PHYSIOLOGICAL SUBSTRATES OF A CIRCADIAN OSCILLATOR

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The suprachiasmatic muclei (SCN) of the hypothalamus are endogenous oscillators that serve a well-defined, critical role in the generation and entrainment of the daily (circadian) oscillations of physiology, metabolism and behavior of mammals. Our broad research objective is to understand the molecular, cellular and neurophysiological mechanisms by which the SCN keep 24 hr time. The model species to be studied is the rat. The SCN pacemaker survives intact in the hypothalamic brain slice where it is accessible to experiments aimed at dissecting cellular mechanisms. Our methodological approach combines brain slice culture with conventional neurophysiological techniques that measure the circadian rhythm of neuronal activity nd biochemical analyses that measure cyclic nucleotide levels, enzyme activities and proteins phosphorylated. The present proposal develops naturally from our finding that the SCN rhythm can be reset in the brain slice by treatments affecting cAMP or cGMP pathways. Further, the pacemaker substrates are changing so that the rhythm is reset by cAMP in the donor's days, and by cGMP or high K+(o) during the donor's night. Our specific aims include: A) to more fully explore the role of cAMP in SCN function )(by examining the level of cAMP action within the SCN, the activity of enzymes regulating cAMP, regulation of the cAMP effect by melatonin, the involvement of cAMP stimulated phosphorylation in phase-shifting, and the involvement of new protein synthesis in cAMP stimulation); B) to examine the nature of the effect of cGMP analogs at night (by examining the specificity of cGMP stimulation, the effect of depolarizing stimuli on cGMP levels, and determining whether protein kinase inhibitors are effective at night); and C) to identify other second messenger pathways underlying the SCN pacemaker (by exploring potential, possibly interactive, roles for Ca++ and phospholipid metabolites). Because the SCN integrates most circadian behaviors and metabolic changes, this Study has basic relevance to understanding many brain and metabolic dysfunctions, including certain forms of mental illness.