The long-term goal of this project is understand basic mechanisms of circadian rhythmicity in vertebrate retina. Many aspects of retinal physiology and metabolism are regulated by circadian clocks. These clocks are set by daily cycles of environmental and physiological stimuli, resulting in appropriately timed daily rhythms of visual system function. Rhythmic retinal activities include the expression of photoreceptor genes, the turnover of photoreceptive membrane, changes in synaptic structure and function, rod-cone dominance, and the synthesis and release of retinal neuromodulators. The ubiquity of circadian rhythmicity in the retina indicates a critical role for the circadian system in maintenance of retinal health and optimal visual function. Previous studies demonstrated that a fully functional circadian clock is located within the retinal photoreceptor layer, and suggested that individual photoreceptor cells are capable of circadian rhythm generation. This clock regulates melatonin synthesis within the photoreceptor cells. The photoreceptor clock can be entrained by cycles of either light or a neuromodulator, dopamine. The circadian clock, the melatonin synthetic and regulatory mechanisms, and the transduction pathways for entrainment of the clock by light and dopamine are all preserved in a cultured photoreceptor layer preparation from the retina of Xenopus laevis. This preparation makes it possible to test hypotheses about photoreceptor clock mechanisms without the complications of interactions with other cell types. The melatonin release rhythm of photoreceptor layers will be used in the proposed experiments as a measure of circadian clock responses to experimental manipulations. The specific goal of this project is to define transduction mechanisms by which light and dopamine entrain the photoreceptor circadian clock. Previous studies showed that light and dopamine have nearly identical ultimate effects on the timing of the clock. However, recent data indicate that the transduction pathways for these signal are distinct at the second messenger level, and suggest specific hypotheses about how components of these pathways interact to entrain retinal rhythms. The experiments proposed here will determine: (1) the characteristics and identities of the dopamine receptors and photopigments that mediate entrainment; (2) the roles of membrane potential and Ca2+ fluxes in the entrainment pathways; and (3) the role of a novel protein kinase mechanism in the entrainment pathways. The results of these studies will define basic mechanisms of retinal circadian rhythmicity, and will provide important information about how therapeutic interventions may affect the retinal circadian clock.
