Effects of acetylcholine on hypoglossal and C4 nerve activity in brainstem-spinal cord preparations from newborn rat.

Effects of acetylcholine (ACh) on respiratory activity have been an intriguing theme especially in relation to central chemoreception and the control of hypoglossal nerve activity. We studied the effects of ACh on hypoglossal and phrenic (C4) nerve activities and inspiratory and pre-inspiratory neurons in the rostral ventrolateral medulla in brainstem-spinal cord preparations from newborn rats. ACh application increased respiratory rhythm, decreased inspiratory hypoglossal and C4 nerve burst amplitude, and enhanced pre-inspiratory hypoglossal activity. ACh induced membrane depolarization of pre-inspiratory neurons that might be involved in facilitation of respiratory rhythm by ACh. Effects of ACh on hypoglossal and C4 nerve activity were partially reversed by a nicotinic receptor blocker, mecamylamine. Further application of a muscarinic receptor antagonist, oxybutynin, resulted in slight increase of hypoglossal (but not C4) burst amplitude. Thus, ACh induced different effects on hypoglossal and C4 nerve activity in the brainstem-spinal cord preparation.

Deficiency of Biogenic Amines Modulates the Activity of Hypoglossal Nerve in the Reserpine Model of Parkinson's Disease.

The underlying cause of respiratory impairments appearing in Parkinson's disease (PD) is still far from being elucidated. To better understand the pathogenesis of respiratory disorders appearing in PD, we studied hypoglossal (HG) and phrenic (PHR) motoneuron dysfunction in a rat model evoked with reserpine administration. After reserpine, a decrease in the baseline amplitude and minute HG activity was noted, and no depressive phase of the hypoxic ventilatory response was observed. The pre-inspiratory time of HG activity along with the ratio of pre-inspiratory time to total respiratory cycle time and the ratio of pre-inspiratory to inspiratory amplitude were significantly reduced during normoxia, hypoxia, and recovery compared to sham rats. We suggest that the massive depletion of not only dopamine, but above all noradrenaline and serotonin in the brainstem observed in our study, has an impact on the pre-inspiratory activity of the HG. The shortening of the pre-inspiratory activity of the HG in the reserpine model may indicate a serious problem with maintaining the correct diameter of the upper airways in the preparation phase for inspiratory effort and explain the development of obstructive sleep apnea in some PD patients. Therapies involving the supplementation of amine depletion other than dopamine should be considered.

The pontine Kölliker-Fuse nucleus gates facial, hypoglossal, and vagal upper airway related motor activity.

The pontine Kölliker-Fuse nucleus (KFn) is a core nucleus of respiratory network that mediates the inspiratory-expiratory phase transition and gates eupneic motor discharges in the vagal and hypoglossal nerves. In the present study, we investigated whether the same KFn circuit may also gate motor activities that control the resistance of the nasal airway, which is of particular importance in rodents. To do so, we simultaneously recorded phrenic, facial, vagal and hypoglossal cranial nerve activity in an in situ perfused brainstem preparation before and after bilateral injection of the GABA-receptor agonist isoguvacine (50-70 nl, 10 mM) into the KFn (n = 11). Our results show that bilateral inhibition of the KFn triggers apneusis (prolonged inspiration) and abolished pre-inspiratory discharge of facial, vagal and hypoglossal nerves as well as post-inspiratory discharge in the vagus. We conclude that the KFn plays a critical role for the eupneic regulation of naso-pharyngeal airway patency and the potential functions of the KFn in regulating airway patency and orofacial behavior is discussed.

Designer Receptors Exclusively Activated by Designer Drugs Approach to Treatment of Sleep-disordered Breathing.

Rationale: Obstructive sleep apnea is recurrent upper airway obstruction caused by a loss of upper airway muscle tone during sleep. The main goal of our study was to determine if designer receptors exclusively activated by designer drugs (DREADD) could be used to activate the genioglossus muscle as a potential novel treatment strategy for sleep apnea. We have previously shown that the prototypical DREADD ligand clozapine-N-oxide increased pharyngeal diameter in mice expressing DREADD in the hypoglossal nucleus. However, the need for direct brainstem viral injections and clozapine-N-oxide toxicity diminished translational potential of this approach, and breathing during sleep was not examined.Objectives: Here, we took advantage of our model of sleep-disordered breathing in diet-induced obese mice, retrograde properties of the adeno-associated virus serotype 9 (AAV9) viral vector, and the novel DREADD ligand J60.Methods: We administered AAV9-hSyn-hM3(Gq)-mCherry or control AAV9 into the genioglossus muscle of diet-induced obese mice and examined the effect of J60 on genioglossus activity, pharyngeal patency, and breathing during sleep.Measurements and Main Results: Compared with control, J60 increased genioglossus tonic activity by greater than sixfold and tongue uptake of 2-deoxy-2-[18F]fluoro-d-glucose by 1.5-fold. J60 increased pharyngeal patency and relieved upper airway obstruction during non-REM sleep.Conclusions: We conclude that following intralingual administration of AAV9-DREADD, J60 can activate the genioglossus muscle and improve pharyngeal patency and breathing during sleep.

Tongue and hypoglossal morphology after intralingual cholera toxin B-saporin injection.

INTRODUCTION: We recently developed an inducible model of dysphagia using intralingual injection of cholera toxin B conjugated to saporin (CTB-SAP) to cause death of hypoglossal neurons. In this study we aimed to evaluate tongue morphology and ultrastructural changes in hypoglossal neurons and nerve fibers in this model. METHODS: Tissues were collected from 20 rats (10 control and 10 CTB-SAP animals) on day 9 post-injection. Tongues were weighed, measured, and analyzed for microscopic changes using laminin immunohistochemistry. Hypoglossal neurons and axons were examined using transmission electron microscopy. RESULTS: The cross-sectional area of myofibers in the posterior genioglossus was decreased in CTB-SAP-injected rats. Degenerative changes were observed in both the cell bodies and distal axons of hypoglossal neurons. DISCUSSION: Preliminary results indicate this model may have translational application to a variety of neurodegenerative diseases resulting in tongue dysfunction and associated dysphagia.

Influences of GABAergic Inhibition in the Dorsal Medulla on Contralateral Swallowing Neurons in Rats.

OBJECTIVES: We aimed to examine the effect of unilateral inhibition of the medullary dorsal swallowing networks on the activities of swallowing-related cranial motor nerves and swallowing interneurons. METHODS: In 25 juvenile rats, we recorded bilateral vagal nerve activity (VNA) as well as unilateral phrenic and hypoglossal activity (HNA) during fictive swallowing elicited by electrical stimulation of the superior laryngeal nerve during control and following microinjection of the GABA agonist muscimol into the caudal dorsal medulla oblongata in a perfused brainstem preparation. In 20 animals, swallowing interneurons contralateral to the muscimol injection side were simultaneously recorded extracellularly and their firing rates were analyzed during swallowing. RESULTS: Integrated VNA and HNA to the injection side decreased to 49.0 ± 16.6% and 32.3 ± 17.9%, respectively. However, the VNA on the uninjected side showed little change after muscimol injection. Following local inhibition, 11 out of 20 contralateral swallowing interneurons showed either increased or decreased of their respective firing discharge during evoked-swallowing, while no significant changes in activity were observed in the remaining nine neurons. CONCLUSION: The neuronal networks underlying the swallowing pattern generation in the dorsal medulla mediate the ipsilateral motor outputs and modulate the contralateral activity of swallowing interneurons, suggesting that the bilateral coordination of the swallowing central pattern generator regulates the spatiotemporal organization of pharyngeal swallowing movements. LEVEL OF EVIDENCE: NA Laryngoscope, 131:2187-2198, 2021.

Muscarinic Inhibition of Hypoglossal Motoneurons: Possible Implications for Upper Airway Muscle Hypotonia during REM Sleep.

Proper function of pharyngeal dilator muscles, including the genioglossus muscle of the tongue, is required to maintain upper airway patency. During sleep, the activity of these muscles is suppressed, and as a result individuals with obstructive sleep apnea experience repeated episodes of upper airway closure when they are asleep, in particular during rapid-eye-movement (REM) sleep. Blocking cholinergic transmission in the hypoglossal motor nucleus (MoXII) restores REM sleep genioglossus activity, highlighting the importance of cholinergic transmission in the inhibition of hypoglossal motor neurons (HMNs) during REM sleep. Glutamatergic afferent input from neurons in the parahypoglossal (PH) region to the HMNs is critical for MoXII respiratory motor output. We hypothesized that state-dependent cholinergic regulation may be mediated by this pathway. Here we studied the effects of cholinergic transmission in HMNs in adult male and female mice using patch-clamp recordings in brain slices. Using channelrhodopsin-2-assisted circuit mapping, we first demonstrated that PH glutamatergic neurons directly and robustly activate HMNs (PHGlut → HMNs). We then show that carbachol consistently depresses this input and that this effect is presynaptic. Additionally, carbachol directly affects HMNs by a variable combination of muscarinic-mediated excitatory and inhibitory responses. Altogether, our results suggest that cholinergic signaling impairs upper airway dilator muscle activity by suppressing glutamatergic input from PH premotoneurons to HMNs and by directly inhibiting HMNs. Our findings highlight the complexity of cholinergic control of HMNs at both the presynaptic and postsynaptic levels and provide a possible mechanism for REM sleep suppression of upper airway muscle activity.SIGNIFICANCE STATEMENT Individuals with obstructive sleep apnea can breathe adequately when awake but experience repeated episodes of upper airway closure when asleep, in particular during REM sleep. Similar to skeletal postural muscles, pharyngeal dilator muscles responsible for maintaining an open upper airway become hypotonic during REM sleep. Unlike spinal motoneurons controlling postural muscles that are inhibited by glycinergic transmission during REM sleep, hypoglossal motoneurons that control the upper airway muscles are inhibited in REM sleep by the combination of monoaminergic disfacilitation and cholinergic inhibition. In this study, we demonstrated how cholinergic signaling inhibits hypoglossal motoneurons through presynaptic and postsynaptic muscarinic receptors. Our results provide a potential mechanism for upper airway hypotonia during REM sleep.

Doxapram stimulates respiratory activity through distinct activation of neurons in the nucleus hypoglossus and the pre-Bötzinger complex.

Doxapram is a respiratory stimulant used for decades as a treatment option in apnea of prematurity refractory to methylxanthine treatment. Its mode of action, however, is still poorly understood. We investigated direct effects of doxapram on the pre-Bötzinger complex (PreBötC) and on a downstream motor output system, the hypoglossal nucleus (XII), in the transverse brainstem slice preparation. While doxapram has only a modest stimulatory effect on frequency of activity generated within the PreBötC, a much more robust increase in the amplitude of population activity in the subsequent motor output generated in the XII was observed. In whole cell patch-clamp recordings of PreBötC and XII neurons, we confirmed significantly increased firing of evoked action potentials in XII neurons in the presence of doxapram, while PreBötC neurons showed no significant alteration in firing properties. Interestingly, the amplitude of activity in the motor output was not increased in the presence of doxapram compared with control conditions during hypoxia. We conclude that part of the stimulatory effects of doxapram is caused by direct input on brainstem centers with differential effects on the rhythm generating kernel (PreBötC) and the downstream motor output (XII). NEW & NOTEWORTHY The clinically used respiratory stimulant doxapram has distinct effects on the rhythm generating kernel (pre-Bötzinger complex) and motor output centers (nucleus hypoglossus). These effects are obliterated during hypoxia and are mediated by distinct changes in the intrinsic properties of neurons of the nucleus hypoglossus and synaptic transmission received by pre-Bötzinger complex neurons.

Hypoglossal Motor Neuron Death Via Intralingual CTB-saporin (CTB-SAP) Injections Mimic Aspects of Amyotrophic Lateral Sclerosis (ALS) Related to Dysphagia.

Amyotrophic lateral sclerosis (ALS) is a devastating disease leading to degeneration of motor neurons and skeletal muscles, including those required for swallowing. Tongue weakness is one of the earliest signs of bulbar dysfunction in ALS, which is attributed to degeneration of motor neurons in the hypoglossal nucleus in the brainstem, the axons of which directly innervate the tongue. Despite its fundamental importance, dysphagia (difficulty swallowing) and strategies to preserve swallowing function have seldom been studied in ALS models. It is difficult to study dysphagia in ALS models since the amount and rate at which hypoglossal motor neuron death occurs cannot be controlled, and degeneration is not limited to the hypoglossal nucleus. Here, we report a novel experimental model using intralingual injections of cholera toxin B conjugated to saporin (CTB-SAP) to study the impact of only hypoglossal motor neuron death without the many complications that are present in ALS models. Hypoglossal motor neuron survival, swallowing function, and hypoglossal motor output were assessed in Sprague-Dawley rats after intralingual injection of either CTB-SAP (25 g) or unconjugated CTB and SAP (controls) into the genioglossus muscle. CTB-SAP treated rats exhibited significant (p ≤ 0.05) deficits vs. controls in: (1) lick rate (6.0 ±â€¯0.1 vs. 6.6 ±â€¯0.1 Hz; (2) hypoglossal motor output (0.3 ±â€¯0.05 vs. 0.6 ±â€¯0.10 mV); and (3) hypoglossal motor neuron survival (398 ±â€¯34 vs. 1018 ±â€¯41 neurons). Thus, this novel, inducible model of hypoglossal motor neuron death mimics the dysphagia phenotype that is observed in ALS rodent models, and will allow us to study strategies to preserve swallowing function.

Traditional Chinese herbal formula relieves snoring by modulating activities of upper airway related nerves in aged rats.

AIM: The present study investigated whether intraperitoneal treatment with the herbal formula B210 ([B210]; a herbal composition of Gastrodia elata and Cinnamomum cassia) can reduce snoring in aged rats. Also, we studied possible neural mechanisms involved in B210 treatment and subsequent reduced snoring in rats. METHODS AND RESULT: We compared pressure and frequency of snoring, activities of phrenic nerve (PNA), activities of recurrent laryngeal nerve (RLNA) and activities of hypoglossal nerve (HNA), inspiratory time (TI) and expiratory time (TE) of PNA, and pre-inspiratory time (Pre-TI) of HNA in aged rats between sham and B210 treatment groups (30 mg/mL dissolved in DMSO). We found that aged rats that received B210 treatment had significantly reduced pressure and frequency of snoring than rats who received sham treatment. Also, we observed that aged rats that received B210 treatment had significantly increased PNA, RLNA, and HNA, extended TI and TE of PNA, and prolonged Pre-TI of HNA compared to rats that received sham treatment. In other words, B210 treatment may relieve snoring through modulating activities and breathing time of upper airway related nerves in aged rats. CONCLUSION: We suggested that the B210 might be a potential herbal formula for snoring remission.

Functional up-regulation of the M-current by retigabine contrasts hyperexcitability and excitotoxicity on rat hypoglossal motoneurons.

KEY POINTS: Excessive neuronal excitability characterizes several neuropathological conditions, including neurodegenerative diseases such as amyotrophic lateral sclerosis. Hypoglossal motoneurons (HMs), which control tongue muscles, are extremely vulnerable to this disease and undergo damage and death when exposed to an excessive glutamate extracellular concentration that causes excitotoxicity. Our laboratory devised an in vitro model of excitotoxicity obtained by pharmacological blockade of glutamate transporters. In this paradigm, HMs display hyperexcitability, collective bursting and eventually cell death. The results of the present study show that pharmacological up-regulation of a K+ current (M-current), via application of the anti-convulsant retigabine, prevented all hallmarks of HM excitotoxicity, comprising bursting, generation of reactive oxygen species, expression of toxic markers and cell death. ○Our data may have translational value to develop new treatments against neurological diseases by using positive pharmacological modulators of the M-current. ABSTRACT: Neuronal hyperexcitability is a symptom characterizing several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). In the ALS bulbar form, hypoglossal motoneurons (HMs) are an early target for neurodegeneration because of their high vulnerability to metabolic insults. In recent years, our laboratory has developed an in vitro model of a brainstem slice comprising the hypoglossal nucleus in which HM neurodegeneration is achieved by blocking glutamate clearance with dl-threo-ß-benzyloxyaspartate (TBOA), thus leading to delayed excitotoxicity. During this process, HMs display a set of hallmarks such as hyperexcitability (and network bursting), reactive oxygen species (ROS) generation and, finally, cell death. The present study aimed to investigate whether blocking early hyperexcitability and bursting with the anti-convulsant drug retigabine was sufficient to achieve neuroprotection against excitotoxicity. Retigabine is a selective positive allosteric modulator of the M-current (IM ), an endogenous mechanism that neurons (comprising HMs) express to dampen excitability. Retigabine (10 µm; co-applied with TBOA) contrasted ROS generation, release of endogenous toxic factors into the HM cytoplasm and excitotoxicity-induced HM death. Electrophysiological experiments showed that retigabine readily contrasted and arrested bursting evoked by TBOA administration. Because neuronal IM subunits (Kv7.2, Kv7.3 and Kv7.5) were expressed in the hypoglossal nucleus and in functionally connected medullary nuclei, we suggest that they were responsible for the strong reduction in network excitability, a potent phenomenon for achieving neuroprotection against TBOA-induced excitotoxicity. The results of the present study may have translational value for testing novel positive pharmacological modulators of the IM under pathological conditions (including neurodegenerative disorders) characterized by excessive neuronal excitability.

Hypoglossal motoneurons are endogenously activated by serotonin during the active period of circadian cycle.

In obstructive sleep apnea patients, contraction of lingual muscles protects the pharyngeal airway from collapse. Hypoglossal (XII) motoneurons innervate the muscles of the tongue and are themselves under wake-related excitatory drives, including that mediated by serotonin (5-HT). Estimates of endogenous 5-HT activation vary among different studies. We tested whether endogenous drive mediated by 5-HT is present in rat XII motoneurons when measured during the active period of the circadian cycle. We monitored sleep-wake states and lingual and nuchal electromyograms (EMGs) while perfusing the XII nucleus with a vehicle or a 5-HT2 receptor antagonist (mianserin, 0.2mM) at the active period onset. EMG levels were measured during each behavioral state and normalized by the mean EMG activity during wakefulness at 4-7am. Wake-related lingual EMG was significantly lower during mianserin perfusion than with the vehicle (53.0±9.7% vs. 84.5±8.7%; p=0.002). Mianserin had no effect on nuchal EMG or sleep-wake behavior. Thus, rat XII motoneurons receive endogenous serotonergic activation during wakefulness when measured during the dark period. This indicates that XII motoneuronal activity is enhanced by 5-HT output during the active period of the circadian cycle.

Phasic inhibition as a mechanism for generation of rapid respiratory rhythms.

Central neural networks operate continuously throughout life to control respiration, yet mechanisms regulating ventilatory frequency are poorly understood. Inspiration is generated by the pre-Bötzinger complex of the ventrolateral medulla, where it is thought that excitation increases inspiratory frequency and inhibition causes apnea. To test this model, we used an in vitro optogenetic approach to stimulate select populations of hindbrain neurons and characterize how they modulate frequency. Unexpectedly, we found that inhibition was required for increases in frequency caused by stimulation of Phox2b-lineage, putative CO2-chemosensitive neurons. As a mechanistic explanation for inhibition-dependent increases in frequency, we found that phasic stimulation of inhibitory neurons can increase inspiratory frequency via postinhibitory rebound. We present evidence that Phox2b-mediated increases in frequency are caused by rebound excitation following an inhibitory synaptic volley relayed by expiration. Thus, although it is widely thought that inhibition between inspiration and expiration simply prevents activity in the antagonistic phase, we instead propose a model whereby inhibitory coupling via postinhibitory rebound excitation actually generates fast modes of inspiration.

Maintenance of Mouse Gustatory Terminal Field Organization Is Disrupted following Selective Removal of Peripheral Sodium Salt Taste Activity at Adulthood.

Neural activity plays a critical role in the development of central circuits in sensory systems. However, the maintenance of these circuits at adulthood is usually not dependent on sensory-elicited neural activity. Recent work in the mouse gustatory system showed that selectively deleting the primary transduction channel for sodium taste, the epithelial sodium channel (ENaC), throughout development dramatically impacted the organization of the central terminal fields of three nerves that carry taste information to the nucleus of the solitary tract. More specifically, deleting ENaCs during development prevented the normal maturation of the fields. The present study was designed to extend these findings by testing the hypothesis that the loss of sodium taste activity impacts the maintenance of the normal adult terminal field organization in male and female mice. To do this, we used an inducible Cre-dependent genetic recombination strategy to delete ENaC function after terminal field maturation occurred. We found that removal of sodium taste neural activity at adulthood resulted in significant reorganization of mature gustatory afferent terminal fields in the nucleus of the solitary tract. Specifically, the chorda tympani and greater superficial petrosal nerve terminal fields were 1.4× and 1.6× larger than age-matched controls, respectively. By contrast, the glossopharyngeal nerve, which is not highly sensitive to sodium taste stimulation, did not undergo terminal field reorganization. These surprising results suggest that gustatory nerve terminal fields remain plastic well into adulthood, which likely impacts central coding of taste information and taste-related behaviors with altered taste experience.SIGNIFICANCE STATEMENT Neural activity plays a major role in the development of sensory circuits in the mammalian brain. However, the importance of sensory-driven activity in maintaining these circuits at adulthood, especially in subcortical structures, appears to be much less. Here, we tested whether the loss of sodium taste activity in adult mice impacts the maintenance of how taste nerves project to the first central relay. We found that specific loss of sodium-elicited taste activity at adulthood produced dramatic and selective reorganization of terminal fields in the brainstem. This demonstrates, for the first time, that taste-elicited activity is necessary for the normal maintenance of central gustatory circuits at adulthood and highlights a level of plasticity not seen in other sensory system subcortical circuits.

Microglia support ATF3-positive neurons following hypoglossal nerve axotomy.

Microglia are essential in developmental processes and maintenance of neuronal homeostasis. Experimental axotomy of motor neurons results in neurodegeneration, and microglia in motor nuclei become activated and migrate towards injured neurons. However, whether these activated microglia are protective or destructive to neurons remains controversial. In the present study, we transected the hypoglossal nerve in BALB/c mice, causing activating transcription factor 3 (ATF3) and growth associated protein 43 (GAP43) induction, and partial neuronal death. Inhibition of microglial accumulation by minocycline administration impaired microglial accumulation, decreased GAP43 mRNA expression, and reduced motor neuron survival. Expression of ATF3 contributed to nerve regeneration, and increased within 6 h after axotomy, prior to microglial migration. Further, microglial contact with neuronal cell bodies was associated with neuronal ATF3 expression. Colchicine administration blocked lesion-induced ATF3 transcription in axotomized neurons and microglial accumulation. In addition, perineuronal microglia-derived ciliary neurotrophic factor (CNTF) increased, indicating that perineuronal microglia in the hypoglossal nucleus protect axotomized motor neurons by releasing trophic factors. We also observed that microglia secrete CNTF and that neurons have CNTFRα and can respond to it in vitro. CNTF promote neurite elongation and neuronal survival of primary cultured neurons. Microglia make contact through unknown neuronal signals that are possibly regulated by ATF3 in hypoglossal nucleus. Moreover, they play important roles in regenerating motor neurons and are potential new therapeutic targets for motor neuron diseases.

Chemogenetic stimulation of the hypoglossal neurons improves upper airway patency.

Obstructive sleep apnea (OSA) is characterized by recurrent upper airway obstruction during sleep. OSA leads to high cardiovascular morbidity and mortality. The pathogenesis of OSA has been linked to a defect in neuromuscular control of the pharynx. There is no effective pharmacotherapy for OSA. The objective of this study was to determine whether upper airway patency can be improved using chemogenetic approach by deploying designer receptors exclusively activated by designer drug (DREADD) in the hypoglossal motorneurons. DREADD (rAAV5-hSyn-hM3(Gq)-mCherry) and control virus (rAAV5-hSyn-EGFP) were stereotactically administered to the hypoglossal nucleus of C57BL/6J mice. In 6-8 weeks genioglossus EMG and dynamic MRI of the upper airway were performed before and after administration of the DREADD ligand clozapine-N-oxide (CNO) or vehicle (saline). In DREADD-treated mice, CNO activated the genioglossus muscle and markedly dilated the pharynx, whereas saline had no effect. Control virus treated mice showed no effect of CNO. Our results suggest that chemogenetic approach can be considered as a treatment option for OSA and other motorneuron disorders.

α2-Adrenergic blockade rescues hypoglossal motor defense against obstructive sleep apnea.

Decreased noradrenergic excitation of hypoglossal motoneurons during sleep causing hypotonia of pharyngeal dilator muscles is a major contributor to the pathogenesis of obstructive sleep apnea (OSA), a widespread disease for which treatment options are limited. Previous OSA drug candidates targeting various excitatory/inhibitory receptors on hypoglossal motoneurons have proved unviable in reactivating these neurons, particularly during rapid-eye-movement (REM) sleep. To identify a viable drug target, we show that the repurposed α2-adrenergic antagonist yohimbine potently reversed the depressant effect of REM sleep on baseline hypoglossal motoneuron activity (a first-line motor defense against OSA) in rats. Remarkably, yohimbine also restored the obstructive apnea-induced long-term facilitation of hypoglossal motoneuron activity (hLTF), a much-neglected form of noradrenergic-dependent neuroplasticity that could provide a second-line motor defense against OSA but was also depressed during REM sleep. Corroborating immunohistologic, optogenetic, and pharmacologic evidence confirmed that yohimbine's beneficial effects on baseline hypoglossal motoneuron activity and hLTF were mediated mainly through activation of pontine A7 and A5 noradrenergic neurons. Our results suggest a 2-tier (impaired first- and second-line motor defense) mechanism of noradrenergic-dependent pathogenesis of OSA and a promising pharmacotherapy for rescuing both these intrinsic defenses against OSA through disinhibition of A7 and A5 neurons by α2-adrenergic blockade.

Nicotinic receptors modulate the onset of reactive oxygen species production and mitochondrial dysfunction evoked by glutamate uptake block in the rat hypoglossal nucleus.

In several neurodegenerative diseases, glutamate-mediated excitotoxicity is considered to be a major process to initiate cell degeneration. Indeed, subsequent to excessive glutamate receptor stimulation, reactive oxygen species (ROS) generation and mitochondrial dysfunction are regarded as two major gateways leading to neuron death. These processes are mimicked in an in vitro model of rat brainstem slice when excitotoxicity is induced by DL-threo-ß-benzyloxyaspartate (TBOA), a specific glutamate-uptake blocker that increases extracellular glutamate. Our recent study has demonstrated that brainstem hypoglossal motoneurons, which are very vulnerable to this damage, were neuroprotected from excitotoxicity with nicotine application through the activation of nicotinic acetylcholine receptors (nAChRs) and subsequent inhibition of ROS and mitochondrial dysfunction. The present study examined if endogenous cholinergic activity exerted any protective effect in this pathophysiological model and how ROS production (estimated with rhodamine fluorescence) and mitochondrial dysfunction (measured as methyltetrazolium reduction) were time-related during the early phase of excitotoxicity (0-4h). nAChR antagonists did not modify TBOA-evoked ROS production (that was nearly doubled over control) or mitochondrial impairment (25% decline), suggesting that intrinsic nAChR activity was insufficient to contrast excitotoxicity and needed further stimulation with nicotine to become effective. ROS production always preceded mitochondrial dysfunction by about 2h. Nicotine prevented both ROS production and mitochondrial metabolic depression with a delayed action that alluded to a complex chain of events targeting these two lesional processes. The present data indicate a relatively wide time frame during which strong nAChR activation can arrest a runaway neurotoxic process leading to cell death.

Electrophysiological characterization of the M-current in rat hypoglossal motoneurons.

The M-current (IM) is a voltage-dependent, persistent K+ current so termed because it is strongly inhibited by the cholinergic agonist muscarine. The IM main function is to limit neuronal excitability by contrasting action potential firing. Although motoneurons are sensitive to acetylcholine, the role of IM in modulating their excitability is still controversial. The aim of the present report was to examine the presence of IM in hypoglossal motoneurons (HMs) and its role in the modulation of firing properties using an in vitro model of rat brainstem slice. For this purpose, we employed the whole-cell patch-clamp technique to record HM responses upon stimulation with either a standard IM deactivation voltage protocol or depolarizing current steps. Voltage commands from depolarized potential induced inward relaxations with the common characteristics of IM, comprising inhibition by either muscarine (10µM) or the selective IM inhibitor linopirdine (30µM). IM was pharmacologically distinguished from the hyperpolarization-activated inward-rectifying current and, within the -20 to -50mV range, deactivated with >100-ms time constant. Current-clamp experiments demonstrated that IM strongly regulated HM action potential firing, since both muscarine and linopirdine increased spike frequency whereas the M-channel opener retigabine (20µM) reduced it. Conversely, IM seemed uninvolved in the generation of the medium afterhyperpolarizing potential. Our results suggest that HMs possess IM, whose pharmacological modulation is an important tool to up- or down-regulate excitability, to be explored in experimental models of neurodegeneration.

Nicotinic receptor activation contrasts pathophysiological bursting and neurodegeneration evoked by glutamate uptake block on rat hypoglossal motoneurons.

KEY POINTS: Impaired uptake of glutamate builds up the extracellular level of this excitatory transmitter to trigger rhythmic neuronal bursting and delayed cell death in the brainstem motor nucleus hypoglossus. This process is the expression of the excitotoxicity that underlies motoneuron degeneration in diseases such as amyotrophic lateral sclerosis affecting bulbar motoneurons. In a model of motoneuron excitotoxicity produced by pharmacological block of glutamate uptake in vitro, rhythmic bursting is suppressed by activation of neuronal nicotinic receptors with their conventional agonist nicotine. Emergence of bursting is facilitated by nicotinic receptor antagonists. Following excitotoxicity, nicotinic receptor activity decreases mitochondrial energy dysfunction, endoplasmic reticulum stress and production of toxic radicals. Globally, these phenomena synergize to provide motoneuron protection. Nicotinic receptors may represent a novel target to contrast pathological overactivity of brainstem motoneurons and therefore to prevent their metabolic distress and death. ABSTRACT: Excitotoxicity is thought to be one of the early processes in the onset of amyotrophic lateral sclerosis (ALS) because high levels of glutamate have been detected in the cerebrospinal fluid of such patients due to dysfunctional uptake of this transmitter that gradually damages brainstem and spinal motoneurons. To explore potential mechanisms to arrest ALS onset, we used an established in vitro model of rat brainstem slice preparation in which excitotoxicity is induced by the glutamate uptake blocker dl-threo-ß-benzyloxyaspartate (TBOA). Because certain brain neurons may be neuroprotected via activation of nicotinic acetylcholine receptors (nAChRs) by nicotine, we investigated if nicotine could arrest excitotoxic damage to highly ALS-vulnerable hypoglossal motoneurons (HMs). On 50% of patch-clamped HMs, TBOA induced intense network bursts that were inhibited by 1-10 µm nicotine, whereas nAChR antagonists facilitated burst emergence in non-burster cells. Furthermore, nicotine inhibited excitatory transmission and enhanced synaptic inhibition. Strong neuroprotection by nicotine prevented the HM loss observed after 4 h of TBOA exposure. This neuroprotective action was due to suppression of downstream effectors of neurotoxicity such as increased intracellular levels of reactive oxygen species, impaired energy metabolism and upregulated genes involved in endoplasmic reticulum (ER) stress. In addition, HMs surviving TBOA toxicity often expressed UDP-glucose glycoprotein glucosyltransferase, a key element in repair of misfolded proteins: this phenomenon was absent after nicotine application, indicative of ER stress prevention. Our results suggest nAChRs to be potential targets for inhibiting excitotoxic damage of motoneurons at an early stage of the neurodegenerative process.