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.

Recent studies have identified an especially important role for basal forebrain GABAergic (BFVGAT) neurons in the regulation of behavioral waking and fast cortical rhythms associated with cognition. However, BFVGAT neurons comprise several neurochemically and anatomically distinct subpopulations, including parvalbumin-containing BFVGAT neurons and somatostatin-containing BFVGAT neurons (BFSOM neurons), and it was recently reported that optogenetic activation of BFSOM neurons increases the probability of a wakefulness to non-rapid-eye movement (NREM) sleep transition when stimulated during the rest period of the animal. This finding was unexpected given that most BFSOM neurons are not NREM sleep active and that central administration of the synthetic somatostatin analog, octreotide, suppresses NREM sleep or increases REM sleep. Here we used a combination of genetically driven chemogenetic and optogenetic activation, chemogenetic inhibition, and ablation approaches to further explore the in vivo role of BFSOM neurons in arousal control. Our findings indicate that acute activation or inhibition of BFSOM neurons is neither wakefulness nor NREM sleep promoting and is without significant effect on the EEG, and that chronic loss of these neurons is without effect on total 24 h sleep amounts, although a small but significant increase in waking was observed in the lesioned mice during the early active period. Our in vitro cell recordings further reveal electrophysiological heterogeneity in BFSOM neurons, specifically suggesting at least two distinct subpopulations. Together, our data support the more nuanced view that BFSOM neurons are electrically heterogeneous and are not NREM sleep or wake promoting per se, but may exert, in particular during the early active period, a modest inhibitory influence on arousal circuitry.SIGNIFICANCE STATEMENT The cellular basal forebrain (BF) is a highly complex area of the brain that is implicated in a wide range of higher-level neurobiological processes, including regulating and maintaining normal levels of electrocortical and behavioral arousal. The respective in vivo roles of BF cell populations and their neurotransmitter systems in the regulation of electrocortical and behavioral arousal remains incompletely understood. Here we seek to define the neurobiological contribution of GABAergic somatostatin-containing BF neurons to arousal control. Understanding the respective contribution of BF cell populations to arousal control may provide critical insight into the pathogenesis of a host of neuropsychiatric and neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, schizophrenia, and the cognitive impairments of normal aging.

Optogenetics and chemogenetics are powerful tools, allowing the specific activation or inhibition of targeted neuronal subpopulations. Application of these techniques to sleep and circadian research has resulted in the unveiling of several neuronal populations that are involved in sleep-wake control, and allowed a comprehensive interrogation of the circuitry through which these nodes are coordinated to orchestrate the sleep-wake cycle. In this review, we discuss six recently described sleep-wake and circadian circuits that show promise as therapeutic targets for sleep medicine. The parafacial zone (PZ) and the ventral tegmental area (VTA) are potential druggable targets for the treatment of insomnia. The brainstem circuit underlying rapid eye movement sleep behavior disorder (RBD) offers new possibilities for treating RBD and neurodegenerative synucleinopathies, whereas the parabrachial nucleus, as a nexus linking arousal state control and breathing, is a promising target for developing treatments for sleep apnea. Therapies that act upon the hypothalamic circuitry underlying the circadian regulation of aggression or the photic regulation of arousal and mood pathway carry enormous potential for helping to reduce the socioeconomic burden of neuropsychiatric and neurodegenerative disorders on society. Intriguingly, the development of chemogenetics as a therapeutic strategy is now well underway and such an approach has the capacity to lead to more focused and less invasive therapies for treating sleep-wake disorders and related comorbidities.

Wakefulness, along with fast cortical rhythms and associated cognition, depend on the basal forebrain (BF). BF cholinergic cell loss in dementia and the sedative effect of anti-cholinergic drugs have long implicated these neurons as important for cognition and wakefulness. The BF also contains intermingled inhibitory GABAergic and excitatory glutamatergic cell groups whose exact neurobiological roles are unclear. Here we show that genetically targeted chemogenetic activation of BF cholinergic or glutamatergic neurons in behaving mice produced significant effects on state consolidation and/or the electroencephalogram but had no effect on total wake. Similar activation of BF GABAergic neurons produced sustained wakefulness and high-frequency cortical rhythms, whereas chemogenetic inhibition increased sleep. Our findings reveal a major contribution of BF GABAergic neurons to wakefulness and the fast cortical rhythms associated with cognition. These findings may be clinically applicable to manipulations aimed at increasing forebrain activation in dementia and the minimally conscious state.

The histaminergic neurons of the tuberomammillary nucleus (TMN^HDC) of the posterior hypothalamus have long been implicated in promoting arousal. More recently, a role for GABAergic signaling by the TMN^HDC neurons in arousal control has been proposed. Here, we investigated the effects of selective chronic disruption of GABA synthesis (via genetic deletion of the GABA synthesis enzyme, glutamic acid decarboxylase 67) or GABAergic transmission (via genetic deletion of the vesicular GABA transporter (VGAT)) in the TMN^HDC neurons on sleep-wake in male mice. We also examined the effects of acute chemogenetic activation and optogenetic inhibition of TMN^HDC neurons upon arousal in male mice. Unexpectedly, we found that neither disruption of GABA synthesis nor GABAergic transmission altered hourly sleep-wake quantities, perhaps because very few TMN^HDC neurons coexpressed VGAT. Acute chemogenetic activation of TMN^HDC neurons did not increase arousal levels above baseline but did enhance vigilance when the mice were exposed to a behavioral cage change challenge. Similarly, acute optogenetic inhibition had little effect upon baseline levels of arousal. In conclusion, we could not identify a role for GABA release by TMN^HDC neurons in arousal control. Further, if TMN^HDC neurons do release GABA, the mechanism by which they do so remains unclear. Our findings support the view that TMN^HDC neurons may be important for enhancing arousal under certain conditions, such as exposure to a novel environment, but play only a minor role in behavioral and EEG arousal under baseline conditions.SIGNIFICANCE STATEMENT The histaminergic neurons of the tuberomammillary nucleus of the hypothalamus (TMN^HDC) have long been thought to promote arousal. Additionally, TMN^HDC neurons may counter-regulate the wake-promoting effects of histamine through co-release of the inhibitory neurotransmitter, GABA. Here, we show that impairing GABA signaling from TMN^HDC neurons does not impact sleep-wake amounts and that few TMN^HDC neurons contain the vesicular GABA transporter, which is presumably required to release GABA. We further show that acute activation or inhibition of TMN^HDC neurons has limited effects upon baseline arousal levels and that activation enhances vigilance during a behavioral challenge. Counter to general belief, our findings support the view that TMN^HDC neurons are neither necessary nor sufficient for the initiation and maintenance of arousal under baseline conditions.

Sudden unexpected environmental changes capture attention and, when perceived as potentially dangerous, evoke defensive behavioral states. Perturbations of the lateral septum (LS) can produce extreme hyperdefensiveness even to innocuous stimuli, but how this structure influences stimulus-evoked defensive responses and threat perception remains unclear. Here, we show that Crhr2-expressing neurons in mouse LS exhibit phasic activation upon detection of threatening but not rewarding stimuli. Threat-stimulus-driven activity predicts the probability but not vigor or type of defensive behavior evoked. Although necessary for and sufficient to potentiate stimulus-triggered defensive responses, LS^Crhr2 neurons do not promote specific behaviors. Rather, their stimulation elicits negative valence and physiological arousal. Moreover, LS^Crhr2 activity tracks brain state fluctuations and drives cortical activation and rapid awakening in the absence of threat. Together, our findings suggest that LS directs bottom-up modulation of cortical function to evoke preparatory defensive internal states and selectively enhance responsivity to threat-related stimuli.

Basic and clinical observations suggest that the caudal hypothalamus comprises a key node of the ascending arousal system, but the cell types underlying this are not fully understood. Here we report that glutamate-releasing neurons of the supramammillary region (SuM^vglut2) produce sustained behavioral and EEG arousal when chemogenetically activated. This effect is nearly abolished following selective genetic disruption of glutamate release from SuM^vglut2 neurons. Inhibition of SuM^vglut2 neurons decreases and fragments wake, also suppressing theta and gamma frequency EEG activity. SuM^vglut2 neurons include a subpopulation containing both glutamate and GABA (SuM^vgat/vglut2) and another also expressing nitric oxide synthase (SuM^Nos1/Vglut2). Activation of SuM^vgat/vglut2 neurons produces minimal wake and optogenetic stimulation of SuM^vgat/vglut2 terminals elicits monosynaptic release of both glutamate and GABA onto dentate granule cells. Activation of SuM^Nos1/Vglut2 neurons potently drives wakefulness, whereas inhibition reduces REM sleep theta activity. These results identify SuM^vglut2 neurons as a key node of the wake-sleep regulatory system.

The precise neural circuitry that mediates arousal during sleep apnea is not known. We previously found that glutamatergic neurons in the external lateral parabrachial nucleus (PBel) play a critical role in arousal to elevated CO2 or hypoxia. Because many of the PBel neurons that respond to CO2 express calcitonin gene-related peptide (CGRP), we hypothesized that CGRP may provide a molecular identifier of the CO2 arousal circuit. Here, we report that selective chemogenetic and optogenetic activation of PBel^CGRP neurons caused wakefulness, whereas optogenetic inhibition of PBel^CGRP neurons prevented arousal to CO2, but not to an acoustic tone or shaking. Optogenetic inhibition of PBel^CGRP terminals identified a network of forebrain sites under the control of a PBel^CGRP switch that is necessary to arouse animals from hypercapnia. Our findings define a novel cellular target for interventions that may prevent sleep fragmentation and the attendant cardiovascular and cognitive consequences seen in obstructive sleep apnea. VIDEO ABSTRACT.

'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.