Sleep is a fundamental behavioral state important for survival and is universal in animals with sufficiently complex nervous systems. As a highly conserved neurobehavioral state, sleep has been described in species ranging from jellyfish to humans. Biogenic amines like dopamine, serotonin and norepinephrine have been shown to be critical for sleep regulation across species but the precise circuit mechanisms underlying how amines control persistence of sleep, arousal and wakefulness remain unclear. The fruit fly, Drosophila melanogaster, provides a powerful model system for the study of sleep and circuit mechanisms underlying state transitions and persistence of states to meet the organisms motivational and cognitive needs. In Drosophila, two neuropils in the central brain, the mushroom body (MB) and the central complex (CX) have been shown to influence sleep homeostasis and receive aminergic neuromodulator input critical to sleep–wake switch. Dopamine neurons (DANs) are prevalent neuromodulator inputs to the MB but the mechanisms by which they interact with and regulate sleep- and wake-promoting neurons within MB are unknown. Here we investigate the role of subsets of PAM-DANs that signal wakefulness and project to wake-promoting compartments of the MB. We find that PAM-DANs are GABA responsive and require GABAA-Rdl receptor in regulating sleep. In mapping the pathways downstream of PAM neurons innervating γ5 and β′2 MB compartments we find that wakefulness is regulated by both DopR1 and DopR2 receptors in downstream Kenyon cells (KCs) and mushroom body output neurons (MBONs). Taken together, we have identified and characterized a dopamine modulated sleep microcircuit within the mushroom body that has previously been shown to convey information about positive and negative valence critical for memory formation. These studies will pave way for understanding how flies balance sleep, wakefulness and arousal.

Diseases associated with impaired sleep regulation negatively impact motor skills, emotional states, and cognitive performance. Neural circuits involved in sleep homeostasis play a critical role in sleep-wake transition but the precise circuit mechanisms underlying persistence of sleep, arousal and wakefulness remain unclear. The fruit fly, Drosophila melanogaster, provides a powerful model system for the study of sleep and circuit mechanisms underlying state transitions and persistence of states to meet the organisms motivational and cognitive needs. Here we investigate the role of specific subsets of dopamine neurons innervating the mushroom body (MB), a region implicated in sleep and associative learning in Drosophila. We find that inhibition of specific subsets of PAM and PPL1 dopamine neurons projecting to the MB increase sleep in the presence of strong wake-inducing stimuli. The wakefulness induced by activity of the PAM neurons is mediated by both Dop1 R1 and Dop1R2 receptors located in downstream Kenyon cells and mushroom body output neurons (MBONs). Further, we find that unlike the activity of the MBONs, whose activity is modulated by sleep-need and sleep-debt, the dopamine neuronal activity is independent of sleep-need. The sleep-regulating dopamine neurons characterized in these studies are also involved in mediating short-term courtship memories, appetitive memories and memory extinction processes. Taken together, we have identified and characterized the dopamine circuit and cellular mechanisms that link motivation-dependent behavioral output and suppression of sleep to coordinate motivated processes with wakefulness.

Sleep is a conserved neurobehavioral state observed in animals with sufficiently complex nervous systems and is critical for survival. While the exact function of sleep remains unknown, the lack of sleep can have a range of physiological and behavioral effects. Studies in invertebrates and vertebrates have identified conserved neural mechanisms and cellular pathways in control of sleep, wakefulness and arousal. Methodologies to measure sleep have ranged from EEG recordings in humans and rodents to in-depth analysis of locomotor patterns in flies, fish and worms. Here we focus on sleep measurements using activity monitoring in the highly versatile experimental model system, Drosophila melanogaster, which is amenable to a number of genetic, physiological and behavioral manipulations. Further, we also describe methods used to manipulate sleep and wakefulness to understand the neural regulation of sleep and how organisms balance sleep, wakefulness and behavioral arousal. Sleep as a behavioral state is regulated by a number of factors including food, environmental conditions, and genetic background. The methodologies described here provide, a highthroughput approach to study neural regulation of sleep and factors that affect this complex behavior.

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