The neurohormone, melatonin, is a primary output signal of the circadian clock in retinal photoreceptors. In the retina, melatonin acts as a paracrine signal of darkness by activating specific receptors that ultimately leads to increased sensitivity to light. It facilitates dark adaptation and likely coordinates other diumal events that occur in the retina such as photoreceptor outer segment disc shedding. In addition, melatonin increases the degree of light-induced photoreceptor cell death. The cellular location of the melatonin receptor subtypes and the physiological responses to melatonin receptor binding are important factors that determine the influence of melatonin on the normal health and function of the retina. The long-term goal of this research is to understand the molecular mechanisms of action and the function of melatonin in the retina and the role of circadian sitnals in photoreceptor physiology and disease. The retina of the frog Xenopus laevis has been an exceptional in vitro model system to study retinal melatonin synthesis and function, and RNA encoding three melatonin receptor subtypes is expressed both in the neural retina, including the photoreceptors, and retinal pigment epithelium (RPE) of Xenopus retina. Also, recent advancements in transgenic Xenopus technology have greatly enhanced the utility of this frog model in the study of cell-specific gene expression. The goals of this proposal are to 1) direct rod photoreceptor-specific over-expression of each of the three melatonin receptor subtypes in transgenic Xenopus, and to compare the cellular responses to applied melatonin, and 2) direct Melic melatonin receptor subtype cell-specific over-expression in transgenic Xenopus, and identify all retinal cells that express the transgene by reporter gene fluorescence combined with immunocytochemistry of known retinal proteins. Disruption of circadian rhythms may contribute significantly to the photoreceptor cell death in some degenerative retinal diseases, and melatonin may play a pivotal role in coordinating retinal circadian events. Discovering the molecular mechanisms of melatonin action may therefore provide a greater understanding of the role of circadian signals in environmentally- and genetically- induced photoreceptor degenerations.
