The long term goal of this project is to understand basic mechanisms of vertebrate circadian rhythmicity. Circadian rhythms are daily changes in biological systems that are timed by endogenous biological clocks. These clocks are biological oscillators that self-generate rhythmicity in the absence of external timing cues. The timing of a circadian oscillator is set through entrainment by the daily cycle of light and darkness. In vertebrates, circadian rhythmicity is generated at the cellular level in a few neural and endocrine structures. These localized clocks drive and coordinate rhythms ranging from gene expression to behavior throughout the animal. Disruption of the human circadian system due to pathology, aging or voluntary disruption of sleep-wake cycles can lead to diminished sensory and motor performance, sleep disorders or affective disorders. An understanding of basic biological clock mechanisms will aid in understanding and treating these disorders. The specific goal of this project is to define cellular and molecular mechanisms of circadian rhythm generation and entrainment in retinal photoreceptor cells from Xenopus laevis. Previous studies have shown that isolated layers of photoreceptor cells (including both rods and cones) retain the capacity to generate circadian rhythms in constant darkness, and that these rhythms can be entrained in vitro by either light or dopamine. Recent technical advances have enabled measurement of melatonin release from dispersed photoreceptor cell cultures. We will determine whether dissociated rods, cones, or both are capable of generating circadian rhythms. Using rhythmic melatonin release as a measure of circadian oscillator function in cultured photoreceptors, we will identify transduction pathways that mediate the resetting effects of light, dopamine, and adenine nucleotides. The sequences of cellular events that lead to entrainment will be determined by pharmacological manipulation of candidate second messengers and kinases and biochemical measurement of their responses. Any stimulus that resets circadian phase must act through cellular pathways that impinge on the oscillator, so it should be possible to identify both entrainment and, eventually, oscillator mechanisms by following these pathways. A major unsolved problem is how to test the functional roles of candidate macromolecular clock components in primary cell cultures. Therefore, a new method for expression of heterologous proteins in postmitotic amphibian cells will be tested in cultured photoreceptor preparations. This research will provide information and tools needed for discovery and analysis of photoreceptor clock molecules.
