This proposal represents an intent to characterize the physiological substrates which modulate the primary circadian oscillator in the brain of the rat, the neurons of the suprachiasmatic nuclei (SCN). These neurons serve a well-defined and critical role in the generation and entrainment of the daily oscillations of physiological, metabolic and behavioral functions. The in vitro hypothalamic brain slice technique will be used to investigate circadian mechanisms which are difficult to address in the intact animal, but for which the brain slice offers unusual advantage. In our hands, the SCN in vitro sustains a circadian rhythm of firing rate and peptide secretion for up to 38 hr, even when reduced to remove peripheral hypothalamic regions normally included in the slice. Further, we have found that the electrical oscillations can be phase-shifted in vitro in a response curve similar to the intact animals. Thus, our preliminary neurophysiological investigations have shown that not only the 24 hr oscillator but also the phase resetting mechanism is endogenous to the SCN neurons in the brain slice. Preliminary biochemical studies demonstrate circadian changes in the phosphorylation state of specific SCN proteins and suggest that phosphorylation mechanisms may modulate circadian oscillations in the activity of these neurons. We propose to characterize the phase responsiveness of the SCN in vitro to stimuli which reset the oscillations in electrical activity using conventional neurophysiological and organ culture techniques. This precise definition of the phase-response curve will be used in physiological studies to characterize metabolic pathways underlying the electrical oscillations, particularly cyclic nucleotide- and Ca++-dependent protein phosphorylation as well as pathways involving synthesis of new mRNA and protein. Concurrently, biochemical studies will be carried out to further identify changes in phosphoproteins and their regulators during the normal circadian cycle and during phase-shifting. Because the SCN integrates most circadian behaviors and metabolic fluxes, this study has basic relevance to understanding many brain and metabolic dysfunctions, including certain forms of mental illness.
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