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Melatonin inhibits metabolic activity in the rat suprachiasmatic nuclei
Neuroscience Letters, 81 (1987) 29 34
29
Elsevier Scientific Publishers Ireland Ltd. NSL 04852
Melatonin inhibits metabolic activity in the rat suprachiasmatic nuclei Vincent M. Cassone I, Michael tt. Roberts l, Robert Y. M o o r e 1'2 Departments a[ JNeurology and 2Neurobiology and Behavior, State University o]'New York. Stony Brook, N Y 11794-8121 (U.S.A.)
(Received 23 April 1987, Revised version received 15 June 1987; Accepted 16 June 1987) Key words." Circadian rhythm; Melatonin; Pineal gland; Suprachiasmatic nucleus
The pineal hormone melatonin has been implicated in the regulation of circadian rhythms and in photoperiodic control of reproduction. The effects of melatonin require the hypothalamic suprachiasmatic nucleus (SCN), a principal pacemaker controlling circadian rhythms. To determine whether SCN activity was directly affected by exogenous melatonin, rats received either melatonin or saline injections 15 min before administration of 2-deoxy-ll-14C]glucose (2-I)G) at two times of day, circadian time (CT) 10 and CT14, and the brains of these rats were processed for autoradiographic determination of 2-DG uptake within the SCN. We report that SCN 2-DG uptake was inhibited by melatonin at CT10. when 2-DG uptake is normally high, and unaffected at CT14, when 2-DG uptake is normally low. This indicates that the SCN may be neural substrates through which melatonin exerts at least some of its effects on mammalian physiology.
C i r c a d i a n r h y t h m s in v e r t e b r a t e s are g e n e r a t e d a n d c o n t r o l l e d by central n e r v o u s system p a c e m a k e r s . In reptiles a n d birds, the pineal g l a n d a p p e a r s to be a principal c i r c a d i a n p a c e m a k e r [5, 21] w h e r e a s in m a m m a l s this function is m e d i a t e d p r i m a r i l y by the h y p o t h a l a m i c s u p r a c h i a s m a t i c nuclei ( S C N ) [10, 17]. The m a m m a l i a n S C N generate c i r c a d i a n r h y t h m s in several functions, including the pineal g l a n d ' s synthesis o f the h o r m o n e m e l a t o n i n [2, 10, 22]. P i n e a l e c t o m y has little effect on m a m m a l i a n circadian r h y t h m s [12, 15], a l t h o u g h several lines o f evidence indicate that the pineal gland, t h r o u g h its daily secretion o f the h o r m o n e m e l a t o n i n , m a y m o d u l a t e the m a m malian c i r c a d i a n system [1]. A m o n g these lines o f evidence is the o b s e r v a t i o n that daily injections o f m e l a t o n i n e n t r a i n the c i r c a d i a n l o c o m o t o r r h y t h m s o f rats held in c o n s t a n t d a r k n e s s ( D D ) [8, 13] a n d s y n c h r o n i z e the d i s r u p t e d activity p a t t e r n s o f rats held in c o n s t a n t light (LL), m a i n t a i n i n g t h e m in similar p h a s e relations as in rats held in D D [6, 9]. These effects o f m e l a t o n i n can be b l o c k e d by S C N a b l a t i o n [6. 7]. This suggests that the sites o f a c t i o n for the e n t r a i n i n g effects o f m e l a t o n i n are at the level o f the S C N or at a level o f o r g a n i z a t i o n which otherwise requires them. Correspondence: V.M. Cassone, Department of Neurology, State University of New York, Stony Brook,
NY 11794-8121, U.S.A. 0304-3940.,87'$ 03.50 © 1987 Elsevier Scientific Publishers Ireland Ltd.
30
We investigated whether rat SCN metabolic activity is directly affected by melatonin using the 2-deoxyglucose (2-DG) method for autoradiographically determining glucose utilization [19]. Using the 2-DG method, Schwartz et al. have shown a circadian rhythm in glucose utilization in the rat SCN such that 2-DG uptake, and hencc local glucose utilization, is high during the day and low during the night [18]. In ore' study, effects of melatonin administration were tested at two circadian phases in intact and blind rats: circadian time 10 (CTI0), 2 h before the beginning of subjective night, when 2-DG uptake is relatively high, and CTI4, 2 h after the beginning of subjective night, when 2-DG uptake is relatively low. These two circadian phases were chosen because daily melatonin injection at CT10 affects the circadian phase of locomotion and drinking in rats [1] and reproductive activity in several species of seasonally breeding rodents [14, 20] but has no effect at CTI4. Blind rats were compared to intact animals because several investigators have noted that melatonin's behavioral and reproductive effects may at least in part depend upon documented actions of melatonin in the retinae [4, 16, 21], which are known to directly innervate the SCN via the retinohypothalamic tract [10]. Adult male Long-Evans rats ( n - 2 4 ; 210-225 g) were maintained in a light:dark regime of LD 12:12 h with food and water continuously available. On the day before the experimental procedures described below, 12 rats were anesthetized with vapors of methoxyfluorane (Metofane) and blinded by bilateral enucleation. Intact and blind rats were then separated into 8 experimental and control groups (n = 3/group; Table I). At CTI0, 2 groups of intact rats received either subcutaneous injections of 1 mg/kg melatonin in 1% ethanol and 0.9% saline, a dosage known to affect rat circadian rhythms [1], or ethanol-saline vehicle only. After 15 min, all rats received intramuscular injections of 150 ktCi/kg 2-deoxy-[i-lac]glucose (Pathfinder Labs., St. Louis, MO) and were allowed to survive 45 min until sacrifice. Rats were anesthetized again with Metofane and killed by decapitation, their brains were rapidly removed and frozen on dry ice. This procedure was repeated at CT14 in an additional two groups of intact rats. In order to control for direct effects of light on the visual elements of the SCN, mentioned above, these procedures were repeated in the blind rats. TABLE 1 U P T A K E OF 2 - D E O X Y G L U C O S E (IN pM/g/h ± S.D.) IN T H E H Y P O T H A L A M I C S U P R A C H I A S M A T I C N U C L E I OF R A T S R E C E I V I N G E I T H E R S A L I N E V E H I C L E O R 1 mg/kg M E L A T O N I N AT CTI0 OR C T I 4 *P<0.01 compared to rats receiving melatonin at any time and rats receiving saline at CTI4. Time
CT10 CT 10 CT14 CTI4
Animal
intact blind intact blind
2-DG uptake Saline
Melatonin
395.0_-+98.7* 386.3 ± 43.1 * 293.8 ± 5.7 289.6_+ 7.6
290.1 +21.5 279.5 ± 18.0 288.4 +__24.6 271.4+ 17.8
31
Brains from rats in all experimental and control groups were sectioned at 20/~m through the rostrocaudal extent of the SCN, and, after they were dried on 'subbed' slides, sections along with calibrated C ~4 standards (Amersham Chemicals) were exposed to Kodak OM-I X-ray film in complete darkness for 30 days. The X-ray
MELATONIN
SALINE
i~,
B
D Fig. 1. Autoradiography and accompanying histological sections of suprachiasmatic nuclei (SCN) from rats receiving either melatonin or saline vehicle at CTI0, 2 h before the onset of subjective night• At this time, 1 mg/kg melatonin inhibits 2-DG uptake• A: cresyl violet-stained histological sections of SCN from intact rats sacrificed at CTI0. B: autoradiographs of SCN depicted in A from rats sacrificed at CT10. C: histological sections from blind rats sacrificed at CTI0. D: autoradiographs of SCN depicted in C.
32 film was developed with K o d a k X-ray developer, fixed and dried. Sections were then stained with Cresyl violet and localization of the SCN and the lateral hypothalamic area (LHA) was determined histologically and compared with autoradiograms. Autoradiograms were analyzed densitometrically through a trinocular microscope, Transmittances of the bilateral SCN and L H A were recorded at identical rostrocaudal levels within the hypothatamus of all rats and subtracted from blank values obtained from surrounding exposed film. These numbers were regressed against a standard curve derived from A m e r s h a m C 14 standards to obtain values to be expressed as nCi/g; these were divided by the Pathfinder Labs. specific activity values to obtain p M 2-DG/g/h. Experimental groups were compared by one-way A N O V A and N e w m a n - K e u l s test for significance. The results of this experiment are summarized in Table I and in Figs. 1 and 2. As expected, 2 - D G uptake was high at C T I 0 (Table I, Fig. 1) and low at CT14 (Table I, Fig. 2) in intact and blind rats received control injections of ethanol-saline vehicle. In both intact and blind rats receiving melatonin at CT10, 2 - D G uptake in the SCN was inhibited (Fig. I B, D) to levels indistinguishable from levels obtained at CT14 (Fig. 2B). No effect of melatonin could be determined at CT14 in either intact or in blind rats (Fig. 2; Table I). No day-night difference or effect of melatonin on 2-DG uptake could be determined in the LHA. These data are the first indication that the rat SCN, a central component in m a m malian circadian organization [10, 16, 17], is directly affected by pharmacological doses of melatonin and, since no effect could be observed at CTI4, that this sensi-
SALINE
MELATONIN
A
B Fig. 2. Autoradiography and accompanying histological sections of suprachiasmatic nuclei (SCN) from rats receivingeither melatonin or saline vehicle at CT14. No effect of 1 mg/kg melatonin could be determined in the already low levels of 2-DG uptake in either intact to blind rats. A: cresyl-violet-stainedhistological sections of SCN from blind rats sacrificed at CT 14. B: autoradiographs of SCN depicted in A.
33
tivity to the hormone is variable depending upon the time of day. This observation is consistent with the hypothesis that melatonin's entraining effects may be due to melatonin's modulation of SCN activity. The fact that several investigators have identified melatonin binding sites within the SCN [4, 11] strongly supports the hypothesis that the SCN are primary sites of action for melatonin rather than sites which are secondarily inhibited by other melatonin sensitive moieties. Since these data were not obtained in a photoperiodic species of rodent, they do not directly address the question whether melatonin affects photoperiodic control of reproduction through modulation of the SCN [16, 20]. The molecular mechanisms for melatonin's action within the SCN are still unknown. Melatonin has been shown to affect several features of neurotransmitter release, uptake and axonal transport [3, 20]. However, there is little evidence that perturbation of any identified neurotransmitter system is effective in blocking the action of melatonin. Rather, only ablation of the SCN blocks the effect of melatonin or, by extension, the pineal gland's behavioral, physiological and reproductive function. The effects of pharmacological doses of melatonin described here on the SCN may suggest a physiological role for pineal melatonin in the control of circadian rhythms. The experimental procedures outlined in this paper and the effects we describe will be useful for the further elucidation of melatonin's mechanism of action. This research was supported by NSF Grant BNS 8519660 to V.M.C. and NIH Grant NS-16304 to R.Y.M. I Armstrong, S.M., Cassone, V.M., Chesworth, M.J., Redman, J.R., Short, R.V., Synchronization of rat circadian rhythms by melatonin, J. Neural. Transm., Suppl. 21 (1986) 375 394. 2 Axelrod, J., The pineal gland: a neurochemical transducer, Science, 184 (1974) 1341 1348. 3 Cardinali, D.P., Melatonin. A pineal hormone, Endocr. Rev., 2 (1981) 327 346. 4 Cardinali, D.P., Vacas, M.I. and Boyer, E.E., Specific binding of melatonin in bovine brain, Endocrinology, 105 (1979) 437~,41. 5 Cassone, V.M. and Menaker, M., Is the avian circadian system a neuroendocrine loop?, J. Exp. Zool., 232 (1984) 539 549. 6 Cassone, V.M., Chesworth, M.J. and Armstrong, S.M., Entrainment of rat circadian rhythms by daily injection of melatonin: synchronization in constant light and dependence upon the suprachiasmatic nuclei, Soc. Neurosci., Abstr., 11 (1985) 1140. 7 Cassone, V.M., Chesworth, M.J. and Armstrong, S.M., Entrainment of rat circadian rhythms by daily injection of melatonin depends upon the hypothalamic suprachiasmatic nuclei, Physiol. Behav., 36 (1986)[111 1121. 8 Cassone, V.M., Chesworth, M.J. and Armstrong, S.M., Dose-dependent entrainment of rat circadian rhythms by daily injection of melatonin, J. Biol. Rhythms, 1 (1986) 219 229. 9 Chesworth, M.J., Cassone, V.M. and Armstrong, S.M., Effects of daily melatonin injections on activity rhythms ot" rats in constant light, Am. J. Physiol. in press. 10 Moore, R.Y., Central nervous control of circadian rhythms. In W.F. Ganong and C. Martinin (Eds.), Frontiers in Neuroendocrinology, Raven, New York, 1978, pp. 185 206. I 1 Niles, L.P., Wong, Y.W., Mishra, R.K. and Brown, G.M., Melatonin receptors in the brain, Eur. J. Pharmacol., 55 (1979) 219-220. 12 Quay, W., lndividuation and lack of pineal effect in the rat's circadian locomotor rhythm, Physiol. Behav., 3 (1968) 109 118. 13 Redman, J., Armstrong, S. and Ng, K.T., Free-running activity rhythms in the rat: entrainment by
34 melatonin, Science, 219 (1983) 1089 1091. 14 Reiter, R.J., Neuroendocrine effects of the pineal gland and melatonin, Front. Neuroendocrinol., 7 (1982) 287 316. 15 Richter, C.P., Sleep and activity: their relation to the 24-hour clock. Proc. Assoc. Res. Nerv. Menl. Dis., 45 (1967) 8 29. 16 Rusak, B., Physiological models of the rodent circadian system. In J. Aschoff, S. Daan and G.A. Groos (Eds.), Vertebrate Circadian Systems, Springer, Berlin, 1982, pp. 62 74. 17 Rusak, B. and Zucker, I., Neural regulation of circadian rhythms, Physiol. Rev., 59 (1979) 449-526. 18 Schwartz, W.J., Davidson, L.C. and Smith, C.B., In vivo metabolic activity of a putative circadian oscillator, the rat suprachiasmatic nucleus, J. Comp. Neurol., 189 (1980) 157 165. 19 Sokoloff, L., Metabolic Probes o1 Central Nervous Activity in Experimental Animals and Man. Sinauer, Sunderland, MA, 1984. 20 Tamarkin, L., Baird, C.J. and Almeida, O F . X , Melatonin: a coordinating signal tbr mammalian reproduclion?, Science, 227 (1985) 714 720. 21 Underwood, H., The pineal and circadian rhythms. In R.J. Reiter (Ed.), The Pineal Gland, Raven, New York, 1984, pp. 227 25[. 22 Wurtman, R.J., Axelrod, J. and Kelley, D.E., The Pineal, Academic, New York, 1968.
29
Elsevier Scientific Publishers Ireland Ltd. NSL 04852
Melatonin inhibits metabolic activity in the rat suprachiasmatic nuclei Vincent M. Cassone I, Michael tt. Roberts l, Robert Y. M o o r e 1'2 Departments a[ JNeurology and 2Neurobiology and Behavior, State University o]'New York. Stony Brook, N Y 11794-8121 (U.S.A.)
(Received 23 April 1987, Revised version received 15 June 1987; Accepted 16 June 1987) Key words." Circadian rhythm; Melatonin; Pineal gland; Suprachiasmatic nucleus
The pineal hormone melatonin has been implicated in the regulation of circadian rhythms and in photoperiodic control of reproduction. The effects of melatonin require the hypothalamic suprachiasmatic nucleus (SCN), a principal pacemaker controlling circadian rhythms. To determine whether SCN activity was directly affected by exogenous melatonin, rats received either melatonin or saline injections 15 min before administration of 2-deoxy-ll-14C]glucose (2-I)G) at two times of day, circadian time (CT) 10 and CT14, and the brains of these rats were processed for autoradiographic determination of 2-DG uptake within the SCN. We report that SCN 2-DG uptake was inhibited by melatonin at CT10. when 2-DG uptake is normally high, and unaffected at CT14, when 2-DG uptake is normally low. This indicates that the SCN may be neural substrates through which melatonin exerts at least some of its effects on mammalian physiology.
C i r c a d i a n r h y t h m s in v e r t e b r a t e s are g e n e r a t e d a n d c o n t r o l l e d by central n e r v o u s system p a c e m a k e r s . In reptiles a n d birds, the pineal g l a n d a p p e a r s to be a principal c i r c a d i a n p a c e m a k e r [5, 21] w h e r e a s in m a m m a l s this function is m e d i a t e d p r i m a r i l y by the h y p o t h a l a m i c s u p r a c h i a s m a t i c nuclei ( S C N ) [10, 17]. The m a m m a l i a n S C N generate c i r c a d i a n r h y t h m s in several functions, including the pineal g l a n d ' s synthesis o f the h o r m o n e m e l a t o n i n [2, 10, 22]. P i n e a l e c t o m y has little effect on m a m m a l i a n circadian r h y t h m s [12, 15], a l t h o u g h several lines o f evidence indicate that the pineal gland, t h r o u g h its daily secretion o f the h o r m o n e m e l a t o n i n , m a y m o d u l a t e the m a m malian c i r c a d i a n system [1]. A m o n g these lines o f evidence is the o b s e r v a t i o n that daily injections o f m e l a t o n i n e n t r a i n the c i r c a d i a n l o c o m o t o r r h y t h m s o f rats held in c o n s t a n t d a r k n e s s ( D D ) [8, 13] a n d s y n c h r o n i z e the d i s r u p t e d activity p a t t e r n s o f rats held in c o n s t a n t light (LL), m a i n t a i n i n g t h e m in similar p h a s e relations as in rats held in D D [6, 9]. These effects o f m e l a t o n i n can be b l o c k e d by S C N a b l a t i o n [6. 7]. This suggests that the sites o f a c t i o n for the e n t r a i n i n g effects o f m e l a t o n i n are at the level o f the S C N or at a level o f o r g a n i z a t i o n which otherwise requires them. Correspondence: V.M. Cassone, Department of Neurology, State University of New York, Stony Brook,
NY 11794-8121, U.S.A. 0304-3940.,87'$ 03.50 © 1987 Elsevier Scientific Publishers Ireland Ltd.
30
We investigated whether rat SCN metabolic activity is directly affected by melatonin using the 2-deoxyglucose (2-DG) method for autoradiographically determining glucose utilization [19]. Using the 2-DG method, Schwartz et al. have shown a circadian rhythm in glucose utilization in the rat SCN such that 2-DG uptake, and hencc local glucose utilization, is high during the day and low during the night [18]. In ore' study, effects of melatonin administration were tested at two circadian phases in intact and blind rats: circadian time 10 (CTI0), 2 h before the beginning of subjective night, when 2-DG uptake is relatively high, and CTI4, 2 h after the beginning of subjective night, when 2-DG uptake is relatively low. These two circadian phases were chosen because daily melatonin injection at CT10 affects the circadian phase of locomotion and drinking in rats [1] and reproductive activity in several species of seasonally breeding rodents [14, 20] but has no effect at CTI4. Blind rats were compared to intact animals because several investigators have noted that melatonin's behavioral and reproductive effects may at least in part depend upon documented actions of melatonin in the retinae [4, 16, 21], which are known to directly innervate the SCN via the retinohypothalamic tract [10]. Adult male Long-Evans rats ( n - 2 4 ; 210-225 g) were maintained in a light:dark regime of LD 12:12 h with food and water continuously available. On the day before the experimental procedures described below, 12 rats were anesthetized with vapors of methoxyfluorane (Metofane) and blinded by bilateral enucleation. Intact and blind rats were then separated into 8 experimental and control groups (n = 3/group; Table I). At CTI0, 2 groups of intact rats received either subcutaneous injections of 1 mg/kg melatonin in 1% ethanol and 0.9% saline, a dosage known to affect rat circadian rhythms [1], or ethanol-saline vehicle only. After 15 min, all rats received intramuscular injections of 150 ktCi/kg 2-deoxy-[i-lac]glucose (Pathfinder Labs., St. Louis, MO) and were allowed to survive 45 min until sacrifice. Rats were anesthetized again with Metofane and killed by decapitation, their brains were rapidly removed and frozen on dry ice. This procedure was repeated at CT14 in an additional two groups of intact rats. In order to control for direct effects of light on the visual elements of the SCN, mentioned above, these procedures were repeated in the blind rats. TABLE 1 U P T A K E OF 2 - D E O X Y G L U C O S E (IN pM/g/h ± S.D.) IN T H E H Y P O T H A L A M I C S U P R A C H I A S M A T I C N U C L E I OF R A T S R E C E I V I N G E I T H E R S A L I N E V E H I C L E O R 1 mg/kg M E L A T O N I N AT CTI0 OR C T I 4 *P<0.01 compared to rats receiving melatonin at any time and rats receiving saline at CTI4. Time
CT10 CT 10 CT14 CTI4
Animal
intact blind intact blind
2-DG uptake Saline
Melatonin
395.0_-+98.7* 386.3 ± 43.1 * 293.8 ± 5.7 289.6_+ 7.6
290.1 +21.5 279.5 ± 18.0 288.4 +__24.6 271.4+ 17.8
31
Brains from rats in all experimental and control groups were sectioned at 20/~m through the rostrocaudal extent of the SCN, and, after they were dried on 'subbed' slides, sections along with calibrated C ~4 standards (Amersham Chemicals) were exposed to Kodak OM-I X-ray film in complete darkness for 30 days. The X-ray
MELATONIN
SALINE
i~,
B
D Fig. 1. Autoradiography and accompanying histological sections of suprachiasmatic nuclei (SCN) from rats receiving either melatonin or saline vehicle at CTI0, 2 h before the onset of subjective night• At this time, 1 mg/kg melatonin inhibits 2-DG uptake• A: cresyl violet-stained histological sections of SCN from intact rats sacrificed at CTI0. B: autoradiographs of SCN depicted in A from rats sacrificed at CT10. C: histological sections from blind rats sacrificed at CTI0. D: autoradiographs of SCN depicted in C.
32 film was developed with K o d a k X-ray developer, fixed and dried. Sections were then stained with Cresyl violet and localization of the SCN and the lateral hypothalamic area (LHA) was determined histologically and compared with autoradiograms. Autoradiograms were analyzed densitometrically through a trinocular microscope, Transmittances of the bilateral SCN and L H A were recorded at identical rostrocaudal levels within the hypothatamus of all rats and subtracted from blank values obtained from surrounding exposed film. These numbers were regressed against a standard curve derived from A m e r s h a m C 14 standards to obtain values to be expressed as nCi/g; these were divided by the Pathfinder Labs. specific activity values to obtain p M 2-DG/g/h. Experimental groups were compared by one-way A N O V A and N e w m a n - K e u l s test for significance. The results of this experiment are summarized in Table I and in Figs. 1 and 2. As expected, 2 - D G uptake was high at C T I 0 (Table I, Fig. 1) and low at CT14 (Table I, Fig. 2) in intact and blind rats received control injections of ethanol-saline vehicle. In both intact and blind rats receiving melatonin at CT10, 2 - D G uptake in the SCN was inhibited (Fig. I B, D) to levels indistinguishable from levels obtained at CT14 (Fig. 2B). No effect of melatonin could be determined at CT14 in either intact or in blind rats (Fig. 2; Table I). No day-night difference or effect of melatonin on 2-DG uptake could be determined in the LHA. These data are the first indication that the rat SCN, a central component in m a m malian circadian organization [10, 16, 17], is directly affected by pharmacological doses of melatonin and, since no effect could be observed at CTI4, that this sensi-
SALINE
MELATONIN
A
B Fig. 2. Autoradiography and accompanying histological sections of suprachiasmatic nuclei (SCN) from rats receivingeither melatonin or saline vehicle at CT14. No effect of 1 mg/kg melatonin could be determined in the already low levels of 2-DG uptake in either intact to blind rats. A: cresyl-violet-stainedhistological sections of SCN from blind rats sacrificed at CT 14. B: autoradiographs of SCN depicted in A.
33
tivity to the hormone is variable depending upon the time of day. This observation is consistent with the hypothesis that melatonin's entraining effects may be due to melatonin's modulation of SCN activity. The fact that several investigators have identified melatonin binding sites within the SCN [4, 11] strongly supports the hypothesis that the SCN are primary sites of action for melatonin rather than sites which are secondarily inhibited by other melatonin sensitive moieties. Since these data were not obtained in a photoperiodic species of rodent, they do not directly address the question whether melatonin affects photoperiodic control of reproduction through modulation of the SCN [16, 20]. The molecular mechanisms for melatonin's action within the SCN are still unknown. Melatonin has been shown to affect several features of neurotransmitter release, uptake and axonal transport [3, 20]. However, there is little evidence that perturbation of any identified neurotransmitter system is effective in blocking the action of melatonin. Rather, only ablation of the SCN blocks the effect of melatonin or, by extension, the pineal gland's behavioral, physiological and reproductive function. The effects of pharmacological doses of melatonin described here on the SCN may suggest a physiological role for pineal melatonin in the control of circadian rhythms. The experimental procedures outlined in this paper and the effects we describe will be useful for the further elucidation of melatonin's mechanism of action. This research was supported by NSF Grant BNS 8519660 to V.M.C. and NIH Grant NS-16304 to R.Y.M. I Armstrong, S.M., Cassone, V.M., Chesworth, M.J., Redman, J.R., Short, R.V., Synchronization of rat circadian rhythms by melatonin, J. Neural. Transm., Suppl. 21 (1986) 375 394. 2 Axelrod, J., The pineal gland: a neurochemical transducer, Science, 184 (1974) 1341 1348. 3 Cardinali, D.P., Melatonin. A pineal hormone, Endocr. Rev., 2 (1981) 327 346. 4 Cardinali, D.P., Vacas, M.I. and Boyer, E.E., Specific binding of melatonin in bovine brain, Endocrinology, 105 (1979) 437~,41. 5 Cassone, V.M. and Menaker, M., Is the avian circadian system a neuroendocrine loop?, J. Exp. Zool., 232 (1984) 539 549. 6 Cassone, V.M., Chesworth, M.J. and Armstrong, S.M., Entrainment of rat circadian rhythms by daily injection of melatonin: synchronization in constant light and dependence upon the suprachiasmatic nuclei, Soc. Neurosci., Abstr., 11 (1985) 1140. 7 Cassone, V.M., Chesworth, M.J. and Armstrong, S.M., Entrainment of rat circadian rhythms by daily injection of melatonin depends upon the hypothalamic suprachiasmatic nuclei, Physiol. Behav., 36 (1986)[111 1121. 8 Cassone, V.M., Chesworth, M.J. and Armstrong, S.M., Dose-dependent entrainment of rat circadian rhythms by daily injection of melatonin, J. Biol. Rhythms, 1 (1986) 219 229. 9 Chesworth, M.J., Cassone, V.M. and Armstrong, S.M., Effects of daily melatonin injections on activity rhythms ot" rats in constant light, Am. J. Physiol. in press. 10 Moore, R.Y., Central nervous control of circadian rhythms. In W.F. Ganong and C. Martinin (Eds.), Frontiers in Neuroendocrinology, Raven, New York, 1978, pp. 185 206. I 1 Niles, L.P., Wong, Y.W., Mishra, R.K. and Brown, G.M., Melatonin receptors in the brain, Eur. J. Pharmacol., 55 (1979) 219-220. 12 Quay, W., lndividuation and lack of pineal effect in the rat's circadian locomotor rhythm, Physiol. Behav., 3 (1968) 109 118. 13 Redman, J., Armstrong, S. and Ng, K.T., Free-running activity rhythms in the rat: entrainment by
34 melatonin, Science, 219 (1983) 1089 1091. 14 Reiter, R.J., Neuroendocrine effects of the pineal gland and melatonin, Front. Neuroendocrinol., 7 (1982) 287 316. 15 Richter, C.P., Sleep and activity: their relation to the 24-hour clock. Proc. Assoc. Res. Nerv. Menl. Dis., 45 (1967) 8 29. 16 Rusak, B., Physiological models of the rodent circadian system. In J. Aschoff, S. Daan and G.A. Groos (Eds.), Vertebrate Circadian Systems, Springer, Berlin, 1982, pp. 62 74. 17 Rusak, B. and Zucker, I., Neural regulation of circadian rhythms, Physiol. Rev., 59 (1979) 449-526. 18 Schwartz, W.J., Davidson, L.C. and Smith, C.B., In vivo metabolic activity of a putative circadian oscillator, the rat suprachiasmatic nucleus, J. Comp. Neurol., 189 (1980) 157 165. 19 Sokoloff, L., Metabolic Probes o1 Central Nervous Activity in Experimental Animals and Man. Sinauer, Sunderland, MA, 1984. 20 Tamarkin, L., Baird, C.J. and Almeida, O F . X , Melatonin: a coordinating signal tbr mammalian reproduclion?, Science, 227 (1985) 714 720. 21 Underwood, H., The pineal and circadian rhythms. In R.J. Reiter (Ed.), The Pineal Gland, Raven, New York, 1984, pp. 227 25[. 22 Wurtman, R.J., Axelrod, J. and Kelley, D.E., The Pineal, Academic, New York, 1968.