Sleep is regulated by a homeostatic process that is activated by and counters the effect of sleep loss and a circadian process that determines the time-of-day sleep preferably occurs. The fine-tuned interaction between the two permits us to stay awake throughout the day and to remain asleep at night. Although these two processes are widely thought to operate independently, recent observations suggest that, at a molecular level, both processes use the same circadian transcription factors. Moreover, in vitro assays demonstrated that the activity of the two main circadian clock genes, Clock and Npas2, depend on intracellular redox potential, suggesting that, like sleep, circadian rhythms are intricately associated with cellular metabolism. The aim of this proposal is to analyze, in vivo, the relationship between sleep, circadian rhythms and cellular energy charge in order to better understand the cellular mechanisms that determine our day-time performance and sleep quality. By using redox-sensitive green fluorescent proteins (roGFPs), we will track redox changes within individual fibroblasts in which robust circadian rhythms can be induced, to establish that circadian rhythmicity is accompanied by changes in redox potential. Subsequently, the sleep-wake dependent and circadian contributions to changes in intracellular redox potential will be quantified in transgenic mice constitutively expressing roGFPs. Because clock-gene expression in the brain seems highly compartmentalized, we will follow changes in redox potential using optic-fibers aimed at the SCN (suprachiasmatic nucleus), the master circadian pacemaker, and in the cortex in freely behaving mice. Finally, we will establish how intracellular redox changes in SCN and cortex relate to the expression of the CLOCK and NPAS2 target gene period (per) 2, by studying per2-luciferase knock-in mice. These mice allow us to investigate how circadian rhythms in per2 expression in the periphery are coordinated by the SCN.
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