Distal Vagal Ganglion Research Articles

Morphological and molecular evolution of the ultimobranchial gland of nonmammalian vertebrates, with special reference to the chicken C cells.

This review summarizes the current understanding of the nonmammalian ultimobranchial gland from morphological and molecular perspectives. Ultimobranchial anlage of all animal species develops from the last pharyngeal pouch. The genes involved in the development of pharyngeal pouches are well conserved across vertebrates. The ultimobranchial anlage of nonmammalian vertebrates and monotremes does not merge with the thyroid, remaining as an independent organ throughout adulthood. Although C cells of all animal species secrete calcitonin, the shape, cellular components and location of the ultimobranchial gland vary from species to species. Avian ultimobranchial gland is unique in several phylogenic aspects; the organ is located between the vagus and recurrent laryngeal nerves at the upper thorax and is densely innervated by branches emanating from them. In chick embryos, TuJ1-, HNK-1-, and PGP 9.5-immunoreactive cells that originate from the distal vagal (nodose) ganglion, colonize the ultimobranchial anlage and differentiate into C cells; neuronal cells give rise to C cells. Like C cells of mammals, the cells of fishes, amphibians, reptiles, and also a subset of C cells of birds, appear to be derived from the endodermal epithelium forming ultimobranchial anlage. Thus, the avian ultimobranchial C cells may have dual origins, neural progenitors and endodermal epithelium. Developmental Dynamics 246:719-739, 2017. © 2017 Wiley Periodicals, Inc.

Extrinsic afferents supplying the ovine duodenum and ileum

The study aimed at establishing the distribution of primary sensory neurons by means of retrograde tracers Diamidino Yellow (DY) and Fast Blue (FB) injected into both the sheep duodenum and ileum, respectively. Many DY-labelled cells were found in both the distal vagal ganglia (DVG) and the spinal ganglia (SG) from T9-L3; on the contrary, the majority of the FB-labelled cells were found in the SG. In the SG, a double immunofluorescence stain was used to reveal Nitric Oxide Synthase-Immunoreactivity (NOS-IR) in association with: substance P (SP), calcitonin gene-related peptide (CGRP), neurofilament 200kDa (NF) and isolectin B(4) (IB4). The labelled neurons, both DY and FB generally ranged in size from medium to large. The majority of the SG duodenal projections were NOS negative; the majority of the SG ileal afferent neurons expressed NOS-IR. Both DY and FB NOS-IR neurons often co-localized IB4, CGRP and SP, but rarely NF.

Extrinsic and intrinsic sources of calcitonin gene-related peptide immunoreactivity in the lamb ileum: a morphometric and neurochemical investigation

To investigate extrinsic origins of calcitonin gene-related peptide immunoreactive (CGRP-IR) nerve fibres in the sheep ileum, the retrograde fluorescent tracer Fast Blue (FB) was injected into the ileum wall. Sections of thoraco-lumbar dorsal root ganglia (DRG) and distal (nodose) vagal ganglia showing FB-labelled neurons were processed for CGRP immunohistochemistry. The distribution of CGRP-IR in fibres and nerve cell bodies in the ileum was also studied. CGRP-IR enteric neurons were morphometrically analysed in myenteric (MP) and submucosal plexuses (SMP) of lambs (2-4 months). Sensory neurons retrogradely labelled with FB were scattered in T5-L4 DRG but most were located at the upper lumbar levels (L1-L3); only a minor component of the extrinsic afferent innervation of the ileum was derived from nodose ganglia. In the DRG, 57% of retrogradely labelled neurons were also CGRP-IR. In cryostat sections, a dense network of CGRP-IR fibres was observed in the lamina propria beneath the epithelium, around the lacteals and lymphatic follicles (Peyer's platches), and along and around enteric blood vessels. Rare CGRP-IR fibres were also present in both muscle layers. Dense pericellular baskets of CGRP-IR fibres were observed around CGRP-negative somata. The only CGRP-IR nerve cells were well-defined Dogiel type II neurons localised in the MP and in the external and internal components of the SMP. CGRP-IR neurons in the myenteric ganglia were significantly larger than those in the submucosal ganglia (mean profile areas: about 1,400 mum(2) for myenteric neurons, 750 mum(2) for submucosal neurons). About 6% of myenteric neurons and 25% of submucosal neurons were CGRP-IR Dogiel type II neurons. The percentages of CGRP-IR neurons that were also tachykinin-IR were about 9% (MP) and 42% (SMP), whereas no CGRP-IR neurons exhibited immunoreactivity for vasoactive intestinal peptide, nitric oxide synthase or tyrosine hydroxylase in either plexus. Thus, CGRP immunoreactivity occurs in the enteric nervous system of the sheep ileum (as in human small intestine and MP of pig ileum) in only one morphologically defined type of neuron, Dogiel type II cells. These are probably intrinsic primary afferent neurons.

Nicotinamide promotes long‐term survival and extensive neurite outgrowth in ultimobranchial C cells cultured from chick embryos

In avian species, the ultimobranchial anlage is populated with neuronal cells derived from the distal vagal ganglion. We found that ultimobranchial C cells of chick embryos cultured in the presence of nicotinamide continued to grow for at least 60 days and exhibited profound morphological changes, resulting in the formation of dense networks of neuronal fibers. Nicotinamide, thus, facilitated the manifestation of neuronal features in C cells. The neuronal phenotypes of cultured C cells were analyzed in detail by both scanning and transmission electron microscopy. Their neural nature was also positively established by immunostaining with monoclonal antibodies to the neuronal markers neuron-specific class III beta-tubulin (TuJ1), microtubule-associated protein (MAP) 2, and synaptophysin. Confocal laser scanning microscopy confirmed that these neuron-specific proteins are colocalized with calcitonin in both the somata and the neuronal processes of C cells. Furthermore, reverse transcriptase-polymerase chain reaction analyses, performed at various times up to 30 days in culture, indicated that the C cells have persistent gene expression of calcitonin, the catecholamine-synthesizing enzyme tyrosine hydroxylase, proenkephalin, proopiomelanocortin, neuron-specific beta-tubulin (cbeta4), SCG10, and Bcl-2. The morphological responses of C cells to nicotinamide treatment were analyzed quantitatively over a period of 60 days. The area of C-cell colonies, number of processes per colony, and length of processes continued to increase until culture day 45. In conclusion, nicotinamide stimulates long-term survival and neuronal differentiation of chick embryo C cells, and this culture system may provide a useful model for studying neuronal differentiation mechanisms.

Macrostructure of the cranial cervical ganglionar complex and distal vagal ganglion during post natal development in dogs

Twelve specimens of head and neck of the domestic dog (Canis familiaris) were dissected to study the situation, arrangements and branches of the distal vagal ganglion and the cranial cervical ganglion. The ganglions showed a fusiforme shape, covered by the M. digastricus. The main branches of the cranial cervical ganglion included the internal carotid and external carotid branches and of distal vagal ganglion included the cranial laryngeal nerve. This study showed that the cranial cervical ganglion and the distal vagal ganglion in dogs are well developed structure. There were no obvious anatomical differences between the same ganglions presented in both antimeres.

Localization and Coexistence of Calcium‐Binding Proteins and Neuropeptides in the Vagal Ganglia of the Goat

This study was performed to investigate the neurochemical characteristics of the vagal ganglia of the goat by immunohistochemical methods using calbindin D-28k (CB), calretinin (CR). parvalbumin (PA), substance P (SP). calcitonin generelated peptide (CGRP) and galanin (GAL) antibodies. In the proximal vagal ganglia (jugular ganglia), CGRP- (57.1%), SP- (48.2%), GAL- (8.6%), PA- (8.7%), CB- (8.5%) and CR-like (5.3%) immunoreactive cells were observed. In the distal vagal ganglia (nodose ganglia), CGRP- (40.5%), SP- (30.20%), CB- (22.0%) and CR-like (18.10%) immunoreactive cells were present. The double immunohistochemical study showed, that in the proximal vagal ganglia, CGRP immunoreactivity was co-localized in SP- (84.8%), GAL-(100%), CB- (5.6%) and CR- (5.7%) immunoreactive cells: SP immunoreactivity was co-localized in the CGRP- (80.0%), GAL- (100%). CB- (5.3%) and CR- (5.6%) immunoreactive cells; GAL immunoreactivity coexisted in the CGRP- (4.4%) and SP- (19.8%) immunoreactive cells, but not in calcium-binding proteins (CBP)-immunoreactive cells; PA immunoreactivity was absent in the CGRP- and SP-immunoreactive cells; CB and CR immunoreactivities were seen in the CGRP-(0.8%) and SP-immunoreactive (0.9%) cells. On the other hand, in the distal vagal ganglia, CGRP immunoreactivity appeared in SP- (66.6%), CB- (1.0%) and CR- (1.2%) immunoreactive cells; SP immunoreactivities were observed in the CGRP- (44.1%), CB- (1.0%) and CR- (1.2%) immunoreactive cells; CB immunoreactivities were present in the CGRP- (0.5%) and SP- (0.8%) immunoreactive cells; CR immunoreactivities were contained in the CGRP- (0.5%) and SP- (0.8%) immunoreactive cells. These findings indicate that the goat is distinct from other mammalian species in the distribution and localization of neurochemical substances in the vagal ganglia. and suggest that these differences may be related to physiological characteristics, particular those of the ruminant digestive system.

Evidence to support the distal vagal ganglion as the origin of C cells of the ultimobranchial gland in the chick

Formation and development of the ultimobranchial anlage were studied in chicken embryos by immunohistochemistry with the antibodies to class III beta-tubulin, TuJ1, human leukemic cell-line (HNK-1), and protein gene product (PGP) 9.5, all of which recognized neurons. Medial to the fourth aortic arch, the ultimobranchial anlage was formed by the extension of the ventral portion of the fourth pharyngeal pouch at 4.5 days of incubation. At 5 days of incubation, TuJ1-immunoreactive cells with long cell processes began to enter the ultimobranchial anlage, which displayed a follicle structure. At 6 days of incubation, numerous neuronal cells that were continuous with the distal vagal ganglion (nodose ganglion) and expressed immunoreactivity for TuJ1, HNK-1, and PGP 9.5 were found to be in direct contact with the peripheral portion of ultimobranchial anlage. The TuJ1 antibody reacted only with the neuronal cells, whereas the HNK-1 and PGP 9.5 antibodies reacted with both endodermal epithelial cells and the neuronal cells in the ultimobranchial anlage. Subsequently, the ultimobranchial anlage rapidly increased in size; the follicle wall was thickened and a central cavity disappeared. The TuJ1-immunoreactive cells were distributed throughout the ultimobranchial parenchyma in 7-day-old embryos. The neuronal cell streams from the distal vagal ganglion to the ultimobranchial anlage were still prominent at 8 days of incubation. Almost all parenchymal cells of the ultimobranchial glands were intensely immunoreactive for TuJ1, HNK-1, and PGP 9.5 between 10 and 16 days of incubation. These results indicate that the neuronal cells from the distal vagal ganglion enter into the ultimobranchial anlage and give rise to C cells, i.e., C cells differentiate along the neuronal lineage. During embryonic development, C cells of the chick ultimobranchial glands transiently express a number of neuronal properties.

Glomus cell differentiation in the carotid body region of chick embryos studied by neuron-specific class III beta-tubulin isotype and Leu-7 monoclonal antibodies.

Development of the carotid body and the glomus cell groups in the wall of the common carotid artery and its branches was examined in chickens at various developmental stages by immunohistochemistry using three different monoclonal antibodies, i.e., anti-neuron-specific class III beta-tubulin isotype (TuJ1), anti-rat brain beta-tubulin, and anti-Leu-7 (HNK-1) antibodies. All the antibodies reacted with neurons. The carotid body anlage was first discerned at 6 days of incubation at the lateral portion of the third branchial artery. The cells and nerve fibers immunoreactive for TuJ1, brain beta-tubulin and Leu-7, which were connected with the distal ganglion of the vagus nerve, were found around the carotid body anlage at this stage. Within the carotid body anlage, no immunoreactivity yet appeared. The immunoreactive cells were accumulated around the carotid body anlage until 8 days of incubation. From 9 days of incubation, the immunoreactive cells continuing with the distal vagal ganglion began to enter into the carotid body anlage and also dispersed widely along the common carotid artery and its branches, giving rise to the glomus cells. At 12 days of incubation, a large portion of the carotid body was occupied by the immunoreactive cells. Thus, the present study evidences that the glomus cells in the carotid body and around the arteries are emigrés that arrive in each residential place from the distal vagal ganglion. Immunoreactivity for TuJ1, brain beta-tubulin, and Leu-7 in the glomus cells started to decrease at late stages of embryonic development. After hatching, no TuJ1-immunoreactive cells were detected in the carotid body region.

Horseradish peroxidase study of the location of extrinsic efferent and afferent neurons innervating the colon of dogs

SUMMARY The abdominal portion of the colon of 13 clinically normal dogs was divided into 5 regions (ascending, transverse, left colic flexure, proximal descending, and distal descending), and each region was injected with 30 mg of horseradish peroxidase (hrp). The injected colonic region, brain stem, L7-Cd1 portion of the spinal cord, sympathetic trunk ganglia, celiacomesenteric ganglia, caudal mesenteric ganglion, pelvic plexi, distal vagal (nodose) ganglia, and Ll-Cdl spinal ganglia were obtained at postinjection hour 48, sectioned, and processed by use of the tetramethylbenzidine method. The entire length of the colon was found to be under extrinsic influence of the parasympathetic nucleus of cranial nerve X (px), with the largest average number of labeled cells resulting from injection of the ascending colon. It was also indicated that the entire colon is under extrinsic influence of the sacral portion of the spinal cord because the pelvic ganglia (second-order neurons) of the pelvic plexi contained labeled cells for all colonic regions. The largest average number of labeled cells in pelvic ganglia was seen after injection of the distal portion of the descending colon. Only after injection of the distal portion of the descending colon were labeled cells found in the S1-S3 portion of the spinal cord. Labeled cells in the px, spinal cord, and pelvic ganglia were found bilaterally. Although the entire abdominal portion of the colon appears to be influenced by cranial and sacral parasympathetic preganglionic (via pelvic ganglia) neurons, the relative importance of the 2 areas seems to be reversed between the ascending colon and distal portion of the descending colon. Sympathetic postganglionic innervation was located in the celiacomesenteric ganglion, caudal mesenteric ganglion, and, inconsistently, in sympathetic trunk ganglia. Sensory innervation for all colonic regions was found bilaterally in the distal vagal ganglia, and in 2 groups of spinal ganglia—a cranial group of LI to L5 and a caudal group of S1 to Cd1 ganglia. It has been reported in the literature that the left colic flexure is subject to colonic perforation in dogs with spinal cord disease treated with corticosteroid administration, but a direct, anatomic reason for this site of susceptibility was not found during this study. It is possible that the left colonic flexure represents the most proximal portion of colon detrimentally affected by loss of sacral influence, because the relative importance of vagal and sacral parasympathetic innervation appears to shift along the length of the colon.

Occurrence of calcitonin-positive C cells within the distal vagal ganglion and the recurrent laryngeal nerve of the chicken.

The chicken ultimobranchial glands are richly supplied with nerve fibers originating from both the main trunk of the vagus nerve and its branch--the recurrent laryngeal nerve. C cells immunoreactive for calcitonin were invariably found in the large nerve bundles distributed throughout the ultimobranchial glands. In addition, these cells were often present within the distal vagal ganglia and the recurrent laryngeal nerves. The frequency of occurrence and the pattern of distribution of the C cells in the distal vagal ganglia and the recurrent laryngeal nerves were determined in chickens of various ages by means of an immunoperoxidase method with anticalcitonin and antineurofilament antisera. The left and right sides of the ultimobranchial region were asymmetrical. The left ultimobranchial gland was in close contact with the vagus nerve trunk, especially with the distal vagal ganglion, but it was separated from the recurrent laryngeal nerve. The right gland contacted the recurrent laryngeal nerves, its medial edge being frequently penetrated by the nerve, but the gland was separated from the distal vagal ganglion. On the left side, C cells were found in 25 out of 39 distal vagal ganglia but they were not distributed in the recurrent laryngeal nerve. On the right side, the cells were present in 28 out of 43 recurrent laryngeal nerves but absent in the distal vagal ganglia. The results indicate that the C cells secreting a hormone calcitonin can enter into nerves, but their occurrence is restricted to the nerves in close proximity to the ultimobranchial glands. Electron microscopic studies revealed that C cells in the nerves received numerous axon clusters enveloped with Schwann cell cytoplasm. Naked axons regarded as axon terminals were found in direct contact with the surface of C cells. They were mainly composed of efferent-type nerve endings showing the accumulation of numerous small clear vesicles and a few large dense-cored vesicles. In addition, C cells were partly covered with the long cytoplasmic processes of Schwann cells and were also in contact with the Schwann cell perikarya. The C cells in nerves appear to be controlled by neural stimulation.

Innervation of the C cells of chicken ultimobranchial glands studied by immunohistochemistry, fluorescence microscopy, and electron microscopy.

Innervation of the ultimobranchial glands in the chicken was investigated by immunohistochemistry, fluorescence microscopy and electron microscopy. The nerve fibers distributed in ultimobranchial glands were clearly visualized by immunoperoxidase staining with antiserum to neurofilament triplet proteins (200K-, 150K- and 68K-dalton) extracted from chicken peripheral nerves. The ultimobranchial glands received numerous nerve fibers originating from both the recurrent laryngeal nerves and direct vagal branches. The left and right sides of the ultimobranchial region were asymmetrical. The left ultimobranchial gland had intimate contact with the vagus nerve trunk, especially with the distal vagal ganglion, but was somewhat separated from the recurrent nerve. The right gland touched the recurrent nerve, the medial edge being frequently penetrated by the nerve, but the gland was separated from the vagal trunk. The left gland was innervated mainly by the branches from the distal vagal ganglion, whereas the right gland received mostly the branches from the recurrent nerve. The carotid body was located cranially near to the ultimobranchial gland. Large nerve bundles in the ultimobranchial gland ran toward and entered into the carotid body. By fluorescence microscopy, nerve fibers in ultimobranchial glands were observed associated with blood vessels. Only a few fluorescent nerve fibers were present in close proximity to C cell groups; the C cells of ultimobranchial glands may receive very few adrenergic sympathetic fibers. By electron microscopy, numerous axons ensheathed with Schwann cell cytoplasm were in close contact with the surfaces of C cells. In addition, naked axons regarded as axon terminals or "en passant" synapses came into direct contact with C cells. The morphology of these axon terminals and synaptic endings suggest that ultimobranchial C cells of chickens are supplied mainly with cholinergic efferent type fibers. In the region where large nerve bundles and complex ramifications of nerve fibers were present, Schwann cell perikarya investing the axons were closely juxtaposed with C cells; long cytoplasmic processes of Schwann cells encompassed large portions of the cell surface. All of these features suggest that C-cell activity, i.e., secretion of hormones and catecholamines, may be regulated by nerve stimuli.

The origins of innervation of the canine caudal pharyngeal muscles: an HRP study

In deglutition, movements of the tongue and oropharynx direct a bolus to the laryngopharynx. The major muscles of this region, which includes the ‘cricopharyngeal sphincter’, must undergo sequential relaxation and contraction for correct swallowing action. The innervation of the caudal pharyngeal muscles involved in this action in the dog have not been determined previously by sensitive neuroanatomical techniques. In this study, the location of efferent and afferent neurons innervating the left cricopharyngeus, thyropharyngeus and hyopharyngeus muscles was determined by horseradish peroxidase (HRP) histochemistry in 7 puppies. Labeled cells were found ipsilaterally in the supraspinal nucleus, nucleus ambiguus (including nucleus retrofacialis) and nucleus intercalatus of all animals, in the parasympathetic nucleus of X (dorsal vagal efferent nucleus) of 6 animals, and in the hypoglossal nucleus of 4 animals. Small numbers of HRP-labeled cells were found contralaterally in the supraspinal nucleus of all animals, and in the rostral nucleus ambiguus, in the nucleus intercalatus and the parasympathetic nucleus of X of fewer animals. This defines a more extensive source of efferent neurons for these muscles than had been reported for the cat. Labeled postganglionic sympathetic neurons were found bilaterally in the cranial (superior) cervical, middle cervical and cervicothoracic (stellate) ganglia. Labeled afferent neurons were seen bilaterally in the proximal vagal (jugular) and distal vagal (nodose) ganglia and in the C1–C4 spinal ganglia. The location of sympathetic and sensory nerve cell bodies of the muscles of the laryngopharynx has not been previously reported.

The origins of innervation of the esophagus of the dog

This study defined the origins of extrinsic efferent and afferent innervation of the normal canine esophagus. When all the layers of the wall of the 3 esophageal regions (cervical, thoracic and abdominal) were injected with horseradish peroxidase (HRP), labeled nerve cells were found in the nucleus ambiguus (NA) and parasympathetic nucleus of X (PX) of the brainstem. Most labeled cells in the NA were located in the compact column (retrofacial nucleus) while labeled cells in the PX were located in separate rostral and caudal areas. There was no somatotopic organization in either the NA or PX. Labeled sympathetic postganglionic neurons were found in the cranial cervical, middle cervical, cervicothoracic, thoracic sympathetic trunk and celiacomesenteric ganglia. The HRP injection of the esophageal wall labeled sensory cell bodies in the glossopharyngeal, proximal and distal vagal, and C2-T6 spinal ganglia. There was no discernible pattern of distribution of labeled cells in the autonomic or sensory ganglia. When the HRP injections were confined to the mucosa-submucosa layers of the thoracic esophagus, a small number of labeled cells were identified in the NA; however, no labeled cells were found in the NA when injections were confined to the mucosa-submucosa of either the cervical or abdominal esophageal regions. With these confined injections, the labeled nerve cells appeared in the rostral part of the PX. Thus, it appeared that the internal tunics of the esophagus (i.e., the mucosa and submucosa) were innervated by neurons in the rostral PX while the muscular tunic was innervated by neurons in the caudal PX and the rostral NA. After mucosa-submucosa injections, labeled sympathetic neurons appeared in the same ganglia that were identified after whole wall injections and these had a similar random distribution. These injections also labeled neurons in the glossopharyngeal, proximal vagal, and distal vagal ganglia, but unlike the whole wall injections there was no labeling in the spinal ganglia. This suggested that the labeled cells of the spinal ganglia seen after whole wall injections conveyed impulses from the tunica muscularis and serosa.

Autoradiographic observations on the innervation of the carotid body of the domestic fowl

Tritiated leucine was injected into the distal vagal ganglion of 11 domestic fowl, which survived for 12–24 h under general anaesthesia. The cells of this ganglion are known to be exclusively afferent. EM autoradiography showed that in all 11 birds the vast majority of the silver grains fell upon the nervous tissues of the carotid body. In 5 of these birds a quantitative analysis was made, using point-counting morphometry. The incidence of silver grains per unit area was found to be 26 times greater in axonal endings than in the non-nervous components, and 15 times greater in axons in transit than in non-nervous components. The difference in incidence per unit area between these nervous and non-nervous components was highly significant ( P < 0.001). Of all the observed axonal endings 77% were labelled, but there is evidence that this is a substantial underestimate of the total population of afferent endings; in one bird 88% of the endings were labelled. Of the axons in transit, 18% were labelled. This low value is believed to be related to transfer of the label to the axonal endings by the fast component of axonal transport. Afferent and reciprocal synapses occured in labelled axonal endings, which were therefore considered to have an afferent function. ‘Efferent’ type synapses also occurred in labelled endings, and therefore belonged to axons which in fact were afferent in function. It is concluded that the innervation of the carotid body of the domestic fowl is almost entirely afferent, the nerve cell bodies being in the distal vagal ganglion. Only very few efferent axonal endings are present. Ultrastructural features, including synaptic morphology, appear to constitute unreliable criteria for distinguishing between afferent and efferent axonal endings in the carotid body.

The origins of the afferent fibers to the lingual muscles of the dog, a retrograde labelling study with horseradish peroxidase.

The origin of the afferent fibers to the lingual muscles of the dog was investigated by means of retrograde transport of horseradish peroxidase (HRP) from injection sites in the tongue and the extrinsic lingual muscles. Intralingual injections were not satisfactory because the enzyme diffused beyond the intrinsic lingual muscles to include virtually all tissues within the tongue. Thus, the resultant retrograde labeling of cell bodies of the trigeminal, geniculate, glossopharyngeal, vagal, and first cervical (C1) spinal ganglia represented a composite of lingual sensory innervation. In order to confine HRP uptake to intramuscular nerve endings, injections were limited to surgically isolated extrinsic lingual muscles, i.e., the genioglossus, hyoglossus, and styloglossus. After these intramuscular injections, labeled neurons appeared ipsilaterally in the C1 spinal ganglion, the proximal vagal (jugular) ganglion, and trigeminal ganglion. Earlier suggestions that the lingual proprioceptive neurons of the dog reside in the distal vagal (nodose) ganglion and hypoglossal ganglia were not confirmed. The mesencephalic nucleus of the trigeminal nerve failed to label after enzyme injections into the tongue or the extrinsic lingual muscles. The retrograde labeling of cell bodies in the C1 spinal ganglion was abolished when HRP injections into the extrinsic lingual muscles were preceded by surgical interruption of the ansa cervicalis or distal section of the hypoglossal nerve. Retrograde labeling of neurons in the proximal vagal ganglion persisted after hypoglossal nerve transections.