation of l e v e l s of NE once again r e s u l t s i n a suppression of the elevated l e v e l s of LH even i n the presence of a high s t e r o i d a l background. This could occur through i n a c t i v a t i o n of the exc i t a t o r y receptor and/or a c t i v a t i o n of i n h i b i t o r y NE inputs to the LHRH neurons. This dynamic i n t e r p l a y of excitatory versus i n h i b i t o r y influences of NE on the neurons may be important i n the f i n e tuning of LHRH and gonadotropin release at any point i n time. The r e s u l t s presented i n t h i s report suggest that the r o l e of NE i n the neuroendocrine regulation of LH release i s one of i n h i b i t i o n and e x c i t a t i o n . I t i s evident that the i n h i b i t o r y and excitatory e f f e c t s of NE on LH release are mediated by both alpha- and beta-adrenergic receptors. The i n h i b i t o r y e f f e c t s of NE appear to be more predominant than previously thought since under most experimental conditions a c t i v a t i o n of alpha- and beta-adrenergic receptors r e s u l t e d i n an i n h i b i t i o n of LH release. 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