PHYSIOLOGICAL SUBSTRATES OF CIRCADIAN OSCILLATOR

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The suprachiasmatic nuclei (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 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 neurophysiological techniques that measure the circadian rhythm of the ensemble neuronal activity and whole cell recordings in the slice as well as biochemical analyses that measure cyclic nucleotide levels, enzyme activities and proteins phosphorylated, and immunocytochemical/in situ hybridization techniques that identify and localize molecules of interest. The present proposal develops naturally from our finding that the SCN rhythm can be reset in the brain slice by treatments affecting cAMP-,cGMP- or pertussis toxin-sensitive pathways. Further, even under the constant conditions in the slice chamber, the pacemaker substrates are changing so that the rhythm is reset by cAMP in subjective day, by cGMP during subjective night and by melatonin during the day/night transition. Our specific aims include: A) to more fully explore the role of cAMP/protein kinase A in SCN function (by examining the activity, concentration and phosphorylation state of protein kinase A (PKA), localizing the sites of cAMP and PKA effects, and the involvement of transcription/translation in cAMP stimulation); B) to more fully explore the role of cGMP/protein kinase G at night (by determining the activity, concentration and phosphorylation state of protein kinase G (PKG), localizing cGMP and PKG effect; C) to examine the regulation of cyclic nucleotide phosphodiesterase (by determining the level and regulation of cAMP and cGMP phosphodiesterase activities) and D) to understand the relationship of other second messenger/kinase systems in generating circadian time (by exploring the interactive role of protein kinases in time-keeping). Because the SCN integrates most circadian behaviors and metabolic changes, this study has basic relevance of understanding many brain and metabolic dysfunctions, including sleep disorders and certain forms of mental illness.