Daily oscillations in mammalian physiology and behavior persist even in a constant environment, and their disruption leads to jet lag, sleep disorders, and other maladies, including mood disorders. Such "circadian" (ca. 24 hr) rhythms depend on a biological clock located within the brain, in the suprachiasmatic nucleus (SCN). Most cells express "clock genes", components of a transcriptional feedback loop comprising the intracellular clock, but the SCN is the master pacemaker: it has access to synchronizing light/dark input from the retina, specialized coupling mechanisms to maintain coherence among its component cellular oscillators and enhance its robustness, and neuronal efferent projections to synchronize cellular oscillators in peripheral tissues throughout the body. Recent work, however, has challenged the simplistic view that SCN neurons are all stable, autonomous, single cell transcriptional feedback oscillators. The objective of this proposal is to define the autonomy, persistence, and precision of SCN and fibroblast circadian clock cells, and to explore the interdependence of intracellular transcriptional, electrical, and calcium rhythms in these cells. This will be accomplished using mechanical, pharmacologic, and genetic approaches to disrupt cell interactions and manipulate membrane potential or intracellular calcium. Effects on the intracellular circadian clock will be assessed in individual cells by using optical methods to measure calcium and clock gene transcription, and multielectrode arrays to monitor neuronal firing. Specifically, we will test the hypotheses that: (1) SCN neurons require tonic (but not rhythmic) input from other neurons to maintain rhythmicity, (2) apparent non-rhythmicity of some SCN neurons is a stochastic event due to membrane hyperpolarization rather than a reflection of a stable non-rhythmic subtype, and (3) cells require a tonic level of calcium (but not rhythmic calcium) for transcriptional or electrical rhythms. Answers to these fundamental questions about the cellular basis of circadian rhythmicity will be essential for an understanding of how circadian clocks contribute to health and disease, and serve as a basis for novel therapeutic approaches.
A biological clock in the human brain keeps track of time of day and orchestrates countless circadian (ca. 24 hr) rhythms throughout the body. By further delineating the mechanisms of this clock at the level of single cells, the experiments proposed here may suggest new therapeutic approaches not only to jet lag, shift work, and other sleep disorders, but also to cancer, diabetes, and depression.
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