Laryngeal Muscle Activity Research Articles

Laryngeal electromyography as a diagnostic tool for Parkinson's disease

To study the laryngeal electromyography pattern in patients with Parkinson's disease (PD) and vocal complaints at different stages of the disease. Cross-sectional cohort study. Ninety-four adults with PD and vocal complaints at different stages of the disease (according to the Hoehn and Yahr scale) underwent laryngeal electromyography. Tremors were not detected on laryngeal electromyography of the cricothyroid and thyroarytenoid muscles even in patients with clinical tremor. Laryngeal electromyography hypercontractility during voice rest was the typical result observed in 91.5% of patients regardless of disease severity. Gender and age of subjects did not correlate with laryngeal electromyography results. Patients with PD presented spontaneous intrinsic laryngeal muscle activity during voice rest, regardless of disease severity. This study was significant because it reported on the use of laryngeal electromyography in a large number of patients with PD and vocal complaints grouped according to PD severity. The patterns observed suggest that laryngeal electromyography is a valuable diagnostic tool for PD even at early phases of the disease.

Laryngeal Muscle Activity in Unilateral Vocal Fold Paralysis Patients Using Electromyography and Coronal Reconstructed Images

Objectives: Assess laryngeal muscle activity in patients with unilateral vocal fold paralysis (UVFP) using laryngeal electromyography (LEMG) and coronal multi-planar reconstructed images (coronal images). Methods: Twenty-one patients suffering from paralytic dysphonia, who had had onset of UVFP more than 6 months previously, underwent LEMG, phonatory function, and coronal images. For LEMG analysis, a four-point scale was employed to grade motor unit (MU) recruitment; 4+: absent, 3+: greatly decreased, 2+: moderately decreased, and 1+: mildly decreased (less than full interference). Maximum phonation time (MPT) and mean airflow rate (MFR) were employed. Coronal images during phonation and inhalation were assessed focusing on differences in thickness and vertical position between the vocal folds during phonation. Results: MU recruitment results were three patients for 1+, five for 2+, six for 3+, and seven for 4+. MPT was positively correlated with the MU recruitments ( P = 0.011). The thinning of the affected fold was evident during phonation in 20 of the 21 subjects. All 8 subjects with MU recruitments 1+ and 2+ showed the affected fold located at an equal level to the healthy fold. Twelve of 13 subjects with MU recruitments 3+ and 4+ showed the affected fold located at a higher level than the healthy fold. There was a significant difference between MU recruitment and vertical position of the affected fold ( P = 0.001). Conclusions: MU recruitments in patients with UVFP may be related to phonatory function and the vertical position of the affected fold. Synkinetic reinnervation may occur in some cases with UVFP.

Influence of baclofen on laryngeal and spinal motor drive during cough in the anesthetized cat

The antitussive properties of (±) baclofen on laryngeal muscle activities have not been determined. The hypothesis of this study was that administration of (±) baclofen would suppress upper airway muscle motor activity in a dose-dependent manner during cough. This is a prospective, preclinical, hypothesis-driven, paired design. Electromyograms of the parasternal, rectus abdominis, thyroarytenoid, posterior cricoarytenoid, and thyrohyoid were measured, along with esophageal pressure. Cough was elicited by mechanical stimulation of the lumen of the intrathoracic trachea in spontaneously breathing cats. Baclofen (±) (3-10 μg kg(-1) i.a.) induced decreases in the electromyogram amplitude of the rectus abdominis motor drive during coughing, the inspiratory and active expiratory (E1) phases of cough, and cough number per epoch. There was no effect of (±) baclofen on the EMG amplitudes of any of the laryngeal muscles, the parasternal, or the duration of the passive expiratory (E2) phase. Results from the present study indicate differential control mechanisms for laryngeal and inspiratory motor drive during cough, providing evidence of a control system regulating laryngeal activity and inspiratory spinal drive that is divergent from the control of expiratory spinal motoneurons.

Intrinsic laryngeal muscle activity in response to autonomic nervous system activation

To determine whether the intrinsic laryngeal muscles exhibit a response to induced autonomic nervous system (ANS) activation, and to characterize responses that may occur. Prospective within-subjects study. A cold pressor (CP) task was used to trigger a whole-body ANS response in eight vocally normal adult females. Surface electromyography of the trapezius muscle and bipolar hook-wire electromyography of intrinsic laryngeal muscles, as well as cardiovascular measures of heart rate (HR) and blood pressure (systolic [SBP] and diastolic [DBP]), were used to characterize participants' response to CP exposure compared to at-rest baseline. Cardiovascular response to the CP task was confirmed for subjects via HR or blood pressure measures. Post hoc Bonferroni analysis confirmed statistically significant increases in HR (P = .027), SBP (P < .001), and DBP (P < .001) during the CP task as compared to baseline. Concurrent increases in muscle activity were generally observed in trapezius, posterior cricoarytenoid, bilateral thyroarytenoid/lateral cricoarytenoid muscle complex, and bilateral cricothyroid muscles for each subject. Three individuals elected to repeat the CP task and displayed the same pattern of laryngeal response as observed in their first exposure. Results for repeated baseline measures obtained after the cessation of the CP task were compared to pre-CP baseline. Overall laryngeal activation remained even after the cardiovascular response was attenuated. Results are consistent with the suggestion that human laryngeal muscles exhibit an elevated level of activation concurrent with ANS activation, and endorse the putative link between laryngeal muscle tension and acute stress. Findings are replicable within a subset of three individuals.

Effect of repetitive coughing on swallowing in the anesthetized cat

Aspiration can trigger repetitive coughing, and swallow can be inter‐laced between successive coughs to clear the pharynx of ejected material. We hypothesized that repetitive coughing would increase swallow drive and change swallow phase durations and amplitudes of pharyngeal or laryngeal muscle activities. Cough was induced by mechanical stimulation of the trachea and swallow was elicited by injection of water into the oropharynx in spontaneously breathing cats. Electromyograms (EMG) were recorded from pharyngeal, laryngeal and respiratory muscles. During coughing bouts, EMG amplitudes of the geniohyoid, cricopharyngeus, thyropharyngeus, thyrohyoid significantly increased compared to swallows during eupnea. Swallow‐related phase durations of specific muscles were unchanged during coughing. However, there was a significant reduction in the duration of laryngeal elevation and the total swallow time indicating reduced onset latencies between different upper airway muscle groups. These results support a theory of a highly cooperative airway protective network which responds to aspiration. This network must be present to ensure timely and efficient execution of swallow. Supported by NIH HL89104, HL103415, HL109025.

Stereotypic laryngeal and respiratory motor patterns generate different call types in rat ultrasound vocalization.

Rodents produce highly variable ultrasound whistles as communication signals unlike many other mammals, who employ flow-induced vocal fold oscillations to produce sound. The role of larynx muscles in controlling sound features across different call types in ultrasound vocalization (USV) was investigated using laryngeal muscle electromyographic (EMG) activity, subglottal pressure measurements and vocal sound output in awake and spontaneously behaving Sprague-Dawley rats. Results support the hypothesis that glottal shape determines fundamental frequency. EMG activities of thyroarytenoid and cricothyroid muscles were aligned with call duration. EMG intensity increased with fundamental frequency. Phasic activities of both muscles were aligned with fast changing fundamental frequency contours, for example in trills. Activities of the sternothyroid and sternohyoid muscles, two muscles involved in vocal production in other mammals, are not critical for the production of rat USV. To test how stereotypic laryngeal and respiratory activity are across call types and individuals, sets of ten EMG and subglottal pressure parameters were measured in six different call types from six rats. Using discriminant function analysis, on average 80% of parameter sets were correctly assigned to their respective call type. This was significantly higher than the chance level. Since fundamental frequency features of USV are tightly associated with stereotypic activity of intrinsic laryngeal muscles and muscles contributing to build-up of subglottal pressure, USV provide insight into the neurophysiological control of peripheral vocal motor patterns.

High-resolution measurement of electrically-evoked vagus nerve activity in the anesthetized dog

Objective. Not fully understanding the type of axons activated during vagus nerve stimulation (VNS) is one of several factors that limit the clinical efficacy of VNS therapies. The main goal of this study was to characterize the electrical recruitment of both myelinated and unmyelinated fibers within the cervical vagus nerve. Approach. In anesthetized dogs, recording nerve cuff electrodes were implanted on the vagus nerve following surgical excision of the epineurium. Both the vagal electroneurogram (ENG) and laryngeal muscle activity were recorded in response to stimulation of the right vagus nerve. Main results. Desheathing the nerve significantly increased the signal-to-noise ratio of the ENG by 1.2 to 9.9 dB, depending on the nerve fiber type. Repeated VNS following nerve transection or neuromuscular block (1) enabled the characterization of A-fibers, two sub-types of B-fibers, and unmyelinated C-fibers, (2) confirmed the absence of stimulation-evoked reflex compound nerve action potentials in both the ipsilateral and contralateral vagus nerves, and (3) provided evidence of stimulus spillover into muscle tissue surrounding the stimulating electrode. Significance. Given the anatomical similarities between the canine and human vagus nerves, the results of this study provide a template for better understanding the nerve fiber recruitment patterns associated with VNS therapies.

Neuromuscular induced phonation in a human ex vivo perfused larynx preparation

Considering differences in laryngeal anatomy, degree of control, and range of voice qualities between animals and humans, investigations of the neuromuscular process of voice control are better conducted using a living human larynx in which parametric stimulation of individual laryngeal muscles is possible. Due to difficulties in access and monitoring of laryngeal muscle activities, such investigations are impossible in living human subject experiments. This study reports the recent success in developing an ex vivo perfused human larynx model, which allows parametric muscle stimulation and observation of its influence on phonation of a virtually living human larynx in a well-controlled laboratory environment.

The Assessment Methods of Laryngeal Muscle Activity in Muscle Tension Dysphonia: A Review

The purpose of this paper is to review the methods used for the assessment of muscular tension dysphonia (MTD). The MTD is a functional voice disorder associated with abnormal laryngeal muscle activity. Various assessment methods are available in the literature to evaluate the laryngeal hyperfunction. The case history, laryngoscopy, and palpation are clinical methods for the assessment of patients with MTD. Radiography and surface electromyography (EMG) are objective methods to provide physiological information about MTD. Recent studies show that surface EMG can be an effective tool for assessing muscular tension in MTD.

An electromyographic study of parkinsonian swallowing and its response to levodopa

Few studies have investigated the effect of levodopa on parkinsonian swallowing with findings thus far being equivocal. We used surface electromyography and accelerometry to investigate submental and laryngeal muscle activation during swallowing in 14 parkinsonian subjects before and after levodopa and in 22 age-matched controls. Our aims were (1) to demonstrate the clinical utility of noninvasive electromyography, (2) to identify electromyographic features of parkinsonian swallowing, and (3) to investigate the effect of levodopa on parkinsonian swallowing. The parkinsonian group showed increased burst amplitudes and durations and increased swallow duration, clearing activity and latency between submental and laryngeal bursts (P < .05) and used more swallows than did controls to consume water boluses (P < .001). Levodopa decreased the latency between submental and laryngeal bursts (P < .05) but did not produce effects on individual muscle bursts. The clinical utility of electrophysiological and biomechanical methods of swallowing assessment was demonstrated. Levodopa tended to normalize the timing of the combined swallow response but not the activity of individual muscles.

Numerical investigation of the influence of thyroarytenoid and cricothyroid muscle contraction on the geometry and biomechanical properties of the vocal folds

It is generally accepted that different voice types can be produced for different stiffness and geometry conditions of the vocal folds. However, it remains unclear how the stiffness and geometry of the vocal folds are regulated through laryngeal muscle activation during phonation. A better understanding of such muscular mechanisms of regulating vocal fold properties would provide important insights into the process of physiological control of phonation. In this study, the influence of the activation of the thyroarytenoid (TA) and cricothyroid (CT) muscles on vocal fold geometry and stress distribution within the vocal fold was investigated in a three-dimensional body-cover continuum model of the vocal folds. The results showed that different combinations of the TA and CT activation levels led to different body-cover stress distributions within the vocal fold. Contraction of the TA muscle also caused the vocal fold to bulge towards the glottal midline and created a medial compression force. The results also showed that, in some conditions, coordination of different laryngeal muscles is required to produce an optimal effect on vocal fold geometry or stiffness. [Work supported by NIH.]

Effects of aging and levodopa on the laryngeal adductor reflex in rats

Dopaminergic neurotransmission plays an essential role in sensorimotor function, and declines with age. Previously, we found the laryngeal adductor reflex (LAR) was increased in excitation by a dopamine receptor antagonist. If this airway-protective reflex is similarly affected by aging, it will interfere with volitional control in older adults. The current study tested whether the LAR was affected by aging, and whether such deficits were reversed by levodopa administration in aging rats. We recorded thyroarytenoid (TA) muscle activity at rest and during elicitation of LAR responses by stimulation of the internal branch of the superior laryngeal nerve (iSLN) in 6-, 18- and 30-month-old rats under alpha-chloralose anesthesia. Using paired stimuli at different inter-stimulus intervals (ISIs), LAR central conditioning, resting muscle activity, and reflex latency and amplitudes were quantified. Numbers of dopaminergic neurons in the substantia nigra pars compacta (SNpc) were measured using tyrosine hydroxylase staining. We found: (1) increased resting TA muscle activity and LAR amplitude occurred with fewer dopaminergic neurons in the SNpc in 18- and 30-month-old rats; (2) decreases in LAR latency and increases in amplitude correlated with reduced numbers of dopaminergic neurons in the SNpc; (3) test responses were greater at 1000ms ISI in 18-month-old rats compared with 6-month-old rats; and (4) levodopa administration further increased response latency but did not alter muscle activity, response amplitude, or central conditioning. In conclusion, increases in laryngeal muscle activity levels and reflex amplitudes accompanied age reductions in dopaminergic neurons but were not reversed with levodopa administration.

Absence of inspiratory laryngeal constrictor muscle activity during nasal neurally adjusted ventilatory assist in newborn lambs

In nonsedated newborn lambs, nasal pressure support ventilation (nPSV) can lead to an active glottal closure in early inspiration, which can limit lung ventilation and divert air into the digestive system, with potentially deleterious consequences. During volume control ventilation (nVC), glottal closure is delayed to the end of inspiration, suggesting that it is reflexly linked to the maximum value of inspiratory pressure. Accordingly, the aim of the present study was to test whether inspiratory glottal closure develops at the end of inspiration during nasal neurally adjusted ventilatory assist (nNAVA), an increasingly used ventilatory mode where maximal pressure is also reached at the end of inspiration. Polysomnographic recordings were performed in eight nonsedated, chronically instrumented lambs, which were ventilated with progressively increasing levels of nPSV and nNAVA in random order. States of alertness, diaphragm, and glottal muscle electrical activity, tracheal pressure, Spo(2), tracheal Pet(CO(2)), and respiratory inductive plethysmography were continuously recorded. Although phasic inspiratory glottal constrictor electrical activity appeared during nPSV in 5 of 8 lambs, it was never observed at any nNAVA level in any lamb, even at maximal achievable nNAVA levels. In addition, a decrease in Pco(2) was neither necessary nor sufficient for the development of inspiratory glottal constrictor activity. In conclusion, nNAVA does not induce active inspiratory glottal closure, in contrast to nPSV and nVC. We hypothesize that this absence of inspiratory activity is related to the more physiological airway pressurization during nNAVA, which tightly follows diaphragm electrical activity throughout inspiration.

Subglottal pressure, tracheal airflow, and intrinsic laryngeal muscle activity during rat ultrasound vocalization

Vocal production requires complex planning and coordination of respiratory, laryngeal, and vocal tract movements, which are incompletely understood in most mammals. Rats produce a variety of whistles in the ultrasonic range that are of communicative relevance and of importance as a model system, but the sources of acoustic variability were mostly unknown. The goal was to identify sources of fundamental frequency variability. Subglottal pressure, tracheal airflow, and electromyographic (EMG) data from two intrinsic laryngeal muscles were measured during 22-kHz and 50-kHz call production in awake, spontaneously behaving adult male rats. During ultrasound vocalization, subglottal pressure ranged between 0.8 and 1.9 kPa. Pressure differences between call types were not significant. The relation between fundamental frequency and subglottal pressure within call types was inconsistent. Experimental manipulations of subglottal pressure had only small effects on fundamental frequency. Tracheal airflow patterns were also inconsistently associated with frequency. Pressure and flow seem to play a small role in regulation of fundamental frequency. Muscle activity, however, is precisely regulated and very sensitive to alterations, presumably because of effects on resonance properties in the vocal tract. EMG activity of cricothyroid and thyroarytenoid muscle was tonic in calls with slow or no fundamental frequency modulations, like 22-kHz and flat 50-kHz calls. Both muscles showed brief high-amplitude, alternating bursts at rates up to 150 Hz during production of frequency-modulated 50-kHz calls. A differentiated and fine regulation of intrinsic laryngeal muscles is critical for normal ultrasound vocalization. Many features of the laryngeal muscle activation pattern during ultrasound vocalization in rats are shared with other mammals.

Laryngeal Muscle Activity and Vocal Fold Adduction During Chest, Chestmix, Headmix, and Head Registers in Females

Commercial singers produce chestmix register by maintaining or increasing adduction ofthe vocal processes (VPs) and by engaging the thyroarytenoid (TA) muscle to a greater degree than they would to produce head register. Prospective cohort study. Simultaneous recordings of TA and cricothyroid (CT) muscle activity, videonasendoscopy, and audio were obtained from seven female singers during production of a variety of midrange pitches in chest, chestmix, headmix, and head registers. Fast Fourier transforms were performed to measure the energy in the fundamental frequency and in mid and upper frequency harmonics to determine if the productions that were judged as perceptually distinct registers also showed distinctive acoustic characteristics. Then, measures of TA and CT muscle activity and vocal fold adduction ratings were obtained to determine how these varied as a function of pitch and register. Spectral tilt increased as subjects shifted from chest to chestmix to headmix and finally into head register. For same pitch phonation, subjects increased TA muscle activity and vocal fold adduction as they shifted register from head to headmix to chestmix to chest, particularly during production of higher frequencies. CT activity appeared to be more related to pitch rather than register control. Nonclassically trained singers were able to produce pitches at the high end of the midrange in chestmix register by increasing TA muscle activity and adduction of the VPs.

Cooperative Regulation of Vocal Fold Morphology and Stress by the Cricothyroid and Thyroarytenoid Muscles

Voice is produced by vibrations of vocal folds that consist of multiple layers. The portion of the vocal fold tissue that vibrates varies depending primarily on laryngeal muscle activity. The effective depth of tissue vibration should significantly influence the vibrational behavior of the tissue and resulting voice quality. However, thus far, the effect of the activation of individual muscles on the effective depth is not well understood. In this study, a three-dimensional finite element analysis is performed to investigate the effect of the activation of two major laryngeal muscles, the cricothyroid (CT) and thyroarytenoid (TA) muscles, on vocal fold morphology and stress distribution in the tissue. Because structures that bear less stress can easily be deformed and involved in vibration, information on the morphology and stress distribution may provide a useful estimate of the effective depth. The results of the analyses indicate that the two muscles perform distinct roles, which allow cooperative control of the morphology and stress. When the CT muscle is activated, the tip region of the vocal folds becomes thinner and curves upward, resulting in the elevation of the stress magnitude all over the tissue to a certain degree that depends on the stiffness of each layer. On the other hand, the TA muscle acts to suppress the morphological change and controls the stress magnitude in a position-dependent manner. Thus, the present analyses demonstrate quantitative relationships between the two muscles in their cooperative regulation of vocal fold morphology and stress.

Midbrain and medullary control of postinspiratory activity of the crural and costal diaphragm in vivo

Studies on brain stem respiratory neurons suggest that eupnea consists of three phases: inspiration, postinspiration, and expiration. However, it is not well understood how postinspiration is organized in the diaphragm, i.e., whether postinspiration differs in the crural and costal segments of the diaphragm and what the influence is of postinspiratory neurons on diaphragm function during eupnea. In this in vivo study we investigated the postinspiratory activity of the two diaphragm segments during eupnea and the changes in diaphragm function following modulation of eupnea. Postinspiratory neurons in the medulla were stereotaxically localized extracellularly and neurochemically stimulated. We used three types of preparations: precollicularly decerebrated unanesthetized cats and rats and anesthetized rats. In all preparations, during eupnea, postinspiratory activity was found in the crural but not in the costal diaphragm. When eupnea was discontinued in decerebrate cats in which stimulation in the nucleus retroambiguus induced activation of laryngeal or abdominal muscles, all postinspiratory activity in the crural diaphragm was abolished. In decerebrate rats, stimulation of the midbrain periaqueductal gray abolished postinspiration in the crural diaphragm but induced activation in the costal diaphragm. In anesthetized rats, stimulation of medullary postinspiratory neurons abolished the postinspiratory activity of the crural diaphragm. Vagal nerve stimulation in these rats increased the intensity of postinspiratory neuronal discharge in the solitary nucleus, leading to decreased activity of the crural diaphragm. These data demonstrate that three-phase breathing in the crural diaphragm during eupnea exists in vivo and that postinspiratory neurons have an inhibitory effect on crural diaphragm function.

Efficiency of laryngeal motor nerve repair is greater with bulbar than with mucosal olfactory ensheathing cells

The real ability of OECs provided by olfactory mucosa cultures (OM-OECs) and those from olfactory bulb cultures (OB-OECs) must be better characterized in order to propose their future clinical application. Therefore, we used a lesion of the vagus nerve (VN), which constitutes a severe motor denervation due to long distance of the muscular targets (4.5 cm). We performed a section/anastomosis surgery of the VN, at the third tracheal ring. Then, OM-OECs and OB-OECs were injected in matrigel around the lesion site. Three months after surgery, laryngeal muscle activity, synkinesis phenomena and latency were evaluated by videolaryngoscopy and electromyography recordings. To complete these procedures, axonal morphometric study of the right recurrent nerve was performed to assess axonal regrowth and tracking of green fluorescent protein positive cells was performed. Recurrent nerve is the motor branch innervating the laryngeal muscles, and is located distally to the lesion, near the muscular targets (0.7 cm). These analyses permitted to compare the ability of these two populations to improve functional recovery and axonal regrowth. Our results show that, OM-OECs improved electrical muscular activity and nervous conduction with significant tissue healing but induced aberrant movement and poor functional recovery. In contrast, OB-OECs induced a partial functional recovery associated with an increase in the number of myelinated fibers and nervous conduction. Our study suggests that, as recently reported in a microarray study, OM-OECs and OB-OECs express different properties. In particular, OM-OECs could regulate inflammation processes and extracellular matrix formation but have a poor regeneration potential, whereas, OB-OECs could improve functional recovery by inducing targeted axonal regrowth.

Respiration-Related Laryngeal Electromyography in Children with Bilateral Vocal Fold Paralysis

We present 2 case reports to demonstrate the relationship between laryngeal muscle activity and respiration in children with bilateral vocal fold paralysis (BVFP) by simultaneous laryngeal electromyography (EMG) with recording of chest wall movement and intercostal muscle EMG. Laryngeal EMG was performed together with recording of chest wall movement in a 55-day-old girl who was undergoing tracheostomy for idiopathic congenital BVFP. Normal phasic activity was observed, i.e., the thyroarytenoid (TA) muscle was active during expiration and the posterior cricoarytenoid (PCA) muscle during inspiration, suggesting a good prognosis for recovery. The child was decannulated at 11 months. Laryngeal EMG together with recording of chest wall movement and intercostal EMG in a 5-year-old girl who was tracheostomy-dependent following tracheoesophageal fistula repair due to BVFP showed phasic activity during expiration for both the TA and PCA muscles, indicating aberrant regeneration of the PCA motor nerve. The timing of laryngeal muscle activity with respiration in the assessment of pediatric congenital BVFP is essential to demonstrate the presence of normal or abnormal medullary respiratory neuronal input to laryngeal motoneurons. In cases in which BVFP is due to recurrent laryngeal nerve injury, respiration-related laryngeal EMG will identify aberrant regeneration. Laryngeal EMG should be combined with intercostal muscle EMG in the evaluation of children with significant vocal fold dyfunction of either central or peripheral origin.

Cholinergic inputs to laryngeal motoneurons functionally identified in vivo in rat: A combined electrophysiological and microscopic study

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.