Many organisms, including humans, express physiological and behavioral rhythms that vary with a 24-hour period. These rhythms are regulated by an internal circadian clock that is synchronized to the physical environment by external stimuli. Efforts to elucidate the physiological mechanisms underlying the generation of circadian rhythms in mammals have focused on the suprachiasmatic nucleus (SCN) of the hypothalamus as a site of a circadian pacemaker. However, little information exists concerning the cellular and molecular mechanisms responsible for the generation of neural circadian rhythms in the SCN. Recent findings have demonstrated that inhibition of protein synthesis can lead to phase shifts in the circadian clock of mammals and can alter the response of the clock to the phase- shifting effects of light. The experiments outlined in this proposal will use in vivo and in vitro approaches to explore in detail the role of protein synthesis in the generation of circadian rhythms and in mediating the effects of both photic and non-photic stimuli on the mammalian circadian clock. In in vivo studies using the golden hamster, protein synthesis inhibitors will be used to determine when protein synthesis is required for light to be able to induce phase shifts in the circadian clock. Using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) to separate out proteins in SCN tissue of hamsters injected with 35-S- methionine, proteins produced at specific times of the circadian day and/or in response to various stimuli that can induce phase shifts in the circadian clock (e.g., pulses of light or darkness, exposure to a novel running wheel or treatment with short-acting benzodiazepines) will be identified. These studies should identify proteins that 1) express circadian oscillations, 2) are produced in response to light or other stimuli that can induce phase shifts in the circadian clock and 3) are actually associated with the phase shifts induced by these stimuli. In addition, a new in vitro system involving dispersed rat SCN cells in culture has demonstrated that SCN cells can produce a circadian rhythm in vasopressin release in vitro. This system will be utilized to identify specific proteins that are produced by SCN cells in culture and to identify common proteins that are produced by the SCN under in vitro and in vivo conditions. Taken together, the results from the proposed studies should provide new information on the role of protein synthesis in the entrainment and generation of circadian rhythms in mammals and should ultimately lead to an understanding of the molecular components of the mammalian circadian clock. An understanding of the biological basis of circadian rhythms in animals may lead to procedures useful in the diagnosis and treatment of pathophysiologic conditions associated with circadian rhythm dysfunction that have been observed in sleep disorders, mental illness and/or endocrine abnormalities.
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