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During rapid eye movement (REM) sleep, anti-gravity muscle tone and bodily movements are mostly absent, because somatic motoneurons are inhibited by descending inhibitory pathways. Recent studies showed that glycine/GABA neurons in the ventromedial medulla (VMM; Gly VMM neurons) play an important role in generating muscle atonia during REM sleep (REM-atonia). However, how these REM-atonia-inducing neurons interconnect with other neuronal populations has been unknown. In the present study, we first identified a specific subpopulation of Gly VMM neurons that play an important role in induction of REM-atonia by virus vector-mediated tracing in male mice in which glycinergic neurons expressed Cre recombinase. We found these neurons receive direct synaptic input from neurons in several brain stem regions, including glutamatergic neurons in the sublaterodorsal tegmental nucleus (SLD; Glu SLD neurons). Silencing this circuit by specifically expressing tetanus toxin light chain (TeTNLC) resulted in REM sleep without atonia. This manipulation also caused a marked decrease in time spent in cataplexy-like episodes (CLEs) when applied to narcoleptic orexin-ataxin-3 mice. We also showed that Gly VMM neurons play an important role in maintenance of sleep. This present study identified a population of glycinergic neurons in the VMM that are commonly involved in REM-atonia and cataplexy. SIGNIFICANCE STATEMENT We identified a population of glycinergic neurons in the ventral medulla that plays an important role in inducing muscle atonia during rapid eye movement (REM) sleep. It sends axonal projections almost exclusively to motoneurons in the spinal cord and brain stem except to those that innervate extraocular muscles, while other glycinergic neurons in the same region also send projections to other regions including monoaminergic nuclei. Furthermore, these neurons receive direct inputs from several brainstem regions including glutamatergic neurons in the sublaterodorsal tegmental nucleus (SLD). Genetic silencing of this pathway resulted in REM sleep without atonia and a decrease of cataplexy when applied to narcoleptic mice. This work identified a neural population involved in generating muscle atonia during REM sleep and cataplexy., (Copyright © 2021 the authors.)

Narcolepsy is characterized by chronic sleepiness and cataplexy-sudden muscle paralysis triggered by strong, positive emotions. This condition is caused by a lack of orexin (hypocretin) signaling, but little is known about the neural mechanisms that mediate cataplexy. The amygdala regulates responses to rewarding stimuli and contains neurons active during cataplexy. In addition, lesions of the amygdala reduce cataplexy. Because GABAergic neurons of the central nucleus of the amygdala (CeA) target brainstem regions known to regulate muscle tone, we hypothesized that these cells promote emotion-triggered cataplexy. We injected adeno-associated viral vectors coding for Cre-dependent DREADDs or a control vector into the CeA of orexin knock-out mice crossed with vGAT-Cre mice, resulting in selective expression of the excitatory hM3 receptor or the inhibitory hM4 receptor in GABAergic neurons of the CeA. We measured sleep/wake behavior and cataplexy after injection of saline or the hM3/hM4 ligand clozapine -N- oxide (CNO) under baseline conditions and under conditions that should elicit positive emotions. In mice expressing hM3, CNO approximately doubled the amount of cataplexy in the first 3 h after dosing under baseline conditions. Rewarding stimuli (chocolate or running wheels) also increased cataplexy, but CNO produced no further increase. In mice expressing hM4, CNO reduced cataplexy in the presence of chocolate or running wheels. These results demonstrate that GABAergic neurons of the CeA are sufficient and necessary for the production of cataplexy in mice, and they likely are a key part of the mechanism through which positive emotions trigger cataplexy. SIGNIFICANCE STATEMENT Cataplexy is one of the major symptoms of narcolepsy, but little is known about how strong, positive emotions trigger these episodes of muscle paralysis. Prior research shows that amygdala neurons are active during cataplexy and cataplexy is reduced by lesions of the amygdala. We found that cataplexy is substantially increased by selective activation of GABAergic neurons in the central nucleus of the amygdala (CeA). We also demonstrate that inhibition of these neurons reduces reward-promoted cataplexy. These results build upon prior work to establish the CeA as a crucial element in the neural mechanisms of cataplexy. These results demonstrate the importance of the CeA in regulating responses to rewarding stimuli, shedding light on the broader neurobiology of emotions and motor control., (Copyright © 2017 the authors 0270-6474/17/373995-12$15.00/0.)

Narcolepsy is characterized by chronic sleepiness and cataplexy, episodes of profound muscle weakness that are often triggered by strong, positive emotions. Narcolepsy with cataplexy is caused by a loss of orexin (also known as hypocretin) signaling, but almost nothing is known about the neural mechanisms through which positive emotions trigger cataplexy. Using orexin knock-out mice as a model of narcolepsy, we found that palatable foods, especially chocolate, markedly increased cataplexy and activated neurons in the medial prefrontal cortex (mPFC). Reversible suppression of mPFC activity using an engineered chloride channel substantially reduced cataplexy induced by chocolate but did not affect spontaneous cataplexy. In addition, neurons in the mPFC innervated parts of the amygdala and lateral hypothalamus that contain neurons active during cataplexy and that innervate brainstem regions known to regulate motor tone. These observations indicate that the mPFC is a critical site through which positive emotions trigger cataplexy.

Cataplexy, a sudden unexpected muscle paralysis, is a debilitating symptom of the neurodegenerative sleep disorder, narcolepsy. During these attacks, the person is paralyzed, but fully conscious and aware of their surroundings. To identify potential neurons that might serve as surrogate orexin neurons to suppress such attacks, the gene for orexin (hypocretin), a peptide lost in most human narcoleptics, was delivered into the brains of the orexin-ataxin-3 transgenic mouse model of human narcolepsy. Three weeks after the recombinant adenoassociated virus (rAAV)-mediated orexin gene transfer, sleep-wake behavior was assessed. rAAV-orexin gene delivery into neurons of the zona incerta (ZI), or the lateral hypothalamus (LH) blocked cataplexy. Orexin gene transfer into the striatum or in the melanin-concentrating hormone neurons in the ZI or LH had no such effect, indicating site specificity. In transgenic mice lacking orexin neurons but given rAAV-orexin, detectable levels of orexin-A were evident in the CSF, indicating release of the peptide from the surrogate neurons. Retrograde tracer studies showed that the amygdala innervates the ZI consistent with evidence that strong emotions trigger cataplexy. In turn, the ZI projects to the locus ceruleus, indicating that the ZI is part of a circuit that stabilizes motor tone. Our results indicate that these neurons might also be recruited to block the muscle paralysis in narcolepsy.

Narcolepsy is a human sleep disorder resulting from the loss of neurons containing the neuropeptide orexin, also known as hypocretin. Cataplexy, which is a sudden loss of muscle tone during waking, is an important diagnostic symptom of narcolepsy. In humans and canines with narcolepsy, cataplexy is considered to be a separate and distinct behavioral state. However, in the mouse model of the disease this issue is not resolved. The present study monitored the activity of forty four neurons in the rostral pons in hypocretin knock-out mice. Majority of the neurons were active during wake and REM sleep, while four neurons were selectively active during REM sleep. All of these neurons were less active during cataplexy compared with REM sleep. Thus, although cataplexy and REM sleep share many common features, including the muscle atonia, cataplexy is a distinct state in mice.

Narcolepsy is caused by a lack of orexin (hypocretin), but the physiologic process that underlies the sleepiness of narcolepsy is unknown. Using orexin knock-out (KO) mice as a model of narcolepsy, we critically tested the three leading hypotheses: poor circadian control of sleep and wakefulness, inadequate activation of arousal regions, or abnormal sleep homeostasis. Compared with wild-type (WT) littermates, orexin KO mice had essentially normal amounts of sleep and wake, but wake and non-rapid eye movement (NREM) bouts were very brief, with many more transitions between all behavioral states. In constant darkness, orexin KO mice had normal amplitude and timing of sleep-wake rhythms, providing no evidence for disordered circadian control. When placed in a new, clean cage, both groups of mice remained awake for approximately 45 min, demonstrating that, even in the absence of orexin, fundamental arousal regions can be engaged to produce sustained wakefulness. After depriving mice of sleep for 2-8 hr, orexin KO mice recovered their NREM and rapid eye movement sleep deficits at comparable rates and to the same extent as WT mice, with similar increases in EEG delta power, suggesting that their homeostatic control of sleep is normal. These experiments demonstrate that the fragmented wakefulness of orexin deficiency is not a consequence of abnormal sleep homeostasis, poor circadian control, or defective fundamental arousal systems. Instead, the fragmented behavior of orexin KO mice may be best described as behavioral state instability, with apparently low thresholds to transition between states.