Laryngeal Motoneurons Research Articles

Temporal Expression of Hox Genes and Phox2b in the Rat Nucleus Ambiguus During Development: Implications on Laryngeal Innervation.

Recurrent laryngeal nerve (RLN) injury results in synkinetic reinnervation and vocal fold paralysis. Investigation of cues expressed in the developing brainstem that influence correct selective targeting of intrinsic laryngeal muscles may elucidate post-injury abnormalities contributing to non-functional reinnervation. Primary targets of interest were Hoxb1 and Hoxb2, members of the Hox family that create overlapping gradients in the developing brain, and their target Phox2b, a transcription factor necessary for cranial nerve branchio- and visceromotoneuron survival. Rat embryos at developmental days E14, E16, E18, and E20 (4 animals/age) were sectioned for RNA in situ hybridization to detect Hoxb1, Hoxb2, and Phox2b mRNA within the brainstem. Slides were costained with Islet1 antibody for identification of the nucleus ambiguus. Results were confirmed using immunohistochemistry. Sections were imaged on a confocal microscope. RNA and protein expressions were quantified using QuPath. Statistical analyses were performed using R. Hoxb1, Hoxb2, and Phox2b expressions varied according to embryologic age. Hoxb1 and Hoxb2 expression peaked at E16, with significant decreases at E18 and E20 (one-way ANOVA p = 0.001 for both). Phox2b expression was highest at E14 and trended downward with increased embryologic age (one-way ANOVA p = 0.005). Peak expression of Hoxb1 and Hoxb2 is observed at time points when the RLN arrives at the larynx and begins to branch toward individual muscles, positioning these gene products to be involved in cueing laryngeal motoneuron identity and target identification. Higher expression of Phox2b earlier in development suggests a role in laryngeal motoneuron formation. NA Laryngoscope, 133:3462-3471, 2023.

Optogenetic stimulation of pre–Bötzinger complex reveals novel circuit interactions in swallowing–breathing coordination

The coordination of swallowing with breathing, in particular inspiration, is essential for homeostasis in most organisms. While much has been learned about the neuronal network critical for inspiration in mammals, the pre-Bötzinger complex (preBötC), little is known about how this network interacts with swallowing. Here we activate within the preBötC excitatory neurons (defined as Vglut2 and Sst neurons) and inhibitory neurons (defined as Vgat neurons) and inhibit and activate neurons defined by the transcription factor Dbx1 to gain an understanding of the coordination between the preBötC and swallow behavior. We found that stimulating inhibitory preBötC neurons did not mimic the premature shutdown of inspiratory activity caused by water swallows, suggesting that swallow-induced suppression of inspiratory activity is not directly mediated by the inhibitory neurons in the preBötC. By contrast, stimulation of preBötC Dbx1 neurons delayed laryngeal closure of the swallow sequence. Inhibition of Dbx1 neurons increased laryngeal closure duration and stimulation of Sst neurons pushed swallow occurrence to later in the respiratory cycle, suggesting that excitatory neurons from the preBötC connect to the laryngeal motoneurons and contribute to the timing of swallowing. Interestingly, the delayed swallow sequence was also caused by chronic intermittent hypoxia (CIH), a model for sleep apnea, which is 1) known to destabilize inspiratory activity and 2) associated with dysphagia. This delay was not present when inhibiting Dbx1 neurons. We propose that a stable preBötC is essential for normal swallow pattern generation and disruption may contribute to the dysphagia seen in obstructive sleep apnea.

Cortical basis for skilled vocalization

Marmosets display remarkable vocal motor abilities. Macaques do not. What is it about the marmoset brain that enables its skill in the vocal domain? We examined the cortical control of a laryngeal muscle that is essential for vocalization in both species. We found that, in both monkeys, multiple premotor areas in the frontal lobe along with the primary motor cortex (M1) are major sources of disynaptic drive to laryngeal motoneurons. Two of the premotor areas, ventral area 6 (area 6V) and the supplementary motor area (SMA), are a substantially larger source of descending output in marmosets. We propose that the enhanced vocal motor skills of marmosets are due, in part, to the expansion of descending output from these premotor areas.

The laryngeal adductor reflex (LAR) is modulated by the serotonin 5‐HT1A agonist 8‐OH‐DPAT

Airway protection is maintained through a constellation of behaviors. The laryngeal adductor reflex (LAR) responds to mechanical or chemical stimulation of the laryngeal orifice with rapid adduction of the vocal folds. While there are abundant serotonin receptors on laryngeal motoneurons, the effects of serotonin agents on the LAR have not been investigated. To this end, a dose response protocol of 8‐OH‐DPAT (5HT1A receptor agonist) was performed on adult cats (n = 2). Animals were anesthetized using an intravenous dose of sodium pentobarbital and then tracheostomized. Electromyogram (EMG) activity of laryngeal muscles was recorded and LARs were induced by infusing water into the larynx during both normocapnia (0% CO2) and hypercapnia (10% CO2) conditions. A series of six cumulative doses (1 – 300 μg/kg) were delivered intra‐venously. 8‐OH‐DPAT is thought to activate the serotonergic raphe neuron autoreceptors at lower doses and the post‐synaptic receptors on target neurons at higher doses. LAR occurred as a response to water stimulus in 50±23% of control trials. LAR occurrence was modulated by 8‐OH‐DPAT in a dose‐dependent manner: LAR frequency at lower doses may have decreased but this was variable, and LAR frequency appeared to be increased at higher doses (LAR occurred in 13±18%, 17±23%, 50±70%, 57±9%, 84±7%, and 60±4% of stimulus trials at 1, 3, 10, 30, 100, and 300 μg/kg doses, respectively). LAR can be used as a strong clinical predictor of swallow impairment; while other airway protective behaviors require activity across multiple cranial nerves, the LAR is regulated by only laryngeal branches of the vagus nerve. Thus, the LAR is a promising biomarker for testing novel therapeutic agents. These results suggest that pharmacological interventions may be capable of facilitating laryngeal function in patients suffering from dysphagia.

Characterization of laryngeal motor neuron properties in the American bullfrog, Lithobates catesbieanus

Motor neurons represent the final output from the central respiratory network. American bullfrogs, Lithobates catesbieanus, have provided insight into development and plasticity of the breathing control system, yet cellular aspects of bullfrog motor neurons are not well-described. In this study, we characterized properties of laryngeal motor neurons that produce motor outflow to the glottal dilator, a muscle that gates airflow to the lungs of anurans. To this end, we measured several intrinsic membrane properties of labeled laryngeal motor neurons in brain slices. Using unsupervised clustering analyses, we identified two broad classes of motor neurons: those with high firing rates and strong adaptation (∼70 %), and those with lower firing rates and less adaptation (∼30 %). These results suggest that two neuronal cell types innervate the glottal dilator, roughly aligning with the composition of fast and slower twitch fibers of this muscle. In sum, these data reinforce the need to consider cell-type when assessing motor neuron function in the respiratory network.

The role of glycinergic inhibition in respiratory pattern formation and cardio-respiratory coupling in rats.

Cardio-respiratory coupling is reflected as respiratory sinus arrhythmia (RSA) and inspiratory-related bursting of sympathetic nerve activity. Inspiratory-related inhibitory and/or postinspiratory-related excitatory drive of cardiac vagal motoneurons (CVMs) can generate RSA. Since respiratory oscillations may depend on synaptic inhibition, we investigated the effects of blocking glycinergic neurotransmission (systemic and local application of the glycine receptor (GlyR) antagonist, strychnine) on the expression of the respiratory motor pattern, RSA and sympatho-respiratory coupling. We recorded heart-rate, phrenic, recurrent laryngeal and thoracic sympathetic nerve activities (PNA, RLNA, t-SNA) in a working-heart-brainstem preparation of rats, and show that systemic strychnine (50–200 ​nM) abolished RSA and triggered a shift of postinspiratory RLNA into inspiration, while t-SNA remained unchanged. Bilateral strychnine microinjection into the ventrolateral medullary area containing CVMs and laryngeal motoneurons (LMNs) of the nucleus ambiguus (NA/CVLM), the nucleus tractus solitarii, pre-Bötzinger Complex, Bötzinger Complex or Kölliker-Fuse nuclei revealed that only NA/CVLM strychnine microinjections mimicked the effects of systemic application. In all other target nuclei, except the Bötzinger Complex, GlyR-blockade attenuated the inspiratory-tachycardia of the RSA to a similar degree while evoking only a modest change in respiratory motor patterning, without changing the timing of postinspiratory-RLNA, or t-SNA. Thus, glycinergic inhibition at the motoneuronal level is involved in the generation of RSA and the separation of inspiratory and postinspiratory bursting of LMNs. Within the distributed ponto-medullary respiratory pre-motor network, local glycinergic inhibition contribute to the modulation of RSA tachycardia, respiratory frequency and phase duration but, surprisingly it had no major role in the mediation of respiratory-sympathetic coupling.

Laryngeal Adductor Reflex Motor Bursts Rapidly Oscillate in the Cat

The laryngeal adductor reflex (LAR) is a protective behavior induced by stimulation of upper airway afferents. Laryngeal afferent information reaches interneurons in the medulla via the superior laryngeal nerve (SLN). Expiratory laryngeal motoneurons (ELMs) are subsequently excited causing contraction of the thyroarytenoid (TA) and posterior cricoarytenoid (PCA) muscles. The LAR consists of a short latency (~10 ms), short duration (~10 ms) burst (R1) and a longer latency (~40ms) burst (R2) in laryngeal electromyogram (EMG) activity. We have shown that R1 LAR magnitude undergoes frequency‐dependent depression in response to electrical stimulation (eStim) of the SLN. Based on preliminary observations, we hypothesized that the R1 component of the LAR induced by SLN eStim oscillates. In anesthetized, spontaneously breathing cats (n=12: 7 males, 5 females), we recorded EMG activity from the TA bilaterally and the PCA unilaterally. The SLN was electrically stimulated at 2 or 20 Hz to evoke TA LAR ipsilateral to stimulation (iTA). No sex differences in TA or PCA LAR EMG magnitude were observed. Consistent with previous findings, TA LAR magnitude differed with breathing phase; normalized iTA LAR magnitude was 10% larger during expiration compared to inspiration. TA LAR contralateral to stimulation (cTA) and ipsilateral PCA LAR magnitudes were not breathing phase dependent. TA and PCA LAR EMG oscillations were highly variable across animals (range: 244 to 1140 Hz). Mean iTA EMG frequency was decreased at 20 Hz (2 to 20 Hz: 616 to 518 Hz, p=0.05). Mean cTA periodicity trended down at 20 Hz (2 to 20 Hz cTA: 647 to 531 Hz, p=0.07). In separate decerebrate, paralyzed cats (n=13), extracellular recordings of neurons in the ventral respiratory column (VRC) with multiple electrode arrays were conducted. Peristimulus time histograms provided evidence that ELMs oscillated at frequencies following SLN eStim similar to the lower range of periodicities in TA EMGs in unparalyzed animals. A subset of neurons recorded in the VRC were excited or inhibited by SLN eStim at short latencies. Spike train analysis suggested that ELM oscillations could be accounted for by synaptic inhibition and excitation from laryngeal‐responsive VRC inspiratory, expiratory, and non‐breathing modulated neurons. We conclude that the LAR is temporally regulated by a complex, anatomically distributed brainstem network that is capable of strong synchrony in response to stimulation of laryngeal afferents. We speculate that the function of this laryngeal‐responsive network is to exert tight regulatory control over LAR patterning.Support or Funding InformationSupported by NIH HL131716 and 3OT2OD023854

Potential mechanisms of opioid depression of the laryngeal adductor reflex: insights from a neuromechanical model of the respiratory control system

Opioid use increases the risk of aspiration and pneumonia, presumably through depression of protective reflexes that prevent intrusion of foreign material into the airways. The laryngeal adductor reflex (LAR) is an airway protective behavior that utilizes bilateral thyroarytenoid (TA) muscles and prevents material entering the trachea and lungs. The operational features and specific neural mechanisms which regulate and coordinate the occurrence of the LAR to prevent aspiration during opioid use are not fully understood. In vivo experiments were conducted using unilateral electrical stimulation of the superior laryngeal nerve (SLN) to induce the LAR. Systemic opioid administration preferentially attenuated the contralateral LAR, which is consistent with dysregulation of airway protection. We speculated that computational modeling of a specific LAR circuit would provide testable hypotheses that could account for this effect of opioids on the LAR. We have developed a predictive computational model which incorporates a distributed neural network that excites expiratory laryngeal motoneurons (ELM). The stochastic neural network simulator consists of discrete “integrate and fire” populations. The model circuit was informed by response patterns during the LAR of neurons in the regions of the nucleus tractus solitarius (NTS), the reticular formation (RF), and the ventrolateral respiratory column (VRC). With our network scale model, we simulated modulation of the LAR during administration of opioids by decreasing input currents to neurons in the RF that provide presynaptic input to ELMs of the VRC and then we activated laryngeal receptors. Simulator outputs were compared to collected in vivo data. Computer simulations reproduced the temporal features of the LAR, and the unilateral effects of opioids on the LAR by increasing potassium conductance of presynaptic neurons, depressing presynaptic terminal excitability, and hyperpolarizing ELM populations. Simulations of the LAR and phasic breathing suggest that an important area of focus for modeling efforts will be (i) refining the spatiotemporal characteristics of the behavior, (ii) the potential role of NTS laryngeal interneuronal networks in the effects of opioids, and (iii) synaptic regulation of oscillatory behavior of the LAR.Support or Funding InformationSupported by NIH HL131716, 3OT2OD023854, and NS 110169.

Neurons in the Intermediate Reticular Nucleus Coordinate Postinspiratory Activity, Swallowing, and Respiratory-Sympathetic Coupling in the Rat.

Breathing results from sequential recruitment of muscles in the expiratory, inspiratory, and postinspiratory (post-I) phases of the respiratory cycle. Here we investigate whether neurons in the medullary intermediate reticular nucleus (IRt) are components of a central pattern generator (CPG) that generates post-I activity in laryngeal adductors and vasomotor sympathetic nerves and interacts with other members of the central respiratory network to terminate inspiration. We first identified the region of the (male) rat IRt that contains the highest density of lightly cholinergic neurons, many of which are glutamatergic, which aligns well with the putative postinspiratory complex in the mouse (Anderson et al., 2016). Acute bilateral inhibition of this region reduced the amplitudes of post-I vagal and sympathetic nerve activities. However, although associated with reduced expiratory duration and increased respiratory frequency, IRt inhibition did not affect inspiratory duration or abolish the recruitment of post-I activity during acute hypoxemia as predicted. Rather than representing an independent CPG for post-I activity, we hypothesized that IRt neurons may instead function as a relay that distributes post-I activity generated elsewhere, and wondered whether they could be a site of integration for para-respiratory CPGs that drive the same outputs. Consistent with this idea, IRt inhibition blocked rhythmic motor and autonomic components of fictive swallow but not swallow-related apnea. Our data support a role for IRt neurons in the transmission of post-I and swallowing activity to motor and sympathetic outputs, but suggest that other mechanisms also contribute to the generation of post-I activity.SIGNIFICANCE STATEMENT Interactions between multiple coupled oscillators underlie a three-part respiratory cycle composed from inspiratory, postinspiratory (post-I), and late-expiratory phases. Central post-I activity terminates inspiration and activates laryngeal motoneurons. We investigate whether neurons in the intermediate reticular nucleus (IRt) form the central pattern generator (CPG) responsible for post-I activity. We confirm that IRt activity contributes to post-I motor and autonomic outputs, and find that IRt neurons are necessary for activation of the same outputs during swallow, but that they are not required for termination of inspiration or recruitment of post-I activity during hypoxemia. We conclude that this population may not represent a distinct CPG, but instead may function as a premotor relay that integrates activity generated by diverse respiratory and nonrespiratory CPGs.

Cluster Analysis of Neuronal Discharge Patterns During Breathing and the Laryngeal Adductor Reflex with Computer Simulations and Dynamic Visualizations

The laryngeal adductor reflex (LAR) is a simple airway protective behavior that closes the vocal folds in response to mechanical or chemical stimulation of sensory afferents in the larynx. Previous work suggested that the intra‐medullary pathway that is responsible for activating laryngeal motoneurons is composed of as little as two synapses. We speculated that the central pathways supporting this behavior are more complex and involve neurons of several different phenotypes. Breathing and non‐breathing modulated neurons (n=632) were recorded with multiple electrode arrays in the ventral respiratory column (ventral respiratory group, pre‐Bötzinger, Bötzinger), pons, raphé, dorsal lateral medulla and spinal cord of decerebrate, paralyzed, vagus‐intact cats (n=13). The LAR was induced by electrical stimulation of the superior laryngeal nerve. We classified brainstem neuron activity phenotypes using a quantitative, reproducible clustering routine during baseline breathing and the LAR. We identified 25 breathing phenotypes that were mapped onto 16 LAR phenotypes. These results were used to produce a simplified model and simulations of the LAR. A 3‐D brainstem atlas displayed neuronal firing rates during periods of baseline breathing and/or the LAR, illuminated in proportion to firing rate. These data support distinct rather than a continuum of phenotypes based on discharge patterns during breathing as well as during the LAR.Support or Funding InformationSupport: OT2OD023854‐01, HL109025, HL131716This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Biology and Evolution of Speech: A Comparative Analysis

I analyze the biological underpinnings of human speech from a comparative perspective. By first identifying mechanisms that are evolutionarily derived relative to other primates, we obtain members of the faculty of language, derived components (FLD). Understanding when and why these evolved is central to understanding the evolution of speech. There is little evidence for human-specific mechanisms in auditory perception, and the hypothesis that speech perception is “special” is poorly supported by comparative data. Regarding speech production, human peripheral vocal anatomy includes several derived characteristics (permanently descended larynx, loss of air sacs), but their importance has been overestimated. In contrast, the central neural mechanisms underlying speech production involve crucial derived characteristics (direct monosynaptic connections from motor cortex to laryngeal motor neurons, derived intracortical dorsal circuitry between auditory and motor regions). Paleo-DNA from fossil hominins provides an exciting new opportunity to determine when these derived speech production mechanisms arose during evolution.

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

Non-chemosensitive parafacial neurons simultaneously regulate active expiration and airway patency under hypercapnia in rats.

Hypercapnia or parafacial respiratory group (pFRG) disinhibition at normocapnia evokes active expiration in rats by recruitment of pFRG late-expiratory (late-E) neurons. We show that hypercapnia simultaneously evoked active expiration and exaggerated glottal dilatation by late-E synaptic excitation of abdominal, hypoglossal and laryngeal motoneurons. Simultaneous rhythmic expiratory activity in previously silent pFRG late-E neurons, which did not express the marker of ventral medullary CO2 -sensitive neurons (transcription factor Phox2b), was also evoked by hypercapnia. Hypercapnia-evoked active expiration, neural and neuronal late-E activities were eliminated by pFRG inhibition, but not after blockade of synaptic excitation. Hypercapnia produces disinhibition of non-chemosensitive pFRG late-E neurons to evoke active expiration and concomitant cranial motor respiratory responses controlling the oropharyngeal and upper airway patency. 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 disinhibition of pFRG late-E neurons, 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, exaggerated late-E discharges in cranial respiratory motor outflows, and glottal dilatation via late-E synaptic excitation of abdominal, hypoglossal and laryngeal motoneurons. Simultaneous rhythmic late-E activity in previously silent pFRG neurons, which did not express the marker of ventral medullary CO2 -sensitive neurons (transcription factor Phox2b), was also evoked by hypercapnia. In addition, hypercapnia-evoked AbN active expiration, neural and neuronal late-E activities were eliminated by pFRG inhibition, but not after blockade of synaptic excitation. On the other hand, pFRG inhibition did not affect either hypercapnia-induced inspiratory increases in respiratory motor outflows or CO2 sensitivity of the more medial Phox2b-positive neurons in the retrotrapezoid nucleus (RTN). 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 produces disinhibition of non-chemosensitive pFRG late-E neurons in in situ preparations of juvenile rats to activate abdominal, hypoglossal and laryngeal motoneurons.

Testing the evolutionary conservation of vocal motoneurons in vertebrates

Medullary motoneurons drive vocalization in many vertebrate lineages including fish, amphibians, birds, and mammals. The developmental history of vocal motoneuron populations in each of these lineages remains largely unknown. The highly conserved transcription factor Paired-like Homeobox 2b (Phox2b) is presumed to be expressed in all vertebrate hindbrain branchial motoneurons, including laryngeal motoneurons essential for vocalization in humans. We used immunohistochemistry and in situ hybridization to examine Phox2b protein and mRNA expression in caudal hindbrain and rostral spinal cord motoneuron populations in seven species across five chordate classes. Phox2b was present in motoneurons dedicated to sound production in mice and frogs (bullfrog, African clawed frog), but not those in bird (zebra finch) or bony fish (midshipman, channel catfish). Overall, the pattern of caudal medullary motoneuron Phox2b expression was conserved across vertebrates and similar to expression in sea lamprey. These observations suggest that motoneurons dedicated to sound production in vertebrates are not derived from a single developmentally or evolutionarily conserved progenitor pool.

Tauopathy in the Ponto‐Medullary Brainstem Nuclei of Aged Tau‐P301L Mice

We recently identified key brainstem areas critically involved in coordinating swallowing and breathing: 1) Nucleus of the solitary tract (NTS), which generates a phasic or rhythmic ‘command’ to produce sequential swallowing in response to sensory stimuli; and 2) Kölliker‐Fuse nucleus (KF), which provides tonic drive for the laryngeal adductors and completely seals the trachea during, and between, swallows. Our aim was to identify whether an underlying pathology exists in the NTS and KF of an established model of neurodegeneration, the Tau‐P301 mouse, and humans that suffered from dementia, which may link dementia and swallowing dysfunction.Tauopathy and neurofibrillary tangle‐related neuropathological morphology was identified by immunohistochemistry using antisera raised against phosphorylated tau. Double‐fluorescence immunohistochemistry was performed to characterise the identity and functionality of cell bodies expressing phosphorylated tau.Cell bodies immunopositive for phosphorylated tau were distributed within the KF, and to a lesser degree, NTS, of Tau‐P301 mice, and the KF of dementia patients with a history of swallowing disorder. This coincided with an upregulated density/intensity of GABA and GAD67 immunoreactivity in fibers/terminals within the KF. Tau positive cell bodies in the KF coexpressed the transcription factor FOXP2.Respiratory‐related neurons in the brainstem are susceptible to neurodegeneration in Tau‐P301 mice and humans suffering dementia, which may disrupt the KF's control of laryngeal adductor motor neurons in the NA and lead to miscoordinated swallowing/breathing patterns.

Electrophysiological properties of laryngeal motoneurones in rats submitted to chronic intermittent hypoxia.

The respiratory control of the glottis by laryngeal motoneurones is characterized by inspiratory abduction and post-inspiratory adduction causing decreases and increases in upper airway resistance, respectively. Chronic intermittent hypoxia (CIH), an important component of obstructive sleep apnoea, exaggerated glottal abduction (before inspiration), associated with active expiration and decreased glottal adduction during post-inspiration. CIH increased the inspiratory and decreased the post-inspiratory laryngeal motoneurone activities, which is not associated to changes in their intrinsic electrophysiological properties. We conclude that the changes in the respiratory network after CIH seem to be an adaptive process required for an appropriated pulmonary ventilation and control of upper airway resistance under intermittent episodes of hypoxia. To keep an appropriate airflow to and from the lungs under physiological conditions a precise neural co-ordination of the upper airway resistance by laryngeal motoneurones in the nucleus ambiguus is essential. Chronic intermittent hypoxia (CIH), an important component of obstructive sleep apnoea, may alter these fine mechanisms. Here, using nerve and whole cell patch clamp recordings in in situ preparations of rats we investigated the effects of CIH on the respiratory control of the upper airway resistance, on the electrophysiological properties of laryngeal motoneurones in the nucleus ambiguus, and the role of carotid body (CB) afferents to the brainstem on the underlying mechanisms of these effects. CIH rats exhibited longer pre-inspiratory and lower post-inspiratory superior laryngeal nerve activities than control rats. These changes produced exaggerated glottal abduction (before inspiration) and decreased glottal adduction during post-inspiration, indicating a reduction of upper airway resistance during these respiratory phases after CIH. CB denervation abolished these changes produced by CIH. Regarding choline acetyltransferase positive-laryngeal motoneurones, CIH increased the firing frequency of inspiratory and decreased the firing frequency of post-inspiratory laryngeal motoneurones, without changes in their intrinsic electrophysiological properties. These data show that the effects of CIH on the upper airway resistance and laryngeal motoneurones activities are driven by the integrity of CB, which afferents induce changes in the central respiratory generators in the brainstem. These neural changes in the respiratory network seem to be an adaptive process required for an appropriated pulmonary ventilation and control of upper airway resistance under intermittent episodes of hypoxia.

Catecholamine inputs to expiratory laryngeal motoneurons in rats.

Many respiration-related interneurons and motoneurons receive a catecholaminergic input, but the extent and distribution of this input to recurrent laryngeal motoneurons that innervate intrinsic muscles of the larynx are not clear. In the present study, we examined the catecholaminergic input to expiratory laryngeal motoneurons in the caudal nucleus ambiguus by combining intracellular labeling of single identified motoneurons, with immunohistochemistry to reveal tyrosine hydroxylase immunoreactive (catecholaminergic) terminal varicosities. Close appositions were found between the two structures, with 18 ± 5 close appositions per motoneuron (n = 7). Close appositions were more frequently observed on distal rather than proximal dendrites. Axosomatic appositions were not seen. In order to determine the source of this input, microinjections of cholera toxin B subunit (1%, 20 nl) were made into the caudal nucleus ambiguus. Retrogradely labeled neurons, located in the ipsilateral nucleus tractus solitarius and the area postrema, were tyrosine hydroxylase-positive. Our results not only demonstrate details of the extent and distribution of potential catecholamine inputs to the expiratory laryngeal motoneuron, but further indicate that the inputs, at least in part, originate from the dorsomedial medulla, providing a potential anatomical basis for previously reported catecholaminergic effects on the laryngeal adductor reflex.

CrossTalk opposing view: The pre‐Bötzinger complex is not essential for respiratory depression following systemic administration of opioid analgesics

Opioid receptors and enkephalinergic nerve terminals are widely distributed throughout respiratory-related regions of the brainstem and in the phrenic motor nucleus of the spinal cord (Xia & Haddad, 1991; Laferriere et al. 1999; Wang et al. 2002; Haji et al. 2003a; Lonergan et al. 2003a,b; Strornetta et al. 2003). Since opiate drugs given systemically will act on opioid receptors with conjoint selectivity in all respiratory regions, respiratory depression is unlikely to be dependent on actions at a single site. Therapeutic doses of opioids given to most mammalian species depress respiratory rate, minute ventilation, alveolar–arterial gas exchange and respiratory responsiveness to hypoxia and hypercapnia (Jaffe & Martin, 1990). Opioid-mediated depression of respiration is due at least in part to direct effects on the brainstem respiratory network, which includes several sites of action in medullary and pontine regions (reviewed by Pattison, 2008; Lalley, 2008). The degree of opioid-mediated respiratory depression depends on agonist dose, opioid receptor density and the subtypes of opioid receptor in various respiratory regions. Species variability and stage of development are also factors (Santiago & Edelman, 1985). In the paragraphs to follow, we review results of studies that indicate that the pre-Botzinger complex (preBotC) is not essential for respiratory depression by systemically administered opioid analgesics.

Reorganization of laryngeal motoneurons after crush injury in the recurrent laryngeal nerve of the rat

Motoneurons innervating laryngeal muscles are located in the nucleus ambiguus (Amb), but there is no general agreement on the somatotopic representation and even less is known on how an injury in the recurrent laryngeal nerve (RLN) affects this pattern. This study analyzes the normal somatotopy of those motoneurons and describes its changes over time after a crush injury to the RLN. In the control group (control group 1, n = 9 rats), the posterior cricoarytenoid (PCA) and thyroarytenoid (TA) muscles were injected with cholera toxin-B. In the experimental groups the left RLN of each animal was crushed with a fine tip forceps and, after several survival periods (1, 2, 4, 8, 12 weeks; minimum six rats per time), the PCA and TA muscles were injected as described above. After each surgery, the motility of the vocal folds was evaluated. Additional control experiments were performed; the second control experiment (control group 2, n = 6 rats) was performed labeling the TA and PCA immediately prior to the section of the superior laryngeal nerve (SLN), in order to eliminate the possibility of accidental labeling of the cricothyroid (CT) muscle by spread from the injection site. The third control group (control group 3, n = 5 rats) was included to determine if there is some sprouting from the SLN into the territories of the RLN after a crush of this last nerve. One week after the crush injury of the RLN, the PCA and TA muscles were injected immediately before the section of the SLN. The results show that a single population of neurons represents each muscle with the PCA in the most rostral position followed caudalwards by the TA. One week post-RLN injury, both the somatotopy and the number of labeled motoneurons changed, where the labeled neurons were distributed randomly; in addition, an area of topographical overlap of the two populations was observed and vocal fold mobility was lost. In the rest of the survival periods, the overlapping area is larger, but the movement of the vocal folds tends to recover. After 12 weeks of survival, the disorganization within the Amb is the largest, but the number of motoneurons is similar to control, and all animals recovered the movement of the left vocal fold. Our additional controls indicate that no tracer spread to the CT muscle occurred, and that many of the labeled motoneurons from the PCA after 1 week post-RLN injury correspond to motoneurons whose axons travel in the SLN. Therefore, it seems that after RLN injury there is a collateral sprouting and collateral innervation. Although the somatotopic organization of the Amb is lost after a crush injury of the RLN and does not recover in the times studied here, the movement of the vocal folds as well as the number of neurons that supply the TA and the PCA muscles recovered within 8 weeks, indicating that the central nervous system of the rat has a great capacity of plasticity.

Expiratory-modulated laryngeal motoneurons exhibit a hyperpolarization preceding depolarization during superior laryngeal nerve stimulation in the in vivo adult rat

Swallowing requires the sequential activation of tongue, pharyngeal and esophageal muscles to propel the food bolus towards the stomach. Aspiration during swallow is prevented by adduction of the vocal cords during the oropharyngeal phase. Expiratory-modulated laryngeal motoneurons (ELM) exhibit a burst of action potentials during swallows elicited by electrical stimulation of the superior laryngeal nerve (SLN). Here we sought to investigate changes in membrane potential in ELM during superior laryngeal nerve stimulation in the anaesthetised, in vivo adult rat preparation. Intracellular recordings of ELM in the caudal nucleus ambiguus (identified by antidromic activation from the recurrent laryngeal nerve) demonstrated that ELM bursting activity following SLN stimulation is associated with a depolarization that is preceded by a small hyperpolarization. During spontaneous ELM bursts, the preceding hyperpolarization separated the bursting activity from its usual post-inspiratory activity. These findings demonstrate that the in vivo adult rat preparation is suitable for the study of swallow-related activity in laryngeal motoneurons.