CELLULAR BASIS OF CIRCADIAN RHYTHMS IN MAMMALS

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The suprachiasmatic nucleus (SCN) of mammals acts as the master circadian pacemaker that exerts temporal control over many behaviors and physiological processes. It is presently unclear whether generation of circadian periodicity and other circadian properties emerge from single cells or from cellular interactions within the SCN. In addition, recent molecular evidence suggests that other areas of the brain may manifest circadian properties as well. The proposed studies directly address these issues by taking advantage of long-duration recording technologies - two different planar electrode arrays - and the unique properties of two mammalian circadian models, the tau mutant hamster and the Clock mutant mouse. The proposed experiments on the SCN are designed to systematically determine the circadian properties of explants, then dispersals and, finally, isolated neurons. The first specific aim tests the hypotheses that period stability is determined at the level of the tissue while temperature compensation and entrainment are intrinsic to individual neurons. The second specific aim tests the hypothesis that individual SCN neurons are competent, autonomous circadian pacemakers that comprise a unique cell class. The strategy is to first characterize the rhythmic ability of fully isolated SCN neurons and then, after fixation, their neurochemical content. The third specific aim tests the hypothesis that a coupling mechanism within the SCN averages the intrinsic periods of individual cells. Using long-term, multisite recording, the periods of co-cultured wild-type and mutant cells will be measured under conditions that couple and uncouple neuronal rhythms. Using specific antagonists, agonists and genetic knockouts, this specific aim goes on to test the hypothesis that nitric oxide is the coupling factor that synchronizes activity within the SCN and restricts the range of expressed periods. The recent discoveries of putative circadian clock genes in mammals have provided a set of cellular markers for potential circadian pacemakers outside the SCN. The fourth specific aim will determine whether four brain areas outside of the SCN can express circadian rhythms in firing rate in vitro. These experiments will, for the first time, identify the circadian pacemakers, their intrinsic properties, and the mechanisms that couple them within the SCN and whether other brain regions may also possess these abilities.