Sleep is thought to be essential to restore brain functions, and converging evidence suggests that a key function may be to rebalance cellular changes triggered by plasticity during wake. This evidence is consistent with the hypothesis that sleep and wake may occur, be regulated, and perform their functions at the level of individual neurons. Recently, using multi-array recordings in freely moving rats, we obtained direct evidence that sleep can occur locally within a group of cortical neurons, while the rest of the brain remains awake, and that such local sleep increases with the duration of wake. If so, many questions arise: does local sleep also occur in species very different from mammals, such as flies? Are the mechanisms underlying the occurrence of local sleep similar to those that are known to regulate sleep need, specifically intense neural plasticity leading to tiredness, which requires sleep to enforce synaptic renormalization? Or does local sleep reflect temporary neuronal fatigue due to intense neural activity and short-lasting depletion of energy or calcium stores, and thus not qualify properly as sleep? Finally, it is unknown whether there are cellular/ultrastructural signatures that sleep has occurred and presumably performed its functions. In this proposal, we will test whether local sleep exists in flies by using in vivo calcium imaging to monitor simultaneously the activity of dozens of neurons in specific neuronal circuits. We will then test whether local sleep, like sleep proper, is regulated by intense synaptic plasticity (tiredness) or instead by mere activity (fatigue). To do so we will first establish if local sleep increases during extended wake in a complex enriched environment (fly mall), expected to lead to tiredness in the mushroom bodies, relative to extended wake in an impoverished environment (single tube). Next, we will induce local dTrpA1-mediated activation during extended wake in single tubes, as well as during sleep. These 2 conditions of intense activity with little plasticity are expected to lead to fatigue, and their effects on local sleep will again be compared with those of extended wake in single tubes. To further decouple the effects of plasticity from those of activity we will repeat the experiments in learning mutants, in which exposure to the fly mall should not lead to plasticity/tiredness. Finally, we will use SBF-SEM to test whether there are ultrastructural signatures that can distinguish neurons of flies that have been awake from those of flies that slept, and compare the effects of wake with plasticity leading to tiredness with those of wake associated with dTrpA1-mediated intense firing leading to fatigue. Altogether, these studies in flies will complement those in mice in Project II, which use similar or the very same methods. Together, they will establish if sleep and wake are regulated homeostatically at the single neuron level, and if they leave ultrastructural signatures that reflect their consequences and functions for individual cells.
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