Healthy aging mammals, including humans, exhibit changes in sleep and in circadian rhythms. These changes contribute to the high incidence of sleep disturbances in the elderly. Sleep is regulated by two interacting processes: an oscillating circadian clock (C) and the homeostatic sleep- promoting system (S). Studies of the molecular basis of C are relatively new in mammals, and process S in poorly understood at a molecular level. It appears that age alters mammalian clock function at a cellular/molecular level. Since the molecular clock mechanisms are known to be well-conserved from Drosophila melanogaster. Along with other advantages of a simple invertebrate model, the genetic clock mechanisms are more completely described than in mammals, and we can exploit many tools to measure and manipulate clock gene function. Our ongoing studies suggest that circadian rest in Drosophila may also share with mammalian sleep, a homeostatic regulatory process akin to S, manifested by a compensatory rebound after rest deprivation. We have found in our preliminary studies that, as in aging mammals, there are age- related changes in behavioral measures of C and also in S (measured by rest rebound), in Drosophila. We therefore propose to study age-related changes in the regulation of rest in Drosophila, using both behavioral and molecular techniques. Our overall hypothesis is that aging changes molecular mechanisms of clock and rest regulation in Drosophila melanogaster. In Protocol 1 we will examine the effect of age on the clock input by measuring the molecular basis of photic resetting (degradation of TIMELESS protein) together with behavioral assays with measures of cyclic expression of the clock genes-period and timeless using both standard molecular assays and bioluminescence patterns emitted from transgenic flies with a period-luciferase fusion gene. The luciferase reporter provides a toll to characterize gene expression patterns in vivo in individual aging animals. To evaluate the aging of the clock output as it influences rest, we will measure age-related changes in rest and also deprive the flies of rest to drive a compensatory rebound (Protocol 3). In Protocol 4, we will separate S from C by measuring rest and rest rebound in arrhythmic mutant flies lacking each clock gene (per0 and tim0). Finally, in Protocol 5 we will evaluate the role of adenosine, a sleep-promoting agent in mammals, in rest regulation and aging changes and the rest of Drosophila.
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