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Parasympathetic motor neurons in the dorsal motor nucleus of the vagus (DMV) innervate the stomach by way of the gastric and hepatic branches of the vagus nerve. To investigate whether single neurons of the DMV provide collateral innervations to various parts of the stomach, we injected the retrograde tracer Fluoro-Gold (FG) into the cardia and the retrograde tracer cholera toxin subunit b (CTb) into the antrum or the pylorus of the same animal. Both retrogradely FG-labeled and CTb-labeled neurons were found throughout the DMV. Almost all CTb-labeled neurons (97%) were double-labeled with FG after injection of FG into the cardia and CTb into the antrum, while only a few CTb-labeled neurons (11%) were double-labeled with FG after injection of FG into the cardia and CTb into the pylorus. Thus, the cardia and the antrum received collateral projections, but the pylorus received projections mainly from different neurons in the DMV. These results indicate that different neurons in the DMV activate either the cardia or the pyloric sphincter muscles. We also labeled, retrogradely, the neurons projecting to the cardia and the pylorus in the DMV with cholera toxin-conjugated horseradish peroxidase (CT-HRP) to examine their ultrastructural characteristics. Although the neurons projecting to the cardia (21.6x15.0 microm, 248.0 microm2 per section) were significantly smaller than the neurons projecting to the pylorus (27.5x15.9 microm, 323.2 microm2 per section), their ultrastructural appearances were similar. Both types of neurons were small-to-medium sized, round or oval in shape, and generally had a small amount of cytoplasm containing a few Nissl bodies and a round nucleus. The average number of axosomatic terminals per section was low in the neurons projecting to the cardia (2.3) and the neurons projecting to the pylorus (3.0). Almost all axon terminals contacting these motor neurons contained round synaptic vesicles and made asymmetric synaptic contacts (Gray's type I).

The central subnucleus of the nucleus tractus solitarii (ceNTS) receives afferent projections from the esophageal wall and projects to the nucleus ambiguus, thus serving as a relay nucleus for peristalsis of the esophagus. Here we examine the synaptic organization of the ceNTS, and its esophageal afferents by using transganglionic anterograde transport of cholera toxin-conjugated horseradish peroxidase (CT-HRP). When CT-HRP was injected into the subdiaphragmatic esophagus, many anterogradely labeled terminals were found only in the ceNTS. The ceNTS was composed of round or oval-shaped, small neurons (14.7x8.7 micro m) containing sparse organelles and an irregularly shaped nucleus. The average number of axosomatic terminals was only 1.3 per section cut through the nucleolus. Most of them (92%) contained round vesicles and formed asymmetric synaptic contacts (Gray's type I), and a few (8%) contained pleomorphic vesicles and formed symmetric synaptic contacts (Gray's type II). All anterogradely labeled terminals contacted dendrites but not the neuronal somata. The labeled terminals were large (2.55+/-0.07 micro m) and exclusively Gray's type I. More than half of them (60%) contacted small dendrites (less than 1 micro m in diameter), and contained dense-cored vesicles. More than 40% of the labeled terminals contacted two to four dendrites, thus forming a synaptic glomerulus. Sometimes a labeled terminal that contacted an unlabeled terminal by an adherent junction was found within the glomerulus. The large terminals and these complex synaptic relations appeared to characterize the esophageal afferent projections in the ceNTS.

The accessory nucleus is composed of neurons in the medial column that innervate the sternocleidomastoid muscle, and neurons in the lateral column that innervate the trapezius muscle. We retrogradely labeled these neurons by injection of cholera toxin conjugated horseradish peroxidase into the sternomastoid (SM) or the clavotrapezius (CT) muscles, and investigated fine structure and synaptology of these neurons. Almost all SM and CT motoneurons had the appearance of alpha-motoneurons, i.e., large, oval or polygonal cells containing well-developed organelles, Nissl bodies, and a prominent spherical nucleus. More than 60% of the somatic membrane was covered with terminals. The SM motoneurons (34.4 x 52.2 microm, 1,363.1 microm(2) in a section) were slightly larger than the CT motoneurons (32.8 x 54.2 microm, 1,180.8 microm(2)). The average number of axosomatic terminals in a section was 52.2 for the SM, and 54.2 for the CT motoneurons. More than half of them (58.0%) contained pleomorphic vesicles and made symmetric synaptic contacts (Gray's type II) with the SM motoneurons, while 57.9% of them contained round vesicles and made asymmetric synaptic contacts (Gray's type I) with the CT motoneurons. A few C-terminals were present on the SM (3.5) and the CT (3.7) motoneurons. About 60% of the axodendritic terminals were Gray's type I in both the SM and the CT motoneurons. A few labeled small motoneurons were also found among the SM and the CT motoneurons. They were small (19.2 x 26.2 microm, 367.0 microm(2)), round cells containing poorly developed organelles with a few axosomatic terminals (9.3). Only 20% of the somatic membrane was covered with the terminals. Thus, these neurons were presumed to be gamma-motoneurons. These results indicate that the motoneurons in the medial and the lateral column of the accessory nucleus have different ultrastructural characteristics.

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The upper cervical esophagus is exerted on swallowing and peristalsis by somatic and visceral motoneurons, whereas the lower esophagus is exerted on only peristalsis by visceral motoneurons. We examined the origin of the esophageal motoneurons and whether there were any differences between the distributions of the upper and the lower esophageal motoneurons in the medulla and the spinal cord using cholera toxin subunit b (CTb) as the retrograde tracer. Following injection of CTb into the cervical esophagus resulted in heavy labeling of the neurons in the nucleus ambiguus including the compact (AmC), semicompact (AmS) and loose (AmL) formations, and the medial column of lamina IX at the C1-C5 levels of the cervical spinal cord corresponding to the spinal accessory nucleus. A few labeled neurons were found in the inferior salivatory nucleus, the rostral division of the dorsal motor nucleus of the vagus (DMX), the accessory facial nucleus and the lateral column of lamina IX at the C2 and C3 levels. All these labeled neurons showed ChAT immunoreactivity. When CTb was injected into the cut end of the unilateral recurrent laryngeal nerve, many labeled neurons were found in the ipsilateral AmC, the AmL, and the bilateral medial column at the C1 and C2 levels. Following injection of CTb into the subdiaphragmatic esophagus resulted in heavy labeling of the neurons only in the AmC and the DMX. When CTb was injected into the sternomastoid muscle, many labeled neurons were found in the medullary reticular formation, the facial nucleus, the medial column at the C1-C3, C5 and C6 levels, and the lateral column at the C2, C3, C5 and C6 levels. Injections of a Fluoro-Gold into the cervical esophagus and a CTb into the sternomastoid muscle or the subdiaphragmatic esophagus in the same animal showed many double labeled neurons in the medial column of the accessory nucleus at the C1 and C2 levels, but no double labeled neurons in the AmC. These results indicated that the upper cervical esophagus is innervated by the visceral medullary vagal motoneurons as well as the somatic spinal accessory motoneurons. The lower esophagus is innervated only by the visceral medullary vagal motoneurons.