Genetic Activation, Inactivation, and Deletion Reveal a Limited And Nuanced Role for Somatostatin-Containing Basal Forebrain Neurons in Behavioral State Control

Recent studies have identified an especially important role for basal forebrain GABAergic (BF(VGAT)) neurons in the regulation of behavioral waking and fast cortical rhythms associated with cognition. However, BF(VGAT) neurons comprise several neurochemically and anatomically distinct subpopulations, including parvalbumin-containing BF(VGAT) neurons and somatostatin-containing BF(VGAT) neurons (BF(SOM) neurons), and it was recently reported that optogenetic activation of BF(SOM) 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 BF(SOM) 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 BF(SOM) neurons in arousal control. Our findings indicate that acute activation or inhibition of BF(SOM) 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 BF(SOM) neurons, specifically suggesting at least two distinct subpopulations. Together, our data support the more nuanced view that BF(SOM) 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\u27s disease, Parkinson\u27s disease, schizophrenia, and the cognitive impairments of normal aging

Reassessing the Role of Histaminergic Tuberomammillary Neurons in Arousal Control

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

Suprachiasmatic VIP neurons are required for normal circadian rhythmicity and comprised of molecularly distinct subpopulations

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

Supramammillary glutamate neurons are a key node of the arousal system

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 (SuMvglut2) produce sustained behavioral and EEG arousal when chemogenetically activated. This effect is nearly abolished following selective genetic disruption of glutamate release from SuMvglut2 neurons. Inhibition of SuMvglut2 neurons decreases and fragments wake, also suppressing theta and gamma frequency EEG activity. SuMvglut2 neurons include a subpopulation containing both glutamate and GABA (SuMvgat/vglut2) and another also expressing nitric oxide synthase (SuMNos1/Vglut2). Activation of SuMvgat/vglut2 neurons produces minimal wake and optogenetic stimulation of SuMvgat/vglut2 terminals elicits monosynaptic release of both glutamate and GABA onto dentate granule cells. Activation of SuMNos1/Vglut2 neurons potently drives wakefulness, whereas inhibition reduces REM sleep theta activity. These results identify SuMvglut2 neurons as a key node of the wake−sleep regulatory system

The Sleep-Promoting Ventrolateral Preoptic Nucleus: What Have We Learned over the Past 25 Years?

For over a century, the role of the preoptic hypothalamus and adjacent basal forebrain in sleep–wake regulation has been recognized. However, for years, the identity and location of sleep- and wake-promoting neurons in this region remained largely unresolved. Twenty-five years ago, Saper and colleagues uncovered a small collection of sleep-active neurons in the ventrolateral preoptic nucleus (VLPO) of the preoptic hypothalamus, and since this seminal discovery the VLPO has been intensively investigated by labs around the world, including our own. Herein, we first review the history of the preoptic area, with an emphasis on the VLPO in sleep–wake control. We then attempt to synthesize our current understanding of the circuit, cellular and synaptic bases by which the VLPO both regulates and is itself regulated, in order to exert a powerful control over behavioral state, as well as examining data suggesting an involvement of the VLPO in other physiological processes

The Sleep-Promoting Ventrolateral Preoptic Nucleus: What Have We Learned over the Past 25 Years?

For over a century, the role of the preoptic hypothalamus and adjacent basal forebrain in sleep-wake regulation has been recognized. However, for years, the identity and location of sleep- and wake-promoting neurons in this region remained largely unresolved. Twenty-five years ago, Saper and colleagues uncovered a small collection of sleep-active neurons in the ventrolateral preoptic nucleus (VLPO) of the preoptic hypothalamus, and since this seminal discovery the VLPO has been intensively investigated by labs around the world, including our own. Herein, we first review the history of the preoptic area, with an emphasis on the VLPO in sleep-wake control. We then attempt to synthesize our current understanding of the circuit, cellular and synaptic bases by which the VLPO both regulates and is itself regulated, in order to exert a powerful control over behavioral state, as well as examining data suggesting an involvement of the VLPO in other physiological processes

The Sleep-Promoting Ventrolateral Preoptic Nucleus: What Have We Learned over the Past 25 Years?

For over a century, the role of the preoptic hypothalamus and adjacent basal forebrain in sleep–wake regulation has been recognized. However, for years, the identity and location of sleep- and wake-promoting neurons in this region remained largely unresolved. Twenty-five years ago, Saper and colleagues uncovered a small collection of sleep-active neurons in the ventrolateral preoptic nucleus (VLPO) of the preoptic hypothalamus, and since this seminal discovery the VLPO has been intensively investigated by labs around the world, including our own. Herein, we first review the history of the preoptic area, with an emphasis on the VLPO in sleep–wake control. We then attempt to synthesize our current understanding of the circuit, cellular and synaptic bases by which the VLPO both regulates and is itself regulated, in order to exert a powerful control over behavioral state, as well as examining data suggesting an involvement of the VLPO in other physiological processes

Changes in extracellular Ca2+ can affect the pattern of discharge in rat thalamic neurons

The aim of this study was to investigate some of the cellular mechanisms involved in the effects caused by changes in extracellular Ca2+ concentration ([Ca2+]o).Current and voltage-clamp experiments were carried out on acutely isolated thalamic neurons of rats.Increasing [Ca2+]o alone induced a transition of the discharge from single spike to burst mode in isolated current-clamped neurons.Increasing [Ca2+]o caused the voltage-dependent characteristics of the low voltage-activated (LVA) transient Ca2+ currents to shift towards positive values on the voltage axis. Changing [Ca2+]o from 0.5 to 5 mM caused the inactivation curve to shift by 21 mV.Extracellular Ca2+ blocked a steady cationic current. This current reversed at −35 mV, was scarcely affected by Mg2+ and was completely blocked by the non-selective cation channel inhibitor gadolinium (10 μM). The effect of [Ca2+]o was mimicked by 500 μM spermine, a polyamine which acts as an agonist for the Ca2+-sensing receptor, and was modulated by intracellular GTP-γ-S.At the resting potential, both the voltage shift and the block of the inward current removed the inactivation of LVA calcium channels and, together with the increase in the Ca2+ driving force, favoured a rise in the low threshold Ca2+ spikes, causing the thalamic firing to change to the oscillatory mode.Our data indicate that [Ca2+]o is involved in multiple mechanisms of control of the thalamic relay and pacemaker activity. These findings shed light on the correlation between hypercalcaemia, low frequency EEG activity and symptoms such as sleepiness and lethargy described in many clinical papers

To eat or to sleep: That is a lateral hypothalamic question

The lateral hypothalamus (LH) is a functionally and anatomically complex brain region that is involved in the regulation of many behavioral and physiological processes including feeding, arousal, energy balance, stress, reward and motivated behaviors, pain perception, body temperature regulation, digestive functions and blood pressure. Despite noteworthy experimental efforts over the past decades, the circuit, cellular and synaptic bases by which these different processes are regulated by the LH remains incompletely understood. This knowledge gap links in large part to the high cellular heterogeneity of the LH. Fortunately, the rapid evolution of newer genetic and electrophysiological tools is now permitting the selective manipulation, typically genetically-driven, of discrete LH cell populations. This, in turn, permits not only assignment of function to discrete cell groups, but also reveals that considerable synergistic and antagonistic interactions exist between key LH cell populations that regulate feeding and arousal. For example, we now know that while LH melanin-concentrating hormone (MCH) and orexin/hypocretin neurons both function as sensors of the internal metabolic environment, their roles regulating sleep and arousal are actually opposing. Additional studies have uncovered similarly important roles for subpopulations of LH GABAergic cells in the regulation of both feeding and arousal. Herein we review the role of LH MCH, orexin/hypocretin and GABAergic cell populations in the regulation of energy homeostasis (including feeding) and sleep-wake and discuss how these three cell populations, and their subpopulations, may interact to optimize and coordinate metabolism, sleep and arousal

GABAergic parafacial zone is a medullary slow–wave–sleep promoting center

Work in animals and humans suggest the existence of a slow–wave sleep (SWS) promoting/EEG synchronizing center in the mammalian lower brainstem. While sleep–active GABAergic neurons in the medullary parafacial zone (PZ) are needed for normal SWS, it remains unclear if these neurons can initiate and maintain SWS or EEG slow wave activity (SWA) in behaving mice. We used genetically targeted activation and optogenetic–based mapping to uncover the downstream circuitry engaged by SWS–promoting PZ neurons, and we show that this circuit uniquely and potently initiates SWS and EEG SWA, regardless of the time of day. PZ neurons monosynaptically innervate and release synaptic GABA onto parabrachial neurons that in turn project to and release synaptic glutamate onto cortically–projecting neurons of the magnocellular basal forebrain; hence a circuit substrate is in place through which GABAergic PZ neurons can potently trigger SWS and modulate the cortical EEG