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 Ca(2+) 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.

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. Because 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.

Neurons use two main schemes to encode information: rate coding (frequency of firing) and temporal coding (timing or pattern of firing). While the importance of rate coding is well established, it remains controversial whether temporal codes alone are sufficient for controlling behavior. Moreover, the molecular mechanisms underlying the generation of specific temporal codes are enigmatic. Here, we show in Drosophila clock neurons that distinct temporal spike patterns, dissociated from changes in firing rate, encode time-dependent arousal and regulate sleep. From a large-scale genetic screen, we identify the molecular pathways mediating the circadian-dependent changes in ionic flux and spike morphology that rhythmically modulate spike timing. Remarkably, the daytime spiking pattern alone is sufficient to drive plasticity in downstream arousal neurons, leading to increased firing of these cells. These findings demonstrate a causal role for temporal coding in behavior and define a form of synaptic plasticity triggered solely by temporal spike patterns.

BACKGROUND: Emerging data suggest an important relationship between sleep and Alzheimers 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.

Free-living animals must not only regulate the amount of food they consume but also choose which types of food to ingest. The shifting of food preference driven by nutrient-specific hunger can be essential for survival, yet little is known about the underlying mechanisms. We identified a dopamine circuit that encodes protein-specific hunger in Drosophila The activity of these neurons increased after substantial protein deprivation. Activation of this circuit simultaneously promoted protein intake and restricted sugar consumption, via signaling to distinct downstream neurons. Protein starvation triggered branch-specific plastic changes in these dopaminergic neurons, thus enabling sustained protein consumption. These studies reveal a crucial circuit mechanism by which animals adjust their dietary strategy to maintain protein homeostasis.

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.

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.

Background

Excess body burden of uric acid promotes gout. Diminished renal clearance of uric acid causes hyperuricemia in most patients with gout, and the renal urate transporter (URAT)1 is important for regulation of serum uric acid (sUA) levels. The URAT1 inhibitors probenecid and benzbromarone are used as gout therapies; however, their use is limited by drug-drug interactions and off-target toxicity, respectively. Here, we define the mechanism of action of lesinurad (Zurampic®; RDEA594), a novel URAT1 inhibitor, recently approved in the USA and Europe for treatment of chronic gout.

Methods

sUA levels, fractional excretion of uric acid (FE_UA), lesinurad plasma levels, and urinary excretion of lesinurad were measured in healthy volunteers treated with lesinurad. In addition, lesinurad, probenecid, and benzbromarone were compared in vitro for effects on urate transporters and the organic anion transporters (OAT)1 and OAT3, changes in mitochondrial membrane potential, and human peroxisome proliferator-activated receptor gamma (PPARγ) activity.

Results

After 6 hours, a single 200-mg dose of lesinurad elevated FE_UA 3.6-fold (p < 0.001) and reduced sUA levels by 33 % (p < 0.001). At concentrations achieved in the clinic, lesinurad inhibited activity of URAT1 and OAT4 in vitro, did not inhibit GLUT9, and had no effect on ABCG2. Lesinurad also showed a low risk for mitochondrial toxicity and PPARγ induction compared to benzbromarone. Unlike probenecid, lesinurad did not inhibit OAT1 or OAT3 in the clinical setting.

Conclusion

The pharmacodynamic effects and in vitro activity of lesinurad are consistent with inhibition of URAT1 and OAT4, major apical transporters for uric acid. Lesinurad also has a favorable selectivity and safety profile, consistent with an important role in sUA-lowering therapy for patients with gout.