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Humans and animals lacking orexin neurons exhibit daytime sleepiness, sleep attacks, and state instability. While the circuit basis by which orexin neurons contribute to consolidated wakefulness remains unclear, existing models posit that orexin neurons provide their wake-stabilizing influence by exerting excitatory tone on other brain arousal nodes. Here we show using in vivo optogenetics, in vitro optogenetic-based circuit mapping, and single-cell transcriptomics that orexin neurons also contribute to arousal maintenance through indirect inhibition of sleep-promoting neurons of the ventrolateral preoptic nucleus. Activation of this subcortical circuit rapidly drives wakefulness from sleep by differentially modulating the activity of ventrolateral preoptic neurons. We further identify and characterize a feedforward circuit through which orexin (and co-released glutamate) acts to indirectly target and inhibit sleep-promoting ventrolateral preoptic neurons to produce arousal. This revealed circuitry provides an alternate framework for understanding how orexin neurons contribute to the maintenance of consolidated wakefulness and stabilize behavioral state.

A role for the brain's serotoninergic (5HT) system in the regulation of sleep and wakefulness has been long suggested. Yet, previous studies employing pharmacological, lesion and genetically driven approaches have produced inconsistent findings, leaving 5HT's role in sleep-wake regulation incompletely understood. Here we sought to define the specific contribution of 5HT neurons within the dorsal raphe nucleus (DRN5HT) to sleep and arousal control. To do this, we employed a chemogenetic strategy to selectively and acutely activate DRN5HT neurons and monitored sleep-wake using electroencephalogram recordings. We additionally assessed indices of anxiety using the open field and elevated plus maze behavioral tests and employed telemetric-based recordings to test effects of acute DRN5HT activation on body temperature and locomotor activity. Our findings indicate that the DRN5HT cell population may not modulate sleep-wake per se, but rather that its activation has apparent anxiolytic properties, suggesting the more nuanced view that DRN5HT neurons are sleep permissive under circumstances that produce anxiety or stress.