Hypoglossal Motoneurons Research Articles

Developmental nicotine exposure alters glycinergic neurotransmission to hypoglossal motoneurons in neonatal rats.

We tested the hypothesis that nicotine exposure in utero and after birth [developmental nicotine exposure (DNE)] disrupts development of glycinergic synaptic transmission to hypoglossal motoneurons (XIIMNs). Glycinergic spontaneous and miniature inhibitory postsynaptic currents (sIPSC/mIPSC) were recorded from XIIMNs in brain stem slices from 1- to 5-day-old rat pups of either sex, under baseline conditions and following stimulation of nicotinic acetylcholine (ACh) receptors with nicotine (i.e., an acute nicotine challenge). Under baseline conditions, there were no significant effects of DNE on the amplitude or frequency of either sIPSCs or mIPSCs. In addition, DNE did not alter the magnitude of the whole cell current evoked by bath application of glycine, consistent with an absence of change in postsynaptic glycine-mediated conductance. An acute nicotine challenge (bath application of 0.5 μM nicotine) increased sIPSC frequency in the DNE cells, but not control cells. In contrast, nicotine challenge did not change mIPSC frequency in either control or DNE cells. In addition, there were no significant changes in the amplitude of either sIPSCs or mIPSCs in response to nicotine challenge. The increased frequency of sIPSCs in response to an acute nicotine challenge in DNE cells reflects an enhancement of action potential-mediated input from glycinergic interneurons to hypoglossal motoneurons. This could lead to more intense inhibition of hypoglossal motoneurons in response to exogenous nicotine or endogenous ACh. The former would occur with smoking or e-cigarette use while the latter occurs with changes in sleep state and with hypercapnia. NEW & NOTEWORTHY Here we show that perinatal nicotine exposure does not impact baseline glycinergic neurotransmission to hypoglossal motoneurons but enhances glycinergic inputs to hypoglossal motoneurons in response to activation of nicotinic acetylcholine (ACh) receptors with acute nicotine. Given that ACh is the endogenous ligand for nicotinic ACh receptors, the latter reveals a potential mechanism whereby perinatal nicotine exposure alters motor function under conditions where ACh release increases, such as the transition from non-rapid-eye movement to rapid-eye movement sleep, and during hypercapnia.

Functional Consequences of the Postnatal Switch From Neonatal to Mutant Adult Glycine Receptor α1 Subunits in the Shaky Mouse Model of Startle Disease.

Mutations in GlyR α1 or β subunit genes in humans and rodents lead to severe startle disease characterized by rigidity, massive stiffness and excessive startle responses upon unexpected tactile or acoustic stimuli. The recently characterized startle disease mouse mutant shaky carries a missense mutation (Q177K) in the β8-β9 loop within the large extracellular N-terminal domain of the GlyR α1 subunit. This results in a disrupted hydrogen bond network around K177 and faster GlyR decay times. Symptoms in mice start at postnatal day 14 and increase until premature death of homozygous shaky mice around 4–6 weeks after birth. Here we investigate the in vivo functional effects of the Q177K mutation using behavioral analysis coupled to protein biochemistry and functional assays. Western blot analysis revealed GlyR α1 subunit expression in wild-type and shaky animals around postnatal day 7, a week before symptoms in mutant mice become obvious. Before 2 weeks of age, homozygous shaky mice appeared healthy and showed no changes in body weight. However, analysis of gait and hind-limb clasping revealed that motor coordination was already impaired. Motor coordination and the activity pattern at P28 improved significantly upon diazepam treatment, a pharmacotherapy used in human startle disease. To investigate whether functional deficits in glycinergic neurotransmission are present prior to phenotypic onset, we performed whole-cell recordings from hypoglossal motoneurons (HMs) in brain stem slices from wild-type and shaky mice at different postnatal stages. Shaky homozygotes showed a decline in mIPSC amplitude and frequency at P9-P13, progressing to significant reductions in mIPSC amplitude and decay time at P18-24 compared to wild-type littermates. Extrasynaptic GlyRs recorded by bath-application of glycine also revealed reduced current amplitudes in shaky mice compared to wild-type neurons, suggesting that presynaptic GlyR function is also impaired. Thus, a distinct, but behaviorally ineffective impairment of glycinergic synapses precedes the symptoms onset in shaky mice. These findings extend our current knowledge on startle disease in the shaky mouse model in that they demonstrate how the progression of GlyR dysfunction causes, with a delay of about 1 week, the appearance of disease symptoms.

0072 A Chronic Animal Model to Study the Neuromechanisms that Control Hypoglossal Motoneuron Activity during Hypoxic REM Sleep

0072 A Chronic Animal Model to Study the Neuromechanisms that Control Hypoglossal Motoneuron Activity during Hypoxic REM Sleep

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.

Impact of Intralingual AAVrh10‐miRSOD1 Injection on Respiratory Function in SOD1G93A Mice

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease in which patients normally succumb to the disease within 5 years of onset. Death is usually a result of impaired respiratory function due to loss of motor neurons that control upper airway muscles and/or the diaphragm. The motor neurons that control upper airway muscles and thereby airway patency are found in the brainstem in a region called the hypoglossal motor nucleus. One cause of ALS is a mutation in the superoxide dismutase‐1 (SOD1) gene that results in the buildup of SOD1 protein in neurons. For this subset of patients, reducing expression of the mutated gene may slow the progression of the disease. The purpose of this study was to determine if delivering a microRNA against SOD1 (miRSOD1) to the hypoglossal motor nucleus of mice that overexpress mutant SOD1 (SOD1G93A) would increase respiratory function. Thus, we hypothesized that intralingual injection of AAV‐miRSOD1 into SOD1G93A mice would knockdown mutant SOD1 in hypoglossal motor neurons and improve respiration. Six‐week‐old male and female mice received intralingual injections of either AAVrh10‐miRSOD1 or vehicle (phosphate buffered saline). Starting at 13 weeks of age, respiratory parameters at baseline and in response to hypoxia (11% O2) + hypercapnia (7% CO2) were studied in miRSOD1‐injected SOD1G93A mice (n=11), vehicle‐injected SOD1G93A mice (n=12), and vehicle injected wild‐type mice (n=17) via whole‐body plethysmography every other week until the SOD1G93A mice reached end‐stage (20% body weight loss). Minute ventilation during hypoxia + hypercapnia and mean inspiratory flow at baseline were significantly reduced (p<0.05) in vehicle‐injected but not miRSOD1‐injected SOD1G93A mice as compared to wild‐type mice. miRSOD1 injections also extended survival in females by ~1 week; although this did not reach statistical significance, we suggest that any increase in survival could be clinically significant. In conclusion, this study indicates that intralingual miRSOD1 treatment restored respiratory function to wild‐type levels (potentially via increasing upper airway patency) and is worthy of further exploration as a possible therapy to preserve respiratory capacity in ALS patients. Future directions include studying the vector genome expression and SOD1 knock down in local and systemic tissues to understand the degree of transduction. In addition, we will measure the impact of intralingual miRSOD1 on swallowing and upper airway function.Support or Funding InformationSupported by NIH R21 NS098131‐02 and K99/R00 HL119606This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Developmental changes in hypoglossal motoneuron morphology in neonatal rat

The autonomic brainstem generates and modifies breathing rhythm by integrating inputs from chemo‐ and mechanosensors in the viscera while coordinating descending outputs from higher CNS structures. Hypoglossal motoneurons (XII MNs) receive inputs from respiratory premotor neurons in the medulla. Previous studies in rodents have demonstrated significant changes in breathing control during the first three weeks of life, with a putative sensitive period at 10 to 13 days post‐birth (P10–P13) characterized by pronounced changes in neurotransmitters, receptors, excitation‐inhibition balance, and breathing physiology [Li et al., J Physiol, 577:957–770, 2006]. However, age‐dependent morphological changes of XII MNs during the first three weeks post‐birth and especially during this sensitive period, have not been thoroughly studied. In this study, we comprehensively characterize and quantify the postnatal morphological changes in rat XII MNs. We hypothesized that morphological changes occur in XII MN morphology and arbor complexity corresponding to the functionally‐defined sensitive period observed at P10–P13. To test this hypothesis, we used innovative statistical approaches to quantify and compare developmental changes in Golgi‐Cox stained XII MNs at nine postnatal ages between P1–P21. Soma size increased ~40% from P1 to P21, with no significant change in shape. However, dendritic arborization increased in extent and complexity. Dendritic branching of developing neurons significantly increased from P1 through P13, with the greatest increase at P10–P13 based on the Sholl method. For many parameters, three age groups 1) P1–P5, 2) P7–P12, and 3) P13–P21 were used as possible windows of development. We also found that at specific ages certain parameters such as soma size and dendrite distance were non‐normally distributed. Our detailed characterization of XII MN morphological development establishes a foundation for the study and elucidation of morphological changes caused by maternal and perinatal conditions using a rigorous statistical approach.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Developmental plasticity of GABAergic neurotransmission to brainstem motoneurons.

Critical homeostatic behaviours such as suckling, swallowing and breathing depend on the precise control of tongue muscle activity. Perinatal nicotine exposure has multiple effects on baseline inhibitory GABAergic neurotransmission to hypoglossal motoneurons (XIIMNs), consistent with homeostatic compensations directed at maintaining normal motoneuron output. Developmental nicotine exposure (DNE) alters how GABAergic neurotransmission is modulated by acute activation of nicotinic acetylcholine receptors, which may provide insight into mechanisms by which nicotine exposure alters motor function under conditions that result in increased release of GABA, such as hypoxia, or endogenous acetylcholine, as occurs in the transition from NREM to REM sleep, or in response to exogenous nicotine. Nicotinic acetylcholine receptor (nAChR) signalling regulates neuronal differentiation and synaptogenesis. Here we test the hypothesis that developmental nicotine exposure (DNE) disrupts the development of GABAergic synaptic transmission to hypoglossal motoneurons (XIIMNs). GABAergic spontaneous and miniature inhibitory postsynaptic currents (sIPSCs/mIPSCs) were recorded from XIIMNs in brainstem slices from control and DNE rat pups of either sex, 1-5days old, at baseline and following acute stimulation of nAChRs with nicotine. At baseline, sIPSCs were less frequent and smaller in DNE cells (consistent with decreased action potential-mediated GABA release), and mIPSCs were more frequent (consistent with increased vesicular GABA release from presynaptic terminals). Acute nicotine challenge increased sIPSC frequency in both groups, though the increase was greater in DNE cells. Acute nicotine challenge did not change the frequency of mIPSCs in either group, though mIPSC amplitude increased significantly in DNE cells, but not control cells. Stimulation of postsynaptic GABAA receptors with muscimol caused a significantly greater chloride current in DNE cells than in control cells. The increased quantal release of GABA, coupled with the rise in the strength of postsynaptic inhibition may be homeostatic adjustments to the decreased action-potential-mediated input from GABAergic interneurons. However, this will exaggerate synaptic inhibition under conditions where the release of GABA (e.g. hypoxia) or ACh (sleep-wake transitions) is increased. These findings reveal a mechanism that may explain why DNE is associated with deficits in the ability to respond appropriately to chemosensory stimuli or to changes in neuromodulation secondary to changes in central nervous system state.

Capsaicin Enhances Glutamatergic Synaptic Transmission to Neonatal Rat Hypoglossal Motor Neurons via a TRPV1-Independent Mechanism.

We investigated whether capsaicin modulated synaptic transmission to hypoglossal motor neurons (HMNs) by acting on transient receptor potential vanilloid type 1 (TRPV1) receptors. Using whole-cell patch clamp recording from neonatal rat HMNs, we found that capsaicin increased spontaneous excitatory post-synaptic current (sEPSC) frequency and amplitude. Interestingly, the only effect of capsaicin on spontaneous inhibitory post-synaptic currents (sIPSCs) was a significant decrease in sIPSC amplitude without altering frequency, indicating a post-synaptic mechanism of action. The frequency of miniature excitatory post-synaptic currents (mEPSCs), recorded in the presence of tetrodotoxin (TTX), was also increased by capsaicin, but capsaicin did not alter mEPSC amplitude, consistent with a pre-synaptic mechanism of action. A negative shift in membrane current (Iholding) was elicited by capsaicin under both recording conditions. The effect of capsaicin on excitatory synaptic transmission remained unchanged in the presence of the TRPV1 antagonists, capsazepine or SB366791, suggesting that capsaicin acts to modulate EPSCs via a mechanism which does not require TRPV1 activation. Capsaicin, however, did not alter evoked excitatory post-synaptic currents (eEPSCs) or the paired-pulse ratio (PPR) of eEPSCs. Repetitive action potential (AP) firing in HMNs was also unaltered by capsaicin, indicating that capsaicin does not change HMN intrinsic excitability. We have demonstrated that capsaicin modulates glutamatergic excitatory, as well as glycinergic inhibitory, synaptic transmission in HMNs by differing pre- and post-synaptic mechanisms. These results expand our understanding regarding the extent to which capsaicin can modulate synaptic transmission to central neurons.

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.

Propofol Protects Rat Hypoglossal Motoneurons in an In Vitro Model of Excitotoxicity by Boosting GABAergic Inhibition and Reducing Oxidative Stress

In brainstem motor networks, hypoglossal motoneurons (HMs) play the physiological role of driving tongue contraction, an activity critical for inspiration, phonation, chewing and swallowing. HMs are an early target of neurodegenerative diseases like amyotrophic lateral sclerosis that, in its bulbar form, is manifested with initial dysphagia and dysarthria. One important pathogenetic component of this disease is the high level of extracellular glutamate due to uptake block that generates excitotoxicity. To understand the earliest phases of this condition we devised a model, the rat brainstem slice, in which block of glutamate uptake is associated with intense bursting of HMs, dysmetabolism and death. Since blocking bursting becomes a goal to prevent cell damage, the present report enquired whether boosting GABAergic inhibition could fulfill this aim and confer beneficial outcome. Propofol (0.5 µM) and midazolam (0.01 µM), two allosteric modulators of GABAA receptors, were used at concentrations yielding analogous potentiation of GABA-mediated currents. Propofol also partly depressed NMDA receptor currents. Both drugs significantly shortened bursting episodes without changing single burst properties, their synchronicity, or their occurrence. Two hours later, propofol prevented the rise in reactive oxygen species (ROS) and, at 4 hours, it inhibited intracellular release of apoptosis-inducing factor (AIF) and prevented concomitant cell loss. Midazolam did not contrast ROS and AIF release. The present work provides experimental evidence for the neuroprotective action of a general anesthetic like propofol, which, in this case, may be achieved through a combination of boosted GABAergic inhibition and reduced ROS production.

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.

Extraocular Motor System Exhibits a Higher Expression of Neurotrophins When Compared with Other Brainstem Motor Systems.

Extraocular motoneurons resist degeneration in diseases such as amyotrophic lateral sclerosis. The main objective of the present work was to characterize the presence of neurotrophins in extraocular motoneurons and muscles of the adult rat. We also compared these results with those obtained from other cranial motor systems, such as facial and hypoglossal, which indeed suffer neurodegeneration. Immunocytochemical analysis was used to describe the expression of nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 in oculomotor, trochlear, abducens, facial, and hypoglossal nuclei of adult rats, and Western blots were used to describe the presence of neurotrophins in extraocular, facial (buccinator), and tongue muscles, which are innervated by the above-mentioned motoneurons. In brainstem samples, brain-derived neurotrophic factor was present both in extraocular and facial motoneuron somata, and to a lesser degree, in hypoglossal motoneurons. Neurotrophin-3 was present in extraocular motor nuclei, while facial and hypoglossal motoneurons were almost devoid of this protein. Finally, nerve growth factor was not present in the soma of any group of motoneurons, although it was present in dendrites of motoneurons located in the neuropil. Neuropil optical density levels were higher in extraocular motoneuron nuclei when compared with facial and hypoglossal nuclei. Neurotrophins could be originated in target muscles, since Western blot analyses revealed the presence of the three molecules in all sampled muscles, to a larger extent in extraocular muscles when compared with facial and tongue muscles. We suggest that the different neurotrophin availability could be related to the particular resistance of extraocular motoneurons to neurodegeneration.

Altered neurobiological function of brainstem hypoglossal neurons in DiGeorge/22q11.2 Deletion Syndrome

DiGeorge/22q11.2 Deletion Syndrome (22q11DS) is a common genetic microdeletion syndrome that underlies several neurodevelopmental disorders including autism, attention deficit/hyperactivity disorder, and schizophrenia. In addition to cognitive impairments, those with 22q11DS have disrupted feeding and swallowing from birth onward. This perinatal dysphagia significantly compromises nutritional status, impairs appropriate weight gain, and can lead to life threatening aspiration-based infections. Appropriately timed excitation and inhibition of brainstem hypoglossal motor neurons, which innervate tongue muscles, is essential for proper feeding and swallowing. In this study we have examined changes in hypoglossal motor neuron function in the LgDel mouse model of 22q11DS. Hypoglossal motor neurons from LgDel mouse pups have action potentials with afterhyperpolarizations, mediated by a large conductance charybdotoxin-sensitive Ca-activated K current, that are significantly shorter in duration and greater in magnitude than those in wild-type pups. In addition, the amplitude, but not frequency, of glutamatergic excitatory glutamatergic postsynaptic currents (EPSCs) is diminished, and GABAergic, but not glycinergic, neurotransmission to hypoglossal motor neurons was reduced in LgDel animals. These observations provide a foundation for understanding the neurological changes in hypoglossal motor neuron function and their contribution to swallowing abnormalities that occur in DiGeorge/22q11.2 Deletion Syndrome.

Neurotoxicity of propofol on rat hypoglossal motoneurons in vitro

Although propofol is a widely used intravenous general anaesthetic, many studies report its toxic potential, particularly on the developing central nervous system. We investigated its action on hypoglossal motoneurons (HMs) that control two critical functions in neonates, namely tongue muscle activity and airway patency. Thus, clinically relevant concentrations of propofol (1 and 5μM) were applied (4h) to neonatal rat brainstem slices to evaluate the expression of apoptosis-inducing factor (AIF) as biomarker of toxicity. This anaesthetic strongly increased AIF in the cytoplasm and the nucleus, without early loss of HMs. Electrophysiological recordings from HMs showed that propofol (5μM) enhanced GABA- and glycine-evoked current amplitude and lengthened GABAergic current decay time. Propofol also depressed NMDA receptor-mediated responses without affecting AMPA receptors. Since GABA and glycine depolarize neonatal HMs, we propose that the damaging action by propofol on these motoneurons might arise from the facilitated action of these transmitters with subsequent cytoplasmic Ca2+ overload. This phenomenon, in turn, may trigger cell death mechanisms manifested as increased expression of AIF and its translocation into the nucleus. Since propofol is also employed for induction and maintenance of paediatric surgery, caution is needed because its potential neurotoxicity might negatively impact neurodevelopment.

Nicotine protects rat hypoglossal motoneurons from excitotoxic death via downregulation of connexin 36

Motoneuron disease including amyotrophic lateral sclerosis may be due, at an early stage, to deficit in the extracellular clearance of the excitatory transmitter glutamate. A model of glutamate-mediated excitotoxic cell death based on pharmacological inhibition of its uptake was used to investigate how activation of neuronal nicotinic receptors by nicotine may protect motoneurons. Hypoglossal motoneurons (HMs) in neonatal rat brainstem slices were exposed to the glutamate uptake blocker DL-threo-β-benzyloxyaspartate (TBOA) that evoked large Ca2+ transients time locked among nearby HMs, whose number fell by about 30% 4 h later. As nicotine or the gap junction blocker carbenoxolone suppressed bursting, we studied connexin 36 (Cx36), which constitutes gap junctions in neurons and found it largely expressed by HMs. Cx36 was downregulated when nicotine or carbenoxolone was co-applied with TBOA. Expression of Cx36 was preferentially observed in cytosolic rather than membrane fractions after nicotine and TBOA, suggesting protein redistribution with no change in synthesis. Nicotine raised the expression of heat shock protein 70 (Hsp70), a protective factor that binds the apoptotic-inducing factor (AIF) whose nuclear translocation is a cause of cell death. TBOA increased intracellular AIF, an effect blocked by nicotine. These results indicate that activation of neuronal nicotinic receptors is an early tool for protecting motoneurons from excitotoxicity and that this process is carried out via the combined decrease in Cx36 activity, overexpression of Hsp70 and fall in AIF translocation. Thus, retarding or inhibiting HM death may be experimentally achieved by targeting one of these processes leading to motoneuron death.

0143 CARBACHOL INHIBITS GLUTAMATERGIC INPUT TO MOUSE HYPOGLOSSAL MOTOR NEURONS - A MECHANISM FOR REM SLEEP SUPPRESSION OF GENIOGLOSSUS ACTIVITY

In REM sleep, the genioglossus (GG) muscle undergoes a dramatic suppression of activity. A current hypothesis is that the loss of GG activity during REM sleep is mediated by a combination of 1) monoaminergic disfacilitation and 2) a cholinergic inhibition of hypoglossal motor neurons. Strikingly, blockade of cholinergic receptors in the hypoglossal motor nucleus fully restores REM sleep tonic and inspiratory-modulated components of GG activity (Grace et al., 2013), suggesting that the cholinergic signal is largely responsible for the REM sleep suppression of GG activity. Respiratory rhythm generator neurons of the pre-Bötzinger complex drive the activation of hypoglossal motor neurons through glutamatergic premotor neurons in the parahypoglossal region (PH). In the current study, we investigate how cholinergic signaling affects the PH glutamatergic input to hypoglossal motor neurons. We stereotaxically injected the PH region of vGluT2-cre mice with a cre-dependent AAV-ChR2-mCherry to expressed channelrhodopsin2 (ChR2) in PH glutamatergic premotor neurons. We then performed whole-cell recordings in hypoglossal neurons while photostimulating PH glutamatergic inputs expressing ChR2. Photostimulation of the glutamatergic PH input evoked AMPA-mediated EPSCs in hypoglossal motor neurons. These photoevoked EPSCs were maintained in TTX, indicating direct connectivity between stimulated terminals and recorded hypoglossal motoneurons. Bath application of carbachol strongly inhibited the PH glutamatergic excitation of hypoglossal neurons via muscarinic receptors. The effect of carbachol on photoevoked EPSCs was maintained when 1) we blocked postsynaptic G-mediated effects by adding GDP-β-S in the recording pipette and 2) when we blocked action potential mediated transmission by adding TTX in the extracellular bath solution. These results indicate that carbachol inhibits PH input to hypoglossal motoneurons through a presynaptic mechanism. Our results provide a possible mechanism for cholinergic inhibition of the hypoglossal motor neurons in REM sleep. We propose that the cholinergic presynaptic suppression of the excitatory drive from the PH premotor neurons can be responsible for the reduction in activity of hypoglossal motor neurons in REM sleep. P01HL095491.

Effect of 4-Aminopyridine on Genioglossus Muscle Activity during Sleep in Healthy Adults.

The reduction in upper airway muscle activity from wakefulness to sleep plays a key role in the development of obstructive sleep apnea. Potassium (K+) channels have been recently identified as the downstream mechanisms through which hypoglossal motoneuron membrane excitability is reduced both in non-rapid eye movement (NREM) sleep and REM sleep. In animal models, the administration of 4-aminopyridine (4-AP), a voltage-gated K+ channel blocker, increased genioglossus activity during wakefulness and across all sleep stages. We tested the hypothesis that administration of a single dose of 4-AP 10 mg extended release would increase genioglossus activity (electromyography of the genioglossus muscle [EMGGG]) during wakefulness and sleep, and thereby decrease pharyngeal collapsibility. We performed a randomized controlled crossover proof-of-concept trial in 10 healthy participants. Participants received active treatment or placebo in randomized order 3 hours before bedtime in the physiology laboratory. EMGGG during wakefulness and NREM sleep and upper airway collapsibility measured during NREM sleep were unchanged between placebo and 4-AP nights. Tonic but not phasic EMGGG during REM sleep was higher on the 4-AP night when measured as a percentage of maximal voluntary activation (median [interquartile range] 0.3 [0.5] on placebo vs. 0.8 [1.9] %max on 4 AP; P = 0.04), but not when measured in μV or as a percentage of wakefulness value. A single dose of 4-AP 10 mg extended release showed only a small increase in tonic EMGGG during REM sleep in this group of healthy subjects. We speculate that a higher dose of 4-AP may further increase EMGGG. However, given the potentially severe, dose-related adverse effects of this drug, including seizures, the administration of 4-AP does not appear to be an effective strategy to increase genioglossus activity during sleep in humans. Clinical Trial registered with clinicaltrials.gov (NCT02656160).

Developmental nicotine exposure increases the response to muscimol in hypoglossal motoneurons.

Prenatal nicotine exposure with continued exposure through breast milk over the first week of life (developmental nicotine exposure, DNE) is known to cause complex changes to the brainstem respiratory circuit including increased sensitivity to inhibitory neurotransmitters and increased staining of GABAAR‐1a subunit in the hypoglossal nucleus. Here we test the hypothesis that DNE alters the response of hypoglossal motoneurons (XII MNs) to the GABAA receptor agonist Muscimol. We obtainedwhole cell patch clamp recordings from XII MNs in medullary slices (700 micron thick) from DNE pups and control pups. Cells were voltage clamped at −75mV. Using a cesium based intracellular solution, and in the presence of the AMPA receptor antagonists 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX), the NMDA receptor antagonist D(‐)‐2‐Amino‐5‐phosphonopentanoic acid (AP‐5), the glycine receptor antagonist Strychnine, and the voltage gated sodium channel blocker Tetrodotoxin, bath application of 0.5 mM Muscimol produces an inward current in these cells. We measured peak inward current from 10 control cells and 10 DNE cells. Our results show that DNE causes in an increased GABAA receptor mediated inward current (Control {n=10} −239.3±30.75 pA, DNE {n=10} −364.4±38.7 pA, P=0.02). This result is consistent with DNE causing increased GABAA receptor expression in XII MNs.Support or Funding InformationNIH RO1H071302 AHA 12GRXIT120S0354

Phox2b‐negative Parafacial Neurons Simultaneously Regulate Active Expiration and Airway Patency Under Hypercapnia in in situ Preparations of Rats

Hypercapnia produces active expiration in rats and the recruitment of late‐expiratory (late‐E) neurons located in the parafacial Respiratory Group (pFRG) of the ventral medullary brainstem. We tested the hypothesis that hypercapnia produces active expiration and concomitant cranial respiratory motor responses controlling the oropharyngeal and upper airway patency by pFRG late‐E neurons disinhibition, but not via synaptic excitation. Phrenic nerve, abdominal nerve (AbN), cranial respiratory motor nerves, subglottal pressure, and medullary and spinal neurons/motoneurons were recorded in in situ preparations of juvenile rats. Hypercapnia evoked AbN active expiration (n=12), exaggerated late‐E discharges in cranial respiratory motor outflows (n=12), and glottal dilatation (n=6) via late‐E synaptic excitation of abdominal (n=8), hypoglossal (n=6) and laryngeal (n=12) motoneurons. Simultaneous rhythmic late‐E activity in previously silent Phox2b‐negative pFRG neurons was also evoked by hypercapnia (n=12). In addition, hypercapnia‐evoked AbN active expiration, neural and neuronal late‐E activities were eliminated by pFRG inhibition (n=18), but not after blockade of synaptic excitation (n=9). On the other hand, pFRG inhibition did not affect either hypercapnia‐induced inspiratory increases in respiratory motor outflows (n=12) or CO2 sensitivity of the more medial Phox2b‐positive neurons in the retrotrapezoid nucleus (RTN; n=8), despite elimination of AbN active expiration. Our data suggest that neither RTN Phox2b‐positive nor other CO2‐sensitive brainstem neurons activate Phox2b‐negative pFRG late‐E neurons under hypercapnia to produce AbN active expiration and concomitant cranial motor respiratory responses controlling the oropharyngeal and upper airway patency. Hypercapnia seems to produce pFRG late‐E neuronal disinhibition in in situ preparations of juvenile rats to activate abdominal, hypoglossal and laryngeal motoneurons.Support or Funding InformationFAPESP: SÃO PAULO RESEARCH FOUNDATION

Activation of the Hypoglossal to Tongue Musculature Motor Pathway by Remote Control

Reduced tongue muscle tone precipitates obstructive sleep apnea (OSA), and activation of the tongue musculature can lessen OSA. The hypoglossal motor nucleus (HMN) innervates the tongue muscles but there is no pharmacological agent currently able to selectively manipulate a channel (e.g., Kir2.4) that is highly restricted in its expression to cranial motor pools such as the HMN. To model the effect of manipulating such a restricted target, we introduced a “designer” receptor into the HMN and selectively modulated it with a “designer” drug. We used cre-dependent viral vectors (AAV8-hSyn-DIO-hM3Dq-mCherry) to transduce hypoglossal motoneurons of ChAT-Cre+ mice with hM3Dq (activating) receptors. We measured sleep and breathing in three conditions: (i) sham, (ii) after systemic administration of clozapine-N-oxide (CNO; 1 mg/kg) or (iii) vehicle. CNO activates hM3Dq receptors but is otherwise biologically inert. Systemic administration of CNO caused significant and sustained increases in tongue muscle activity in non-REM (261 ± 33% for 10 hrs) and REM sleep (217 ± 21% for 8 hrs), both P < 0.01 versus controls. Responses were specific and selective for the tongue with no effects on diaphragm or postural muscle activities, or sleep-wake states. These results support targeting a selective and restricted “druggable” target at the HMN (e.g., Kir2.4) to activate tongue motor activity during sleep.