The mechanisms of the homeostatic regulation of sleep are largely unknown, although a role for gene expression seems likely based on genetic and molecular studies. In this proposal we suggest that several of the genes identified as critical components of the circadian pacemaker in the SCN also have a separate, non-circadian role in the cerebral cortex (and perhaps other brain regions) in sleep homeostasis. We found that mice lacking the circadian 'clock'-genes cryptochrome (cry1,2-/-), not only lack circadian rhythms but also exhibit all the electrocortical hallmarks of high sleep pressure. This unexpected phenotype was associated with elevated brain expression of the transcriptional regulators period (per)l, and per2, which are transcriptionally inhibited by CRY1,2. Wild-type mice following sleep deprivation also have elevated levels of per1,2 mRNA in the cortex specifically. Here we propose to confirm and further characterize the relationship between sleep-wake and these molecular changes at both mRNA and protein levels. Determining this relationship and the neuro-anatomical specificity of these changes is critical to test the hypothesis that a transcriptional network of interacting clock genes outside the SCN underlies the sleep homeostat. To separate homeostatic from circadian sleep regulatory aspects, SCN lesions will be performed. We expect that in the absence of circadian output provided by the SCN, the cortical oscillation in per expression will be driven by sleep and wake, as in intact animals. We will directly manipulate this clock-gene network by examining sleep regulation in mice lacking cry1, cry2, per1, per2, clock, and npas2. Mice lacking either cry1 or cry2 will be recorded to assess whether inactivation of only one cry gene can replicate the cry1,2-/-sleep phenotype. CLOCK and NPAS2 are positive transcriptional regulators of per1,2, cry1,2, and other genes, and should provide an important contrast to the cry1,2-/- phenotype. Given the partial redundancy in these gene families, we expect comparisons of mice lacking both per1,2, or both npas2,clock, to be especially informative. An improved understanding of the molecular substrate of sleep homeostasis could be helpful to a wide variety of individuals with acute or chronic sleep problems.
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