Differences of GDNF expression are linked to the differential timing of RLN axon regeneration and individual muscle reinnervation. The present finding suggests the need to further investigate the role of GDNF and other neurotrophic factors in the timing of reinnervation, axon guidance, and neuromuscular junction formation as it relates to synkinetic and nonsynkinetic RLN reinnervation. Future experimental results may provide insight to therapeutic options that could stimulate appropriate neuromuscular junction formation and nonsynkintic functional reinnervation following RLN injury.

No abstract

The larynx serves respiratory, protective, and phonatory functions. The motor and sensory innervation to the larynx controlling these functions is provided by the superior laryngeal nerve (SLN) and the recurrent laryngeal nerve (RLN). Classical studies state that the SLN innervates the cricothyroid muscle and provides sensory innervation to the supraglottic cavity, whereas the RLN supplies motor innervation to the remaining intrinsic laryngeal muscles and sensory innervation to the infraglottic cavity, but recent data suggest a more complex anatomical and functional organisation. The current neuroanatomical tracing study was undertaken to provide a comprehensive description of the central brainstem connections of the axons within the SLN and the RLN, including those neurons that innervate the larynx. The study has been carried out in 41 adult male Sprague-Dawley rats. The central projections of the laryngeal nerves were labelled following application of biotinylated dextran amines onto the SLN, the RLN or both. The most remarkable result of the study is that in the rat the RLN does not contain any afferent axons from the larynx, in contrast to the pattern observed in many other species including man. The RLN supplied only special visceromotor innervation to the intrinsic muscles of the larynx from motoneurons in the nucleus ambiguus (Amb). All the afferent axons innervating the larynx are contained within the SLN, and reach the nucleus of the solitary tract. The SLN also contained secretomotor efferents originating from motoneurons in the dorsal motor nucleus of the vagus, and special visceral efferent fibres from the Amb. In conclusion, the present study shows that in the rat the innervation of the larynx differs in significant ways from that described in other species.

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.

Neurons innervating the intrinsic muscles of the larynx are located within the nucleus ambiguus but the precise distribution of the neurons for each muscle is still a matter for debate. The purpose of this study was to finely determine the position and the number of the neurons innervating the intrinsic laryngeal muscles cricothyroid, posterior cricoarytenoid, and thyroarytenoid in the rat. The study was carried out in a total of 28 Sprague Dawley rats. The B subunit of the cholera toxin was employed as a retrograde tracer to determine the locations, within the nucleus ambiguus, of the neurons of these intrinsic laryngeal muscles following intramuscular injection. The labelled neurons were found ipsilaterally in the nucleus ambiguus grouped in discrete populations with reproducible rostrocaudal and dorsoventral locations among the sample of animals. Neurons innervating the cricothyroid muscle were located the most rostral of the three populations, neurons innervating the posterior cricoarytenoid were found more caudal, though there was a region of rostrocaudal overlap between these two populations. The most caudal were the neurons innervating the thyroarytenoid muscle, presenting a variable degree of overlap with the posterior cricoarytenoid muscle. The mean number (6SD) of labelled neurons was found to be 41 6 9 for the cricothyroid, 39 6 10 for the posterior cricoarytenoid and 33 6 12 for the thyroarytenoid.

NA Laryngoscope, 126:E337-E342, 2016.

Objective: An injury of the recurrent laryngeal nerve (RLN) triggers axonal regeneration but results in a poor functional recovery. Netrin-1 and glial cell-derived neurotrophic factor (GDNF) expression are up-regulated in laryngeal muscles during RLN regeneration, but the role of their receptors produced in the nucleus ambiguus is unknown. The aim of this work was to determine the timing of the production of Netrin-1 and GDNF receptors during RLN regeneration and correlate this with the previously identified timing of up-regulation of their trophic factors in the laryngeal muscles.Study Design: Laboratory experiment with rat model. Methods: The right RLN was transected and dextran amine tracer applied. At 7, 14, and 21 days postinjury (DPI), brainstems were removed and harvested. Immunostaining was performed for Netrin-1 (deleted in colorectal carcinoma [DCC], UNC5A) and GDNF receptors (rearranged during transfection [Ret], glycosylphosphatidylinositol-linked cell surface receptors [GFRα1, GFRα2, GFRα3]). The timing and type of receptor production relative to injury as well as their position in the nucleus ambiguus was analyzed.Results: Netrin-1 UNC5A receptors were minimal in the nucleus ambiguus during RLN regeneration. DCC, the receptor that plays an attract role, was immunopositive from 7 to 21 DPI. All GDNF receptors, except GFRα2, were clearly positive from 7 to 14 DPI. No differences of production were observed according to the position of the motor neurons in the nucleus ambiguus.Conclusion: An injury of the RLN leads to a higher production of Netrin-1 DCC and GDNF receptors in the nucleus ambiguus. The timing of receptor production is similar to up-regulation of their trophic factors in the laryngeal muscles.

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. Anat Rec, 297:955-963, 2014. V C 2014 Wiley Periodicals, Inc.Key words: peripheral nerve injury; somatotopy; motoneurons; larynx