Our long term goal is to understand the role of the hormone melatonin and its receptors in coordinating behavioral and physiological responses to daily and seasonal changes in the environment. Here, we propose to use functional, immunohistochemical and molecular tools, in order: I) To determine the subtype and location of neuronal putative melatonin receptor subtype (ML1) in the central nervous system. The retina and brain visual system will be used as models to determine the location and subtype of melatonin receptor(s) using a) functional responses (i.e., modulation of the dopamine release from retina and glutamate from optic tectum and ectostriatum, b) immunohistochemical localization of melatonin receptor subtype immunoreactivity in the retina and brain, and colocalization with D1 dopamine receptors in single retinal ganglion cells, and c) molecular approaches to amplify melatonin receptor subtypes in the tectofugal pathway by RT-PCR and coexpression of melatonin and D1 dopamine receptors in single retinal ganglion cells; II) To determine the effect of physiological activation of human recombinant ML1A and ML1B melatonin receptors on signalling and gene expression. This hypothesis will be tested in CHO cells expressing human recombinant ML1A and ML1B melatonin receptors and in neuronal cultures of embryonic retinas. Cells will be exposed to physiological concentrations of melatonin for a length of time that mimic either long (16/8 L/D) or short (8/16 L/D) photoperiods. At different times after melatonin withdrawal we will measure a) expression of melatonin receptor protein and mRNA, b) supersensitization of cAMP-mediated signalling (i.e., foreskolin stimulated cAMP formation, protein kinase A activity, phosphorylation of the cAMP response element binding protein), and c) regulation of CRE-mediated gene expression using the luciferase reporter gene system; III) To determine the role of light in regulating melatonin receptor expression we will measure receptor density by quantitative autoradiography with 2-[125I]- iodomelatonin, and mRNA expression by in situ hybridization histochemistry in brain visual pathways that are exposed to various light intensities, and in animals with unilateral eye patches. We will also examine modulation of melatonin receptor protein and mRNA expression in tectofugal pathways by light and activation of retinal D1-dopamine receptors. These studies will enhance our understanding on how environmental light and endogenous melatonin regulate melatonin receptor density and the expression of genes involved in circadian rhythms and visual function. Furthermore our studies are aimed to discover subtype selective melatonin receptor agonists and antagonists which may lead to new therapies for the treatment of visual function and circadian rhythms disturbances of sleep and mood.
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