Vasopressin neurons in the paraventricular hypothalamus promote wakefulness via lateral hypothalamic orexin neurons.

The sleep-wakefulness cycle is regulated by complicated neural networks that include many different populations of neurons throughout the brain. Arginine vasopressin neurons in the paraventricular nucleus of the hypothalamus (PVHAVP) regulate various physiological events and behaviors, such as body-fluid homeostasis, blood pressure, stress response, social interaction, and feeding. Changes in arousal level often accompany these PVHAVP-mediated adaptive responses. However, the contribution of PVHAVP neurons to sleep-wakefulness regulation has remained unknown. Here, we report the involvement of PVHAVP neurons in arousal promotion. Optogenetic stimulation of PVHAVP neurons rapidly induced transitions to wakefulness from both NREM and REM sleep. This arousal effect was dependent on AVP expression in these neurons. Similarly, chemogenetic activation of PVHAVP neurons increased wakefulness and reduced NREM and REM sleep, whereas chemogenetic inhibition of these neurons significantly reduced wakefulness and increased NREM sleep. We observed dense projections of PVHAVP neurons in the lateral hypothalamus with potential connections to orexin/hypocretin (LHOrx) neurons. Optogenetic stimulation of PVHAVP neuronal fibers in the LH immediately induced wakefulness, whereas blocking orexin receptors attenuated the arousal effect of PVHAVP neuronal activation drastically. Monosynaptic rabies-virus tracing revealed that PVHAVP neurons receive inputs from multiple brain regions involved in sleep-wakefulness regulation, as well as those involved in stress response and energy metabolism. Moreover, PVHAVP neurons mediated the arousal induced by novelty stress and a melanocortin receptor agonist melanotan-II. Thus, our data suggested that PVHAVP neurons promote wakefulness via LHOrx neurons in the basal sleep-wakefulness and some stressful conditions.

Optogenetic Manipulation of Neural Circuits During Monitoring Sleep/wakefulness States in Mice.

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 (ChR2), is expressed by a virus vector in a particular type of neuronal cells in various Cre-driver mice. Activation of these opsins is triggered by application of light pulses which are delivered by laser or LED through optic cables, and the effect of activation is observed with very high time resolution. Experimenters are able to acutely stimulate neurons while monitoring behavior or another physiological outcome in mice. Optogenetics can enable useful strategies to evaluate function of neuronal circuits in the regulation of sleep/wakefulness states in mice. Here we describe a technique for examining the effect of optogenetic manipulation of neurons with a specific chemical identity during electroencephalogram (EEG) and electromyogram (EMG) monitoring to evaluate the sleep stage of mice. As an example, we describe manipulation of GABAergic neurons in the bed nucleus of the stria terminalis (BNST). Acute optogenetic excitation of these neurons triggers a rapid transition to wakefulness when applied during NREM sleep. Optogenetic manipulation along with EEG/EMG recording can be applied to decipher the neuronal circuits that regulate sleep/wakefulness states.

Excitation of GABAergic Neurons in the Bed Nucleus of the Stria Terminalis Triggers Immediate Transition from Non-Rapid Eye Movement Sleep to Wakefulness in Mice.

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 (GABABNST 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 GABABNST 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 GABABNST neurons evoked a sustained wakefulness state, but this arousal effect was markedly attenuated by DORA-22. These observations suggest that GABABNST 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 (GABABNST neurons) during nonrapid eye movement (NREM) sleep in mice resulted in immediate transition to a wakefulness state without function of orexins. Prolonged excitation of GABABNST 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 animals' 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.