Melatonin Receptors as Therapeutic Targets in Primates

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The overall goal of our research is to understand the mechanism(s) by which melatonin regulates neuroendocrine, circadian, visual, and cardiovascular functions and to apply this knowledge to the design of new therapeutic strategies. The goal of this study is to determine the distribution of melatonin receptors [MT1 (formerly Mel1a), MT2 (formerly Mel1b)] in the circadian and limbic system of a diurnal non-human primate. Furthermore, we will test the hypothesis that melatonin promotes sleep independently of time of day and induces phase shifts of circadian rhythms of activity, sleep and temperature through activation of the MT1 and MT2 melatonin receptors, respectively. The experiments described here will be conducted in the diurnal non-human Primate, Macaca mulata which is a well established model for studies of sleep and circadian rhythms and is sensitive to the sleep promoting effects of melatonin. The effects of melatonin on sleep parameters, and circadian phase of motor activity and core body temperature will be assessed by actigraphy and radiotelemetry. We will first determine the distribution, affinity, density and mRNA expression of MT1 and MT2 melatonin receptors in the suprachiasmatic nucleus and areas of the limbic system by quantitative receptor autoradiography with the radioligands 2-[125I] iodomelatonin (MT1/MT2) and 3H-4P-PDOT (MT2). The mRNA expression will be determined by in situ hybridization using digoxigenin and 33P-UTP label oligoprobes. We will then assess the periods of circadian sensitivity for melatonin to promote sleep and to phase shift circadian rhythms (CT2, CT6, CT10) of motor activity and core body temperature. Using, the specific but non-selective melatonin receptor antagonist luzindole to block melatonin receptors, and the beta- adrenoceptor antagonist propranolol to inhibit pineal melatonin production, we will assess whether endogenous nocturnal melatonin promotes sleep by melatonin receptor activation. Finally, using the non selective (MT1/MT2) melatonin receptor antagonist luzindole and the selective MT2 antagonist, 4P-PDOT we will assess whether exogenous melatonin mediates the sleep promoting effect at CT 6 and CT 10 by activation of the MT1 receptor and the phase advance at CT 10 by activation of the MT2 melatonin receptor. Results from these studies will provide, for the first time, proof of the principle that activation of distinct melatonin receptors can regulate sleep and phase shift of circadian rhythms in a diurnal species showing physiological and behavioral responses similar to man. Such knowledge would stimulate the design of novel therapeutic strategies for the treatment of insomnia, circadian disorders of sleep and mood and the discovery of receptor-selective and therapeutically effective melatonin receptor analogues.