THE JOURNAL OF COMPARATIVE NEUROLOGY 311520-530 (1991)

Afferent and Efferent Projections of the Glossopharyngeal-Vagal Nerve in the Hagfish HIDEKI MATSUDA, RICHARD C. GORIS, AND REIJI KISHIDA Department of Anatomy, Yokohama City University School of Medicine, Kanazawa-ku, Yokohama 236 (H.M.,R.C.G.),and Department of Anatomy, Yamaguchi University School of Medicine, Ube-shi 755 (R.K.),Japan

ABSTRACT Anterograde and retrograde transport of horseradish peroxidase was used to examine the afferent and efferent projections of the glossopharyngeal-vagalnerve in the hagfish Eptatretus burgeri. Anterogradely labeled ganglion cells are scattered in the glossopharyngeal-vagal nerve trunk, in the saccular ganglion, and in the brainstem. Afferent fibers of the glossopharyngealvagal nerve terminate in both the vagal lobe and the fasciculus communis. Close observation showed no morphological differentiation between these two structures, indicating that they are not separate entities, but a single, continuous structure that is homologous with the nucleus and tractus solitarius of other vertebrates. The median part of this structure (the commissura infima) is displaced more rostrally than the same part of the solitary nucleus in many other vertebrates. Some of the afferent fibers invade the ventral portion of the trigeminal sensory nucleus, which receives the maxillo-mandibular nerve fibers, and terminate there. Our study showed that the hagfish has only one nucleus in the vagal motor system, i.e., the vagal motor nucleus, which contains both parasympathetic and branchiomotor neurons. The dendrites of the vagal motor neurons in the hagfish are more highly developed than those in other vertebrates. This suggests that the motor reflex arc of the glossopharyngeal-vagal nerve in hagfishes may be simpler than in other vertebrates. Key words: solitary complex, visceral motor system, horseradish peroxidase

Hagfishes and lampreys are the only living members of the cyclostomes,which preserve some primitive features of vertebrates (Romer, '74). According to paleontological studies (Janvier, '81; Forey, '84; Aldridge et al., '86), hagfishes derive earlier than lampreys from the stem of the phylogenetical tree of vertebrates. The present analysis in the hagfish may provide a basis for understanding early features of the vertebrate brain. In our laboratory we have been able to provide fairly detailed descriptions of the primitive state of some cranial nerve systems (Vth, VIIth, and VIIIth) of the hagfish Eptatretus burgeri (Amemiya et al., '85; Kishida et al., '86, '87; Nishizawa et al., '88), but to date such detailed information has not been reported for the IXth and Xth cranial nerves of this animal. There are many unsolved problems regarding the glossopharyngeal-vagal nerves of the hagfish. The very existence of the IXth nerve is controversial (Johnston, '08; Jansen, '30). The position of the vagal lobe (the solitary nucleus of other Vertebrates) is quite different from that in other vertebrates (Jansen, '30; Nishizawa, '88). Is the caudal motor nucleus of the vagus of Addens ('33) the real vagal motor nucleus? o 1991 WILEY-LISS, INC.

We performed anterograde and retrograde -.orsera ish peroxidase (HRP) studies on the glossopharyngeal-vagal nerves in hagfishes Eptatretus burgeri to solve these problems.

MATERIALS AND METHODS A total of 13adult hagfish, Eptatretus burgeri, were used: 10 for HRP applicationto the glossopharyngeal-vagalnerves, one for HRP application to the spino-occipital nerve, and two as normal preparations. All fish were first anesthetized with 1%ethyl carbamate (Urethane) in artificial seawater. Each experimental fish was then wrapped in a polyvinyl sheet and buried in shaved ice with only the head protruding. An incision was made in the appropriate location on the head, and the main trunk of the vagal nerve or the spino-occipital nerve was exposed. The left vagal nerve of 5 animals was cut about 3 mm distal to the brain capsule, and Accepted June 5,1991. Address reprint requests to Dr. Reiji Kishida, Dept. of Anatomy, Yamaguchi University School of Medicine, Kogushi 1144, Ube-shi, 755 Japan.

HAGFISH GLOSSOPHARYNGEAL-VAGAL SYSTEM the proximal stump was sucked into a short length (about 5 mm) of plastic catheter. The proximal end of the catheter was first sealed with quick-drying glue. Then the catheter was filled with 50% horseradish peroxidase (HRP, Toyobo Go., Japan) in distilled water containing 2.5%L-a-lysophosphatidylcholine (Sigma). The distal end of the catheter was then sealed with the same glue. In the remaining 5 animals, one-half of a split plastic catheter was put under the nerve trunk, 5-10 mm distal to the brain capsule, and sponge containing HRP was put around the nerve trunk, covered with the other half of the catheter, and sealed with quickdrying glue. HRP was also applied by this method to the spino-occipitalnerve. After applying antibiotics, the wound was sutured and the fish was allowed to recover. One to 3 weeks after the operation, the fish were reanesthetized and perfused, and each brain with its enveloping structures was removed, Frontal frozen serial sectionswere cut at 40 km and mounted alternately on two series of slide glasses. The sections were processed for HRP with a modified DAB method (Adams, ’81),and alternate sections were counterstained with cresyl violet. The fish for normal preparations were each perfused through the heart after anesthesia, first with 200 cc of physiological saline containing 1,000 I.U. heparin and then with 10% formalin. After removal as above, the brain structures were embedded in celloidin, and frontal serial sections were cut at 40 pm. These were stained with cresyl violet and hematoxylin-eosin.

521 dendrites of neurons in the vagal motor nucleus were also well labeled (see Fig. 4).In contrast, when the thin fibers were well stained, sensory fibers and terminals in the vagal lobe were well stained (see Fig. 3). Inside the brain capsule, nIX-Xjoined the saccular nerve, but without merging. This was clear from the fact that the saccular nerve remained unlabeled after HRP application to the vagal trunk (Fig. 1D-F). A few labeled ganglion cells (35-50 pm in diameter) were scattered (Fig. 1E) among the labeled fibers. At the entrance of the brain, nIX-X touched the saccular ganglion (Fig. 1D,E). In the normal preparations there were two types of cells, large (35-50 pm) and small (15-20 pm), in this ganglion. In labeled preparations, the large cells were labeled but the small cells were not.

Afferent fibers in the brain

Anterogradely labeled fibers from nIX-X passed through the lateral part of the vagal motor nucleus and reached the vagal lobe, which is situated at the dorsomedial portion of the vagal motor nucleus (Fig. 1E). Some fibers terminated in the vagal lobe. Labeled fibers and terminals in the vagal lobe were thin and contained many varicosities (see Figs. 3B,D). Several labeled ganglion cells found in the vagal lobe were of the same size (35-50 pm) as those which existed in the bundle of the vagal nerve (Fig. 1D). Some of the labeled fibers in the vagal lobe extended to the “fasciculus communis” (Jansen, ’30), which was situated ventrolaterally to column 5 of the trigeminal sensory RESULTS nucleus (Nishizawa, ‘88), and ascended and terminated in Root of the glossopharyngeal-vagal nerve this structure (Figs. 1A-E, 2). Throughout this structure, Two types of labeled fibers were distinguished, thick we found many small Nissl-stained cell bodies along the (8-10 pm) and thin (1-2 pm), in the root of the glossopha- HRP labeled fibers (Figs. 2, 3). In the normal preparations ryngeal-vagal nerve (nIX-X). Near the point where HRP these cells were scattered from the vagal lobe to the was applied, thick fibers were situated in the lateral portion “fasciculus communis.” Ascending fibers of this structure and thin fibers were situated in the medial portion. Inside (Fig. 1B,C) turned medially at the entrance of the trigemithe brain capsule, thick fibers shifted to the ventral area nal sensory and motor nerves and ran ventrally to the area and thin fibers to the dorsal area (Fig. 1F). In some cases acousticolateralis (Figs. lA, 2B). Laterally they overlapped the thick fibers were stained more strongly than the thin the ventral portion of column 5 (Fig. 1A). The fasciculus fibers, and in other cases the reverse was true. When the communis descended in the medial margin of the medullary thick fibers were strongly stained, cell bodies, axons, and horn and reached a position dorsal to the ventricle at the

Abbreviations A

AAL

AD AN

cc

Ce ci D DMX DV fc gs gu

Mes Met

MO mV mVII

mx

mXC ns nsom nsos nvm

nucleus A of Kusunoki (’82) area acousticolateralis aqueduct ambiguus nucleus central canal cerebellum commissura infima diencephalon dorsal motor nucleus of vagus descendingtrigeminal sensory nucleus fasciculus communis saccular ganglion utricular ganglion mesencephalon metencephalon medulla oblongata trigeminal motor nucleus facial motor nucleus vagal motor nucleus caudal vagal motor nucleus saccular nerve motor root of spino-occipitalnerve sensory root of spino-occipitalnerve trigeminal motor nerve

nvs nvii nviii nix-x OT PrV

sc

SM SN

so ss sv sx

TSN V

VM

vs v1

V2 v3 v-111 V-IV 1-5

trigeminal sensory nerve facial nerve acoustic nerve glossophaqngeal-vagal nerve optic tectum principal nucleus of trigeminal sensory nucleus spinal cord somatic motor solitary nucleus spino-occipitalnucleus somatic sensory trigeminal sensory nucleus vagal lobe of Jansen trigeminal sensory nucleus ventricle visceral motor visceral sensory ophthalmic division of SV maxillary division of sV mandibular division of sV third ventricle forth ventricle nuclear columns 1-5of the sV of Nishizawa et al. (’88)

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OT

C Fig. 1. Schematic representation of the distribution patterns of HRP labeled glossopharyngeal-vagal cells and fibers. Sections at six levels are shown. A. Optic tectum. B. Trigeminal motor nucleus. C. Facial motor nucleus. Asterisk shows axons of vagal motor neurons which reverse course in a hairpin turn. D. Vagal motor nucleus and vagal lobe. E. Caudal vagal motor nucleus. F. Caudal end of medulla

oblongata. Dashes show labeled fibers of the vagal nerve, and dots, labeled terminals. Triangles indicate labeled cell bodies of the vagal motor nerve, and circles, ganglion cells of the vagal sensory nerve. Note that the labeled terminals are observed not only in sX and fc but also in column 5 of the sV of Nishizawa et al. ('88). Scale bar = 500 pm.

level of the caudal end of the optic tectum. Here, labeled fibers crossed the midline of the brain and continued to the contralateral side (Fig. 1B). Throughout their course, adjacent to column 5, many of the labeled fibers invaded the column, where they terminated, mainly in the ventrolateral part (Fig. 1A-D).

of the vagus of Addens ('33) (Fig. 1E). Labeled cells were large and multipolar with well-developeddendrites (Figs. 3, 4). Many labeled dendrites extended outside the vagal motor nucleus dorsally, medially, and ventrally; laterally, only a few dendrites extended outside the nucleus. Some of these dendrites had many varicosities. Dorsally, some of the dendrites entered the vagal lobe (Figs. lD,E, 3A-D, 4). Dorsomedially, some of them reached the deep portion in the reticular formation (Figs. 1D,E, 4). Ventromedially, there was a dense plexus of the dendrites just beneath the

Vagal motor neurons Most cells in the vagal motor nucleus were retrogradely labeled, but none were labeled in the caudal motor nucleus

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a m

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Fig. 3. HRP-labeled vagal lobe and the vagal motor nucleus. Cross sections. A. Rostral part of sX and mX. ~ 4 0B. . Magnified view of A. X 125. C. caudal part of sX and mX. ~ 4 0D. . Magnified view of C. x 125. Arrows in B and D indicate varicosities. x 125.

ventral surface of the brain (Figs. 1D,E, 4). Some of the labeled dendrites which extended ventromedially passed through the ventral half of the caudal motor nucleus of the vagus of Addens ('33)where some dendritic branches with varicosities seemed to contact cell bodies directly (Fig. 5).

There were also a few dendrites in the dorsal half of the caudal nucleus (Fig. 4). Some of the axons of labeled neurons first ran mediodorsally and then reversed course in a hairpin turn before exiting from the brainstem (Fig. 1C-E).

HAGFISH GLOSSOPHARYNGEAL-VAGAL SYSTEM

Fig. 4. Cell bodies and well-developed dendrites of HRP-labeled neurons in the vagal motor nucleus. Cross section. x 150.

Fig. 5. A. HRP-labeled neurons in the vagal motor nucleus and Nissl stained neurons in the caudal . Magnified view of A. Labeled dendrites o f the vagal motor nucleus. Cresyl violet counterstaining. ~ 4 0B. vagal motor neurons seem to contact neurons of the caudal vagal motor nucleus (arrow). ~ 3 0 0 .

525

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Spino-occipital nucleus When HRP was applied to the vagal trunk, no neurons in the spino-occipital nucleus were labeled, but when it was applied to the spino-occipitalnerves, neurons in the spinooccipital nucleus were retrogradely labeled (Fig. 6). Caudally, this nucleus led to the anterior horn of the spinal cord. These neurons were very large and multipolar with well-developed dendrites. Labeled dendrites extended in all directions and were smooth without varicosities. Some dendrites extended medially to near the midline of the medulla. Axons of these neurons ran laterally and exited from the brainstem rostrally to the first spinal nerve. Anterogradely labeled spino-occipital fibers entered the brain from the ventral margin of the trigeminal sensory nucleus at the same level as the motor roots of this nerve. Then they ran medially along the margin of the nucleus to the medial margin of column 4 and ascended to a point rostral to the spino-occipitalmotor nucleus. These results are summarized in Figure 7.

DISCUSSION There is controversy about the presence (Worthington, ’05; Ayers, ’11; Holmgren, ’19; Jansen, ’30;Addens, ’33) or absence (Johnston, ’08; Rothing and Kappers, ’14; Black, ’17) of the glossopharyngeal nerve (the IX nerve) in the hagfish. Authors that argue for the presence of the IX nerve hold that it has fused with the vagal nerve (the X nerve). Jansen (’30)described two separate rootlets and a few cells rostral to the X nerve, which he took to represent a tiny IX nerve, but we could identify neither the rootlets nor the cells in our preparations. The gills of the hagfish are situated more caudally than those of other fishes, and the fused IX-X nerve forms a single trunk for a long distance. Because we applied HRP to the rostral part of this trunk, our results did not add any new evidence for the solution of this problem.

Sensory system In our preparations we found anterogradely labeled sensory neurons in the IX-X nerve trunk, in the IX-X nerve root within the medulla, and in the saccular ganglion of the VIII nerve. Peters (’63) reported that many sensory neurons of the IX-X nerve were scattered throughout its length but did not mention any sensory neurons in the medulla nor in the saccular ganglion. Our labeled neurons in the medulla and in the saccular ganglion seemed to be displaced neurons from the nerve trunk, because their round shape and size were similar to labeled neurons in the nerve trunk. In the saccular ganglion, we found large labeled vagal sensory neurons and small unlabeled vestibular neurons. Nishizawa et al. (’88)reported a similar situation in the utricular ganglion, where large trigeminal sensory neurons and small vestibular neurons were found. They suggested that these neurons were related to sensation in the oral mucous membranes. In our opinion, the large labeled vagal sensory neurons in the saccular ganglion, as well as those in the nerve trunk and in the medulla, convey sensation from the mucous membranes in the pharynx and gill pouch. In our normal preparations, many small Nissl-stained cell bodies were scattered from the vagal lobe to the fasciculus communis. From these data, we concluded that the fasciculus communis was not simply a fasciculus but a structure consisting of both nucleus and tract.

Jansen (’30) reported that the IX-X sensory nerve fibers entered the brain to form the “vagal lobe,” but that most of them did not end there, continuing on instead to the “fasciculus communis.” He did not, however, state what difference there was between the vagal lobe and the fasciculus communis. Our present work shows that the vagal lobe and the fasciculus communis are not, as supposed by Jansen (’30), separate entities, but a single, continuous structure with no morphological differentiation within its entirety. We consider this structure to be homologous with the nucleus and tractus solitarius of other vertebrates, the only significant difference being its orientation. In other words, the caudally positioned vagal lobe of the hagfish corresponds to the rostral end of the solitary complex of other vertebrates, a configuration unique in the vertebrate central nervous system. In other vertebrates, the solitary complex has a V-shape in a dorsal view, but in the hagfish, it is M-shaped (Fig. 8A,C). This difference may indicate that the brain of the hagfish is more primitive than the brains of other vertebrates. According to Nishizawa et al. (’881, the topological organization of the trigeminal sensory system of the hagfish is not inverted, although it is inverted in other vertebrates. They considered that the hagfish brain represents the most primitive features among those vertebrates. There are two facts which suggest the possibility that the brain of the hagfish is the most primitive in vertebrates. First, the hagfish is the only animal lacking a cerebellum, and second, its 4th ventricle is poorly developed. But the poorly developed 4th ventricle of this animal may not be due simply to lack of the cerebellum. In lampreys, urodeles, anurans, and snakes, the cerebellum is quite small, but their 4th ventricle has the same proportions as in other vertebrates (Ariens Kappers et al., ‘67, Larsell, ‘67, our observation). Therefore, it seems that the size of the 4th ventricle and the cerebellum developed independently. However, the lack of a cerebellum and the poorly developed state of the 4th ventricle may be due t o degeneration instead of being primitive characters. In its most primitive condition, the solitary complex probably had the configuration of an inverted “U” (Fig. 8B). In the hagfish, this has become distorted into the form of an “M” by the hyperdevelopment of the trigeminal nucleus pushing upward on the arms of the inverted “U”. In the course of evolution in other vertebrates, the root of the Xth nerve seems to have retained its primitive location, whereas the cerebellum and/or the 4th ventricle developed. The effect of this development was to displace caudally the central point of the “M”-shape solitary complex, resulting in the “V” shape that we see today in most other vertebrates. Most teleosts have a well-developedcerebellum, but the solitary complex of some teleosts does not have either a “V” shape or an “M” shape (Morita et al., ’85a,b).The reason, we think, is that their extremely developed vagal lobe gives a special shape to the solitary complex. In other vertebrates (mammals, Altschuler et al., ’89; birds, Dubbeldam et al., ’79; reptiles, Barbas-Henry and Lohman, ’84; amphibians, Matesz and Szekely, ’78; teleosts, Morita et al., ’85a,b; elasmobranchs, Barry, ’871, the rostral end of the solitary complex receives fibers from the upper alimentary tract. In the hagfish, the upper alimentary tract, including the inner branchial duct, branchial sac, pharyngo-cutaneous duct, and oesophageo-cutaneousduct,

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Fig. 7. Schematic representation of the distribution patterns of HRP labeled spino-occipital nerve fibers and cells. A. Rostralmost distribution of the fibers. B. Level where the nucleus is largest. C. Level where the motor fibers enter the brain. D. Level where the sensory fibers enter the brain. Scale bar = 500 km.

@ VM

SM

Fig. 8. Sketch of the afferent and efferent projections of the vagal nerve. A. Dosal view in the hagfish. B. Dorsal view of a generalized embryonic brainstem. C. Dorsal view in other vertebrates. D. Cross section in the hagfish. E. Cross section of a generalized embryonic

E

medulla oblongata. F. Cross section in other vertebrates. Dots indicate terminals of the vagal sensory nerve. Asterisks show the obex (C) or its equivalent level (A, B).

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is much larger than in other vertebrates except some teleosts. Therefore, visceral sensory input from the upper alimentary tract via the IX-X nerve would be greater than in other vertebrates except some teleosts, and this could explain the enlarged lobe at the beginning of solitary complex in the hagfish. Our HRP study showed that the IX-X sensory fibers terminated not only in the solitary complex but also in the trigeminal sensory nucleus. IX and X nerve fibers terminating in the trigeminal nucleus have also been found in other vertebrates (mammals, Torvik, '56; birds, Arends and Dubbeldam, '84; reptiles, Barbas-Henry and Lohman, '84; amphibians, Matesz and Szekely, '78) and they consistently terminate in the dorsal part of the trigeminal sensory nucleus, i.e., the mandibular division. In the hagfish, they terminate in the ventral part of the trigeminal nucleus (column 5), which receives the maxillo-mandibular nerve fibers in this species (Nishizawa et al., '88). In humans, the IX and X nerve fibers projecting to the trigeminal sensory nucleus convey cutaneous information from the skin of the external ear (Carpenter, '76). In other mammals and in birds, lizards, and frogs, the trigeminal projections of the IX and X nerve fibers probably also come from the region of the external ear. Although the hagfish lacks an external ear, the relation between the sensory V nucleus and the IX and X nerve fibers is the same as in other animals.

(Shapiro and Miselis, '85). In that study, they found that the dendrites of cells in the DMX extended to the solitary nucleus and suggested the possibility of "monosynaptic vagovagal interaction.'' In the hagfish, we also found that the dorsal dendrites of the mX neurons extended into the vagal lobe of the solitary complex, where they were even more highly developed than the rat DMX neurons in the work of Shapiro and Miselis ('85). This suggests two functional aspects of the hagfish. First, the motor reflex arc of the IX and the X nerve in hagfishes may be simpler than in other vertebrates. In fact, the medial and ventromedial dendritic fields are in the same location as the reticular terminal area of the direct spinal afferent fibers (Kishida et al., "9,and this indicates the possibility of monosynaptic or bisynaptic motor reflex connections with the primary afferents from the viscera and the skin of the body. Other vertebrates have one or more interneurons in such reflex arcs and the neurons in the DMX and the AN have less-developed dendrites. Second, the mX may have some mechanism for synchronization with the spinal cord motor neurons, because some of the axon-like dendrites from cells in the mX seem to have direct contacts with cells in the mXc, which project to the spinal cord (Fig. 5 ) .

Motor system

Adams, J.C. (1981) Heavy metal intensification of DAB-based reaction product. J. Histchem. Cytochem. 29775. Addens, J.L. (1933) The motor nuclei and roots of the cranial and first spinal nerves o f vertebrates. Z. Anat. Entw. Gesch. 101:307-410. Aldridge, R.J., D.E.G. Briggs, E.N.K. Clarkson, and M.P. Smith (1986) The affinities of conodonts - new evidence from the carboniferous of Edinburgh, Scotland. Lethaia. 19279-291. Altschuler, S.M., X. Bao, D. Bieger, D.A. Hopkins, and R.R. Miselis (1989) Viscerotopic representation of the upper alimentary tract in the rat: Sensory ganglia and nuclei of the solitary and spinal trigeminal tracts. J. Comp. Neurol. 283t248-268. Amemiya, F., R. Kishida, T. Kobayashi, and T. Kusunoki (1979)Anatomical studies on the brain of the hagfish 11. Distribution of the spinal projecting neurons in the hrainstem. Acta Ant. Nippon. (in Japanese) 54: 114. Amemiya, F., R. Kishida, R.C. Goris, H. Onishi, and T. Kusunoki (1985) Primary vestibular projections in the hagfish, Eptatretus burgeri. Brain Res. 337r73-79. Arends, J.J.A., and J.L. Dubbeldam (1984) The subnuclei and primary afferents of the descending trigeminal system in the mallard (Anas platyrhynchos L.). Neuroscience 13:781-795. Ariens Kappers, C.U., G.C. Huber, and E.C. Crosby (1967) The Comparative Anatomy of the Nervous System of Vertebrates, including Man. New York Hafner. Ayers, H., and J. Worthington (1911) The finer anatomy of the brain of Bdellostoma dombeyi. J. Comp. Neurol. 21:593-617. Barbas-Henry, H.A., and A.H.M. Lohman (1984) The motor nuclei and primary projections of the IXth, Xth, XIth, and XIIth cranial nerves in the monitor lizard, Varanus exanthematicus. J. Comp. Neurol. 226:565579. Barry, M.A. (1987) Central connections of the IXth and Xth cranial nerves in the clearnose skate, Raja eglanteria. Brain Res. 425:159-166. Black, D. (1917) The motor nuclei of the cerebral nerves in phylogeny; a study of the phenomena of neurobiotaxis, Part I. Cyclostomi and Pisces. J. Comp. Neurol. 27:467-564. Carpenter, M.B. (1976) Human Neuroanatomy. Baltimore: Williams and Wilkins. Dubbeldam, J.L., E.R. Brus, S.B.J. Menken, and S. Zeilstra (1979) The central projections of the glossopharyngeal and vagus ganglia in the mallard, Anasplatyrhynchos L. J. Comp. Neurol. 183:149-168. Forey, P.L. (1984) Yet more reflections on agnathan-gnathostome relationships. J. Vert. Paleont. 4~330-343. Holmgren, N. (1919) Zur Anatomie des Gehirns von Myxine. Kungl. Svensk vet. Akad. Handl. 60, pt. 7:l-96.

Addens ('33) assumed that the caudal vagal motor nucleus (mXc) was the parasympathetic nucleus of the vagal nerve and that the vagal motor nucleus (mX) was the branchiomotor nucleus. However, our work showed that the mXc had nothing to do with the vagal nerve because it remained completely unlabeled after application of HRP to the vagal nerve trunk. In fact, the mXc of Addens projects to the spinal cord, because it is the same as the densely packed, spinal-projectingneurons observed by Amemiya et al. ('79) and Ronan ('89). There was another motor nucleus, the spino-occipital, in the vicinity of the mX, but our work showed that this also had nothing to do with the vagal system. Instead it was labeled when HRP was applied to the spino-occipital nerve, which we believe to be a well-developed first spinal nerve. In conclusion, the hagfish has only one nucleus in the vagal motor system, the mX, which contains both parasympathetic and branchiomotor neurons. The number of axons that reverse in hairpin turns is less than half the number of the cell bodies in the mX. Many axons may exit directly from the brain. In other vertebrates, the axons of the neurons in the dorsal motor nucleus of the vagus (DMX) exit directly from the brain, and the axons in the ambiguus nucleus (AN) reverse in hairpin turns (Fig. 8F). In the hagfish, the neurons with hairpin-turn axons may correspond to the neurons of the AN in other vertebrates, and the neurons with direct axons may correspond to the neurons of the DMX in other vertebrates. The dendrites of the neurons of the mX in the hagfish are more highly developed than those of the DMX in other vertebrates (mammals, Kalia and Mesulam, '80; birds, Katz and Karten, '85; reptiles, Barbas-Henry and Lohman, '84; amphibians, Stuesse et al., '84; elasmobranchs, Barry, '87). Recently, the cholera toxin-horseradish peroxidase method, which labels arborizing dendrites better than other HRP methods, has been used for the vagal nerve in the rat

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Afferent and efferent projections of the glossopharyngeal-vagal nerve in the hagfish.

Anterograde and retrograde transport of horseradish peroxidase was used to examine the afferent and efferent projections of the glossopharyngeal-vagal...
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