Orexin neurons inhibit sleep to promote arousal.

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.

A Focal Inactivation and Computational Study of Ventrolateral Periaqueductal Gray and Deep Mesencephalic Reticular Nucleus Involvement in Sleep State Switching and Bistability.

Neurons of the ventrolateral periaqueductal gray (vlPAG) and adjacent deep mesencephalic reticular nucleus (DpMe) are implicated in the control of sleep-wake state and are hypothesized components of a flip-flop circuit that maintains sleep bistability by preventing the overexpression of non-rapid eye movement (NREM)/REM sleep intermediary states (NRt). To determine the contribution of vlPAG/DpMe neurons in maintaining sleep bistability we combined computer simulations of flip-flop circuitry with focal inactivation of vlPAG/DpMe neurons by microdialysis delivery of the GABAA receptor agonist muscimol in freely behaving male rats (n = 25) instrumented for electroencephalographic and electromyographic recording. REM sleep was enhanced by muscimol at the vlPAG/DpMe, consistent with previous studies; however, our analyses of NRt dynamics in vivo and those produced by flop-flop circuit simulations show that current thinking is too narrowly focused on the contribution of REM sleep-inactive populations toward vlPAG/DpMe involvement in REM sleep control. We found that much of the muscimol-mediated increase in REM sleep was more appropriately classified as NRt. This loss of sleep bistability was accompanied by fragmentation of REM sleep, as evidenced by an increased number of short REM sleep bouts. REM sleep fragmentation stemmed from an increased number and duration of NRt bouts originating in REM sleep. By contrast, NREM sleep bouts were not likewise fragmented by vlPAG/DpMe inactivation. In flip-flop circuit simulations, these changes could not be replicated through inhibition of the REM sleep-inactive population alone. Instead, combined suppression of REM sleep active and inactive vlPAG/DpMe subpopulations was required to replicate the changes in NRt dynamics.

Neural memory of the genioglossus muscle during sleep is stage-dependent in healthy subjects and obstructive sleep apnoea patients.

KEY POINTS: In most patients with obstructive sleep apnoea (OSA), there is a spontaneous resolution of the breathing disorders during slow wave sleep (SWS) for yet unknown reasons related to non-anatomical factors. Some recently identified forms of neural memory specific of upper airway muscles may play a role in this phenomenon. In the present study, we show for the first time that a form of memory of the genioglossus (tongue) muscle is greatly enhanced during SWS compared to non-rapid eye movement stage 2 sleep. The present study represents a step forward in understanding the mechanisms responsible for the spontaneous development of stable breathing during SWS in OSA patients and may help the discovery of novel therapeutic strategies for this disease. ABSTRACT: Several studies have shown that obstructive sleep apnoea (OSA) improves during slow wave sleep (SWS) for reasons that remain unclear. Recent studies have identified forms of neural memory such as short-term potentiation or after-discharge that can occur in response to upper airway obstruction. Neural memory may play a role in the development of stable breathing during SWS by increasing upper airway muscles activity in this sleep stage. We hypothesize that the after-discharge of the genioglossus muscle following upper airway obstruction is enhanced during SWS compared to non-rapid eye movement stage 2 (N2). During sleep, we performed five-breath drops in continuous positive airway pressure (CPAP-drop) to simulate obstructive events and reflexively activate the genioglossus. Immediately afterwards, CPAP was returned to an optimal level. Once the post-drop ventilation returned to eupnoea, the genioglossus after-discharge was measured as the time it took for genioglossus activity to return to baseline levels. In total, 171 CPAP-drops were analysed from a group of 16 healthy subjects and 19 OSA patients. A mixed-model analysis showed that after-discharge duration during SWS was 208% (95% confidence interval = 112% to 387%, P = 0.022) greater than during N2 after adjusting for covariates (ventilatory drive, CPAP levels). There was also a non-significant trend for a -35% reduction in after-discharge duration following an arousal vs. no-arousal from sleep (95% confidence interval = -59.5% to 5%, P = 0.08). Genioglossus after-discharge is two-fold greater in SWS vs. N2, which could partly explain the breathing stabilization described in OSA patients during this sleep stage.

A resource of potential drug targets and strategic decision-making for obstructive sleep apnoea pharmacotherapy.

There is currently no pharmacotherapy for obstructive sleep apnoea (OSA) but there is no principled a priori reason why there should not be one. This review identifies a rational decision-making strategy with the necessary logical underpinnings that any reasonable approach would be expected to navigate to develop a viable pharmacotherapy for OSA. The process first involves phenotyping an individual to quantify and characterize the critical predisposing factor(s) to their OSA pathogenesis and identify, a priori, if the patient is likely to benefit from a pharmacotherapy that targets those factors. We then identify rational strategies to manipulate those critical predisposing factor(s), and the barriers that have to be overcome for success of any OSA pharmacotherapy. A new analysis then identifies candidate drug targets to manipulate the upper airway motor circuitry for OSA pharmacotherapy. The first conclusion is that there are two general pharmacological approaches for OSA treatment that are of the most potential benefit and are practically realistic, one being fairly intuitive but the second perhaps less so. The second conclusion is that after identifying the critical physiological obstacles to OSA pharmacotherapy, there are current therapeutic targets of high interest for future development. The final analysis provides a tabulated resource of 'druggable' targets that are relatively restricted to the circuitry controlling the upper airway musculature, with these candidate targets being of high priority for screening and further study. We also emphasize that a pharmacotherapy may not cure OSA per se, but may still be a useful adjunct to improve the effectiveness of, and adherence to, other treatment mainstays.

Neuroligin 3 R451C mutation alters electroencephalography spectral activity in an animal model of autism spectrum disorders.

Human studies demonstrate that sleep impairment is a concurrent comorbidity of autism spectrum disorders (ASD), but its etiology remains largely uncertain. One of the prominent theories of ASD suggests that an imbalance in synaptic excitation/inhibition may contribute to various aspects of ASD, including sleep impairments. Following the identification of Nlgn3R451C mutation in patients with ASD, its effects on synaptic transmission and social behaviours have been examined extensively in the mouse model. However, the contributory role of this mutation to sleep impairments in ASD remains unknown. In this study, we showed that Nlgn3R451C knock-in mice, an established genetic model for ASD, exhibited normal duration and distribution of sleep/wake states but significantly altered electroencephalography (EEG) power spectral profiles for wake and sleep.

Evaluating the Evidence Surrounding Pontine Cholinergic Involvement in REM Sleep Generation.

Rapid eye movement (REM) sleep - characterized by vivid dreaming, motor paralysis, and heightened neural activity - is one of the fundamental states of the mammalian central nervous system. Initial theories of REM sleep generation posited that induction of the state required activation of the "pontine REM sleep generator" by cholinergic inputs. Here, we review and evaluate the evidence surrounding cholinergic involvement in REM sleep generation. We submit that: (i) the capacity of pontine cholinergic neurotransmission to generate REM sleep has been firmly established by gain-of-function experiments, (ii) the function of endogenous cholinergic input to REM sleep generating sites cannot be determined by gain-of-function experiments; rather, loss-of-function studies are required, (iii) loss-of-function studies show that endogenous cholinergic input to the PTF is not required for REM sleep generation, and (iv) cholinergic input to the pontine REM sleep generating sites serve an accessory role in REM sleep generation: reinforcing non-REM-to-REM sleep transitions making them quicker and less likely to fail.

Endogenous cholinergic input to the pontine REM sleep generator is not required for REM sleep to occur.

Initial theories of rapid eye movement (REM) sleep generation posited that induction of the state required activation of the pontine subceruleus (SubC) by cholinergic inputs. Although the capacity of cholinergic neurotransmission to contribute to REM sleep generation has been established, the role of cholinergic inputs in the generation of REM sleep is ultimately undetermined as the critical test of this hypothesis (local blockade of SubC acetylcholine receptors) has not been rigorously performed. We used bilateral microdialysis in freely behaving rats (n = 32), instrumented for electroencephalographic and electromyographic recording, to locally manipulate neurotransmission in the SubC with select drugs. As predicted, combined microperfusion of D-AP5 (glutamate receptor antagonist) and muscimol (GABAA receptor agonist) in the SubC virtually eliminated REM sleep. However, REM sleep was not reduced by scopolamine microperfusion in this same region, at a concentration capable of blocking the effects of cholinergic receptor stimulation. This result suggests that transmission of REM sleep drive to the SubC is acetylcholine-independent. Although SubC cholinergic inputs are not majorly involved in REM sleep generation, they may perform a minor function in the reinforcement of transitions into REM sleep, as evidenced by increases in non-REM-to-REM sleep transition duration and failure rate during cholinergic receptor blockade. Cholinergic receptor antagonism also attenuated the normal increase in hippocampal θ oscillations that characterize REM sleep. Using computational modeling, we show that our in vivo results are consistent with a mutually excitatory interaction between the SubC and cholinergic neurons where, importantly, cholinergic neuron activation is gated by SubC activity.

Mechanisms of REM sleep in health and disease.

PURPOSE OF REVIEW: Our understanding of rapid eye movement (REM) sleep and how it is generated remains a topic of debate. Understanding REM sleep mechanisms is important because several sleep disorders result from disturbances in the neural circuits that control REM sleep and its characteristics. This review highlights recent work concerning how the central nervous system regulates REM sleep, and how the make up and breakdown of these REM sleep-generating circuits contribute to narcolepsy, REM sleep behaviour disorder and sleep apnea. RECENT FINDINGS: A complex interaction between brainstem REM sleep core circuits and forebrain and hypothalamic structures is necessary to generate REM sleep. Cholinergic activation and GABAergic inhibition trigger the activation of subcoeruleus neurons, which form the core of the REM sleep circuit. SUMMARY: Untimely activation of REM sleep circuits leads to cataplexy - involuntary muscle weakness or paralysis - a major symptom of narcolepsy. Degeneration of the REM circuit is associated with excessive muscle activation in REM sleep behaviour disorder. Inappropriate arousal from sleep during obstructive sleep apnea repeatedly disturbs the activity of sleep circuits, particularly the REM sleep circuit.

Identification of a pharmacological target for genioglossus reactivation throughout sleep.

STUDY OBJECTIVES: Obstructive sleep apnea (OSA) is a significant public health problem caused by repeated episodes of upper airway closure that occur only during sleep. Attempts to treat OSA pharmacologically have been unsuccessful because there has not been identification of a target operating at cranial motor nuclei, blockade of which can reactivate pharyngeal muscle activity throughout sleep. Increasing potassium conductance is a common mechanism by which state-dependent neuromodulators reduce motoneuron excitability. Therefore, we aimed to determine if potassium channel blockade is an effective strategy to reactivate the pharyngeal musculature throughout sleep. DESIGN PARTICIPANTS AND INTERVENTIONS: In rats chronically instrumented for recording sleep-wake states and respiratory motor activities, we locally microperfused pharmacological agents into the hypoglossal motor pool to modulate potassium channels of three major classes: inwardly rectifying, two-pore domain, and voltage-gated. MEASUREMENTS AND RESULTS: Microperfusion of the inwardly rectifying potassium channel blocker, barium, as well as the voltage-gated potassium channel blockers, tetraethylammonium and 4-aminopyridine, increased tonic and respiratory-related genioglossus activities throughout nonrapid eye movement (non-REM) and rapid eye movement (REM) sleep to 133-300% of levels present during baseline wakefulness. In contrast, microperfusion of methanandamide (TWIK-related acid-sensitive potassium [TASK] channel blocker/cannabinoid receptor agonist) activated genioglossus in wakefulness but not in sleep. CONCLUSIONS: These findings establish proof-of-principle that targeted blockade of certain potassium channels at the hypoglossal motor pool is an effective strategy for reversing upper airway hypotonia and causing sustained reactivation of genioglossus throughout nonrapid eye movement and rapid eye movement sleep. These findings identify an important new direction for translational approaches to the pharmacological treatment of obstructive sleep apnea.

K+ channel modulation causes genioglossus inhibition in REM sleep and is a strategy for reactivation.

Rapid eye movement (REM) sleep is accompanied by periods of upper airway motor suppression that cause hypoventilation and obstructive apneas in susceptible individuals. A common idea has been that upper airway motor suppression in REM sleep is caused by the neurotransmitters glycine and γ-amino butyric acid (GABA) acting at pharyngeal motor pools to inhibit motoneuron activity. Data refute this as a workable explanation because blockade of this putative glycine/GABAergic mechanism releases pharyngeal motor activity in all states, and least of all in REM sleep. Here we summarize a novel motor-inhibitory mechanism that suppresses hypoglossal motor activity largely in REM sleep, this being a muscarinic receptor mechanism linked to G-protein-coupled inwardly rectifying potassium (GIRK) channels. We then outline how this discovery informs efforts to pursue therapeutic targets to reactivate hypoglossal motor activity throughout sleep via potassium channel modulation. One such target is the inwardly rectifying potassium channel Kir2.4 whose expression in the brain is almost exclusive to cranial motor nuclei.

Identification of the mechanism mediating genioglossus muscle suppression in REM sleep.

RATIONALE: Inhibition of pharyngeal motoneurons accompanies REM sleep and is a cause of hypoventilation and obstructive sleep apnea in humans. One explanation posits that the neurotransmitters glycine and γ-aminobutyric acid are responsible for REM sleep motor inhibition. However, blockade of that mechanism at cranial motor nuclei increases motor activity in all sleep-wake states, and least of all in REM sleep, arguing against it as a major mechanism of REM sleep pharyngeal motor inhibition. OBJECTIVES: To identify the mechanism of REM sleep inhibition at the hypoglossal motor pool. METHODS: Genioglossus and diaphragm activities were recorded in 34 rats across sleep-wake states. Microdialysis probes were implanted into the hypoglossal motor pool. MEASUREMENTS AND MAIN RESULTS: Here we show that muscarinic receptor antagonism at the hypoglossal motor pool prevents the inhibition of genioglossus activity throughout REM sleep; likewise, with G-protein-coupled inwardly rectifying potassium (GIRK) channel blockade. Importantly, the genioglossus activating effects of these interventions were largest in REM sleep and minimal or often absent in other sleep-wake states. Finally, we showed that muscarinic inhibition of the genioglossus is functionally linked to GIRK channel activation. CONCLUSIONS: We identify a powerful cholinergic-GIRK channel mechanism operating at the hypoglossal motor pool that has its largest inhibitory influence in REM sleep and minimal or no effects in other sleep-wake states. This mechanism is the major cause of REM sleep inhibition at a pharyngeal motor pool critical for effective breathing.

5-HT1A receptor-responsive pedunculopontine tegmental neurons suppress REM sleep and respiratory motor activity.

Serotonin type 1A (5-HT(1A)) receptor-responsive neurons in the pedunculopontine tegmental nucleus (PPTn) become maximally active immediately before and during rapid eye movement (REM) sleep. A prevailing model of REM sleep generation indicates that activation of such neurons contributes significantly to the generation of REM sleep, and if correct then inactivation of such neurons ought to suppress REM sleep. We test this hypothesis using bilateral microperfusion of the 5-HT(1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, 10 µm) into the PPTn; this tool has been shown to selectively silence REM sleep-active PPTn neurons while the activity of wake/REM sleep-active PPTn neurons is unaffected. Contrary to the prevailing model, bilateral microperfusion of 8-OH-DPAT into the PPTn (n = 23 rats) significantly increased REM sleep both as a percentage of the total recording time and sleep time, compared with both within-animal vehicle controls and between-animal time-controls. This increased REM sleep resulted from an increased frequency of REM sleep bouts but not their duration, indicating an effect on mechanisms of REM sleep initiation but not maintenance. Furthermore, an increased proportion of the REM sleep bouts stemmed from periods of low REM sleep drive quantified electrographically. Targeted suppression of 5-HT(1A) receptor-responsive PPTn neurons also increased respiratory rate and respiratory-related genioglossus activity, and increased the frequency and amplitude of the sporadic genioglossus activations occurring during REM sleep. These data indicate that 5-HT(1A) receptor-responsive PPTn neurons normally function to restrain REM sleep by elevating the drive threshold for REM sleep induction, and restrain the expression of respiratory rate and motor activities.

State-dependent vs. central motor effects of ethanol on breathing.

Ethanol, one of the most widely used drugs in Western society, worsens obstructive sleep apnea in humans. No studies, however, have distinguished between two primary mechanisms that could mediate suppression of genioglossus (GG) activity with ethanol. We test the hypothesis that ethanol suppresses GG activity by effects at the hypoglossal motor pool and/or by state-dependent regulation of motor activity via independent influences on sleep/arousal processes. Intraperitoneal injections of ethanol (1.25 g/kg, n = 6 rats) resulted in maximum blood levels of 125.5 +/- 15.8 mg/dl, i.e., physiologically relevant levels for producing behavioral impairment in rats and humans. Ethanol decreased wakefulness, reduced sleep latency, and increased non-rapid eye movement sleep (P < 0.001, n = 10 rats) and significantly reduced postural muscle tone and electroencephalogram frequencies, consistent with sedation. Ethanol also caused a state-dependent (wakefulness only) decrease in respiratory-related GG activity (P = 0.018) but did not affect diaphragm amplitude or rate, with the magnitude of GG decrease related to baseline activity (P < 0.0002). Ethanol did not alter GG activity when applied to the hypoglossal motor pool (0.025-1 M, n = 16 isoflurane-anesthetized rats). In conclusion, ethanol promoted sleep and altered electroencephalogram and postural motor activities, indicative of sedation. The lack of effect on GG with ethanol at the hypoglossal motor pool indicates that the GG and postural motor suppression following systemic administration was mediated via effects on state-dependent/arousal-related processes. These data show that ethanol can suppress GG by primary influences on state-dependent aspects of central nervous system function independent of effects on the respiratory network per se, a distinction that has not previously been identified experimentally.