Circadian rhythms regulate the function of living systems at virtually every level of organization - from molecular to organismal. A fundamental question in the field concerns the molecular mechanism of circadian oscillators. How are circadian oscillations generated, and what components at the tissue, cellular or subcellular level are required for circadian properties? Our long-term objectives are to understand the cellular and biochemical events that underlie circadian rhythms among vertebrates. The mechanisms that generate circadian rhythms will be studied in vitro using dissociated check pineal cells as a model system. Chick pineal cells contain circadian oscillators, with photoreceptive input, which regulate the rhythmic synthesis of melatonin. The phase-shifting effects of light on the circadian oscillator will be used as a physiological stimulus to study this pathway. Identification of the steps in the photic entrainment pathway should ultimately aid in identifying components of the oscillating mechanism because the pathway must end upon some component of the biological clock. The potential entraining effects of norepinephrine will be analyzed to determine whether an additional input pathway to the oscillator exists. If adrenergic input entrains the rhythm, then tracing this pathway will also aid in identifying components of the oscillator. The role of """"""""G- proteins"""""""" in controlling the melatonin rhythm will be investigated. These regulatory proteins are in a pivotal position to regulate the spontaneous oscillation in cyclic AMP in chick pineal cells and therefore are prime candidates for generating these oscillations. The hypothesis that single pinealocytes are circadian pacemaker cells will be tested directly using single-cell measurement. These experiments will determine for the first time whether circadian oscillations in vertebrates are a cellular property. The possible role of calcium in mediating various processes within pineal cells will be explored. Finally, we will attempt to develop cell strains and cell lines of pinealocytes. These cell lines would provide a powerful new model system for studying the cell biology of circadian rhythms. Establishment of cell lines would enable biochemical, molecular and genetic approaches to be feasible in a vertebrate preparation. An understanding of the biological basis of circadian rhythms in vertebrates may lead to procedures useful in the diagnosis and treatment of pathophysiologic conditions associated with circadian rhythm dysfunctions such as sleep disorders, mental health and endocrine abnormalities.
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