Motoneurons In Nucleus Ambiguus Research Articles

Adenoviral gene transfer of BDNF and GDNF synergistically prevent motoneuron loss in the nucleus ambiguus

We have previously shown that neuroprotective effects of an adenoviral glial cell line-derived neurotrophic factor (GDNF) gene transfer on the lesioned adult rat motoneurons in the nucleus ambiguus. In the present study, we examined neuroprotective effects of adenoviral gene transfer of brain-derived neurotrophic factor (BDNF) or/and GDNF to motoneurons in nucleus ambiguus using an adult rat vagal nerve avulsion model. The animals avulsed and inoculated with adenoviral vectors encoding BDNF (AxCAmBDNFME) or/and GDNF (AxCAhGDNF) showed immunolabeling for BDNF or/and GDNF in the nucleus ambiguus on the treated side, respectively, and expression of virus-induced BDNF or/and GDNF mRNA transcripts in the brainstem tissue that contained the nucleus ambiguus of the treated side. The treatment with AxCAhGDNF or AxCAmBDNFME significantly prevented the loss of vagal motoneurons in comparison to the control; the protective effect of AxCAmBDNFME was greater than that of AxCAhGDNF. The combined treatment with AxCAmBDNFME and AxCAhGDNF acted synergistically and significantly larger number of vagal motoneurons was preserved as compared to either AxCAmBDNFME treatment or AxCAhGDNF treatment. The treatment with AxCAmBDNFME or/and AxCAhGDNF after avulsion also suppressed the activity of nitric oxide synthase in lesioned motoneurons in the nucleus ambiguus. These results indicate that adenovirus-mediated BDNF and GDNF gene transfer may prevent the degeneration of motoneurons in humans after either vagal nerve injury or recurrent laryngeal nerve injury.

Molecular Changes in Neurons of Rat Nucleus Ambiguus after Axotomy, As Revealed by a Novel Method of In Vivo Fluorescence Neuronal Labeling Combined with Single-cell RT-PCR

To elucidate the molecular changes of motoneurons which innervate laryngeal muscles after transection of rat recurrent laryngeal nerve, we have devised a new method of DiI labeling combined with single-cell RT-PCR, by which nucleus ambiguus (NA) neurons projecting to the intrinsic laryngeal muscles were labeled retrogradely by injecting the fluorescent dye DiI into bilateral thyroarytenoid muscles; then either one of the recurrent laryngeal nerves was transected, and the fluorescence-bright single neurons were microdissected, followed by single-cell reverse transcription (RT)-PCR. Immunohistochemically, many DiI-labeled motoneurons in NA were immunoreactive to phosphorylated Stat3 in the nucleus on 1, 7, 10 and 14 days after axotomy. Thirty μm-thick frozen sections of brain stem were mounted onto membrane slide, and DiI-labeled single neurons in NA were microdissected under a fluorescence microscope. By real-time quantitative RT-PCR for single motoneurons, the expression levels of GAP-43 and nNOS genes were upregulated at 7 days and 1 day after axotomy, respectively. The expression levels of Stat3, Reg-2, and Bcl-2 genes were upregulated at 7 days, whereas that of Bax was downregulated at 1 and 7 days after axotomy as compared with control levels. With this novel method in which single-cell RT-PCR was combined with neurotracer DiI labeling we could demonstrate a part of the molecular cascades involving Stat3 in neurons projecting to laryngeal muscles after axotomy.

T-588 prevents motoneuron loss after recurrent laryngeal nerve injury

Problem: Unsuccessful reinnervation or neurofunctional recovery after recurrent laryngeal nerve injury may attribute to the loss of motoneurons in nucleus ambiguus. To assess the possibility of new drug therapy for injury of these motoneurons, we examined the neuroprotective effect of a novel neuroprotective compound, T-588 on the motoneuron loss in nucleus ambiguus after adult rat vagal nerve avulsion. Methods: The left vagal nerves of 12-week-old Sprague-Dawley rats were avulsed and removed at the level of jugular foramen. After the operation, the animals were freely administrated water containing 0.05% T-588 (Toyama Chemical Co Ltd, Toyama, Japan) solution. Four weeks after the operation, the number of surviving motoneurons in nucleus ambiguus was counted after Nissl staining to evaluate the neuroprotective effect of T-588. Results: In T-588-treated animals, a significantly larger number of surviving motoneurons in nucleus ambiguus was observed compared to control animals 4 weeks after the operation. Conclusion: Oral administration of T-588 after vagal nerve avulsion improved the survival of motoneurons in nucleus ambiguus. Significance: Oral administration of T-588 may prevent the degeneration of motoneurons in nucleus ambiguus in adult humans with recurrent laryngeal nerve injury, and could be a new treatment modality for recurrent laryngeal nerve paralysis. Support: None reported.

Neurofunctional recovery after GDNF gene transfer in the recurrent laryngeal nerve-injured rat

Problem: To assess the possibility of gene therapy for recurrent laryngeal nerve (RLN) injury, we examined the neuroprotective effect after glial cell line-derived neurotrophic factor (GDNF) gene transfer into nucleus ambiguus in a rat vagal nerve avulsion model. Morphological preservation of motoneurons has previously been demonstrated (Brain Res 962: 61–67, 2003). However, functional recovery of RLN after gene therapy has not been demonstrated. In the present study, we examined neurofunctional recovery after GDNF gene transfer in a rat RLN injury model. Methods: The left RLN of Sprague-Dawley rats was crushed for 60 seconds with forceps. Immediately after the crush and confirmation of left fixed vocal fold, an adenoviral vector encoding beta-galactosidase gene (AxCALacZ) or GDNF (AxCAhGDNF) was directly injected into the crushed site of RLN. Transgene expression by AxCALacZ in nucleus ambiguus was examined by X-gal histochemistry. Measurement of conduction velocity (CV) of RLN and laryngoscopy to check motion recovery of the left vocal fold were performed 2 and 4 weeks postoperation. The cross-section of RLN was histologically examined after epon/toluidine blue staining. Results: Motoneurons and their neurites in nucleus ambiguus were labeled with X-gal staining 4 days after AxCALacZ injection, indicating successful induction of foreign gene into the nucleus ambiguus by retrogradely transported adenoviral vector from the crushed site. In comparison between AxCAhGDNF-injected, AxCALacZ-injected, and noninjected animals, significantly better CV of RLN and better vocal fold motion recovery rate were observed in AxCAhGDNF-injected animals 2 and 4 weeks postoperation. RLN cross section of AxCAhGDNF-injected animals showed better myelination compared to the controls. Conclusion: Injection of adenoviral vectors into the crushed site of RLN successfully induced the foreign gene into motoneurons in nucleus ambiguus. Adenoviral GDNF gene transfer after RLN crush improved the CV of RLN and motion recovery of the paralyzed vocal fold. Significance: Adenoviral GDNF gene therapy may provide neurofunctional recovery in humans with a paralyzed vocal fold. Support: None reported.

Growth Associated Protein-43 mRNA Expression in Nucleus Ambiguus Motoneurons after Recurrent Laryngeal Nerve Injury in the Rat

Objective—To investigate the time-dependent differences in growth associated protein-43 (GAP-43) mRNA expression in the nucleus ambiguus (NA) motoneurons after recurrent laryngeal nerve (RLN) transection in the rat. Material and Methods—Animals were sacrificed on Days 1, 7, 14 and 21 after axotomy. The sections were then processed for in situ hybridization of GAP-43 mRNA. Results—GAP-43 mRNA transiently increased after axotomy, reaching a peak on postoperative Day 7, and then decreased gradually at 14 and 21 days after axotomy. The peak period of GAP-43 expression in the NA is different from those in the facial and hypoglossal nuclei after axotomy. Conclusion—We have already reported that neuronal nitric oxide synthase (nNOS) is induced in the NA motoneurons after axotomy and reaches a peak on postoperative Day 14. This study shows that the expressions of GAP-43 and nNOS are chronologically different.

Developmental study of cytochrome oxidase activity in the brain stem respiratory nuclei of postnatal rats.

We utilized cytochrome oxidase (CO) as a marker of neuronal functional activity to examine metabolic changes in brain stem respiratory nuclei of rats from newborn to 21 day of age. The pre-Bötzinger complex (PBC), upper airway motoneurons of nucleus ambiguus (NA(UAM)), ventrolateral nucleus of solitary tract (NTS(VL)), and medial and lateral parabrachial nuclei (PB(M) and PB(L), respectively) were examined at postnatal days (P) 0, 1, 2, 3, 4, 5, 7, 14, and 21. CO histochemistry was performed, and the intensity of CO reaction product was quantitatively analyzed by optical densitometry. In addition, CO histochemistry was combined with neurokinin-1 receptor (NK1R) immunogold-silver staining to doubly label neurons of PBC in P14 animals. The results showed that levels of CO activity generally increased with age in all of the nuclei examined. However, a significant decrease was found in NA(UAM) at P3 (P < 0.01), and a distinct plateau of CO activity was noted at P3 in PBC and at P3 and P4 in NTS(VL), PB(M), and PB(L). Of the neurons examined in PBC, 83% were doubly labeled with CO and NK1R. Of these, CO activity was high in 33.9%, moderate in 27.3%, and light in 38.8% of neurons, suggesting different energy demands in these metabolic groups that may be related to their physiological or synaptic properties. The transient decrease or plateau in CO activity at P3 and P4 implies a period of synaptic adjustment or reorganization during development, when there may be decreased excitatory synaptic drive or increased inhibitory synaptic drive, or both, in these brain stem respiratory nuclei. The adjustment, in turn, may render the system less responsive to respiratory insults. This may bear some relevance to our understanding of pathological events during postnatal development, such as occurs in sudden infant death syndrome.

Induction of Nitric Oxide Synthase Activity in Nucleus Ambiguus Motoneurons after Injury to the Rat Recurrent Laryngeal Nerve

Recent studies have implicated nitric oxide (NO) in neuronal degeneration and plasticity in the motor nervous system. In the present study, we investigated the induction of nitric oxide synthase (NOS) in the motoneurons in the nucleus ambiguus (NA) after injury to the rat recurrent laryngeal nerve (RLN) using nicotinamide-adenine-dinucleotide-phosphate-diaphorase (NADPH-d) histochemistry. NADPH-d reactivity was clearly induced in motoneurons in the ipsilateral NA after transection or avulsion of the RLN, compared with control animals. This finding suggests that NO may play an important role in the pathogenesis of RLN paralysis. Another interesting finding in the present study was the induction of NADPH-d reactivity in nerve terminals of the NA after RLN injury. This finding suggests that RLN injury has some effect on nitrergic input to the NA and a direct effect on the motoneurons.

Nucleus ambiguus and bulbospinal ventral respiratory group neurons in the neonatal rat

The in vitro brainstem-spinal cord preparation of the neonatal rat is an important model system for studies of the respiratory control system, yet there have not been studies to anatomically characterize respiratory neuron populations in the neonate. Fluorescent retrograde tracers were used to identify bulbospinal neurons of the ventral respiratory group and motoneurons of nucleus ambiguus in neonatal rats. Fluoro-Gold injections into the C 4 ventral horn labeled bulbospinal neurons within a densely packed column within the ventrolateral intermediate reticular nucleus from the level of the pyramidal decussation to the facial nucleus. This cell column corresponded closely to the location of the ventral respiratory group of the adult rat. In particular, neurons were labeled in regions corresponding to the rostral ventral respiratory group and the Bötzinger complex. Unlike adult rats, the preBötzinger complex also contained many bulbospinal neurons. Fluoro-Gold–labeled neurons were also located in the medial reticular nuclei, raphe pallidus, and obscurus and spinal vestibular nucleus. As in adult rats, bulbospinal ventral respiratory group neurons overlapped with cervical vagal motoneurons in the external formation, and partially with those in the loose formation, but not with those in the semicompact or compact formation of nucleus ambiguus. These results indicate that the distribution of bulbospinal ventral respiratory group neurons corresponds with that observed in physiological studies of neonatal rats.

Bidirectional electrical coupling between inspiratory motoneurons in the newborn mouse nucleus ambiguus.

Some spinal and brain stem motoneurons are electrically coupled in the early postnatal period. To test whether respiratory motoneurons in the brain stem are electrically coupled, we performed single and dual whole cell patch recordings from presumptive motoneurons in the nucleus ambiguus in a rhythmically active brain stem slice from newborn mice. Two of eight (25%) biocytin-injected neurons showed dye-coupling and 4 of 11 (36%) of intracellularly recorded pairs of neurons showed evidence of bidirectional electrical coupling. Impulse activity in one cell elicited small spikelets in the other and hyperpolarization of one cell led to hyperpolarization of the other with a coupling ratio (DeltaV2:DeltaV1) of 0.03-0.14. We conclude that inspiratory ambiguus motoneurons in the newborn mouse brain stem are bidirectionally electrically coupled, which may serve to transmit or coordinate signals, chemical or electrical.

Thyrotropin-releasing hormone inputs are preferentially directed towards respiratory motoneurons in rat nucleus ambiguus.

In the present study, we assessed the extent of the thyrotropin-releasing hormone (TRH) input to motoneurons in the ambigual, facial, and hypoglossal nuclei of the rat using a combination of intracellular recording, dye filling, and immunohistochemistry. Twelve motoneurons in the rostral nucleus ambiguus were labelled by intracellular injection in vivo of Neurobiotin (Vector). Seven out of 12 ambigual motoneurons displayed rhythmic fluctuations of their membrane potential in phase with phrenic nerve discharge, whereas the other five had no modulations of any kind. Seven facial motoneurons and seven hypoglossal motoneurons were also filled with Neurobiotin. All three motor nuclei contained TRH-immunoreactive varicosities, with the largest numbers found in the nucleus ambiguus. Close appositions were seen between TRH-immunoreactive boutons and every labelled motoneuron. Respiratory-related motoneurons in the nucleus ambiguus received the largest number of TRH appositions with 74 +/- 38 appositions/neuron (mean +/- S.D.; n = 7). In contrast, nonrespiratory ambigual motoneurons received significantly fewer TRH appositions (11 +/- 5; n = 5; P < 0.05; Mann-Whitney U test). Facial motoneurons received about the same number of TRH appositions as nonrespiratory ambigual motoneurons, with 13 +/- 4 (n = 7). Hypoglossal motoneurons received the fewest appositions from TRH-containing boutons, with 8 +/- 2 (n = 7). There were no differences in the TRH inputs to respiratory and nonrespiratory motoneurons in the facial and hypoglossal nuclei. These results demonstrate that, among motoneurons in the medulla, respiratory motoneurons in the rostral nucleus ambiguus are preferentially innervated by the TRH-immunoreactive boutons.

Calcitonin gene-related peptide in nucleus ambiguus motoneurons in rat: viscerotopic organization.

Calcitonin gene-related peptide has been reported in the rat nucleus ambiguus. This nucleus comprises a dorsal division that is the source of special visceral efferents innervating the striated muscle of the upper alimentary tract and a ventral division supplying general visceral efferents primarily to the heart. The distribution of calcitonin gene-related peptide immunoreactive neurons in the two divisions was determined by using a combination of immunocytochemical techniques and fluorescent retrograde tracing. In 22 rats, injections of Fluoro-Gold were made into either the supranodosal vagus nerve, palatopharynx, larynx, esophagus, or heart. Following colchicine injection, medullary sections were processed immunocytochemically for calcitonin gene-related peptide. Injection of Fluoro-Gold into the supranodosal vagus resulted in prominent labeling of neurons in the dorsal and ventral divisions of the nucleus ambiguus. The majority of fluorescent labeled neurons in the dorsal division were found to be immunoreactive for calcitonin gene-related peptide, while those labeled neurons in the ventral division were unreactive for the peptide. With esophageal, and palatopharyngeal and cricothyroid injections, many fluorescent labeled neurons that were immunoreactive for calcitonin gene-related peptide were found respectively in the compact and semicompact formations of the dorsal division. In contrast, injections of the heart resulted in fluorescent labeled neurons, which were unreactive for calcitonin gene-related peptide, localized to the external formation. The results demonstrate that calcitonin gene-related peptide immunoreactive neurons are localized entirely to the dorsal division of the nucleus ambiguus and that all striated muscular areas of the alimentary tract are innervated by calcitonin gene-related peptide containing motoneurons. The localization of calcitonin gene-related peptide to vagal motoneurons also known to contain acetylcholine and the increase in acetylcholine receptor synthesis caused by this peptide suggest that calcitonin gene-related peptide acts as a cotransmitter with acetylcholine in special visceral efferent vagal motoneurons.

Dendritic architecture of nucleus ambiguus motoneurons projecting to the upper alimentary tract in the rat

The motor innervation for palatal, pharyngeal, laryngeal, and esophageal muscles originates within the nucleus ambiguus. Although the viscerotopic organization of the upper alimentary tract in the nucleus ambiguus has been extensively studied, little information concerning the dendritic arborization of nucleus ambiguus motoneurons is available. The neural tracer cholera toxin-horseradish peroxidase, which is particularly effective at revealing dendrites of retrogradely labeled neurons, was used to determine the dendritic architecture and organization of nucleus ambiguus motoneurons. In 72 rats, cholera toxin-horseradish peroxidase in volumes of 1.0-18 microliters was directly applied under pressure to the musculature of various sites along the upper alimentary tract. Motoneurons innervating the soft palate, pharynx, cricothyroid muscle, and cervical esophagus were all found to have extensive dendrites that extended into the adjacent reticular formation with a distinct pattern for each muscle group. In contrast, the dendrites of motoneurons innervating the thoracic and subdiaphragmatic esophagus were confined to the compact formation of the nucleus ambiguus. Dendritic bundling within the confines of the nucleus ambiguus was prominent following injection of tracer into the soft palate, pharynx, and esophagus. The bundles were primarily oriented in a rostrocaudal direction. These data suggest that the extensive extranuclear dendritic arborization of motoneurons innervating the soft palate, pharynx, larynx, and cervical esophagus provide a wide ranging target for multiple central afferents that may be involved in the differential control of muscles that participate in multiple complex motor functions. The lack of extensive extranuclear dendrites of motoneurons innervating the distal esophagus suggest that they receive focused central afferents. The prominent bundling of dendrites within the nucleus ambiguus may provide for synchronization of motoneurons innervating a specific muscle and perhaps for synchronization of motoneurons innervating different muscles acting in sequence.

Subnuclear organization of the lateral tegmental field of the rat. I: Nucleus ambiguus and ventral respiratory group

Three classes of neurons within the lateral tegmental field of the rat medulla having different target projections were identified by retrograde labelling with three different fluorescent tracers. Labelled bulbospinal premotor and propriobulbar interneurons of the ventral respiratory group and vagal motoneurons of nucleus ambiguus formed partially intermingled longitudinal columns encompassed within a common region of the lateral tegmental field. Labelled neurons of each class were organized in a nonuniform distribution within these columns forming subdivisions distinguished by neuron morphology, orientation, and target projection. The three major rostrocaudal divisions of the ventral respiratory group (VRG) previously identified in the cat and rabbit were identified here in the rat, suggesting a common pattern of VRG organization among these species.

Electrophysiological evidence for axonal branching of ambiguous laryngeal motoneurons

Laryngeal motoneurons in the nucleus ambiguus (NA) were identified antidromically by stimulation of the ipsilateral superior laryngeal nerve (S) and/or the recurrent laryngeal nerve (R). In some NA motoneurons, antidromic spikes elicited by both S and R stimulation collided with the spontaneously occurring discharges. In the same neuron, spikes evoked antidromically by stimulation of one laryngeal nerve always collided with antidromic spikes elicited by stimulation of the other laryngeal nerve. Of 105 NA neurons activated by S and R stimulation, 36 neurons satisfied the criteria, and were classified as NA neurons with branching axons (branching NA (B-NA) neurons). Those neurons activated by either S or R stimulation but not both were classified as NA neurons without branching axons (unbranched NA (UB-NA) neurons). Mean antidromic latencies of B-NA neurons were 0.79 ± 0.20 ms to S stimulation and 1.91 ± 0.45 ms to R stimulation and those values for UB-NA neurons were 0.84 ± 0.17 ms to S stimulation and 2.10 ± 0.53 ms to R stimulation respectively. None of these mean values were significantly different from one another. Conduction time in the unbranched portion of the branching axon was estimated according to the equation reported by Anderson and Yoshida. The mean conduction time for 20 B-NA neurons was 0.45 ± 0.35 ms. The branching point in B-NA neurons was estimated on the basis of the conduction time in the unbranched stem portion and those times in two branches of a branching axon measured electrophysiologically. The results suggest that the majority of B-NA neurons bifurcate within a half axonal length. Recording sites of 16 B-NA and 14 UB-NA neurons were localized within or close to NA. Most B-NA neurons tended to be located toward the margin of NA, while many UB-NA neurons were situated at the central part of NA.

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.