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.

The largest synaptic input to the sleep-promoting ventrolateral preoptic area (VLPO) [1] arises from the lateral hypothalamus [2], a brain area associated with arousal [3-5]. However, the neurochemical identity of the majority of these VLPO-projecting neurons within the lateral hypothalamus (LH), as well as their function in the arousal network, remains unknown. Herein we describe a population of VLPO-projecting neurons in the LH that express the vesicular GABA transporter (VGAT; a marker for GABA-releasing neurons). In addition to the VLPO, these neurons also project to several other established sleep and arousal nodes, including the tuberomammillary nucleus, ventral periaqueductal gray, and locus coeruleus. Selective and acute chemogenetic activation of LH VGAT(+) neurons was profoundly wake promoting, whereas acute inhibition increased sleep. Because of its direct and massive inputs to the VLPO, this population may play a particularly important role in sleep-wake switching.

'Sundowning' in dementia and Alzheimer's disease is characterized by early-evening agitation and aggression. While such periodicity suggests a circadian origin, whether the circadian clock directly regulates aggressive behavior is unknown. We demonstrate that a daily rhythm in aggression propensity in male mice is gated by GABAergic subparaventricular zone (SPZ^GABA) neurons, the major postsynaptic targets of the central circadian clock, the suprachiasmatic nucleus. Optogenetic mapping revealed that SPZ^GABA neurons receive input from vasoactive intestinal polypeptide suprachiasmatic nucleus neurons and innervate neurons in the ventrolateral part of the ventromedial hypothalamus (VMH), which is known to regulate aggression. Additionally, VMH-projecting dorsal SPZ neurons are more active during early day than early night, and acute chemogenetic inhibition of SPZ^GABA transmission phase-dependently increases aggression. Finally, SPZ^GABA-recipient central VMH neurons directly innervate ventrolateral VMH neurons, and activation of this intra-VMH circuit drove attack behavior. Altogether, we reveal a functional polysynaptic circuit by which the suprachiasmatic nucleus clock regulates aggression.

The hypothalamic suprachiasmatic (SCN) clock contains several neurochemically defined cell groups that contribute to the genesis of circadian rhythms. Using cell-specific and genetically targeted approaches we have confirmed an indispensable role for vasoactive intestinal polypeptide-expressing SCN (SCN^VIP) neurons, including their molecular clock, in generating the mammalian locomotor activity (LMA) circadian rhythm. Optogenetic-assisted circuit mapping revealed functional, di-synaptic connectivity between SCN^VIP neurons and dorsomedial hypothalamic neurons, providing a circuit substrate by which SCN^VIP neurons may regulate LMA rhythms. In vivo photometry revealed that while SCN^VIP neurons are acutely responsive to light, their activity is otherwise behavioral state invariant. Single-nuclei RNA-sequencing revealed that SCN^VIP neurons comprise two transcriptionally distinct subtypes, including putative pacemaker and non-pacemaker populations. Altogether, our work establishes necessity of SCN^VIP neurons for the LMA circadian rhythm, elucidates organization of circadian outflow from and modulatory input to SCN^VIP cells, and demonstrates a subpopulation-level molecular heterogeneity that suggests distinct functions for specific SCN^VIP subtypes.

During obstructive sleep apnea, elevation of CO₂ during apneas contributes to awakening and restoring airway patency. We previously found that glutamatergic neurons in the external lateral parabrachial nucleus (PBel) containing calcitonin gene related peptide (PBel^CGRP neurons) are critical for causing arousal during hypercapnia. However, others found that genetic deletion of serotonin (5HT) neurons in the brainstem also prevented arousal from hypercapnia. To examine interactions between the two systems, we showed that dorsal raphe (DR) 5HT neurons selectively targeted the PBel. Either genetically directed deletion or acute optogenetic silencing of DR^Sert neurons dramatically increased the latency of mice to arouse during hypercapnia, as did silencing DR^Sert terminals in the PBel. This effect was mediated by 5HT_2a receptors which are expressed by PBel^CGRP neurons. Our results indicate that the serotonergic input from the DR to the PBel via 5HT_2a receptors is critical for modulating the sensitivity of the PBel^CGRP neurons that cause arousal to rising levels of blood CO₂.