Posterior Cricoarytenoid Motoneurons Research Articles

Somatotopic Changes in the Nucleus Ambiguus After Section and Regeneration of the Recurrent Laryngeal Nerve of the Rat

Changes in motoneurons innervating laryngeal muscles after section and regeneration of the recurrent laryngeal nerve (RLN) are far from being understood. Here, we report the somatotopic changes within the nucleus ambiguus (Amb) after the nerve injury and relates it to the resulting laryngeal fold impairment. The left RLN of each animal was transected and the stumps were glued together using surgical fibrin glue. After several survival periods (1, 2, 4, 8, 12, 16 weeks; at least six rats at each time point) the posterior cricoarytenoid (PCA) and thyroarytenoid (TA) muscles were injected with fluorescent-conjugated cholera toxin and the motility of the vocal folds evaluated. After section and subsequent repair of the RLN, no movement of the vocal folds could be detected at any of the survival times studied and the somatotopy and the number of labeled motoneurons changed. From 4 wpi award, the somatotopy was significantly disorganized, with the PCA motoneurons being located rostrally relative to their normal location. A rostrocaudal overlap between the two pools of motoneurons supplying the PCA and TA muscles was observed from 2 wpi onwards. Hardly any labeled neurons were found in the contralateral Amb in any of the experimental groups. An injury of the RLN leads to a reinnervation of the denervated motor endplates of PCA and TA. However, misdirected axons sprout and regrowth from the proximal stump to the larynx. As a result, misplaced innervation of muscles results in a lack of functional recovery of the laryngeal folds movement following a RLN injury.

Serotonin Inputs to Laryngeal Constrictor Motoneurons in the Rat

The objective was to demonstrate close appositions between serotonin-immunoreactive boutons and laryngeal constrictor (LCon) motoneurons in Sprague-Dawley rats. Animal experimental. 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. Serotonin appositions were found on all 6 LCon motoneurons, with an average number of 17 +/- 6 close appositions per neuron. 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.

Anatomic effect of electrical stimulation on the reinnervation of the paralyzed canine larynx

Problem: The larynx relies on correct innervation to mediate its normal functions. The aberrantly reinnervated larynx is compromised in its vocalization and airway-protective functions. Methods: Six canines were implanted with electrical stimulation devices, and their recurrent laryngeal nerve sectioned and re-anastomosed on one side. The effects of interrupted chronic muscle stimulation of experimental animals were compared with nonstimulated control animals. Physiologic measures of electromyography of thyroarytenoid (TA) and posterior cricoarytenoid (PCA) muscles demonstrated enhanced native reinnervation and reduced foreign reinnervation at 180 days in experimental animals. Fluorescent retrograde tracers true blue (TB) and fluorogold (FG) were injected into the PCA and TA, respectively. The identity and relative locations of labeled motoneurons in the nucleus ambiguus were analyzed for segregation and compared to normal animals. Results: PCA and TA muscles that appeared to become selectively reinnervated based on physiologic measurements retained the normal localization of motoneuron cell bodies in the nucleus ambiguus. Significant segregation was observed across the mediolateral axis. Animals with greater foreign reinnervation based on physiologic measurements showed more random distribution of PCA and TA motoneurons, with variable double-labeling of some neurons. Conclusion: This study provides the first direct anatomic evidence that stimulation of a denervated laryngeal muscle promotes selective reinnervation by native over foreign reinnervation. The native reinnervation appears to be from the original motoneuron projections to the PCA or TA located within the nucleus ambiguus of the brainstem. Significance: Laryngeal paralysis is a significant problem with decreased function and increased morbidity for affected individuals. This study demonstrates anatomically that stimulation of the larynx by an implantable device leads to improved reinnervation. Thus, implantable devices may be useful in the rehabilitation of patients with laryngeal paralysis. Support: Supported by NIH grant R01-DC001149.

Serotonin inputs to inspiratory laryngeal motoneurons in the rat.

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.

Monosynaptic inputs from the nucleus tractus solitarii to the laryngeal motoneurons in the nucleus ambiguus of the rat.

The cricothyroid (CT) and the posterior cricoarytenoid (PCA) muscles in the larynx are activated by the laryngeal motoneurons located within the nucleus ambiguus; these motoneurons receive the laryngeal sensory information from the nucleus tractus solitarii (NTS) during respiration and swallowing. We investigated whether the neurons in the NTS projected directly to the laryngeal motoneurons, and what is the synaptic organization of their nerve terminals on the laryngeal motoneurons using the electron microscope. When wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) was injected into the NTS after cholera toxin subunit B-conjugated HRP (CT-HRP) was injected into the CT muscle or the PCA muscle, the anterogradely WGA-HRP-labeled terminals from the NTS were found to directly contact the retrogradely CT-HRP-labeled dendrites and soma of both the CT and the PCA motoneurons. The labeled NTS terminals comprised about 4% of the axosomatic terminals in a section through the CT motoneurons, and about 9% on both the small (PCA-A) and the large (PCA-B) PCA motoneurons. The number of labeled axosomatic terminals containing round vesicles and making asymmetric synaptic contacts (Gray's type I) was almost equal to that of the labeled terminals containing pleomorphic vesicles and making symmetric synaptic contacts (Gray's type II) on the CT motoneurons. The labeled axosomatic terminals were mostly Gray's type II on the PCA-A motoneurons, while the majority of them were Gray's type I on the PCA-B motoneurons. These results indicate that the laryngeal CT and PCA motoneurons receive a few direct excitatory and inhibitory inputs from the neurons in the NTS.

Intracellular recording from posterior cricoarytenoid motoneurons in the rat.

The ability to maintain coordinated vocal cord abduction and upper airway patency is dependent on the integrity of the posterior cricoarytenoid (PCA) motoneurons and their multiple neural connections. Study of the PCA motoneurons represents the initial step in understanding the complex mechanisms responsible for coordinated vocal cord abduction and may provide an insight into the possible pathological processes underlying the various clinical presentations of vocal cord dysfunction. Intracellular recordings were made from 11 PCA motoneurons in Sprague-Dawley rats, which all showed an inspiratory augmenting discharge pattern that is also characteristic of phrenic nerve activity. The resting membrane potential was -56+/-11 mV. Two PCA motoneurons were injected with Neurobiotin to demonstrate neuronal morphology, which was found to be similar to that obtained by retrograde labeling with cholera toxin B subunit. The technique described for intracellular recording of PCA motoneurons should allow more detailed morphological, electrophysiological, and immunohistochemical information to be obtained, to thereby identify some of the factors responsible for maintaining normal function of the PCA muscle.

Identification of posterior cricoarytenoid motoneurons in the rat.

The posterior cricoarytenoid (PCA) muscle is the sole abductor of the larynx and is controlled by motoneurons located in the nucleus ambiguus. These motoneurons receive inputs from a variety of interneurons, including those that impart respiratory modulation, and are responsible for the phasic inspiratory activity of the PCA muscle. Identification of PCA motoneurons is therefore an essential initial step in understanding the mechanisms responsible for coordinated vocal cord abduction. We identified PCA motoneurons in the rat model by retrograde labeling, and following antidromic activation. A total of 194 neurons were identified by retrograde labeling with cholera toxin B subunit (CTB). Labeling was exclusively ipsilateral where the contralateral vagus and superior laryngeal nerves had been divided. The neurons were multipolar, with dimensions of 33.2 +/- 6.4 microm (mean +/- standard deviation) in length and 22.4 +/- 3.4 microm in width. The neurons were located within a range of 0.6 to 2.4 mm caudal to the caudal pole of the facial nerve, 1.2 to 1.7 mm lateral to the midline, and 1.5 to 2.3 mm deep to the dorsal surface of the medulla. The PCA motoneurons were antidromically activated by focal stimulation of the PCA muscle. The extracellular field was recorded in 5 rats, and the PCA motoneurons were found within a range of 0.8 to 1.7 mm caudal to the caudal pole of the facial nerve, 1.5 to 2.0 mm lateral to the midline, and 1.9 to 2.4 mm deep to the dorsal surface of the medulla. The mean conduction velocity ranged from 37.0 +/- 5.8 to 68.6 +/- 5.0 m/s. An extracellular antidromic field potential, which corresponds to the distribution of the PCA motoneuron pool demonstrated by retrograde labeling with CTB, can be reliably obtained in a rat model following focal PCA muscle stimulation.

Respiratory activity of the rat posterior cricoarytenoid muscle.

An anatomic and electrophysiological study of the rat posterior cricoarytenoid (PCA) muscle is described. The intramuscular nerve distribution of the PCA branch of the recurrent laryngeal nerve was demonstrated by a modified Sihler's stain. The nerve to the PCA was found to terminate in superior and inferior branches with a distribution that appeared to be confined to the PCA muscle. Electromyography (EMG) recordings of PCA muscle activity in anesthetized rats were obtained under stereotaxic control together with measurement of phrenic nerve discharge. A total of 151 recordings were made in 7 PCA muscles from 4 rats. Phasic inspiratory activity with a waveform similar to that of phrenic nerve discharge was found in 134 recordings, while a biphasic pattern with both inspiratory and post-inspiratory peaks was recorded from random sites within the PCA muscle on 17 occasions. The PCA EMG activity commenced 24.6 +/- 2.2 milliseconds (p < .0001) before phrenic nerve discharge. The results are in accord with findings of earlier studies that show that PCA muscle activity commences prior to inspiratory airflow and diaphragmatic muscle activity. The data suggest that PCA and diaphragm motoneurons share common or similar medullary pre-motoneurons. The earlier onset of PCA muscle activity may indicate a role for medullary pre-inspiratory neurons in initiating PCA activity.

Serotonergic cells in nucleus raphe pallidus provide tonic drive to posterior cricoarytenoid motoneurons via 5-hydroxytryptamine 2 receptors in cats

Microinjection of serotonin and 5-hydroxytryptamine 2 (5-HT 2) agonist 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM) in nucleus ambiguus caused excitation of posterior cricoarytenoid (PCA) muscles of the larynx in anesthetized, spontaneously breathing cats. Intravenous administration of 5-HT 2 antagonist ketanserin produced complete block of excitatory effect of DOM injection. Electrical stimulation of nucleus raphe pallidus caused excitation of PCA activity, that was blocked by pretreatment of ketanserin. These results indicate that serotonergic cells in the-raphe pallidus provide tonic drive to medullary PCA motoneurons through 5-HT 2 receptors.

Misdirected regeneration of injured recurrent laryngeal nerve in the cat

Introduction: Misdirected regeneration (MR) frequently occurs following injury to the recurrent laryngeal nerve (RLN) resulting in neurotmesis or axonotmesis. Physiological and anatomic parameters involved in the functional recovery of the larynx following freezing injury or neurorrhaphy of the RLN were studied. A multi-facilitated approach is undertaken to clarify the functional abnormalities caused by the MR after recurrent laryngeal nerve injury. Materials and Methods: Three groups of adult cats were studied. These included controls, cats with recurrent laryngeal neurorrhaphy, and cats with recurrent laryngeal nerve freeze injuries. From 2 weeks to 9 months after the nerve injury, the animals were studied endoscopically and with electromyography (EMG). Using the same animal, the number and location of motoneurons supplying the ipsilateral posterior cricoarytenoid (PCA) muscle were examined with horseradish peroxidase (HRP). Animals were subsequently sacrificed to study the pattern of reinnervation. Results: Following neurorrhaphy all cats had vocal cord paralysis. After neurorrhaphy, effective motion function did not return in the affected vocal cord and it remained fixed in the paramedian position. Although EMG of the laryngeal muscles of the affected side showed interference voltage, the pattern of activities was markedly different from that of the unaffected side, and reciprocity among the laryngeal muscles was not restored. The number of PCA motoneurons recovered to the normal range, but a considerable number of neuronal bodies were dispersed outside the normal PCA area. This indicates misdirected reinnervation to the PCA muscle by motoneurons that originally served other laryngeal muscles. In the freezing injury, effective vocal cord movement finally recovered after 6 months. At this time, EMG showed a normal pattern, although a relatively small amount of misdirected neurons was observed. Discussion: Functional recovery of vocal cord motion does not occur following neurorrhaphy. Prominently disorganized arrangement of laryngeal motor neurons was observed in the horseradish peroxidase study. This suggests that inappropriate reinnervation develops in spite of reapproximation and suturing. Altered central organization of the motor nucleus is a significant pathogenic factor in the loss of laryngeal muscular coordination following recurrent laryngeal nerve lesions. The degree of recovery is related to the mechanism of injury.

Laryngeal Synkinesis following Reinnervation in the Rat: Neuroanatomic and Physiologic Study Using Retrograde Fluorescent Tracers and Electromyography

The functional organization of laryngeal motoneurons in the nucleus ambiguous (NA) was evaluated in adult male rats before and after recurrent laryngeal nerve section and reinnervation. Using retrograde double labeling techniques with fluorescent probes, we obtained the number and position of labeled neurons by using the Bioquant 3-D imaging system. Reinnervation was documented by electromyography. In nine control animals vector analysis revealed significant (p less than .05) separation of the posterior cricoarytenoid (PCA) muscle motoneurons and the thyroarytenoid and lateral cricoarytenoid (TA/LCA) muscle motoneurons. The PCA motoneurons were positioned ventromedially in the NA, and TA/LCA motoneurons were found dorsolaterally in the NA. Rostral-caudal separation was not significant. Electromyography revealed phasic electrical activity synchronous with respiration in the PCA, and activity synchronous with deglutition in the TA/LCA. In four animals surviving 15 weeks following recurrent laryngeal nerve section and primary neurorrhaphy, functional organization within the NA was lost and phasic motor unit activity synchronous with respiration was seen in the TA/LCA muscle as well as the PCA. Vector analysis revealed the reinnervating motoneurons for both the PCA and TA/LCA to be positioned dorsolaterally, similar to the control group TA/LCA motoneurons. These findings demonstrate a shift in the topographic organization of laryngeal motoneurons within the NA following reinnervation, with random organization occurring at the neurorrhaphy site.

Localization of Laryngeal Motoneurons in the Medulla Oblongata of Guinea Pigs

The distribution of motoneurons in the brain stem innervating the laryngeal muscles cricothyroid, posterior cricoarytenoid, and thyroarytenoid-lateral cricoarytenoid in the adult guinea pig was investigated. Horseradish peroxidase was injected into individual muscles or applied to the cut end of the superior laryngeal nerve and the recurrent laryngeal nerve. The laryngeal motoneuron pool of guinea pig is divided into two separate areas: the rostral retrofacial nucleus and the caudal nucleus ambiguus. Superior laryngeal and cricothyroid motoneurons were present only in the retrofacial nucleus, while recurrent laryngeal motoneurons were present in both the nucleus ambiguus and the retrofacial nucleus. Posterior cricoarytenoid motoneurons were located in the rostral and ventrolateral part of the recurrent laryngeal motoneuron pool in the nucleus ambiguus. The thyroarytenoid-lateral cricoarytenoid motoneurons partially overlapped the posterior cricoarytenoid pool and extended more dorsomedially and caudally in the nucleus. Our findings on the distribution of perikarya in the laryngeal motoneuron of the guinea pig are essentially the same as those of other mammals.

Fibers supplying the laryngeal musculature in the cranial root of the rabbit accessory nerve: Nucleus of origin, peripheral course, and innervated muscles

The location of the rabbit laryngeal motoneurons whose axons traverse the cranial root of the accessory nerve was studied with injection of HRP or nuclear yellow into the laryngeal muscles in combination with the intracranial severing of either the rootlets of the vagus nerve or those of the cranial root. The motoneurons were located in the diffuse cell group that forms a subnucleus occupying the caudal two-thirds of the nucleus ambiguus and sending fibers to the inferior laryngeal nerve. The caudal one-third of the diffuse cell group supplying the lateral cricoarytenoid muscle, was occupied only by these motoneurons, whereas in its rostral two-thirds, they were intermingled with motoneurons having axons that traversed the vagal rootlets. The thyroarytenoid and posterior cricoarytenoid motoneurons are present in the rostral two-thirds of the diffuse cell group; axons of the former traversed the rootlets of the cranial root, and of the latter traversed the vagal rootlets. On the other hand, the medial scattered cell group, located in the rostral one-third of the nucleus ambiguus and sending fibers to the cricothyroid muscle via the superior laryngeal nerve, contained only motoneurons with axons traversing the vagal rootlets. The above findings clarified that fibers of the cranial root enter the inferior laryngeal nerve after joining the vagus, and then reach the adductor muscles for the vocal fold, with their neurons of origin in a caudal portion of the nucleus ambiguus. The vagal rootlet fibers, with their neurons of origin situated more rostrally in the nucleus, innervate the tensor and abductor muscles via the superior and inferior laryngeal nerve, respectively.

Localization of rabbit laryngeal motoneurons in the nucleus ambiguus

The localization of rabbit laryngeal motoneurons in nucleus ambiguus was investigated using injection of a fluorescent labeling substance, i.e., nuclear yellow, into the individual laryngeal muscles. The nucleus ambiguus of the rabbit comprises four subnuclei, CoG, SGm, SGl, and DiG. The CoG is a group of compactly arranged neurons, and is situated in the rostral one-half of the nucleus. The SG, situated in its rostral one-third, is scattered around the CoG, with a subdivision into SGm and SGl. These subdivisions are medial and lateral to the CoG, respectively. The DiG is formed by diffusely arranged neurons, and is located in the caudal two-thirds of the nucleus. All labeled motoneurons were found in the ipsilateral nucleus ambiguus. The motoneurons supplying the cricothyroid muscle, which is innervated by the superior laryngeal nerve, were present in the SGm, with a clear rostralward segregation from the other motoneurons. The motoneurons supplying the muscles innervated by the inferior laryngeal nerve were located in the DiG, where they displayed a rostrocaudal myotopical arrangement in the order posterior cricoarytenoid, thyroarytenoid, and lateral cricoarytenoid. The posterior cricoarytenoid motoneurons were intermingled with the thyroarytenoid motoneurons in the rostral two-thirds of the DiG, and the former tended to be concentrated more rostrally than the latter. The lateral cricoarytenoid motoneurons were confined to the most caudal one-third of the DiG.

Cytoarchitectonic organization of laryngeal motoneurons within the nucleus ambiguus of the cat

The central distribution of laryngeal motoneurons was studied in the cat by retrograde axonal transport of horseradish peroxidase. The enzyme was injected selectively into the cricothyroid (CT), lateral cricoarytenoid and thyroarytenoid (LCA-TA), and posterior cricoarytenoid (PCA) muscles of the larynx with or without the previous sectioning of the left laryngeal recurrent nerve (LR) or the left superior laryngeal nerve (SL). The CT motoneurons appeared as a compact group of medium-size cells located in the rostral one-third of the nucleus ambiguus (nA). The LCA-TA motoneurons were found in the caudal two-thirds of the nA, constituting a loose group of large motoneurons. The PCA motoneurons were located throughout the whole extend of the nA, the cells being large in the caudal pole and smaller in the rostral one-third of nA. Laryngeal muscle innervation was exclusively of ipsilateral origin. Axonal projections in the brain stem were different depending on the nerve (LR or SL) by which the efferent fibers were sent.