Hibernating mammals actively lower their body temperature to reduce energy expenditure when facing food scarcity¹. This ability to induce a hypometabolic state has evoked great interest owing to its potential medical benefits^2,3. Here we show that a hypothalamic neuronal circuit in rodents induces a long-lasting hypothermic and hypometabolic state similar to hibernation. In this state, although body temperature and levels of oxygen consumption are kept very low, the ability to regulate metabolism still remains functional, as in hibernation⁴. There was no obvious damage to tissues and organs or abnormalities in behaviour after recovery from this state. Our findings could enable the development of a method to induce a hibernation-like state, which would have potential applications in non-hibernating mammalian species including humans.

The sleep cycle is characterized by alternating non–rapid eye movement (NREM) and rapid eye movement (REM) sleeps. The mechanisms by which this cycle is generated are incompletely understood. We found that a transient increase of dopamine (DA) in the basolateral amygdala (BLA) during NREM sleep terminates NREM sleep and initiates REM sleep. DA acts on dopamine receptor D2 (Drd2)–expressing neurons in the BLA to induce the NREM-to-REM transition. This mechanism also plays a role in cataplectic attacks—a pathological intrusion of REM sleep into wakefulness—in narcoleptics. These results show a critical role of DA signaling in the BLA in initiating REM sleep and provide a neuronal basis for sleep cycle generation.

Emotionally salient information activates orexin neurons in the lateral hypothalamus, leading to increase in sympathetic outflow and vigilance level. How this circuit alters animals’ behavior remains unknown. Here we report that noradrenergic neurons in the locus coeruleus (NA^LC neurons) projecting to the lateral amygdala (LA) receive synaptic input from orexin neurons. Pharmacogenetic/optogenetic silencing of this circuit as well as acute blockade of the orexin receptor-1 (OX1R) decreases conditioned fear responses. In contrast, optogenetic stimulation of this circuit potentiates freezing behavior against a similar but distinct context or cue. Increase of orexinergic tone by fasting also potentiates freezing behavior and LA activity, which are blocked by pharmacological blockade of OX1R in the LC. These findings demonstrate the circuit involving orexin, NA^LC and LA neurons mediates fear-related behavior and suggests inappropriate excitation of this pathway may cause fear generalization sometimes seen in psychiatric disorders, such as PTSD.

Orexins are hypothalamic neuropeptides that were initially identified in the rat brain as endogenous ligands for an (previously) orphan G-protein-coupled receptor (GPCR). They are multitasking peptides involved in many physiological functions, including regulation of feeding behavior, wakefulness and autonomic/neuroendocrine functions, and sleep/wakefulness states in mammals. There are two isopeptides of orexin, orexin A and orexin B, which are produced from a common precursor peptide, prepro-orexin. Structures of orexins, as well as orexin genes, are highly conserved throughout mammalian species, suggesting strong evolutionary pressure that maintains the structures. Their lengths and structure suggested that orexin B is the ancestral form of the orexin neuropeptide. In mammals, orexins bind to two subtypes of GPCRs, i.e., orexin 1 receptor (OX1R) and orexin 2 receptor (OX2R). Phylogenetically, the orexin system is present exclusively in vertebrates. In genomes of species outside mammals, there is only one orexin receptor, which is similar to OX2R, suggesting that OX2R is the prototype receptor for orexins. OX1R is likely to have evolved during early mammalian evolution. Orexin-producing neurons (orexin neurons) are mainly located in the lateral hypothalamic area (LHA) in mammals and are also found in hypothalamic regions in many other vertebrates. Orexins are likely to be closely related to the regulation of active, motivated behavior in many species. The orexin system seems to have evolved as a system that supports active and purposeful behavior which is closely related with wakefulness.

Emotionally salient situations usually trigger arousal along with autonomic and neuroendocrine reactions. To determine whether the extended amygdala plays a role in sleep–wakefulness regulation, we examined the effects of optogenetic and pharmacogenetic excitation of GABAergic neurons in the bed nucleus of the stria terminalis (GABA^BNST neurons). Acute optogenetic excitation of these cells during nonrapid eye movement (NREM) sleep resulted in an immediate state transition to wakefulness, whereas stimulation during REM sleep showed no effect on sleep–wakefulness states in male mice. An anterograde tracing study suggested GABA^BNST neurons send axonal projections to several brain regions implicated in arousal, including the preoptic area, lateral hypothalamus, periaqueductal gray, deep mesencephalic nucleus, and parabrachial nucleus. A dual orexin receptor antagonist, DORA-22, did not affect the optogenetic transition from NREM sleep to wakefulness. Chemogenetic excitation of GABA^BNST neurons evoked a sustained wakefulness state, but this arousal effect was markedly attenuated by DORA-22. These observations suggest that GABA^BNST neurons play an important role in transition from NREM sleep to wakefulness without the function of orexin neurons, but prolonged excitation of these cells mobilizes the orexin system to sustain wakefulness. SIGNIFICANCE STATEMENT We examined the role of the bed nucleus of the stria terminalis (BNST) in the regulation of wakefulness. Optogenetic excitation of GABAergic neurons in the BNST (GABA^BNST neurons) during nonrapid eye movement (NREM) sleep in mice resulted in immediate transition to a wakefulness state without function of orexins. Prolonged excitation of GABA^BNST neurons by a chemogenetic method evoked a longer-lasting, sustained wakefulness state, which was abolished by preadministration of a dual orexin receptor antagonist, DORA-22. This study revealed a role of the BNST GABAergic system in sleep–wakefulness control, especially in shifting animals9 behavioral states from NREM sleep to wakefulness, and provides an important insight into the pathophysiology of insomnia and the role of orexin in arousal regulation.

The hypothalamus plays an important role in the regulation of sleep/wakefulness states. While the ventrolateral preoptic nucleus (VLPO) plays a critical role in the initiation and maintenance of sleep, the lateral posterior part of the hypothalamus contains neuronal populations implicated in maintenance of arousal, including orexin-producing neurons (orexin neurons) in the lateral hypothalamic area (LHA) and histaminergic neurons in the tuberomammillary nucleus (TMN). During a search for neurons that make direct synaptic contact with histidine decarboxylase-positive (HDC+), histaminergic neurons (HDC neurons) in the TMN and orexin neurons in the LHA of male mice, we found that these arousal-related neurons are heavily innervated by GABAergic neurons in the preoptic area including the VLPO. We further characterized GABAergic neurons electrophysiologically in the VLPO (GABA^VLPO neurons) that make direct synaptic contact with these hypothalamic arousal-related neurons. These neurons (GABA^VLPO→HDC or GABA^{VLPO→orexin} neurons) were both potently inhibited by noradrenaline and serotonin, showing typical electrophysiological characteristics of sleep-promoting neurons in the VLPO. This work provides direct evidence of monosynaptic connectivity between GABA^VLPO neurons and hypothalamic arousal neurons and identifies the effects of monoamines on these neuronal pathways. SIGNIFICANCE STATEMENT Rabies-virus-mediated tracing of input neurons of two hypothalamic arousal-related neuron populations, histaminergic and orexinergic neurons, showed that they receive similar distributions of input neurons in a variety of brain areas, with rich innervation by GABAergic neurons in the preoptic area, including the ventrolateral preoptic area (VLPO), a region known to play an important role in the initiation and maintenance of sleep. Electrophysiological experiments found that GABAergic neurons in the VLPO (GABA^VLPO neurons) that make direct input to orexin or histaminergic neurons are potently inhibited by noradrenaline and serotonin, suggesting that these monoamines disinhibit histamine and orexin neurons. This work demonstrated functional and structural interactions between GABA^VLPO neurons and hypothalamic arousal-related neurons.