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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. Supramammillary nucleus (SuM) neurons have been studied in the context of REM sleep but their possible role in mediating wakefulness is not known. Here the authors elucidate the distinct functional contributions of three subpopulations in the SuM on electrographical and behavioral arousal in mice using genetically targeted approaches.

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 mammalian basal forebrain controls cortical rhythm and wake-sleep. Anaclet et al. use genetically-targeted chemogenetic systems to activate or inhibit cholinergic, glutamatergic or GABAergic neurons in this region, and reveal their contributions to behavioral and electrocortical arousal in behaving mice.

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. Sleep and wakefulness is stabilized by a population of orexin-expressing neurons. In this study, the authors demonstrate how these neurons drive arousal by silencing sleep-promoting neurons in the ventrolateral preoptic nucleus.

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. Cell groups in the hypothalamic suprachiasmatic clock contribute to the genesis of circadian rhythms. The authors identified two populations of vasoactive intestinal polypeptide-expressing neurons in the suprachiasmatic nucleus which regulate locomotor circadian rhythm in mice.

During obstructive sleep apnea, elevation of CO 2 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 CO 2 . Dorsal raphe 5HT(DR Sert ) neurons regulate arousal from hypercapnia by their projections to the neurons in the external lateral parabrachial nucleus (PBel) that are glutamatergic and also express calcitonin gene related peptide (PBel CGRP ). The DR Sert input to the PBel modulates the arousal system to rising levels of blood CO 2 , and may be mediated by 5HT 2a receptors on the PBel CGRP neurons.

Abstract 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.

‘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 (SPZGABA) neurons, the major postsynaptic targets of the central circadian clock, the suprachiasmatic nucleus. Optogenetic mapping revealed that SPZGABA 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 SPZGABA transmission phase-dependently increases aggression. Finally, SPZGABA-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.

Introduction Optogenetic and chemogenetic studies have shown that activation of basal forebrain (BF) GABAergic neurons rapidly wakes up mice from non-REM (NREM) sleep. These wake-promoting responses have been attributed to BF GABAergic neurons projecting to the cerebral cortex and more specifically to the inhibition of cortical fast-spiking interneurons. Tracing studies have however found that BF GABAergic neurons also densely innervate the lateral hypothalamus (LH) perifornical area, although the role of this pathway in behavioral state control remains mostly unexplored. Methods We conducted in vivo and in vitro optogenetic studies. We selectively expressed channelrhodopsin-2 (ChR2) in BF GABAergic neurons by injecting a cre-dependent viral vector encoding for ChR2 into the BF of VGAT-cre mice. We photostimulated the BF GABAergic input to the LH with optical fibers placed into the LH of EEG instrumented mice. For in vitro recordings we expressed ChR2 in BF GABAergic neurons and we fluorescently labeled orexin or LH GABAergic neurons. We recorded in brain slices from identified orexin neurons or GABA neurons while photostimulating the BF GABAergic input. Results Optogenetic stimulation of the BF GABAergic fibers in the LH produced rapid arousals from NREM sleep. The same stimulation however did not wake up the mice if they were in REM sleep. We conducted additional studies in brain slices to identify the postsynaptic neurons in the LH targeted by the BF GABAergic input. We found that while optogenetic stimulation of the BF GABAergic input did not produce opto-evoked synaptic responses in the orexin neurons, it produced short-latency opto-evoked inhibitory postsynaptic currents (IPSCs) in LH GABAergic neurons. These opto-evoked IPSCs were GABAA receptor-mediated and were maintained in tetrodotoxin (TTX) indicating monosynaptic connectivity. We have previously found that orexin neurons are inhibited by local LH GABAergic neurons. Our hypothesis is that these local GABAergic interneurons are the target of the BF GABAergic arousal input. Conclusion BF GABAergic neurons drive arousal through projections to the LH. We propose that this arousal response is due to the inhibition of local GABAergic interneurons which in turn disinhibit the LH wake-promoting neurons including the orexin neurons. Support (if any) NS091126 and HL149630

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.

Mammalian circadian rhythms, which orchestrate the daily temporal structure of biological processes, including the sleep-wake cycle, are primarily regulated by the circadian clock in the hypothalamic suprachiasmatic nucleus (SCN). The SCN clock is also implicated in providing an arousal 'signal,' particularly during the wake-maintenance zone (WMZ) of our biological day, essential for sustaining normal levels of wakefulness in the presence of mounting sleep pressure. Here we identify a role for SCN Neuromedin-S (SCN ) neurons in regulating the level of arousal, especially during the WMZ. We used chemogenetic and optogenetic methods to activate SCN neurons in vivo, which potently drove wakefulness. Fiber photometry confirmed the wake-active profile of SCN neurons. Genetically ablating SCN neurons disrupted the sleep-wake cycle, reducing wakefulness during the dark period and abolished the circadian rhythm of body temperature. SCN neurons target the dorsomedial hypothalamic nucleus (DMH), and photostimulation of their terminals within the DMH rapidly produces arousal from sleep. Presynaptic inputs to SCN neurons were also identified, including regions known to influence SCN clock regulation. Unexpectedly, we discovered strong input from the preoptic area (POA), which itself receives substantial inhibitory input from the DMH, forming a possible arousal-promoting circuit (SCN->DMH->POA->SCN). Finally, we analyzed the transcriptional profile of SCN neurons via single-nuclei RNA-Seq, revealing three distinct subtypes. Our findings link molecularly-defined SCN neurons to sleep-wake patterns, body temperature rhythms, and arousal control.