The experimental setup included sampling CO 2 from an empty chamber to assess baseline levels, alongside five behavioral chambers, each measuring CO 2 production of a single fly. A stop-flow, push-through respirometry setup was constructed using Sable Systems equipment (Sable Systems International). MR was measured at 25☌ through indirect calorimetry, measuring the CO 2 production of individual flies with a Li-7000 CO 2 analyzer (LI-COR), which was calibrated with pure CO 2 before each run. 15 Unless noted in the figures, all experiments are performed in 3- to 5-day-old mated female flies. 15 This allele removes the entire coding region of the gene and represents a null mutation that has been outcrossed to the w 1118 background and has previously been described as Δ trsn. The trsn null allele is an excision of the trsn EY06981 locus derived from mobilizing the EPgy2 insertion. The wild-type line used in this manuscript is the w 1118 fly strain (Bloomington Stock #5905). Flies were maintained in incubators (Powers Scientific Dros52) at 25☌ on a 12:12 light/dark cycle, with humidity set to 55%–65%. MethodS Drosophila Maintenance and Fly Stocksįlies were grown and maintained on standard food (Bloomington Recipe, Genesee Scientific). These findings suggest that sleep-dependent reductions in MR previously observed in mammals are conserved in the fruit fly and further support the notion that sleep provides a mechanism for energy conservation. Further, we find that starvation inhibits sleep-associated reductions in MR, suggesting feeding state influence physiological changes associated with sleep. Our findings reveal that MR is reduced when flies sleep, and uninterrupted sleep bouts of ~30 minutes or greater are associated with an additional reduction in MR, indicating that flies exhibit sleep stages that are physiologically distinct. 14 Here, we describe a system to simultaneously measure sleep and MR in individual fruit flies. 11–13 In flies and other small insects, stop-flow respirometry can be used to monitor CO 2 production, a by-product of oxidative metabolism and a proxy for MR. In rodents and humans, MR is elevated in response to sleep deprivation and reduced during sleep, supporting the notion that metabolic processes are acutely regulated by sleep state. 7 Although these behavioral metrics of sleep have been studied extensively, significantly less is known about physiological changes associated with sleep in flies. 6, 7 Sleep in Drosophila is typically defined by 5 minutes of behavioral quiescence because this correlates with other behavioral characteristics used to define sleep. 9, 10 Additionally, flies display all the behavioral hallmarks of sleep including extended periods of behavioral quiescence, rebound following deprivation, increased arousal threshold, and species-specific posture. 8 Flies, like mammals, display distinct electrophysiological patterns that correlate with wake and rest. Sleep is characterized by physiological changes in brain activity or through the behavioral correlates that accompany these changes. 2, 6, 7 Here, we describe a novel single-fly respirometry assay in the fruit fly, designed to simultaneously measure sleep and whole-body MR that allows for genetic interrogation of the mechanisms regulating interactions between these processes. The fruit fly, Drosophila melanogaster, displays all the behavioral characteristics of sleep and provides a powerful system for genetic investigation of interactions between sleep and diverse physiological processes. 3 Although a reduction in MR and energy expenditure during sleep has been documented in mammalian and avian species, 4, 5 little is known about the genetic and neural mechanisms governing the effects of sleep on MR. 1, 2 In mammals, metabolic rate (MR) is reduced during sleep raising the possibility that sleep provides a mechanism of energy conservation or partitioning. INTRODUCTIONĭysregulation of sleep is strongly linked to metabolism-related pathologies, and reciprocal interactions between sleep and metabolism suggest these processes are integrated at the cellular and molecular levels. Use of this system, combined with existing genetic tools in Drosophila, will facilitate identification of novel sleep genes and neurons, with implications for understanding the relationship between sleep loss and metabolic disease. Our findings reveal that uninterrupted sleep bouts of 30 minutes or greater are associated with a reduction in metabolic rate providing a physiological readout of sleep. Here, we describe a novel system to simultaneously record sleep and metabolic rate in single Drosophila. Metabolic disorders are associated with sleep disturbances, yet our understanding of the mechanisms underlying interactions between sleep and metabolism remains limited.
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