Computational model of brain-stem circuit for state-dependent control of hypoglossal motoneurons.

In patients with obstructive sleep apnea (OSA), the pharyngeal muscles become relaxed during sleep, which leads to a partial or complete closure of upper airway. Experimental studies suggest that withdrawal of noradrenergic and serotonergic drives importantly contributes to depression of hypoglossal motoneurons and, therefore, may contribute to OSA pathophysiology; however, specific cellular and synaptic mechanisms remain unknown. In this new study, we developed a biophysical network model to test the hypothesis that, to explain experimental observations, the neuronal network for monoaminergic control of excitability of hypoglossal motoneurons needs to include excitatory and inhibitory perihypoglossal interneurons that mediate noradrenergic and serotonergic drives to hypoglossal motoneurons. In the model, the state-dependent activation of the hypoglossal motoneurons was in qualitative agreement with in vivo data during simulated rapid eye movement (REM) and non-REM sleep. The model was applied to test the mechanisms of action of noradrenergic and serotonergic drugs during REM sleep as observed in vivo. We conclude that the proposed minimal neuronal circuit is sufficient to explain in vivo data and supports the hypothesis that perihypoglossal interneurons may mediate state-dependent monoaminergic drive to hypoglossal motoneurons. The population of the hypothesized perihypoglossal interneurons may serve as novel targets for pharmacological treatment of OSA. NEW & NOTEWORTHY In vivo studies suggest that during rapid eye movement sleep, withdrawal of noradrenergic and serotonergic drives critically contributes to depression of hypoglossal motoneurons (HMs), which innervate the tongue muscles. By means of a biophysical model, which is consistent with a broad range of empirical data, we demonstrate that the neuronal network controlling the excitability of HMs needs to include excitatory and inhibitory interneurons that mediate noradrenergic and serotonergic drives to HMs.

Chronic intermittent hypoxia attenuates noradrenergic innervation of hypoglossal motor nucleus.

The state-dependent noradrenergic activation of hypoglossal motoneurons plays an important role in the maintenance of upper airway patency and pathophysiology of obstructive sleep apnea (OSA). Chronic intermittent hypoxia (CIH), a major pathogenic factor of OSA, contributes to the risk for developing neurodegenerative disorders in OSA patients. Using anterograde tracer, channelrhodopsin-2, we mapped axonal projections from noradrenergic A7 and SubCoeruleus neurons to hypoglossal nucleus in DBH-cre mice and assessed the effect of CIH on these projections. We found that CIH significantly reduced the number of axonal projections from SubCoeruleus neurons to both dorsal (by 68%) and to ventral (by73%) subregions of the hypoglossal motor nucleus compared to sham-treated animals. The animals' body weight was also negatively affected by CIH. Both effects, the decrease in axonal projections and body weight, were more pronounced in male than female mice, which was likely caused by less sensitivity of female mice to CIH as compared to males. The A7 neurons appeared to have limited projections to the hypoglossal nucleus. Our findings suggest that CIH-induced reduction of noradrenergic innervation of hypoglossal motoneurons may exacerbate progression of OSA, especially in men.

Activity of pontine A7 noradrenergic neurons is suppressed during REM sleep.

The activity of hypoglossal motoneurons plays an important role in the maintenance of upper airway patency. Both withdrawal of noradrenergic excitatory drive and increase of cholinergic inhibition markedly decrease excitability of hypoglossal motoneurons during sleep and especially during rapid-eye-movement (REM) stage. This leads to increased collapsibility of upper airway during sleep, which is the major neurological factor of obstructive sleep apnea (OSA) pathophysiology. Anatomical and functional data suggest that noradrenergic A7 neurons are the main source of noradrenergic drive to hypoglossal motoneurons. However, it is unknown whether the behavior of A7 neurons during sleep-wake cycle is in accord with their proposed involvement in sleep-related depression of hypoglossal motoneuron activity. Therefore, we sought to assess the behavior of A7 neurons during sleep and wakefulness in naturally sleeping head-restrained rats. We have found that, similar to other pontine noradrenergic neurons, the putative A7 noradrenergic neurons fired with relatively long-lasting action potentials with a low-frequency regular discharge. Importantly, noradrenergic A7 neurons were predominantly silent during REM sleep. The REM-off activity of the A7 neurons supports our hypothesis that these neurons may significantly contribute to the withdrawal of excitatory noradrenergic drive from upper airway motoneurons during REM sleep and, consequently, play an essential role in maintaining upper airway patency and pathophysiology of OSA. Therefore, noradrenergic A7 neurons may serve as an additional target for designing pharmacological approaches to treat OSA.NEW & NOTEWORTHY Noradrenergic A7 neurons are mostly silent during REM sleep. This is in accord with their role in the control of upper airway muscles and important contribution to OSA pathophysiology. Therefore, a modulation of A7 neuron activity can serve as a novel therapeutic target for pharmacological treatment of OSA.

Chronic intermittent hypoxia alters density of aminergic terminals and receptors in the hypoglossal motor nucleus.

RATIONALE: Patients with obstructive sleep apnea (OSA) adapt to the anatomical vulnerability of their upper airway by generating increased activity in upper airway-dilating muscles during wakefulness. Norepinephrine (NE) and serotonin (5-HT) mediate, through α1-adrenergic and 5-HT2A receptors, a wake-related excitatory drive to upper airway motoneurons. In patients with OSA, this drive is necessary to maintain their upper airway open. We tested whether chronic intermittent hypoxia (CIH), a major pathogenic factor of OSA, affects aminergic innervation of XII motoneurons that innervate tongue-protruding muscles in a manner that could alter their airway-dilatory action. OBJECTIVES: To determine the impact of CIH on neurochemical markers of NE and 5-HT innervation of the XII nucleus. METHODS: NE and 5-HT terminal varicosities and α1-adrenergic and 5-HT2A receptors were immunohistochemically visualized and quantified in the XII nucleus in adult rats exposed to CIH or room air exchanges for 10 h/d for 34 to 40 days. MEASUREMENTS AND MAIN RESULTS: CIH-exposed rats had approximately 40% higher density of NE terminals and approximately 20% higher density of 5-HT terminals in the ventromedial quadrant of the XII nucleus, the region that controls tongue protruder muscles, than sham-treated rats. XII motoneurons expressing α1-adrenoceptors were also approximately 10% more numerous in CIH rats, whereas 5-HT2A receptor density tended to be lower in CIH rats. CONCLUSIONS: CIH-elicited increase of NE and 5-HT terminal density and increased expression of α1-adrenoceptors in the XII nucleus may lead to augmentation of endogenous aminergic excitatory drives to XII motoneurons, thereby contributing to the increased upper airway motor tone in patients with OSA.

Hypoglossal premotor neurons of the intermediate medullary reticular region express cholinergic markers.

The inspiratory drive to hypoglossal (XII) motoneurons originates in the caudal medullary intermediate reticular (IRt) region. This drive is mainly glutamatergic, but little is known about the neurochemical features of IRt XII premotor neurons. Prompted by the evidence that XII motoneuronal activity is controlled by both muscarinic (M) and nicotinic cholinergic inputs and that the IRt region contains cells that express choline acetyltransferase (ChAT), a marker of cholinergic neurons, we investigated whether some IRt XII premotor neurons are cholinergic. In seven rats, we applied single-cell reverse transcription-polymerase chain reaction to acutely dissociated IRt neurons retrogradely labeled from the XII nucleus. We found that over half (21/37) of such neurons expressed mRNA for ChAT and one-third (13/37) also had M2 receptor mRNA. In contrast, among the IRt neurons not retrogradely labeled, only 4 of 29 expressed ChAT mRNA (P < 0.0008) and only 3 of 29 expressed M2 receptor mRNA (P < 0.04). The distributions of other cholinergic receptor mRNAs (M1, M3, M4, M5, and nicotinic alpha4-subunit) did not differ between IRt XII premotor neurons and unlabeled IRt neurons. In an additional three rats with retrograde tracers injected into the XII nucleus and ChAT immunohistochemistry, 5-11% of IRt XII premotor neurons located at, and caudal to, the area postrema were ChAT positive, and 27-48% of ChAT-positive caudal IRt neurons were retrogradely labeled from the XII nucleus. Thus the pre- and postsynaptic cholinergic effects previously described in XII motoneurons may originate, at least in part, in medullary IRt neurons.

Neuroanatomical Basis of State-Dependent Activity of Upper Airway Muscles.

Obstructive Sleep Apnea (OSA) is a common sleep-related respiratory disorder that is associated with cognitive, cardiovascular, and metabolic morbidities. The major cause of OSA is the sleep-related reduction of upper airway muscle tone that leads to airway obstructions in individuals with anatomically narrow upper airway. This reduction is mainly due to the suppressant effect of sleep on hypoglossal motoneurons that innervate upper airway muscles. The hypoglossal motoneurons have state-dependent activity, which is decreased during the transition from wakefulness to non-rapid eye movement sleep and is further suppressed during rapid eye movement sleep. Multiple neurotransmitters and their receptors have been implicated in the control of hypoglossal motoneuron activity across the sleep-wake states. However, to date, the results of the rigorous testing show that withdrawal of noradrenergic excitation and cholinergic inhibition essentially contribute to the depression of hypoglossal motoneuron activity during sleep. The present review will focus on origins of noradrenergic and cholinergic innervation of hypoglossal motoneurons and the functional role of these neurons in the state-dependent activity of hypoglossal motoneurons.

Differential pontomedullary catecholaminergic projections to hypoglossal motor nucleus and viscerosensory nucleus of the solitary tract.

In individuals with a narrow or collapsible upper airway, sleep-related hypotonia of upper airway muscles leads to recurrent airway obstructions. Brainstem noradrenergic neurons reduce their activity during slow-wave sleep and become silent during rapid eye movement sleep; this may cause state-dependent changes in the motor output and reflexes. The loss of noradrenergic excitation is a major cause of sleep-related depression of activity in upper airway muscles innervated by the hypoglossal nerve. Our goal was to identify and compare the pontomedullary sources of catecholaminergic (CA) projections to the hypoglossal motor nucleus (Mo12) and the adjacent viscerosensory nucleus of the solitary tract (NTS). In 10 Sprague-Dawley rats, retrograde tracers, Fluoro-Gold or B sub-unit of cholera toxin, were microinjected (5-20nl) into the Mo12, NTS, or both nuclei. Tyrosine hydroxylase (TH) was used as a marker for CA neurons. Following tracer injections into the Mo12, retrogradely labeled and TH-positive neurons were found in the A1/C1 (18.5%), A5 (43.5%), A7 (15.0%), and sub-coeruleus (21.0%) regions, and locus coeruleus (1.7%). In contrast, following injections into the NTS, these proportions were: 48.0, 46.5, 0.2, 0.9, and 4.3%, respectively. The projections to both nuclei were bilateral, with a 3:2 ipsilateral predominance. In four animals with one tracer injected into the Mo12 and the other in NTS, TH-positive cells containing both tracers were found only in the A5 region. Thus, the pontomedullary sources of CA projections to the Mo12 and NTS differ, with only A1/C1 and A5 groups having significant projections to these two functionally distinct targets.

Mesopontine cholinergic projections to the hypoglossal motor nucleus.

Mesopontine cholinergic (ACh) neurons have increased discharge during wakefulness, rapid eye movement (REM) sleep, or both. Hypoglossal (12) motoneurons, which play an important role in the control of upper airway patency, are postsynaptically excited by stimulation of nicotinic receptors, whereas muscarinic receptors presynaptically inhibit inputs to 12 motoneurons. These data suggest that ACh contributes to sleep/wake-related changes in the activity of 12 motoneurons by acting within the hypoglossal motor nucleus (Mo12), but the origins of ACh projections to Mo12 are not well established. We used retrograde tracers to assess the projections of ACh neurons of the mesopontine pedinculopontine tegmental (PPT) and laterodorsal tegmental (LDT) nuclei to the Mo12. In six Sprague-Dawley rats, Fluorogold or B subunit of cholera toxin, were pressure injected (5-20nl) into the Mo12. Retrogradely labeled neurons, identified as ACh using nitric oxide synthase (NOS) immunohistochemistry, were found bilaterally in discrete subregions of both PPT and LDT nuclei. Most retrogradely labeled PPT cells (96%) were located in the PPT pars compacta region adjacent to the ventrolateral tip of the superior cerebellar peduncle. In the LDT, retrogradely labeled neurons were located exclusively in its pars alpha region. Over twice as many ACh neurons projecting to the Mo12 were located in the PPT than LDT. The results demonstrate direct mesopontine ACh projections to the Mo12. These projections may contribute to the characteristic of wakefulness and REM sleep increases, as well as REM sleep-related decrements, of 12 motoneuronal activity.

Kölliker-Fuse GABAergic and glutamatergic neurons project to distinct targets.

The Kölliker-Fuse nucleus (KF) is known primarily for its respiratory function as the "pneumotaxic center" or "pontine respiratory group." Considered part of the parabrachial (PB) complex, KF contains glutamatergic neurons that project to respiratory-related targets in the medulla and spinal cord (Yokota, Oka, Tsumori, Nakamura, & Yasui, 2007). Here we describe an unexpected population of neurons in the caudal KF and adjacent lateral crescent subnucleus (PBlc), which are γ-aminobutyric acid (GABA)ergic and have an entirely different pattern of projections than glutamatergic KF neurons. First, immunofluorescence, in situ hybridization, and Cre-reporter labeling revealed that many of these GABAergic neurons express FoxP2 in both rats and mice. Next, using Cre-dependent axonal tracing in Vgat-IRES-Cre and Vglut2-IRES-Cre mice, we identified different projection patterns from GABAergic and glutamatergic neurons in this region. GABAergic neurons in KF and PBlc project heavily and almost exclusively to trigeminal sensory nuclei, with minimal projections to cardiorespiratory nuclei in the brainstem, and none to the spinal cord. In contrast, glutamatergic KF neurons project heavily to the autonomic, respiratory, and motor regions of the medulla and spinal cord previously identified as efferent targets mediating KF cardiorespiratory effects. These findings identify a novel, GABAergic subpopulation of KF/PB neurons with a distinct efferent projection pattern targeting the brainstem trigeminal sensory system. Rather than regulating breathing, we propose that these neurons influence vibrissal sensorimotor function.

Catecholaminergic A1/C1 neurons contribute to the maintenance of upper airway muscle tone but may not participate in NREM sleep-related depression of these muscles.

Neural mechanisms of obstructive sleep apnea, a common sleep-related breathing disorder, are incompletely understood. Hypoglossal motoneurons, which provide tonic and inspiratory activation of genioglossus (GG) muscle (a major upper airway dilator), receive catecholaminergic input from medullary A1/C1 neurons. We aimed to determine the contribution of A1/C1 neurons in control of GG muscle during sleep and wakefulness. To do so, we placed injections of a viral vector into DBH-cre mice to selectively express the hMD4i inhibitory chemoreceptors in A1/C1 neurons. Administration of the hM4Di ligand, clozapine-N-oxide (CNO), in these mice decreased GG muscle activity during NREM sleep (F1,1,3=17.1, p<0.05); a similar non-significant decrease was observed during wakefulness. CNO administration had no effect on neck muscle activity, respiratory parameters or state durations. In addition, CNO-induced inhibition of A1/C1 neurons did not alter the magnitude of the naturally occurring depression of GG activity during transitions from wakefulness to NREM sleep. These findings suggest that A1/C1 neurons have a net excitatory effect on GG activity that is most likely mediated by hypoglossal motoneurons. However, the activity of A1/C1 neurons does not appear to contribute to NREM sleep-related inhibition of GG muscle activity, suggesting that A1/C1 neurons regulate upper airway patency in a state-independent manner.

Lingual muscle activity across sleep-wake States in rats with surgically altered upper airway.

Obstructive sleep apnea (OSA) patients have increased upper airway muscle activity, including such lingual muscles as the genioglossus (GG), geniohyoid (GH), and hyoglossus (HG). This adaptation partially protects their upper airway against obstructions. Rodents are used to study the central neural control of sleep and breathing but they do not naturally exhibit OSA. We investigated whether, in chronically instrumented, behaving rats, disconnecting the GH and HG muscles from the hyoid (H) apparatus would result in a compensatory increase of other upper airway muscle activity (electromyogram, EMG) and/or other signs of upper airway instability. We first determined that, in intact rats, lingual (GG and intrinsic) muscles maintained stable activity levels when quantified based on 2 h-long recordings conducted on days 6 through 22 after instrumentation. We then studied five rats in which the tendons connecting the GH and HG muscles to the H apparatus were experimentally severed. When quantified across all recording days, lingual EMG during slow-wave sleep (SWS) was modestly but significantly increased in rats with surgically altered upper airway [8.6 ± 0.7% (SE) vs. 6.1 ± 0.7% of the mean during wakefulness; p = 0.012]. Respiratory modulation of lingual EMG occurred mainly during SWS and was similarly infrequent in both groups, and the incidence of sighs and central apneas also was similar. Thus, a weakened action of selected lingual muscles did not produce sleep-disordered breathing but resulted in a relatively elevated activity in other lingual muscles during SWS. These results encourage more extensive surgical manipulations with the aim to obtain a rodent model with collapsible upper airway.

Respiratory modulation of lingual muscle activity across sleep-wake states in rats.

In obstructive sleep apnea (OSA) patients, inspiratory activation (IA) of lingual muscles protects the upper airway from collapse. We aimed to determine when rats' lingual muscles exhibit IA. In 5 Sprague-Dawley and 3 Wistar rats, we monitored cortical EEG and lingual, diaphragmatic and nuchal electromyograms (EMGs), and identified segments of records when lingual EMG exhibited IA. Individual segments lasted 2.4-269 s (median: 14.5 s), most (89%) occurred during slow-wave sleep (SWS), and they collectively occupied 0.3-6.1% of the total recording time. IA usually started to increase with a delay after SWS onset and ended with an arousal, or declined prior to rapid eye movement sleep. IA of lingual EMG was not accompanied by increased diaphragmatic activity or respiratory rate changes, but occurred when cortical EEG power was particularly low in a low beta-1 frequency range (12.5-16.4 Hz). A deep SWS-related activation of upper airway muscles may be an endogenous phenomenon designed to protect the upper airway against collapse.

Rats subjected to chronic-intermittent hypoxia have increased density of noradrenergic terminals in the trigeminal sensory and motor nuclei.

Rodents subjected to chronic intermittent hypoxia (CIH) are used to investigate the mechanisms underlying the consequences of the obstructive sleep apnea (OSA) syndrome. Following CIH, rats have an increased density of noradrenergic terminals in the hypoglossal motor nucleus which innervates lingual muscles that protect the upper airway from collapse in OSA patients. Here, we investigated whether such an increase also occurs in other brainstem nuclei. Six pairs of male Sprague-Dawley rats were exposed to CIH or sham treatment for 10h/day for 35 days, with O(2) level oscillating between 24% and 7% every 3min. Brainstem sections were immunohistochemically processed for dopamine-ß-hydroxylase, a marker for norepinephrine. Noradrenergic terminal varicosities were counted in the center of the trigeminal motor nucleus (Mo5) and the interpolar part of the spinal trigeminal sensory nucleus (Sp5). In the Mo5, noradrenergic varicosities tended to be 9% more numerous in CIH- than sham-treated rats, and in the Sp5 they were 18% more numerous in CIH rats (184±9 vs. 156±8 per 100×100µm counting box; p=0.03, n=18 section pairs).These data suggest that CIH elicits sprouting of noradrenergic terminals in multiple motor and sensory regions of the lower brainstem. This may alter motor and cardiorespiratory outputs and the transmission of cardiorespiratory and motor reflexes in CIH rats and, by implication, in OSA patients.

Antagonism of alpha1-adrenergic and serotonergic receptors in the hypoglossal motor nucleus does not prevent motoneuronal activation elicited from the posterior hypothalamus.

The perifornical (PF) region of the posterior hypothalamus plays an important role in the regulation of sleep-wake states and motor activity. Disinhibition of PF neurons by the GABA(A) receptor antagonist, bicuculline, has been used to study the mechanisms of wake- and motor activity-promoting effects that emanate from the PF region. Bicuculline activates PF neurons, including the orexin-containing cells that have major excitatory projections to brainstem noradrenergic and serotonergic neurons. Since premotor aminergic neurons are an important source of motoneuronal activation, we hypothesized that they mediate the excitation of motoneurons that results from disinhibition of PF neurons with bicuculline. In urethane-anesthetized, paralyzed and artificially ventilated rats, we found that PF bicuculline injections (1mM, 20 nl) made after combined microinjections into the hypoglossal (XII) nucleus of alpha(1)-adrenergic and serotonergic receptor antagonists (prazosin and methysergide) increased XII nerve activity by 80+/-16% (SE) of the control activity level. Thus, activation of XII motoneurons originating in the hypothalamic PF region was not abolished despite effective elimination by the aminergic antagonists of the endogenous noradrenergic and serotonergic excitatory drives to XII motoneurons and abolition of XII motoneuronal activation by exogenous serotonin or phenylephrine. These results show that a major component of XII motoneuronal activation originating in the posterior hypothalamus is mediated by pathways other than the noradrenergic and serotonergic projections to motoneurons.

Disinhibition of perifornical hypothalamic neurones activates noradrenergic neurones and blocks pontine carbachol-induced REM sleep-like episodes in rats.

Studies in behaving animals suggest that neurones located in the perifornical (PF) region of the posterior hypothalamus promote wakefulness and suppress sleep. Among such cells are those that synthesize the excitatory peptides, orexins (ORX). Lack of ORX, or their receptors, is associated with narcolepsy/cataplexy, a disorder characterized by an increased pressure for rapid eye movement (REM) sleep. We used anaesthetized rats in which pontine microinjections of a cholinergic agonist, carbachol, can repeatedly elicit REM sleep-like episodes to test whether activation of PF cells induced by antagonism of endogenous, GABA(A) receptor-mediated, inhibition suppresses the ability of the brainstem to generate REM sleep-like state. Microinjections of the GABA(A) receptor antagonist, bicuculline (20 nl, 1 mm), into the PF region elicited cortical and hippocampal activation, increased the respiratory rate and hypoglossal nerve activity, induced c-fos expression in ORX and other PF neurones, and increased c-fos expression in pontine A7 and other noradrenergic neurones. The ability of pontine carbachol to elicit any cortical, hippocampal or brainstem component of the REM sleep-like response was abolished during the period of bicuculline-induced activation. The activating and REM sleep-suppressing effect of PF bicuculline was not attenuated by systemic administration of the ORX type 1 receptor antagonist, SB334867. Thus, activation of PF neurones that are endogenously inhibited by GABA(A) receptors is sufficient to turn off the brainstem REM sleep-generating network; the effect is, at least in part, due to activation of pontine noradrenergic neurones, but is not mediated by ORX type 1 receptors. A malfunction of the pathway that originates in GABA(A) receptor-expressing PF neurones may cause narcolepsy/cataplexy.