Circadian rhythms are daily changes in biological systems that are regulated and timed by endogenous biological clocks. This rhythmicity is ubiquitous at all levels of biological organization, from gene expression to behavior. The circadian system appears to be particularly important in maintenance of human mental health. Disruption of the circadian system due to pathology or to voluntary shifting of sleep-wake cycles can lead to diminished sensory and motor performance, sleep disorders or affective disorders. An understanding of the basic biological mechanisms of circadian rhythmicity may aid in the understanding and treatment of these and other disorders. The long term goal of this project is to understand circadian mechanisms at the cellular level. The strategy that will be used is based oh the input-output properties that are characteristic of all circadian systems. Circadian clocks are biological oscillators that self-generate rhythmicity in the absence of external timing cues. The timing of a circadian clock is 'set' by environmental time cues, particularly the daily cycle of light and darkness. These timing stimuli must act through input pathways that impinge on the oscillator, so it should be possible to identify oscillator mechanisms by following these pathways. In order to do this at the cellular level, it is necessary to identify the cells that are involved and develop reduced preparations that preserve circadian function in vitro. The proposed research focuses on the circadian oscillator in the retina, one of only three known circadian oscillators in vertebrate central nervous systems. Recently developed organ and tissue culture preparations of the frog retina have been shown to retain the basic properties of a circadian clock. Circadian rhythms of retinal melatonin release in vitro can be measured to monitor circadian oscillator function. Preliminary results suggest that circadian functions are localized in retinal photoreceptor cells. The first specific aim of this proposal is to determine whether this-is true and to further refine the retinal culture system for analysis of circadian mechanisms. The timing of the retinal oscillator can be reset either by light or by the neuromodulator dopamine. The pathways that mediate these effects are distinct, and provide dual routes to the oscillator mechanism. The sequences of cellular events that make up these pathways will be determined by manipulating candidate pathways and determining the effects on oscillator timing. The roles of protein synthesis and phosphorylation in generation and entrainment of the retinal circadian rhythms will be determined. Finally, specific proteins that meet multiple criteria as candidate oscillator components in this system will be identified. An understanding of the cellular mechanisms of circadian rhythmicity in this model system should aid in the understanding of circadian systems in general.
