J Neural Transm [-GenSect] (1993) 94:43-53
m Journal o f Neural Transmission 9 Springer-Verlag 1993 Printed in Austria
Extrahypothalamic effects of melatonin administration on serotonin and norepinephrine synthesis in female Syrian hamsters N. A. M . A l e x i u k a n d J. P. Vriend
Department of Anatomy, University of Manitoba, Winnipeg, MB, Canada Accepted May 6, 1993
Summary. The effects of daily late afternoon injections of melatonin for 10 weeks on the metabolism of serotonin (5-HT) and norepinephrine (NE) were examined in regional brain extracts of intact and ovariectomized (GX) Syrian hamsters. Accumulation of 5-HT and NE after administration of the monoamine oxidase inhibitor pargyline was used as a measure of the rate of neurotransmitter synthesis - with concentrations determined by HPLC with electrochemical detection. Daytime 5-HT synthesis was significantly decreased in the amygdala of melatonin-treated hamsters that had been GX (to 50% of GX controls). No significant effect on 5-HT synthesis could be detected in the mediobasal hypothalamus (MBH), however, a significant increase was demonstrated in the pontine brain stem of both intact and GX hamsters treated with melatonin. Daytime NE synthesis was decreased to levels not significantly different from zero in the amygdala of GX hamsters treated with melatonin, while in the brain stem, melatonin reduced NE synthesis in both intact and GX animals. The present data demonstrate that these melatonin effects on 5-HT and NE metabolism are not limited to the MBH and are not secondary to melatonin-induced changes in circulating levels of the ovarian steroids. Keywords: Melatonin, serotonin, norepinephrine, amygdala, pons, mediobasal hypothalamus, hamster.
Introduction Melatonin (N-acetyl-5-methoxytryptamine), a serotonin derivative synthesized and released in response to noradrenergic stimulation of pinealocytes during darkness, has been studied primarily for its effects upon mammalian reproductive cycles (Tamarkin et al., 1976; Reiter, 1980; Reiter et al., 1981). Daily administration of melatonin late in the light period or short photoperiod exposure for several weeks, results in marked testicular involution and arrested
44
N . A . M . Alexiuk and J. P. Vriend
spermatogenesis in the male Syrian hamster. In the female similarly treated, disruption occurs in the four-day estrous cycle with suppression of ovulation (Tamarkin et al., 1976). Although melatonin's effects upon mammalian reproductive cycles is not disputed, the site(s) and mechanism by which melatonin produces its effects remains the subject of much controversy. The endocrine hypothalamus is generally considered a probable site at which melatonin produces its effects upon reproductive hormones and circadian rhythm regulation (Reiter etal., 1981). It is hypothesized that melatonin induces these changes via modulation of the metabolism of the neurotransmitters regulating hormone secretion (Alexiuk and Vriend, 1991). However, there remains a paucity of data on the effects of melatonin on brain regions outside the hypothalamus, particularly those involved in the modulation of gonadotropin secretion but lacking high-affinity binding sites (Kennaway and Hugel, 1992). The first objective of the present study was to determine whether melatonin administration influences serotonin (5-HT) and norepinephrine (NE) synthesis in various extrahypothalamic brain regions. In this experiment, we examined the effects of melatonin on 5-HT and N E metabolism in the amygdala, the pontine brain stem, and the striatum as well as in the mediobasal hypothalamus (MBH). A second objective was to determine whether the extrahypothalamic effects of melatonin depend on the presence of the ovaries. In the present study, the gonadectomized female hamster served as a model for examining the effects of melatonin administration on neurotransmitter metabolism that were not secondary to changes in circulating ovarian steroids. Materials and methods
Animals Sixty-four 9 week-old female Syrian hamsters (strain Lak; LVG, Charles River, St. Constance, Quebec) were utilized in this study. They were maintained under controlled lighting (14 L/10 D) and temperature conditions (22 + 2 ~ The daily photoperiod began at 04.00 h and ended when the lights were shut off at 18.00 h. The intensity of the light was approximately 200 footcandles at the level of the cages. The hamsters received food (Teklad rodent diet) and water ad libitum and were housed as 4 per cage.
Experimental design The hamsters were acclimatized to the laboratory conditions for one week prior to their assignment to one of the four experimental groups. Following this, 32 of the hamsters were bilaterally ovariectomized (GX) via a midline linea alba incision. An additional 32 hamsters were subjected to sham operations. Both GX and sham-operated hamsters were further divided into two groups of 16. One group received daily subcutaneous injections of 0.1 ml of physiological saline; while the other group received daily injections of 25 micrograms of melatonin (N-acetyl-5-methoxytryptamine) in 0.1 ml of saline. All injections were administered between 16.00 and 17.00 h. Following daily treatments for 10 weeks, both the saline-injected and the melatonintreated groups of hamsters were subdivided into two groups (n = 8), which were sacrificed
Extrahypothalamic effects of melatonin on 5-HT and NE
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via decapitation two hours after the subcutaneous administration of either 0.2 ml of saline or 20 mg pargyline (a monoamine oxidase inhibitor) in 0.2 ml saline. All animals were killed between 12.00 and 16.00 h. The brains were removed and immediately frozen on dry ice.
Serotonin and norepinephrine determination At the time of their dissection the brains were partially thawed. A one mm deep slice of the mediobasal hypothalamus was dissected and a circular punch (2 mm diameter) containing the median eminence and arcuate nucleus were removed. The mean frozen tissue weight was 2.76 rag. A tissue punch of the striatum (caudate nucleus) was taken from a coronal section of the female hamster b r a i n - t h e average tissue weight was 2.65 mg. A tissue punch of amygdala was also harvested from coronal brain sections with an average frozen weight (of punch) of 3.50 rag. The mean weight of the pontine brain stem was 126 mg. All tissue samples were frozen until they were processed for high performance liquid chromatography with electrochemical detection (HPLC-EC). At this time, the tissues were homogenized in 0.1 N perchloric acid containing the internal standard dihydroxybenzylamine (DHBA 10ng/ml) and centrifuged at 12,000g for 5rain. The supernatants were filtered with HPLC nylon filters (0.45 micron pore size) prior to injection into the HPLC system. The monoamines, norepinephrine (NE) and serotonin (5-HT), as well as the 5-HT metabolite, 5-hydroxyindoleacetic acid (5-HIAA), were separated and assayed by HPLCEC. The synthesis of the monoamines serotonin (5-HT) and norepinephrine (NE) was estimated as the accumulation of monoamine (per hour) after inhibition of monoamine oxidase with pargyline (Spector etal., 1963)-an irreversible, non-selective inhibitor of monoamine oxidase. The accumulation of monoamines was calculated via subtraction of the mean monoamine concentrations of hamsters not receiving pargyline from the monoamine concentrations of animals injected with pargyline two hours prior to sacrifice. The HPLC system consisted of a Beckman solvent delivery system (Model 114 M), an Altex injector (Model 210A), and a 10cm C-18 column (Chromatography Sciences Co., Canada). The electrochemical detector (ESA Model 5100A) was equipped with a high sensitivity cell (ESA Model 5011). The detector was set at an oxidation potential of + 0.35 volts. For extracts of hypothalamus, the detector was set to the redox mode (guard cell at + 0.39; detector 2 at -0.35). A Shimadzu integrator (Model C-R3A) was used to record and integrate peak areas, and to calculate the content of monoamines and metabolites. The mobile phase consisted of 60raM sodium acetate, 122nM EDTA, 762nM octane sulphonate, and 7% methanol. The mobile phase was brought to a pH of 4.25 with glacial acetic acid.
Statistical analysis All of the data was subjected to analysis of variance (ANOVA). Two-way ANOVA (Treatment • Surgery) was used to analyze the data on monoamine accumulation after pargyline administration. The data were then subjected to Student's t-tests. Statistical significance was considered as a p-value of less than 0.05. The following levels of significance were distinguished: p < 0.05; p < 0.01; p < 0.001.
Results
Serotonin metabolism D a i l y m e l a t o n i n injections decreased serotonin (5-HT) a c c u m u l a t i o n after pargyline in the a m y g d a l a o f G X h a m s t e r s , ( F = 6.66; p < 0.05) b u t n o t in the intact h a m s t e r s (Fig. 1). N o significant effects o f either m e l a t o n i n a d m i n i s t r a t i o n
46
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Fig. 1. Effects of melatonin on 5-HT accumulation in amygdala after administration of pargyline. ** p < 0.01 compared to ovariectomized controls. C controls; M melatonin; G X ovariectomy; S saline 13
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Fig. 2. Effects ofmelatonin on 5-HT accumulation in pontine brain stem after administration of pargyline. ** p < 0.01 compared to ovariectomized controls. C controls; M melatonin; G X ovariectomy; S saline
or ovariectomy on serotonin (5-HT) synthesis was demonstrated in the median eminence/arcuate region of the MBH (Controls-401 4-21pg/mg/h; Melat o n i n - 4 4 9 + 82; GX/Saline-429 -4- 60; GX/Melatonin-390 4- 33). In the pontine brain stem, melatonin administration increased the 5-HT synthesis in both intact and GX hamsters (F = 6.81; p < 0.05) (Fig. 2). In both the MBH and the striatum of hamsters not treated with pargyline (Table 1), melatonin administration increased the concentrations of the 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA), in both GX and intact anim a l s - (F = 5.22; p < 0.05 by ANOVA) for the MBH and (F = 8.95; p < 0.01 by ANOVA) for the striatum. The increase in pontine brain stem 5-HIAA after melatonin injections, however, was not significant (Table 1). No significant effects of ovariectomy were observed in these regions. In the amygdala, however,
Extrahypothalamic effects of melatonin on 5-HT and NE
47
Table 1.5-HIAA content in hypothalamic and extrahypothalamic brain regions Region Group
Pons (pg/mg)
Amygdala (pg/mg)
MBH (pg/mg)
Striatum (pg/mg)
Controls Melatonin GX/Saline GX/Melatonin
663 741 620 646
267 280 338 294
398 455 418 468
489 577 451 521
4- 56 + 32 4- 15 4- 43
4- 28 4- 20 • 09~ • 17"
4- 25 4- 13 4- 25 • 28
• 18 • 41 4- 21 4- 17"
Values are means 4- SE. * p < 0.05 compared to ovariectomized controls; ~ p < 0.05 compared to saline-injected, intact controls
Table 2. 5-HIAA/5-HT ratios in hypothalamic and extrahypothalamic brain regions Region Group
Pons 5-HIAA/5-HT Ratio x 1,000
MBH 5-HIAA/5-HT Ratio x 1,000
Striatum 5-HIAA/5-HT Ratio x 1,000
Controls Melatonin GX/Saline GX/Melatonin
409 473 391 411
356 398 492 455
898 905 862 816
+ 41 4-45 + 19 4-30
4- 20 4- 31 + 39** -4- 54
+ 67 4- 66 + 44 • 24
Values are means + SE. ** p < 0.01 compared to saline-injected, intact controls
A N O V A d e m o n s t r a t e d an increase in 5 - H I A A c o n t e n t in G X hamsters n o t treated with pargyline (F = 4.85; p < 0.05). O v a r i e c t o m y increased the ratio o f 5 - H I A A / 5 - H T in the m e d i a n e m i n e n c e / arcuate region o f the M B H o f b o t h saline a n d m e l a t o n i n - t r e a t e d hamsters (F = 6.48; p < 0.05) (Table 2). N o significant effect o f either m e l a t o n i n or ovariectomy on the 5 - H I A A / 5 - H T ratio was d e m o n s t r a t e d in either the pont• brain stem or the striatum. In the a m y g d a l a , an overall significant decrease in the ratio was detected by A N O V A (F = 5.11; p < 0.05) in b o t h intact a n d G X hamsters treated with m e l a t o n i n (Fig. 3).
Norepinephrine metabolism The a c c u m u l a t i o n o f n o r e p i n e p h r i n e (NE) after a d m i n i s t r a t i o n o f pargyline was significantly decreased by m e l a t o n i n in G X hamsters in the a m y g d a l a (F = 5.06; p < 0.05) (Fig. 4). In fact, in the a m y g d a l o i d tissue, there was no increase in the a c c u m u l a t i o n o f N E after pargyline in G X hamsters treated with melatonin. A n overall inhibitory effect o f m e l a t o n i n on the a c c u m u l a t i o n o f N E after pargyline a d m i n i s t r a t i o n was also d e m o n s t r a t e d in the pontine brain stem (F = 4.86; p < 0.05) (Fig. 5). In contrast, o v a r i e c t o m y resulted in an increase in N E a c c u m u l a t i o n (F = 5.60; p < 0.05) (Fig.5).
48
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Fig. 4. Effects of melatonin on NE accumulation in amygdala after administration of pargyline. ** p < 0.01 compared to ovariectomized controls. C controls; M melatonin; G X ovariectomy; S saline Discussion Previous data from this laboratory have shown significant effects of melatonin administration on daytime catecholamine metabolism in the mediobasal hypothalamus (MBH) of the female Syrian hamster (Alexiuk and Vriend, 1991). Substantial melatonin-induced decreases in mediobasal hypothalamic N E and in tuberoinfundibular dopamine (DA) synthesis were found, independent of changes in serum concentrations of the gonadal steroids (Alexiuk and Vriend, 1991). The present study demonstrates significant effects of melatonin on serotonergic and noradrenergic metabolism in extrahypothalamic brain regions of both intact and ovariectomized (GX) hamsters. These melatonin-induced changes occurred during the daylight hours, at a time when the endogenous
Extrahypothalamic effects of melatonin on 5-HT and NE O)
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Fig. 5. Effects of melatonin on NE accumulation in pontine brain stem after administration of pargyline. ** p < 0.01 compared to intact controls. C controls; M melatonin; G X ovariectomy; S saline synthesis and secretion of melatonin from the pineal gland is normally suppressed. Daily melatonin administration for 10 weeks was shown to decrease amygdaloid 5-HT synthesis (Fig. 1.) as demonstrated by the significant inhibitory effects of melatonin on 5-HT accumulation after pargyline in the amygdala of GX hamsters. Decreases in the 5-HIAA/5-HT ratios in the amygdala (Fig. 3) of both intact and GX hamsters suggests a reduction in the oxidative metabolism in the brains of melatonin-treated animals. Decreased oxidative metabolism is often associated with reduced transmitter turnover (Smythe et al., 1982; Kuhn et al., 1986). Even though the amygdala is well-recognized for its role in the modulation of gonadotropin secretion (Velasco and Taleisnik, 1969; Arai, 1971; Demaine, 1983; Stopa etal., 1991), to our knowledge, the present data are the first to show that melatonin may be involved in the regulation of amygdaloid neurotransmitter metabolism. The role of the amygdala in the daily surges of gonadotropins which occur in melatonin-treated hamsters (Tamarkin et al., 1976), (as well as in animals under short photoperiod) and in ovariectomized hamsters (Jorgenson and Schwartz, 1984) has not yet been established. Although no significant effects of melatonin on 5-HT synthesis were demonstrated in the MBH, melatonin administration induced significant increases in the synthesis of 5-HT in the pontine brain stem of both intact and GX animals (Fig.2). The present data suggests that there are regional differences in the effects of melatonin on daytime 5-HT synthesis. One could predict that melatonin-induced changes in brain 5-HT metabolism would also occur during the dark phase since it has been demonstrated that hamster brain 5-HT is driven by the photic cycle (Ferraro and Steger, 1990). In the present investigation, increases in the content of the 5-HT metabolite, 5-HIAA, were demonstrated in the MBH and in the striatum of both intact
50
N.A.M. Alexiuk and J. P. Vriend
and GX hamsters treated with melatonin (Table 1). In the amygdala, a significant effect of melatonin was observed only in GX hamsters - while ovariectomy significantly increased 5-HIAA content, this increase was partially reversed by melatonin (p < 0.05). These data could be interpreted as evidence that regional differences in the effects of melatonin on 5-HT metabolism may be related to differences in steroid sensitivity of various brain tissues. A recent study of Naranjo-Rodriguez et al. (1991) lends support to the view of regional differences in brain response to melatonin. These investigators reported the effects of acute melatonin administration on spontaneous multiunit activity in various brain regions of the rat. The most significant inhibitory changes were observed in the amygdala and the rostral hypothalamus (NaranjoRodriguez et al., 1991)- all doses of melatonin administered elicited a decrease in electrical activity in both of these areas. Other brain regions, especially the mesencephalic reticular formation and the caudate nucleus, were shown to have an increase in electrical activity in response to low doses of melatonin, while a decrease in response was demonstrated with higher doses. The occurrence in the present study of a melatonin-induced decrease in NE accumulation (Fig. 4) after pargyline administration in the amygdala of GX hamsters demonstrates that melatonin inhibited the synthesis of NE in this region. Inhibition of NE synthesis was also observed in the MBH of GX hamsters treated with melatonin (Alexiuk and Vriend, 1991). It is well documented that NE in the principal neurotransmitter involved in gonadotropin release (Weiner and Ganong, 1978; Meites and Sonntag, 1981). The present data is consistent with the interpretation that melatonin interferes with normal cycles of LH secretion via inhibition of NE synthesis. In the current investigation, inhibitory effects of melatonin on NE synthesis were demonstrated only in GX hamsters, therefore the detection of this effect in the intact hamsters may have been masked by the presence of the ovarian steroids and their effects on NE synthesis. Since sex steroid binding sites are concentrated in the amygdala as well as in the hypothalamus (Pfaff and Keiner, 1973; McEwen, 1976; MacLusky and McEwen, 1980), estrogen and progesterone can influence the synthesis and metabolism of neurotransmitters in these tissues (Crowley, 1982) - acting as modulators in the amygdaloid-hypothalamicpituitary circuit (Schiess etal., 1988). In intact hamsters, melatonin administration for several weeks reduces circulating levels of estradiol and results in abnormal progesterone surges (Bridges and Goldman, 1975; Jorgenson and Schwartz, 1987). In the pontine brain stem, melatonin administration inhibited NE synthesis in intact as well as in GX hamsters (Fig. 5). The ports has a less dense population of steroid receptors than other brain areas such as the amygdala and the hypothalamus; therefore melatonin-induced changes in NE synthesis in the pons appear to be less influenced by alterations in steroid concentrations than in these tissues. Since melatonin was demonstrated to have an inhibitory effect on NE synthesis in several brain regions examined, one interpretation is that
Extrahypothalamic effects of melatonin on 5-HT and NE
51
this indoleamine may be acting on brain stem noradrenergic cell bodies which distribute fibers throughout the brain. Melatonin may also be influencing the activity of adjacent brain stem raphe nuclei and be involved in the serotonergic-noradrenergic interactions occurring in this region (Clement et al., 1992). Stimulation of serotonergic raphe neurons has been shown to enhance L H secretion in rats (Waloch etal., 1981). A serotonergic-noradrenergic interaction, sensitive to melatonin, could be responsible for photoperiodic regulation of ovulation in hamsters. The question of melatonin's site(s) of action has not yet been completely resolved. Investigators have reported high-affinity binding sites in the rostral and mediobasal hypothalamus (Gitler et al., 1990; Laitinen and Saavedra, 1990), as well as in the pars tuberalis/median eminence region of the pituitary-hypothalamic complex (Vanecek etal., 1987; Weaver and Reppert, 1990; Gauer et al., 1992). Reports of extrahypothalamic melatonin binding include sites in the cingulate gyrus, the hippocampus, the amygdala, the midbrain, the pons and the frontal cortex (Pickering and Niles, 1990; Stankov etal., 1991). The relationship of melatonin binding sites to the physiological effects of melatonin on monoaminergic systems remains to be determined. References Alexiuk NA, Vriend J (1991) Effects of daily afternoon melatonin administration on monoamine accumulation in median eminence and striatum of ovariectomized hamsters receiving pargyline. Neuroendocrinology 54:55-61 Arai Y (1971) Effect of electrochemical stimulation of the amygdala on induction of ovulation in different types of persistent estrous rats and castrated male rats with an ovarian transplant. Endocrinol Jpn 18:211-214 Bridges RS, Goldman BD (1975) Diurnal rhythms in gonadotropins and progesterone in lactating and photoperiod-induced acyclic hamsters. Biol Reprod 13:617-622 Clement HW, Gemsa D, Wesemann W (1992) Serotonin-norepinephrine interactions: a voltammetric study on the effect of serotonin receptor stimulation followed in the n. raphe dorsalis and the locus ceruleus of the rat. J Neural Transm 88:11-23 Crowley WR (1982) Effects of ovarian hormones on norepinephrine and dopamine turnover in individual hypothalamic and extrahypothalamic nuclei. Neuroendocrinology 34: 381-386 Demaine C (1983) Modification of hypothalamic electrical activity by pineal indoles. In: Axelrod J, Fraschini F, Velo GP (eds) The pineal gland and its endocrine role. Plenum Press, New York London, pp417~436 Ferraro JS, Steger RW (1990) Diurnal variations in brain serotonin are driven by the photic cycle and are not circadian in nature. Brain Res 512:121-124 Gauer F, Masson-Pevet M, Pevet P (1992) Pinealectomy and constant illumination increase the density of melatonin binding sites in the pars tuberalis of rodents. Brain Res 575: 32-38 Gitler MS, Zeeberg BR, John C, Reba RC (1990) Specific in vivo binding of 125 I-iodomelatonin to melatonin receptors in rat brain. Neuropharmacology 29:603-608 Jorgenson KL, Schwartz NB (1984) Effect of steroid treatment on tonic and surge secretion of LH and FSH in the female golden hamster: effect of photoperiod. Neuroendocrinology 39:549-554 Jorgenson KL, Schwartz NB (1987) Dynamic pituitary and ovarian changes occurring
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during the anestrus to estrus transition in the golden hamster. Endocrinology 120: 3442 Kennaway DJ, Hugel HM (1992) Melatonin binding sites: are they receptors? Mol Cell Endocrinol 88:1-9 Kuhn DM, Wolf WA, Youdim MBH (1986) Serotonin neurochemistry revisited: a new look at some old axioms. Neurochem Int 8:141-154 Laitinen JT, Saavedra JM (1990) Characterization of melatonin receptors in rat suprachiasmatic nuclei: modulation of affinity with cations and guanine nucleotides. Endocrinology 126:2110-2115 MacLusky NJ, McEwen BS (1980) Progestin receptors in rat brain: distribution and properties of cytoplasmic progestin-binding sites. Endocrinology 106:192-202 McEwen BS (1976) Interactions between hormones and nerve tissue. Sci Am 235(1): 4858 Meites J, Sonntag WE (1981) Hypothalamic hypophysiotropic hormones and neurotransmitters regulation: current views. Annu Rev Pharmacol Toxicol 21:295-322 Naranj o-Rodriguez EB, Prieto-Gomez B, Reyes-Vazquez C (1991) Melatonin modifies the spontaneous multi-unit activity recorded in several brain nuclei of freely behaving rats. Brain Res Bull 27:595-600 Pfaff DW, Keiner M (1973) Atlas of estradiol-concentrating cells in the central nervous systems of the female rat. J Comp Neurol 151:121-158 Pickering DS, Niles LP (1990) Pharmacological characterization of melatonin binding sites in Syrian hamster hypothalamus. Eur J Pharmacol 175:71-77 Reiter RJ (1980) The pineal and its hormones in the control of reproduction in mammals. Endocr Rev 1:109-132 Reiter RJ, Dinh DT, del los Santos R, Guerra JC (1981) Hypothalamic cuts suggest a brain site for the antigonadotrophic action ofmelatonin in the Syrian hamster. Neurosci Lett 23:315-318 Schiess MC, Joels M, Shinnick-Gallagher P (1988) Estrogen priming affects active membrane properties of medial amygdala neurons. Brain Res 440:380-385 Smythe GA, Duncan MW, Bradshaw JE, Cai WY (1982) Serotonergic control of growth hormone secretion and hypothalamic dopamine, norepinephrine, and serotonin levels and metabolism in three hyposomatotropic rat models and in normal rats. Endocrinology 1I0:376-383 Spector S, Hirsch CW, Brodie BB (1963) Association of behavioral effects of pargyline, a non-hydrazide MAO inhibitor with increase in brain norepinephrine. Int J Neuropharmacol 2:81-93 Stankov B, Cozzi B, Lucini V, Capsoni S, Fauteck J, Fumagalli P, Fraschini F (1991) Localization and characterization of melatonin binding sites in the brain of the rabbit (Oryctolagus cuniculus) by autoradiography and in vitro ligand-receptor binding. Neurosci Lett 133:68-72 Stopa EG, Koh ET, Svendsen CN, Rogers WT, Schwaber JS, King JC (1991) Computerassisted mapping of immunoreactive mammalian gonadotropin-releasing hormone in adult human basal forebrain and amygdala. Endocrinology 128(6): 3199-3207 Tamarkin L, Westrom WK, Hamill AI, Goldman BD (t976) Effect of melatonin on the reproductive systems of male and female Syrian hamsters: a diurnal rhythm in sensitivity to melatonin. Endocrinology 99:1534-1541 Vanecek J, Pavlik A, Illnerova H (1987) Hypothalamic melatonin receptor sites revealed by autoradiography. Brain Res 435:359-363 Velasco ME, Taleisnik S (1969) Release of gonadotropins induced by amygdaloid stimulation in the rat. Endocrinology 84:132-139 Waloch M, Gilman D, Whitmoyer D, Sawyer CH (1981) The effects of stimulation and
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lesion of raphe nuclei on luteinizing hormone release in estrogen-progesterone-treated ovariectomized rats. Brain Res 217:305-313 Weaver DR, Reppert SM (1990) Melatonin receptors are present in the ferret pars tuberalis and pars distalis, but not in brain. Endocrinology 127:2607 2609 Weiner RI, Ganong WF (1978) Role of brain monoamines and histamine in regulation of anterior pituitary secretion. Physiol Rev 58:905-976 Authors' address: N. Alexiuk, Department of Anatomy, University of Manitoba, 730 William Avenue, Winnipeg, Manitoba, Canada R3E OW3. Received August 11, 1992