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Ramalingam Vetrivelan, Clifford B Saper, Patrick M Fuller Department of Neurology, Harvard Medical School and Beth Israel Deaconess Medical Center, Boston, MA, USA Abstract: Armodafinil is the pharmacologically active R-enantiomer of modafinil, a widely prescribed wake-promoting agent used to treat several sleep-related disorders including excessive daytime sleepiness associated with narcolepsy, shift work sleep disorder, and obstructive sleep apnea/hypopnea syndrome. Remarkably, however, the neuronal circuitry through which modafinil exerts its wake-promoting effects remains unresolved. In the present study, we sought to determine if the wake-promoting effects of armodafinil are mediated, at least in part, by inhibiting the sleep-promoting neurons of the ventrolateral preoptic (VLPO) nucleus. To do so, we measured changes in waking following intraperitoneal administration of armodafinil (200 mg/kg) or the psychostimulant methamphetamine (1 mg/kg) in rats with cell-body specific lesion of the VLPO. Rats with histologically confirmed lesions of the VLPO demonstrated a sustained increase in wakefulness at baseline, but the increase in wakefulness following administration of both armodafinil and methamphetamine was similar to that of intact animals. These data suggest that armodafinil increases wakefulness by mechanisms that extend beyond inhibition of VLPO neurons. Keywords: EEG, sleep, orexin-saporin, methamphetamine

Antibodies are used in many areas of biomedical and clinical research, but many of these antibodies have not been adequately characterized, which casts doubt on the results reported in many scientific papers. This problem is compounded by a lack of suitable control experiments in many studies. In this article we review the history of the 'antibody characterization crisis', and we document efforts and initiatives to address the problem, notably for antibodies that target human proteins. We also present recommendations for a range of stakeholders - researchers, universities, journals, antibody vendors and repositories, scientific societies and funders - to increase the reproducibility of studies that rely on antibodies., Competing Interests: RK, HV, CL, DH, NP, RM, AL, CS, TE, RT, JT, LF, FF, AB, SL No competing interests declared, AB Co-founder and serving as CEO of SciCrunch Inc, a company that works with publishers to improve the scientific literature; the terms of this agreement have been approved by the COI office at the University of California at San Diego, MR Employed by Addgene, a company that may be affected financially by the opinions reported in this article, HC Employed by Abcam, a for-profit company that sells antibodies and which may be affected financially by the opinions reported in this article, (© 2024, Kahn et al.)

The secretion of cortisol in humans and corticosterone (Cort) in rodents follows a daily rhythm which is important in readying the individual for the daily active cycle and is impaired in chronic depression. This rhythm is orchestrated by the suprachiasmatic nucleus (SCN) which governs the activity of neurons in the paraventricular nucleus of the hypothalamus that produce the corticotropin-releasing hormone (PVH CRH neurons). The dorsomedial nucleus of the hypothalamus (DMH) serves as a crucial intermediary, being innervated by the SCN both directly and via relays in the subparaventricular zone, and projecting axons to the PVH, thereby exerting influence over the cortisol/corticosterone rhythm. However, the role and synaptic mechanisms by which DMH neurons regulate the daily rhythm of Cort secretion has not been explored. We found that either ablating or acutely inhibiting the DMH glutamatergic (DMH Vglut2 ) neurons resulted in a 40-70% reduction in the daily peak of Cort. Deletion of the Vglut2 gene within the DMH produced a similar effect, highlighting the indispensable role of glutamatergic signaling. Chemogenetic stimulation of DMH Vglut2 neurons led to an increase of Cort levels, and optogenetic activation of their terminals in the PVH in hypothalamic slices directly activated PVH CRH neurons through glutamate release (the DMH Vglut2 → PVH CRH pathway). Similarly, ablating, inhibiting, or disrupting GABA transmission by DMH GABAergic (DMH Vgat ) neurons diminished the circadian peak of Cort, particularly under constant darkness conditions. Chemogenetic stimulation of DMH Vgat neurons increased Cort, although with a lower magnitude compared to DMH Vglut2 neuron stimulation, suggesting a role in disinhibiting PVH CRH neurons. Supporting this hypothesis, we found that rostral DMH Vgat neurons project directly to GABAergic neurons in the caudal ventral part of the PVH and adjacent peri-PVH area (cvPVH), which directly inhibit PVH CRH neurons, and that activating the DMH Vgat terminals in the cvPVH in brain slices reduced GABAergic afferent input onto the PVH CRH neurons. Finally, ablation of cvPVH Vgat neurons resulted in increased Cort release at the onset of the active phase, affirming the pivotal role of the DMH Vgat → cvPVH Vgat → PVH CRH pathway in Cort secretion. In summary, our study delineates two parallel pathways transmitting temporal information to PVH CRH neurons, collectively orchestrating the daily surge in Cort in anticipation of the active phase. These findings are crucial to understand the neural circuits regulating Cort secretion, shedding light on the mechanisms governing this physiological process and the coordinated interplay between SCN, DMH, and PVH., Competing Interests: Competing interests The authors declare no competing interests.

About half of the neurons in the parabrachial nucleus (PB) that are activated by CO 2 are located in the external lateral (el) subnucleus, express calcitonin gene-related peptide (CGRP), and cause forebrain arousal. We report here, in male mice, that most of the remaining CO 2 -responsive neurons in the adjacent central lateral (PBcl) and Kölliker-Fuse (KF) PB subnuclei express the transcription factor FoxP2 and many of these neurons project to respiratory sites in the medulla. PBcl FoxP2 neurons show increased intracellular calcium during wakefulness and REM sleep and in response to elevated CO 2 during NREM sleep. Photo-activation of the PBcl FoxP2 neurons increases respiration, whereas either photo-inhibition of PBcl FoxP2 or genetic deletion of PB/KF FoxP2 neurons reduces the respiratory response to CO 2 stimulation without preventing awakening. Thus, augmenting the PBcl/KF FoxP2 response to CO 2 in patients with sleep apnea in combination with inhibition of the PBel CGRP neurons may avoid hypoventilation and minimize EEG arousals., (© 2024. The Author(s).)

The "dorsal pons", or "dorsal pontine tegmentum" (dPnTg), is part of the brainstem. It is a complex, densely packed region whose nuclei are involved in regulating many vital functions. Notable among them are the parabrachial nucleus, the Kölliker Fuse, the Barrington nucleus, the locus coeruleus, and the dorsal, laterodorsal, and ventral tegmental nuclei. In this study, we applied single-nucleus RNA-seq (snRNA-seq) to resolve neuronal subtypes based on their unique transcriptional profiles and then used multiplexed error robust fluorescence in situ hybridization (MERFISH) to map them spatially. We sampled ~1 million cells across the dPnTg and defined the spatial distribution of over 120 neuronal subtypes. Our analysis identified an unpredicted high transcriptional diversity in this region and pinpointed the unique marker genes of many neuronal subtypes. We also demonstrated that many neuronal subtypes are transcriptionally similar between humans and mice, enhancing this study's translational value. Finally, we developed a freely accessible, GPU and CPU-powered dashboard ( http://harvard.heavy.ai:6273/ ) that combines interactive visual analytics and hardware-accelerated SQL into a data science framework to allow the scientific community to query and gain insights into the data., (© 2024. The Author(s).)

Lesion localization is the basis for understanding neurologic disease, which is predicated on neuroanatomical knowledge carefully cataloged from histology and imaging atlases. However, it is often difficult to correlate clinical images of brainstem injury obtained by MRI scans with the details of human brainstem neuroanatomy represented in atlases, which are mostly based on cytoarchitecture using Nissl stain or a single histochemical stain, and usually do not include the cerebellum. Here, we report a high-resolution (200 μm) 7T MRI of a cadaveric male human brainstem and cerebellum paired with detailed, coregistered histology (at 2 μm single-cell resolution) of the immunohistochemically stained cholinergic, serotonergic, and catecholaminergic (dopaminergic, noradrenergic, and adrenergic) neurons, in relationship to each other and to the cerebellum. These immunohistochemical findings provide novel insights into the spatial relationships of brainstem cell types and nuclei, including subpopulations of melanin and TH + neurons, and allows for more informed structural annotation of cell groups. Moreover, the coregistered MRI-paired histology helps validate imaging findings. This is useful for interpreting both scans and histology, and to understand the cell types affected by lesions. Our detailed chemoarchitecture and cytoarchitecture with corresponding high-resolution MRI builds on previous atlases of the human brainstem and cerebellum, and makes precise identification of brainstem and cerebellar cell groups involved in clinical lesions accessible for both laboratory scientists and clinicians alike. SIGNIFICANCE STATEMENT Clinicians and neuroscientists frequently use cross-sectional anatomy of the human brainstem from MRI scans for both clinical and laboratory investigations, but they must rely on brain atlases to neuroanatomical structures. Such atlases generally lack both detail of brainstem chemical cell types, and the cerebellum, which provides an important spatial reference. Our current atlas maps the distribution of key brainstem cell types (cholinergic, serotonergic, and catecholaminergic neurons) in relationship to each other and the cerebellum, and pairs this histology with 7T MR images from the identical brain. This atlas allows correlation of the chemoarchitecture with corresponding MRI, and makes the identification of cell groups that are often discussed, but rarely identifiable on MRI scan, accessible to clinicians and clinical researchers., (Copyright © 2023 Agostinelli et al.)

Competing Interests: TES has received research grants of consulting fees from National Institutes of Health (NIH), Wake Up Narcolepsy, Takeda Pharmaceuticals, Merck, Jazz Pharmaceuticals, Harmony Biosciences, Avadel, Consynance, Idorsia, and Woolsey Pharmaceuticals, and royalties from UpToDate, McGraw Hill, and Springer. CBS declares research grants from NIH, Japan AMED, and the Mathers Foundation, royalties from Oxford Press and McGraw-Hill, and payments from the American Neurological Association as Editor-in-Chief of Annals of Neurology.

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., (© 2022. The Author(s).)

Previous studies suggest that the median preoptic nucleus (MnPO) of the hypothalamus plays an important role in regulating the wake-sleep cycle and, in particular, homeostatic sleep drive. However, the precise cellular phenotypes, targets, and central mechanisms by which the MnPO neurons regulate the wake-sleep cycle remain unknown. Both excitatory and inhibitory MnPO neurons innervate brain regions implicated in sleep promotion and maintenance, suggesting that both cell types may participate in sleep control. Using genetically targeted approaches, we investigated the role of the MnPO GABAergic (MnPO Vgat ) and glutamatergic (MnPO Vglut2 ) neurons in modulating wake-sleep behavior of mice. We found that both neuron populations differentially participate in wake-sleep control, with MnPO Vgat neurons being involved in sleep homeostasis and MnPO Vglut2 neurons facilitating sleep during allostatic (stressful) challenges., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Nearly 14% of Americans experience chronic circadian disruption due to shift work, increasing their risk of obesity, diabetes, and other cardiometabolic disorders. These disorders are also exacerbated by modern eating habits such as frequent snacking and consumption of high-fat foods., Methods: We investigated the effects of recurrent circadian disruption (RCD) on glucose metabolism in C57BL/6 mice and in human participants exposed to non-24-h light-dark (LD) schedules vs. those on standard 24-h LD schedules. These LD schedules were designed to induce circadian misalignment between behaviors including rest/activity and fasting/eating with the output of the near-24-h central circadian pacemaker, while minimizing sleep loss, and were maintained for 12 weeks in mice and 3 weeks in humans. We examined interactions of these circadian-disrupted schedules compared to control 24-h schedules with a lower-fat diet (LFD, 13% in mouse and 25-27% in humans) and high-fat diet (HFD, 45% in mouse and 45-50% in humans). We also used young vs. older mice to determine whether they would respond differently to RCD., Results: When combined with a HFD, we found that RCD caused significant weight gain in mice and increased body fat in humans, and significantly impaired glucose tolerance and insulin sensitivity in both mice and humans, but this did not occur when RCD was combined with a LFD. This effect was similar in both young and older mice., Conclusion: These results in both humans and a model organism indicate that circadian disruption has an adverse effect on metabolism among individuals eating a high-fat Western-style diet, even in the absence of significant sleep loss, and suggest that reducing dietary fat may protect against the metabolic consequences of a lifestyle (such as shift work) that involves chronic circadian disruption., (Copyright © 2022. Published by Elsevier Inc.)

There has been an explosion recently in our understanding of the neuronal populations in the preoptic area involved in thermoregulation of mice. Recent studies have identified several genetically specified populations of neurons predominantly in the median preoptic nucleus (MnPO) but spreading caudolaterally into the preoptic area that regulate body temperature. . These include warm-responsive neurons that express the peptides PACAP, BDNF, or QRFP; and receptors for temperature, leptin, estrogen, or prostaglandin E2 (PGE2). These neurons are predominantly glutamatergic and driving them opto- or chemogenetically can cause profound hypothermia, and in some cases, periods of torpor or a hibernation-like state. Conversely, fever response is likely to depend upon inhibiting the activity of these neurons through the PGE2 receptor EP3. Another cell group, the Brs3-expressing MnPO neurons, are apparently cold-responsive and cause increases in body temperature. MnPO-QRFP neurons cause hypothermia via activation of their terminals in the region of the dorsomedial nucleus of the hypothalamus (DMH). As the MnPO-QRFP neurons are essentially glutamatergic, and the DMH largely uses glutamatergic projections to the raphe pallidus to increase body temperature, this model suggests the existence of local inhibitory interneurons in the DMH region between the MnPO-QRFP glutamatergic neurons that cause hypothermia and the DMH glutamatergic neurons that cause hyperthermia. The new genetically targeted studies in mice provide a way to identify the precise neuronal circuitry that is responsible for our physiological observations in this species, and will suggest critical experiments that can be undertaken to compare these with the thermoregulatory circuitry in other species., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2022 Informa UK Limited, trading as Taylor & Francis Group.)

Hypothalamic hamartomas (HH) are rare, basilar developmental lesions with widespread comorbidities often associated with refractory epilepsy and encephalopathy. Imaging advances allow for early, even prenatal, detection. Genetic studies suggest mutations in GLI3 and other patterning genes are involved in HH pathogenesis. About 50%-80% of children with HH have severe rage and aggression and a majority of patients exhibit externalizing disorders. Behavioral disruption and intellectual disability may predate epilepsy. Neuropsychological, sleep, and endocrine disorders are typical. The purpose of this article is to provide a summary of the current understanding of HH and to highlight opportunities for future research., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Recent studies have shown that the median preoptic area contains a population of neurons expressing an array of fast neurotransmitters and receptors that collectively cause a fall in body temperature in response to environmental warming or depleted energy stores. In this issue of Cell Metabolism, Piñol et al. (2021) identify a separate population of median preoptic neurons that are responsible for cold defense and cause stress-related hyperthermia., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The intermediate nucleus of Brockhaus (INH), also known as the interstitial nucleus of the anterior hypothalamus-1 of Allen and Gorski (INAH-1), the sexually dimorphic nucleus of Swaab and colleagues (SDN), and the ventrolateral preoptic nucleus of Saper and colleagues (VLPO), is a cluster of largely galanin-expressing neurons in the lateral preoptic area, at the level of the crossing of the anterior commissure and dorsal to the supraoptic nucleus. The number of Nissl-stained neurons in the INH has been reported to be larger in men than women and to decrease with aging, although these findings have been controversial, in part because of differences in patient populations and methods used to assess the nucleus. However, recent studies have confirmed that the number of galanin-immunoreactive INH neurons is larger in men than women and decreases with age and have reported further loss with Alzheimer disease. The galanin-immunoreactive VLPO neurons have been thought to drive sleep behavior in many species, and their numbers in older humans correlate with the amount of consolidated sleep they experience. Sleep differences between men and women, during aging, and with Alzheimer disease may also depend upon the integrity of this nucleus., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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 .

Fever is a common phenomenon during infection or inflammatory conditions. This stereotypic rise in body temperature (Tb) in response to inflammatory stimuli is a result of autonomic responses triggered by prostaglandin E2 action on EP3 receptors expressed by neurons in the median preoptic nucleus (MnPO EP3R neurons). To investigate the identity of MnPO EP3R neurons, we first used in situ hybridization to show coexpression of EP3R and the VGluT2 transporter in MnPO neurons. Retrograde tracing showed extensive direct projections from MnPO VGluT2 but few from MnPO Vgat neurons to a key site for fever production, the raphe pallidus. Ablation of MnPO VGluT2 but not MnPO Vgat neurons abolished fever responses but not changes in Tb induced by behavioral stress or thermal challenges. Finally, we crossed EP3R conditional knock-out mice with either VGluT2-IRES-cre or Vgat-IRES-cre mice and used both male and female mice to confirm that the neurons that express EP3R and mediate fever are glutamatergic, not GABAergic. This finding will require rethinking current concepts concerning the central thermoregulatory pathways based on the MnPO EP3R neurons being GABAergic. SIGNIFICANCE STATEMENT Body temperature is regulated by the CNS. The rise of the body temperature, or fever, is an important brain-orchestrated mechanism for fighting against infectious or inflammatory disease, and is tightly regulated by the neurons located in the median preoptic nucleus (MnPO). Here we demonstrate that excitatory MnPO neurons mediate fever and examine a potential central circuit underlying the development of fever responses., (Copyright © 2020 the authors.)

Accumulating evidence supports the role of sleep in synaptic plasticity and memory consolidation. One line of investigation, the synaptic homeostasis hypothesis, has emphasized the increase in synaptic strength during waking, and compensatory downsizing of (presumably less frequently used) synapses during sleep. Conversely, other studies have reported downsizing and loss of dendritic spines following sleep deprivation. We wanted to determine the effect of sleep deprivation on dendritic spines of hippocampal CA1 neurons using genetic methods for fluorescent labeling of dendritic spines. Male Vglut2-Cre mice were injected with an AAV-DIO-ChR2-mCherry reporter in CA1 hippocampus. Gentle handling was used to sleep deprive mice for 5 hr, from lights on (7 am) to 12 noon. Control and sleep-deprived mice were euthanized at 12 noon and processed for quantification of dendritic spines. We used confocal microscope imaging and three-dimensional (3D) analysis to quantify thin, mushroom, and stubby spines from CA1 dendrites, distinguishing between branch segments. We observed significantly greater density of spines in CA1 of sleep-deprived mice, driven primarily by greater numbers of thin spines, and significantly larger spine volume and head diameter. Branch and region-specific analysis revealed that spine volume was greater in primary dendrites of apical and basal segments, along with proximal segments on both apical and basal dendrites, and spine density was increased in secondary branches and distal segments on apical dendrites following sleep deprivation. Our 3D quantification suggests sleep contributes to region- and branch-specific synaptic downscaling in the hippocampus, supporting the theory of broad but selective synaptic downscaling during sleep., (© 2019 Wiley Periodicals, Inc.)

Origin and functions of intermittent transitions among sleep stages, including brief awakenings and arousals, constitute a challenge to the current homeostatic framework for sleep regulation, focusing on factors modulating sleep over large time scales. Here we propose that the complex micro-architecture characterizing sleep on scales of seconds and minutes results from intrinsic non-equilibrium critical dynamics. We investigate θ- and δ-wave dynamics in control rats and in rats where the sleep-promoting ventrolateral preoptic nucleus (VLPO) is lesioned (male Sprague-Dawley rats). We demonstrate that bursts in θ and δ cortical rhythms exhibit complex temporal organization, with long-range correlations and robust duality of power-law (θ-bursts, active phase) and exponential-like (δ-bursts, quiescent phase) duration distributions, features typical of non-equilibrium systems self-organizing at criticality. We show that such non-equilibrium behavior relates to anti-correlated coupling between θ- and δ-bursts, persists across a range of time scales, and is independent of the dominant physiologic state; indications of a basic principle in sleep regulation. Further, we find that VLPO lesions lead to a modulation of cortical dynamics resulting in altered dynamical parameters of θ- and δ-bursts and significant reduction in θ-δ coupling. Our empirical findings and model simulations demonstrate that θ-δ coupling is essential for the emerging non-equilibrium critical dynamics observed across the sleep-wake cycle, and indicate that VLPO neurons may have dual role for both sleep and arousal/brief wake activation. The uncovered critical behavior in sleep- and wake-related cortical rhythms indicates a mechanism essential for the micro-architecture of spontaneous sleep-stage and arousal transitions within a novel, non-homeostatic paradigm of sleep regulation. SIGNIFICANCE STATEMENT We show that the complex micro-architecture of sleep-stage/arousal transitions arises from intrinsic non-equilibrium critical dynamics, connecting the temporal organization of dominant cortical rhythms with empirical observations across scales. We link such behavior to sleep-promoting neuronal population, and demonstrate that VLPO lesion (model of insomnia) alters dynamical features of θ and δ rhythms, and leads to significant reduction in θ-δ coupling. This indicates that VLPO neurons may have dual role for both sleep and arousal/brief wake control. The reported empirical findings and modeling simulations constitute first evidences of a neurophysiological fingerprint of self-organization and criticality in sleep- and wake-related cortical rhythms; a mechanism essential for spontaneous sleep-stage and arousal transitions that lays the bases for a novel, non-homeostatic paradigm of sleep regulation., (Copyright © 2020 the authors.)

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., (Copyright © 2019 the authors.)