Summary Prolonged wakefulness leads to an increased pressure for sleep, but how this homeostatic drive is generated and subsequently persists is unclear. Here, from a neural circuit screen in Drosophila, we identify a subset of ellipsoid body (EB) neurons whose activation generates sleep drive. Patch-clamp analysis indicates these EB neurons are highly sensitive to sleep loss, switching from spiking to burst-firing modes. Functional imaging and translational profiling experiments reveal that elevated sleep need triggers reversible increases in cytosolic Ca2+ levels, NMDA receptor expression, and structural markers of synaptic strength, suggesting these EB neurons undergo “sleep need”-dependent plasticity. Strikingly, the synaptic plasticity of these EB neurons is both necessary and sufficient for generating sleep drive, indicating that sleep pressure is encoded by plastic changes within this circuit. These studies define an integrator circuit for sleep homeostasis and provide a mechanism explaining the generation and persistence of sleep drive.

SUMMARY Background The neuronal circuitry underlying sleep is poorly understood. Although dopamine (DA) is thought to play a key role in sleep/wake regulation, the identities of the individual DA neurons and their downstream targets required for this process are unknown. Results Here, we identify a DA neuron in each PPL1 cluster that promotes wakefulness in Drosophila. Imaging data suggest that the activity of these neurons is increased during wakefulness, consistent with a role in promoting arousal. Strikingly, these neurons project to the dorsal fan-shaped body, which has previously been shown to promote sleep. The reduced sleep caused by activation of DA neurons can be blocked by loss of DopR, and restoration of DopR expression in the fan-shaped body can rescue the wake-promoting effects of DA in a DopR mutant background. Conclusions These experiments define a novel arousal circuit at the single cell level. Since the dorsal fan-shaped body promotes sleep, these data provide a key link between wake and sleep circuits. Furthermore, these findings suggest that inhibition of sleep centers via monoaminergic signaling is an evolutionarily conserved mechanism to promote arousal.

SUMMARY How the circadian clock regulates the timing of sleep is poorly understood. Here, we identify a Drosophila mutant, wide awake (wake), that exhibits a marked delay in sleep onset at dusk. Loss of WAKE in a set of arousal-promoting clock neurons, the large ventrolateral neurons (l-LNvs), impairs sleep onset. WAKE levels cycle, peaking near dusk, and the expression of WAKE in l-LNvs is Clock dependent. Strikingly, Clock and cycle mutants also exhibit a profound delay in sleep onset, which can be rescued by restoring WAKE expression in LNvs. WAKE interacts with the GABAA receptor Resistant to Dieldrin (RDL), upregulating its levels and promoting its localization to the plasma membrane. In wake mutant l-LNvs, GABA sensitivity is decreased and excitability is increased at dusk. We propose that WAKE acts as a clock output molecule specifically for sleep, inhibiting LNvs at dusk to promote the transition from wake to sleep.

SUMMARY Background Emerging data suggest an important relationship between sleep and Alzheimer’s Disease (AD), but how poor sleep promotes the development of AD remains unclear. Results Here, using a Drosophila model of AD, we provide evidence suggesting that changes in neuronal excitability underlie the effects of sleep loss on AD pathogenesis. β-amyloid (Aβ) accumulation leads to reduced and fragmented sleep, while chronic sleep deprivation increases Aβ burden. Moreover, enhancing sleep reduces Aβ deposition. Increasing neuronal excitability phenocopies the effects of reducing sleep on Aβ, and decreasing neuronal activity blocks the elevated Aβ accumulation induced by sleep deprivation. At the single neuron level, we find that chronic sleep deprivation, as well as Aβ expression, enhances intrinsic neuronal excitability. Importantly, these data reveal that sleep loss exacerbates Aβ–induced hyperexcitability and suggest that defects in specific K+ currents underlie the hyperexcitability caused by sleep loss and Aβ expression. Finally, we show that feeding levetiracetam, an anti-epileptic medication, to Aβ-expressing flies suppresses neuronal excitability and significantly prolongs their lifespan. Conclusions Our findings directly link sleep loss to changes in neuronal excitability and Aβ accumulation and further suggest that neuronal hyperexcitability is an important mediator of Aβ toxicity. Taken together, these data provide a mechanistic framework for a positive feedback loop, whereby sleep loss and neuronal excitation accelerate the accumulation of Aβ, a key pathogenic step in the development of AD.

SummaryThe shoot apical meristem (SAM) of angiosperms comprises a group of undifferentiated cells which divide to maintain the meristem and also give rise to all the above-ground structures of the plant. Previous studies revealed that the Arabidopsis ARGONAUTE10 [AGO10, also called PINHEAD (PNH) or ZWILLE (ZLL)] gene is one of the critical SAM regulators, but the mechanism by which AGO10 modulates the SAM is unknown. In the present study we show that AGO10 genetically represses microRNA165/166 (miR165/166) for SAM maintenance as well as establishment of leaf adaxial-abaxial polarity. Levels of miR165/166 in leaves and embryonic SAMs of pnh/zll/ago10 mutants are abnormally elevated, leading to a reduction in the quantity of homeodomain-leucine zipper (HD-ZIP) III gene transcripts, the targets of miR165/166. This reduction is the primary cause of pnh/zll SAM and leaf defects, because the aberrant pnh/zll phenotypes were partially rescued by either increasing levels of HD-ZIP III transcripts or decreasing levels of miR165/166 in the SAM and leaf. Furthermore, plants with an abnormal apex were more frequent among pnh/zll rdr6 and pnh/zll ago7 double mutants and increased levels of miR165/166 were detected in rdr6 apices. These results indicate that AGO10 and RDR6/AGO7 may act in parallel in modulating accumulation of miR165/166 for normal plant development.