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Neurological respiratory deficits are serious outcomes of West Nile virus (WNV) disease. WNV patients requiring intubation have a poor prognosis. We previously reported that WNV-infected rodents also appear to have respiratory deficits when assessed by whole-body plethysmography and diaphragmatic electromyography. The purpose of this study was to determine if the nature of the respiratory deficits in WNV-infected rodents is neurological and if deficits are due to a disorder of brainstem respiratory centers, cervical spinal cord (CSC) phrenic motor neuron (PMN) circuitry, or both. We recorded phrenic nerve (PN) activity and found that in WNV-infected mice, PN amplitude is reduced, corroborating a neurological basis for respiratory deficits. These results were associated with a reduction in CSC motor neuron number. We found no dramatic deficits, however, in brainstem-mediated breathing rhythm generation or responses to hypercapnia. PN frequency and pattern parameters were normal, and all PN parameters changed appropriately upon a CO 2 challenge. Histological analysis revealed generalized microglia activation, astrocyte reactivity, T cell and neutrophil infiltration, and mild histopathologic lesions in both the brainstem and CSC, but none of these were tightly correlated with PN function. Similar results in PN activity, brainstem function, motor neuron number, and histopathology were seen in WNV-infected hamsters, except that histopathologic lesions were more severe. Taken together, the results suggest that respiratory deficits in acute WNV infection are primarily due to a lower motor neuron disorder affecting PMNs and the PN rather than a brainstem disorder. Future efforts should focus on markers of neuronal dysfunction, axonal degeneration, and myelination.

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., (© 2014 Wiley Periodicals, Inc.)

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., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

A striking effect of stimulating the superior laryngeal nerve (SLN) is its ability to inhibit central inspiratory activity (cause ‘phrenic apnoea'), but the mechanism underlying this inhibition remains unclear. Here we demonstrate, by stimulating the SLN at varying frequencies, that the evoked non-respiratory burst activity recorded from expiratory laryngeal motoneurons (ELMs) has an intimate temporal relationship with phrenic apnoea. During 1–5 Hz SLN stimulation, occasional absences of phrenic nerve discharge (PND) occurred such that every absent PND was preceded by an ELM burst activity. During 10–20 Hz SLN stimulation, more bursts were evoked together with more absent PNDs, leading eventually to phrenic apnoea. Interestingly, subsequent microinjections of isoguvacine (10 mm, 20–40 nl) into ipsilateral Bötzinger complex (BötC) and contralateral nucleus tractus solitarii (NTS) significantly attenuated the apnoeic response but not the ELM burst activity. Our results suggest a bifurcating projection from NTS to both the caudal nucleus ambiguus and BötC, which mediates the closely related ELM burst and apnoeic response, respectively. We believe that such an intimate timing between laryngeal behaviour and breathing is crucial for the effective elaboration of the different airway protective behaviours elicited following SLN stimulation, including the laryngeal adductor reflex, swallowing and cough.

The intrinsic laryngeal muscles are differentially modulated during respiration as well as other states and behaviors such as hypocapnia and sleep. Previous anatomical and pharmacological studies indicate a role for acetylcholine at the level of the nucleus ambiguus in the modulation of laryngeal motoneuron (LMN) activity. The present study investigated the anatomical nature of cholinergic input to inspiratory- (ILM) and expiratory-modulated (ELM) laryngeal motoneurons in the loose formation of the nucleus ambiguus. Using combined in vivo intracellular recording, dye filling, and immunohistochemistry, we demonstrate that LMNs identified in Sprague-Dawley rat receive several close appositions from vesicular acetylcholine transporter-immunoreactive (VAChT-ir) boutons. ELMs receive a significantly greater number of close appositions (mean ± standard deviation [SD]: 47 ± 11; n = 5) than ILMs (32 ± 9; n = 8; t-test P < 0.05). For both LMN types, more close appositions were observed on the cell soma and proximal dendrites compared to distal dendrites (two-way analysis of variance [ANOVA], P < 0.0001). Using fluorescence confocal microscopy, almost 90% of VAChT-ir close appositions (n = 45 boutons on n = 4 ELMs) were colocalized with the synaptic marker synaptophysin. These results support a strong influence of cholinergic input on LMNs and may have implications in the differential modulation of laryngeal muscle activity., (© 2010 Wiley-Liss, Inc.)

Breathing is a vegetative function that is altered during more complex behaviours such as exercise, vocalisation and respiratory protective reflexes. Recent years have seen recognition of the importance of respiratory pattern generation in addition to rhythm generation. Respiratory-modulated cranial motoneurons (laryngeal, pharyngeal, hypoglossal, facial) offer a unique insight into the control of respiration since: (1) they receive rhythmic respiratory inputs but; (2) their respiratory-modulated firing pattern differs to that of phrenic neurons to suit their function, (for example, hypoglossal motoneurons begin firing and thus the tongue depresses before the onset of phrenic nerve discharge and diaphragmatic during inspiration) and; (3) their activity is often altered in parallel with changes in respiration during stereotypical non-respiratory behaviours such as coughing, swallowing and sneeze. Here we review some mechanisms that modulate the respiratory-related activity of laryngeal motoneurons with an emphasis on the generation of post-inspiratory activity.

Laryngeal constrictor motoneurons (LCMN) are activated during post-inspiration and act to slow expiratory airflow. However, little is known about how this phasic activity is generated. Here, we investigated the electrophysiological responses of identified LCMN to local application of GABA and bicuculline methiodide (BIC) in 14 anaesthetised Sprague-Dawley rats. During extracellular recordings, GABA iontophoresis (0.5M) strongly inhibited LCMN (n=6). Interestingly, BIC iontophoresis (5 mM) reduced, rather than increased, LCMN post-inspiratory activity (5 out of 6). Furthermore, intracellular recording revealed that BIC reduced not only the hyperpolarisation of the LCMN during inspiration (2.5+/-1.4 mV before and 1.5+/-0.4 mV after the BIC, P=0.05, n=5), but also the depolarisation during post-inspiration (3.0+/-1.3 mV before and 1.6+/-0.4 mV after the BIC, P=0.02, n=5). Our results demonstrate for the first time that the inspiratory inhibition of LCMN is primarily mediated by GABA(A) receptors. A possible involvement of a post-inhibitory rebound mechanism is discussed to explain how blockade of an inspiratory inhibition would affect LCMN excitability during post-inspiration.

Objectives: We sought to modify normal laryngeal constrictor (LC) motoneuron activity to induce a pattern of aberrant LC muscle function that may serve as a model of congenital bilateral vocal cord paralysis., Methods: Single unit extracellular recordings of functionally identified LC motoneurons were made in anesthetized Sprague-Dawley rats, and the response to both intravenous and iontophoretic application of the glycine antagonist strychnine was studied., Results: The postinspiratory firing pattern of LC motoneurons became inspiratory after intravenous injection of strychnine (4 of 5 rats), but no change was recorded in response to strychnine iontophoresis (7 of 8 rats)., Conclusions: Blockade of glycinergic inhibitory neurotransmission by strychnine, acting above the level of the LC motoneuron, causes LC motoneurons to fire during inspiration rather than after inspiration. This observation suggests that impaired glycine neurotransmission may be an underlying mechanism that explains the clinical manifestations of congenital bilateral vocal cord paralysis.

Central apnea is common in the premature newborn. To explain the upper airway findings in different clinical conditions characterized by central apnea, we made single unit recordings from laryngeal motoneurons during normal and hyperventilation. Posterior cricoarytenoid (n = 4) and cricothyroid (n = 4) motoneurons displayed an inspiratory pattern during normal ventilation and remained synchronous with phrenic nerve discharge (PND) during hyperventilation. Laryngeal constrictor motoneurons (LCon) displayed a post-inspiratory pattern during normal ventilation, but lost phasic activity during early hyperventilation (the period after the onset of hyperventilation but before cessation of PND; n = 12). There was a nearly linear relationship between the post-inspiratory activity and strength of PND. Six LCon motoneurons remained silent throughout hyperventilation, while the other six developed a tonic activity during cessation of PND. Further analysis suggested that the silent and tonic LCon motoneurons are likely to share a similar mechanism in their post-inspiratory pattern generation, but differ from each other in their responses to CO2 inputs. In addition, strong inhibition of the LCon tonic activity by the early return of PND could be an important factor in recovery following a period of apnea. Failure of this inspiratory inhibition to occur might explain certain clinical situations, where obstructive apnea supervenes following a period of central apnea.

Objectives/hypothesis: The objective was to demonstrate close appositions between serotonin-immunoreactive boutons and laryngeal constrictor (LCon) motoneurons in Sprague-Dawley rats., Study Design: Animal experimental., Methods: LCon motoneurons were identified functionally by their antidromic responses to stimulation of the recurrent laryngeal nerve and postinspiratory modulation and were filled by intracellular injection of biotin amide (n = 6). The medulla was sectioned and, using immunohistochemical analysis, examined by light microscopy., Results: Serotonin appositions were found on all 6 LCon motoneurons, with an average number of 17 +/- 6 close appositions per neuron., Conclusion: In comparison with the authors' previous study of inspiratory laryngeal motoneurons, the number of serotonin close appositions with LCon motoneurons was similar to that found with posterior cricoarytenoid motoneurons, but significantly less than that found with cricothyroid motoneurons. This finding may represent a basis for differences in tonic activity of laryngeal muscles observed in relation to the sleep-wake cycle.

Substance P terminals have previously been demonstrated around retrogradely labelled posterior cricoarytenoid (PCA) motoneurons, but little is known regarding substance P inputs to other functionally identified laryngeal motoneurons. In the present study, we determined the number and distribution of close appositions between substance P immunoreactive boutons and three types of laryngeal motoneuron by using a combination of intracellular recording, dye-filling and immunocytochemistry in the rat. Cricothyroid (CT) motoneurons received 15+/-5 substance P appositions/neuron (mean+/-S.D., n = 6), PCA motoneurons received 13+/-5 (n = 6), and laryngeal constrictor (LCS) motoneurons received 11+/-4 (n = 5). In contrast to our previous finding of a preferential serotonin innervation of CT motoneurons, we found no significant difference between the substance P inputs to CT, PCA and LCS motoneurons. Our results indicate a modest role for substance P in control of laryngeal motoneuronal function.

Serotonergic neurons are distributed widely throughout the central nervous system and exert a tonic influence on a range of activities in relation to the sleep-wake cycle. Previous morphologic and functional studies have indicated a role for serotonin in control of laryngeal motoneurons. In the present study, we used a combination of intracellular recording, dye-filling, and immunocytochemistry in rats to demonstrate close appositions between serotonin immunoreactive boutons and posterior cricoarytenoid (PCA) and cricothyroid (CT) motoneurons, both of which are located in the nucleus ambiguus and exhibit phasic inspiratory activity. PCA motoneurons received 29 +/- 5 close appositions/neuron (mean +/- SD, n = 6), with the close appositions distributed more frequently on the distal dendrites, less frequently on the proximal dendrites, and sparsely on the axons and somata. CT motoneurons received 56 +/- 15 (n = 6), with close appositions found on both the somata and dendrites, especially proximal dendrites. Close appositions on the axons were only seen on one CT motoneuron. These results demonstrate a significant serotonin input to inspiratory laryngeal motoneurons, which is more prominent on CT compared with PCA motoneurons, and may reflect the different functional role of the muscles that they innervate during the sleep-wake cycle., (Copyright 2002 Wiley-Liss, Inc.)

Objectives: Electromyographic studies of the laryngeal adductor reflex, glottal closure occurring in response to laryngeal stimulation, have demonstrated an early ipsilateral response (Rl) and a late bilateral response (R2). To better define the physiologic properties of these responses, we recorded responses from expiratory laryngeal motoneurons (ELMs) in rats during stimulation of the superior laryngeal nerve (SLN). Methods: Single unit extracellular recordings were obtained from 5 ELMs, identified by their antidromic responses to recurrent laryngeal nerve stimulation and postinspiratory firing pattern, in 4 Sprague-Dawley rats. Results: Unilateral stimulation of the SLN (at 20 Hz) stopped both phrenic nerve inspiratory activity and ELM postinspiratory activity. However, the ELMs displayed robust tonic firing, consisting of non-respiratory burst activity and single action potentials. The single action potentials were identified as short-latency ones (5 to 10 ms) activated by ipsilateral SLN stimulation, with an occurrence rate of 90%, and long-latency ones (20 to 50 ms) activated by bilateral SLN stimulation, with occurrence rates of 47% on the ipsilateral side and 58% on the contralateral side. Conclusions: The Rl response appears to be the result of the short-latency action potentials, orthodromically activated by ipsilateral stimulation of the SLN. The R2 response is likely to be a result of the long-latency action potentials that can be recorded from ELMs on both sides. [ABSTRACT FROM AUTHOR]

1. Cardiovascular sympathetic nerve activity at rest is grouped into waves, or bursts, that are generally, although not exclusively, related to the heart rate and to respiration. In addition, activity is also generated in response to central commands and to environmental stimuli. 2. Responsibility for the integration of all these different elements of sympathetic activity rests with pre-motoneurons in the rostral ventrolateral medulla oblongata. These pre-motoneurons are glutamatergic and spinally projecting where they form synapses with sympathetic preganglionic neurons. 3. Pre-motoneurons also contain and presumably release, neurotransmitters other than glutamate, including amines and neuropeptides that act on metabotropic receptors with long-term effects on cell function. 4. Similarly, in the rostral ventrolateral medulla oblongata the pre-motoneurons are mainly regulated by excitatory influences from glutamate and inhibitory influences from γ-aminobutyric acid (GABA). Major focuses of recent studies are the interactions between non-glutamatergic and GABAergic systems and reflexes that regulate the activity of the sympathetic nervous system. 5. The results indicate that neurotransmitters acting at metabotropic receptors selectively affect different reflexes in the rostral ventrolateral medulla. It is suggested that this differential activation or attenuation of reflexes by different neurotransmitters is a mechanism by which the organism can fine-tune its responses to different homeostatic requirements. [ABSTRACT FROM AUTHOR]

Abstract: Laryngeal constrictor motoneurons (LCMN) are activated during post-inspiration and act to slow expiratory airflow. However, little is known about how this phasic activity is generated. Here, we investigated the electrophysiological responses of identified LCMN to local application of GABA and bicuculline methiodide (BIC) in 14 anaesthetised Sprague–Dawley rats. During extracellular recordings, GABA iontophoresis (0.5M) strongly inhibited LCMN (n =6). Interestingly, BIC iontophoresis (5mM) reduced, rather than increased, LCMN post-inspiratory activity (5 out of 6). Furthermore, intracellular recording revealed that BIC reduced not only the hyperpolarisation of the LCMN during inspiration (2.5±1.4mV before and 1.5±0.4mV after the BIC, P =0.05, n =5), but also the depolarisation during post-inspiration (3.0±1.3mV before and 1.6±0.4mV after the BIC, P =0.02, n =5). Our results demonstrate for the first time that the inspiratory inhibition of LCMN is primarily mediated by GABAA receptors. A possible involvement of a post-inhibitory rebound mechanism is discussed to explain how blockade of an inspiratory inhibition would affect LCMN excitability during post-inspiration. [Copyright &y& Elsevier]

Abstract: Substance P (SP), tyrosine hydroxylase (TH) and serotonin inputs onto laryngeal motoneurons (LMNs) are known to exist, but the distribution of their terminals in the caudal nucleus ambiguus (NA), remains unclear. Using immunofluorescence and confocal microscopy, we assessed simultaneously the distribution of SP, TH, serotonin and synaptophysin immunoreactive (ir) terminals in the caudal NA. SP, TH and serotonin-ir varicosities were considered to represent immunoreactive synapses if, using confocal microscopy, they were co-localized with the presynaptic protein, synaptophysin. Relative to the total number of synapses, we found only a modest number of SP, TH or serotonin-ir synaptic terminals in the caudal NA. The density of SP-ir synaptic terminals was higher than that of TH-ir and serotonin-ir synaptic terminals. Our results suggest that SP, TH, and serotonin-ir inputs may play only a modest role in regulating the activity of LMN. We conclude that SP, TH and serotonin are not always co-localized in terminals forming inputs with LMN and that they arise from separate sub-populations of neurons. [Copyright &y& Elsevier]