- Email: [email protected]
Organization of the trigeminal and facial motor nuclei in the hagfish, Eptatretus burgeri: a retrograde HRP study
Brain Research, 385 (1986) 263-272
263
Elsevier BRE 12081
Organization of the Trigeminal and Facial Motor Nuclei in the Hagfish, Eptatretus burgeri: a Retrograde HRP Study REIJI KISHIDA, HIDEKI ONISH1, HIDEO NISHIZAWA, TETSUO KADOTA, RICHARD C. GORIS and TOYOKAZU KUSUNOKI
Department of Anatomy, Yokohama City University, School of Medicine, 2-33 Urafune-cho, Minami-ku, Yokohama (Japan) (Accepted 18 March 1986)
Key words: Trigeminal nerve - - Facial nerve - - Motor nucleus - - Jaw muscle - - Hagfish - Retrograde horseradish peroxidase transport
We studied the trigeminal and facial motor nuclei of the hagfish by the retrograde HRP method. We distinguished 4 components in a single column of the motor nuclei of the trigeminal nerve and the facial nerve, viz., the pars magnocellularis of the trigeminal motor nucleus (mVm), the anterior part of the pars parvocellularis of the trigeminal motor nucleus (mVpl), the posterior part of the pars parvocellularis of the trigeminal motor nucleus (mVp2) and the facial motor nucleus (mVII). Although in Nissl preparations only the mVm could be distinguished from the rest of the nucleus, the boundaries of the other 3 components were clearly demarcated in HRP preparations. Intramuscular injections into two representative antagonistic jaw muscles revealed that there was no apparent topological organization of the neurons pertaining to the opening and closing muscles in the mVm and mVpl, but both antagonistic muscles were innervated bilaterally. Although the hagfish does possess a cartilaginous jaw, the organization pattern of the motor nuclei of the jaw muscles seems to be the most primitive of all living vertebrates.
INTRODUCTION In gnathostomes (jawed vertebrates), the jaw muscles are innervated by the motor n e u r o n s of the nV and the nVII (in teleosts 16, reptiles 19'2°, birds 23, and mammalslS). Despite their classification as agnathans, hagfishes do possess a cartilaginous jaw, which differs in shape and direction of m o v e m e n t from the jaw of gnathostomes and is capable of bitings. In the brain of hagfishes, the motor nuclei of nV and n V I l are found in a single column. Attempts have been made to distinguish the respective nuclei in this column on the basis of cytoarchitecture, but with only moderate success. A t the rostral end of the column a region of large cells belonging to the m V m has been distinguished and behind it a region of smaller cells, which has been described as the combined m V - V l l p 6'11. Further, an extra group of cells lateral to the m V - V I I p t5 has been described whose affiliation with these motor nuclei is not clear.
In the present work we have used retrograde H R P transport in an effort to identify with certainty the nuclei of the two motor nerves and to compare the organization pattern of the jaw muscles innervating motor nuclei in an agnathan with the organization patterns of those in gnathostomes, as described in the literature. MATERIALS AND METHODS A total of 21 adult hagfish, Eptatretus burgeri were used, 11 for H R P application to the nerves, 6 for H R P intramuscular injection and 4 for controls. Each fish operated on was first anesthetized with 1% ethyl carbamate (Urethane) in artificial seawater. It was then wrapped in a polyvinyl sheet and buried in shaved ice, with only the head protruding. For application to the nerves, an incision was made in the skin of the head and a nerve rma-re (n = 3) of the nV the rmp (n = 4) of the nV or the n V l I , (n = 4), was exposed (Fig. 1). The nerve was cut just distal to the
Correspondence: R. Kishida, Department of Anatomy, Yokohama City University School of Medicine, 2-33 Urafune-cho, Minamiku, Yokohama, 232 Japan. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
264 rma
were injected per muscle. A f t e r 7 - 1 3 days of survival, the fish were perfused and sections m a d e as above. As controls, p a r a f f i n - e m b e d d e d brains from the remaining 4 animals were used. These were sectioned at 15tim, 3 frontally and 1 horizontally, and all were stained with Cresyl violet. The sections were used to obtain a normal picture of the trigeminal and facial m o t o r nuclei and the other cell groups a r o u n d it. RESULTS
Fig. 1. Sketch of a dorsal view of the trigeminal and facial nuclei and nerves of Eptatretus burgeri reconstructed from standard preparations. Triangles indicate the location of cell bodies seen in Nissl preparations. Dotted portions indicate sites of HRP application. Scale, 500/~m.
brain capsule and the short end protruding from the brain capsule was sucked into a short length of plastic catheter. The proximal end of the catheter was first sealed with quick-drying glue. Then the catheter was filled with 50% H R P ( T o y o b o , Japan) in distilled water containing 2.5% L-a-lysophosphatidylcholine (Sigma). The distal end of the catheter was then sealed with the same glue, the wound was sutured and the fish were allowed to recover. F i v e - 7 days after the operation the fish were perfused and the brain with its enveloping structures removed. F r o n t a l frozen serial sections were cut at 40 tim and m o u n t e d alternately on two series of slide glasses. The sections were reacted for H R P with a modified D A B m e t h o d ~ and alternate sections were counterstained with Cresyl violet. Other details of the p r o c e d u r e were as described by A m e m i y a et ai. 3. For intramuscular injection, the anesthetized fish was turned belly up and a midline incision was m a d e in the skin from the mouth to the gills, exposing the underlying musculature. H R P was injected unilaterally into either of two muscles controlling jaw movement, viz., the m. protractor dentium profundus and one of its antagonists, the m. tubulatus. T h r e e fish
In normal, Nissl-stained sections, the m V - V l l p a p p e a r e d as sketched in Fig. 1. A t the rostral end, there was the pars magnocellularis of the mVm. Under this and extending caudally was the pars parvocellularis of the m V - V l l p . Lateral to the m V - V l l p was a separate cell group, nucleus A of Kusunoki 15. W h e n H R P was applied to the nerves, the axons, cell bodies and even fine details of dendrites were stained heavily and clearly. H R P t r a n s p o r t e d retrogradely from the rmp was found only in the m o t o r nucleus of this branch, but H R P from the rma-re and
Jl ',.2..,/ Fig. 2. Sketch of a dorsal view showing the borders of the trigeminal and facial nuclei. Broken lines with numbers indicate the borders of the dendritic fields of (1) the mVm, (2) the mVpl and mVp2 and (3) the mVII. Black triangles indicate the areas which contained HRP-labeled neurons in the present work. The areas containing white triangles, in contrast, were not labeled. Scale, 500/~m.
265 from nVII was found in both m o t o r and sensory nuclei. No neurons outside the m o t o r and sensory nuclei were stained. The m o t o r and sensory nuclei could be clearly distinguished from one a n o t h e r , a s could the pathways to each from the nerve root. F o r this reason, we have chosen to ignore the sensory systems in this work, and to confine our descriptions to the m o t o r nuclei and pathways. In the animals that received intramuscular injection of H R P , only the p e r i k a r y o n of neurons in the area of distribution of the r m p (see below) was labeled and the labeling was light. No axons or dendrites were labeled.
H R P application to the ramus muscularis posterior When H R P was applied to the rmp, neurons in the
region of large cells of the V nucleus at the rostral end of the column were labeled. This is the pars magnocellularis of Jansen 11 (mVm). In the m V - V I I p immediately caudal to the m V m , a few neurons were labeled in the rostral one-third, which we have n a m e d the anterior part of the pars parvocellularis of nV ( m V p l , Fig. 2). The rostral end of the m V p l is located medially to the m V m (Figs. 2 and 3C, D). There was no labeling in the caudal two-thirds of the m V - V I I p , or in the trigeminal and utricular ganglia. Also, among the labeled neurons, there a p p e a r e d to be no sensory neurons, for none had the characteristically r o u n d e d shape of sensory cells. L a b e l e d neurons of the m V m were large and multipolar, with relatively few and short dendrites (Figs. 2 and 3). Some of the dendrites had varicosities, mostly
Fig. 3. Retrograde HRP labeling in the brainstem after application of the enzyme to the rmp of the nV. Frontal sections. A: rostral part of the mVm. ×30. Only the ventral part of the rV is labeled; the same holds for C below. B: magnified view of A. x75. C: caudal part of the mVm. x30. The mVpl appears at this point medially to the mVm. D: magnified view of C. x75. Small triangles, the small neurons of the mVpl. Arrows, axonal loops of the mVm.
266
Fig. 4. HRP labeling after simultaneous application to the rma and the re (a sensory branch). Cresyl violet counterstaining. A" section through the center of the mVm. x 30. B: magnified view of the same section. Triangles, labeled mVpl neurons, x 112.5. Asterisks, unlabeled mVm neurons.
Fig. 5. The same HRP application as in Fig. 4. A: center of the mVp2. x30. Dorsally to the mVp2, part of the sV has been labeled anterogradely. Nucleus A of Kusunoki (a) is not labeled. B - D : magnified views of the section adjacent to A. B: some of the dorsal dendrites extend to the sV. Dots indicate the border of the sV. x 87.5. C: the ventral dendrites are highly developed at the ventral surface of the brain, x 87.5. D: magnified view of the mVp2. × 75. White triangles indicate the cell sketched in Fig. 6. Black triangles in C and D indicate the ventral surface of the brain.
267 along their distal parts. The m V m borders on the sV, and some of its dendrites entered this nucleus (Fig. 3B, D). Most of the dendrites extended fanwise into the medial reticular formation. Some of the axons of this nucleus first ran mediodorsally and then reversed course in a hairpin turn before exiting from the brain. Of the axons showing this reverse of course, some were thick and some were thin (Fig. 3C, D). H R P application to the rma-re
Although H R P was applied to the rma-re as a single unit, the two fiber tracts could be distinguished clearly in the sections. Large numbers of labeled neurons were found throughout the m V - V I I p , i.e., in both the m V p l and mVp2 parts of the mVp (Fig. 2), except the caudal one-fifth (mVII), but there was no labeling in the mVm. Labeled neurons were apparent also along the ventral border of the mVm (Fig. 4), and in both the trigeminal and utricular ganglia (Fig. 5A). It could be clearly seen that the neurons in these two ganglia appertained to the sensory fibers of the re. The m V p l and mVp2 neurons were small and multipolar, with long and profusely branched dendrites. Many dendrites extended outside the complex dorsally, medially and ventrally; laterally, only a few dendrites extended outside the complex (Figs. 2 and 5A, D). Two types of dendrites could be distinguished: one type gradually grew slender as it branched repeatedly in its course from the cell body; the other type became abruptly and extremely slender, to the extent that it could almost be taken for an axon. Of these slender dendrites, again, two types could be' distinguished. One type remained inside the complex and was without varicosities. The other type extended well outside the complex and had many varicosities. Fig. 6 shows a sketch of a typical neuron from the complex. Dorsally, some of the dendrites entered the sV (Fig. 5B). Medially, some of them reached the region of the Miiller cells in the reticular formation (Fig. 7). Ventrally, there was a dense plexus of the slender dendrites just beneath the ventral surface of the brain (Fig. 5C). As was seen in the mVm, some of the axons of the m V p l first ran mediodorsally and then reversed course in a hairpin turn before exiting from the brain. The nucleus A of Kusunoki, situated laterally to the m V p l and mVp2, was not labeled either by applica-
tion to the rma or to the ramus muscularis posterior (Fig. 5A). H R P application to the n V I I
Labeled neurons were situated in the caudal fifth of the m V - V I I p , the area labeled mVII in Fig. 2; i.e., in the area which was not labeled by application of H R P to the rma. There were also a few labeled neurons in the utricular ganglion. Although in the dorsal view shown in Fig. 2 the m V I I seems to be overlapping a part of the mVp2 and in Nissl staining seems to be continuous with the mVp2, in frontal sections of H R P labeled specimens, the boundary between the two is clearly demarcated (Fig. 8). The dendritic field of the mVII was much smaller than that of the m V p l and mVp2 (Fig. 2), and the dendrites ran dorsomedially and ventromedially, mostly outside the mVp2 (Fig. 8B). H R P injection into the muscles
There was only a light scattering of H R P granules in the perikaryon of neurons (Fig. 9). Labeled perikarya were found only in the m V m and the m V p l , but bilaterally, in spite of the unilateral injection, although there was a tendency for the labeling to be slightly heavier on the injected side. The distribution of labeling was the same for both the m. protractor dentium profundus and its antagonist, the m. tubulatus, in spite of the fact that they were injected separately in different animals. In other words, there was no apparent topological organization of the neurons pertaining to either muscle. But there was a significant difference between the two muscles in the number of neurons labeled (Table I).
TABLE I Percentage of labeled neurons after i.m. injection of HRP
Best case of 3 shown for each of the two muscles. Muscle
Labeled nucleus mVm injected side
m. pro. dent. prof. 18.6% m. tubulatus 72.7%
mVpl contralateral side
injected contraside lateral side
17.0% 71.4%
13.8% 40.0%
10.1% 35.7%
268
~
D
~/_ /.
•
•
•
•
,$
A
/ ,P
. IP
Fig. 6. Sketch of a single HRP-labeled neuron from the rma. Squares indicate the border of the mVp2. Fine arrows indicate slender dendrites with varicosities. H e a v y arrow indicates one of these running toward the ventral surface. Triangles indicate slender dendrites without varicosities. Scale, 200 ~ m .
269
Fig. 7. A: frontal section through the center of the mVp2 labeled with HRP applied to the rma and counterstained with Cresyl violet. x 75. B: magnifiedview of the square in A. x 225. Dendrites of labeled neurons from the mVp2 (arrows) extend into the area occupied by Miiller cells in the reticular formation.
DISCUSSION We have clearly identified the border between the motor nuclei of nV and nVII and we also found subdivisions in the mVp, namely, the rostral mVpl and the caudal mVp2. The mVp and mVIIm have been treated together in some previous papers 6'11, because, on the basis of cytoarchitecture, only two components could be distinguished in the motor V - V I I nuclei column: the mVm and the m V - V I I p . We have distinguished 4 components: the mVm, mVpl, mVp2 and mVII. In his study on Bdellostoma and Myxine, Addens 2 proposed that there are two nVII roots, the rVIIml and rVIIm2, and that the rVIIml fuses with the nV roots in adult animals. However, judging from his Figs. 73 and 74, the rVIIml of Addens corresponds to the rma of our study, which is clearly a branch of nV on the basis of its peripheral location. Addens examined the nerves only at their intracranial roots, where the rma is indeed located slightly posterior to the rmp of nV (see Fig. 1). Addens also proposed that there are 3 nuclei in the motor V - V I I nuclei column: the nucl.Vm.lat (our mVm), the nucl.Vm.med (our mVpl), and the nucl.VIIm (our mVp2 and mVII). The fact that he was able to distinguish the mVpl on the sole basis of the normal preparations which he used in his study was a notable accomplishment. He was not, however, able to distinguish the mVp2 from the mVII,
which we have done very clearly in this HRP study. Further, Addens was of the opinion that the rma pertained only to the neurons of the mVp2, but we have shown that this nerve contains axons from the entire m V p l - m V p 2 complex. The hairpinlike reversals of direction seen in some of the axons of the mVm and the mVpl have been reported by Jansen 11 for Myxine, but they have not been reported from the trigeminal system of any other animal. In higher animals axons of the nVI1 have been reported to make a hairpin turn around the nucleus of the nVI before exiting from the brain 5, but the axons of the nV all exit directly, except in some teleosts 13,24. In the hagfish nVI is lacking, and the axons of nVII all exit directly. We have no idea what significance these hairpin turns in the hagfish trigeminal axons might have. The 'turns' observed in the nVII and nV in higher animals all seem to be detouring around some other brain structure, such as the nucleus of the nVI, but in the hagfish there is no apparent reason for the sharp reversal of direction, and there is no set location for the turning point of the individual axons (Fig. 3C, D). Our HRP study has also elucidated the precise configurations of the motor neurons of nV and nVII, because the HRP filled not only the somas but also almost all of the dendrites, as in Golgi preparations. This excellent labeling could be due to a reduced rate of metabolism in the hagfishes as compared with higher vertebrates, permitting the HRP to remain
270 unchanged in the nervous system for a longer time. H o w e v e r , even taking into account the better-thanusual labeling, our H R P - s t a i n e d m o t o r neurons seem to possess a m o r e d e v e l o p e d dendritic system than most j a w e d vertebrates in the literature that have been studied with H R P methods (teleosts 16, amphibians 9, reptiles 4'2°, birds 23, mammalslS). This suggests
Fig. 8. Sections through the border region of the mVp2 and the mVII. Cresyl violet counterstaining. ×75. A: when HRP was applied to the rma, the neurons of the mVp2 (medial side of the section) were labeled, but not the neurons of the mVII (lateral side). B: when HRP was applied to the nVII, rVII and the mVII were labeled, but not the mVp2. The dendrites of the mVII extend medially, but do not pass through the mVp2.
two functional aspects of this animal. First, the m o t o r reflex arch of nV and n V I I in hagfishes may be simpler than in higher vertebrates. In fact, the dorsal dendrites extend into the sensory nucleus of nV, and there is a strong possibility that they m a k e direct m o n o s y n a p t i c connections with the p r i m a r y afferents from the skin of the head. F u r t h e r , the medial and v e n t r o m e d i a l dendritic fields c o r r e s p o n d to the reticular terminal areas of the direct spinal afferent fibers (unpublished data) and this indicates the existence of m o n o s y n a p t i c or bisynaptic m o t o r reflex arches with the primary afferents from the skin of the body. H i g h e r vertebrates have one or m o r e interneurons in these reflex arches and the associated neurons have only a few short dendrites. Second, the m V p l and mVp2 may have some mechanism for synchronization with the spinal cord or the o t h e r brainstem areas, because some of the axon-like dendrites with varicosities from cells in the m V p l and m V p 2 seem to have direct contacts with some Mtiller cell bodies and with ascending and descending axons in the flm (Fig. 7). On the other hand, the axon-like dendrites without varicosities that remain within the nucleus may function as mediators of synchronization with other neurons in the nucleus. Both in H R P application through the r m p and in
Fig. 9. HRP labeling after injection into the m. tubulatus. Light Cresyl violet counterstaining. A: frontal section through the caudal part of the mVm. ×75. B: magnified view of the mVpl in A. ×225. C: magnified view of the mVm in A. ×225. Arrows indicate cell bodies containing HRP granules.
271 that done through intramuscular injection, only motor neurons were labeled. This indicates a probable lack of even a primitive mesencephalic trigeminal nucleus or other neurons mediating proprioception in the jaw muscles or associated structures. This is in accord with the lack of any reference to muscle spindles in the literature. In our preparations, we could distinguish clearly two types of cells: large multipolar cells with relatively few and short dendrites in the mVm, and small multipolar cells with long and profusely branched dendrites in the m V p l , mVp2. This is true of cells in the motor nucleus of nV in most jawed vertebrates (teleosts 16, amphibians 17, reptiles 19'2°, birds 23 and mammalslS). In other cyclostomes (i.e., the lampreysl°'22), the existence in this nucleus of large and small cells had not been reported until recently, when it was noted by Koyama et alJ 4. Regarding reptiles 2°, birds 23 and mammals TM, it has been speculated that the large cells are alpha motoneurons and that at least some of the small cells are gamma motoneurons innervating the jaw closing muscles. We do not believe that the small cells in our preparations are gamma cells, mainly because there are no muscle spindles in either the opening or closing muscles, there is no evidence of any other kind of proprioception involved and both the opening and closing muscles are innervated by both large and small cells, in contrast to the vertebrates cited above, which have alpha neurons only in the opening muscles. However, Jansen and Andersen 12 have reported fast muscle fibers innervated by large diameter axons and slow muscle fibers innervated by small diameter axons in another species of hagfish. We therefore propose that our large cells are neurons controlling the fast muscle fibers and the small cells neurons controlling the slow muscle fibers of the jaw muscles of Eptatretus burge-
ity of the muscles. The location of this branching can be inferred from the fact that H R P application to the rmp labels only ipsilateral neurons, indicating that the branching must take place distally to the point of application. Again referring to Table I, the number of neurons labeled in the mVm is about double those labeled in the m V p l . This is because the m V m sends all of its axons to the rmp, whereas the m V p l sends a good proportion of its axons also to the rma, as shown by the results of the H R P application to the rma, where neurons are labeled in both the m V p l and the mVp2. The proposal that individual neurons branch to innervate simultaneously several muscles is underscored well by the total amount of neurons labeled in the mVm (Table I). Although the two injected muscles, the m. protractor dentium profundus and the m. tubulatus, make up less than 50% of the total mass of the jaw muscles (there are 5 muscles altogether 8, all of which are innervated by the rmp21), ca. 90% of the neurons in the mVm were labeled by the injection. This means that at least a part of these neurons branch to innervate the other >50% of the jaw muscles not injected. A similar case of polyneuronal innervation of skeletal muscles has been reported by Brown et al. 7 in newborn rats, a presumably primitive condition which disappears as the pups grow, whereas it persists even in adult hagfish, which are the most primitive of vertebrates. In conclusion, the jaw mechanism of the hagfish is a very simple one, characterized by several muscles (two openers and 3 closers) whose individual components are incapable of independent movement, but function as a single unit under the control of bilateral neurons in both opening and closing movements.
ri.
ACKNOWLEDGEMENTS
As shown in Table I, the number of neurons labeled by unilateral intramuscular injection was nearly equal on both the injected side and the contralateral side. This indicates that each neuron on whichever side controls both ipsilateral and contralateral muscles by appropriate branching in the immediate vicin-
We wish to thank Prof. S. Kinoshita, Director of Misaki Marine Biological Station, Faculty of Science, University of Tokyo, for the courtesy of supplying the hagfish. We also thank Dr. F. Amemiya and Dr. H. Koyama for discussing our results.
272 ABBREVIATIONS
trigeminal motor nucleus posterior part of the pars parvocellularis of the trigeminal motor nucleus mV-VIIp pars parvocellularis of the trigeminal and facial motor nucleus mVII facial motor nucleus nV trigeminal nerve nVII facial nerve ot optic tectum re ramus externus of nV rma ramus muscularis anterior of nV rmp ramus muscularis posterior of nV rs ramus sensorius of nV rV root of nV rVII root of nVII sV trigeminal sensory nucleus V ventral vent reduced ventricles mVp2
A a
aal com.v D d DAB fai tim gu gV HRP M mVm mVpl
axon nucleus A of Kusunoki area acoustico-lateralis commissura ventralis dorsal diencephalon diaminobenzidine fibrae arcuatae internae fasciculus longitudinalis medialis ganglion utriculare ganglion of nV horseradish peroxidase Miiller cell pars magnocellularis of the trigeminal motor nucleus anterior part of the pars parvocellularis of the
REFERENCES 1 Adams, J.C., Heavy metal intensification of DAB-based HRP reaction product, J. Histochem. Cytochem., 29 (1981) 775. 2 Addens, J.L., The motor nuclei and roots of the cranial and first spinal nerves of vertebrates. Part 1. Introduction. Cyclostomes, Z. Anat. Entwickl.-Gesch., 101 (1933) 307-410. 3 Amemiya, F., Kishida, R., Goris, R.C., Onishi, H. and Kusunoki, T., Primary vestibular projections in the hagfish, Eptatretus burgeri, Brain Research, 337 (1985) 73-79. 4 Barbas-Henry, H.A., The motor nuclei and primary projections of the facial nerve in the monitor lizard Varanus exanthematicus, J. Comp. Neurol., 207 (1982) 105-113. 5 Barnard, J.W., A phylogenetic study of the visceral afferent areas associated with the facial, glossopharyngeal, and vagus nerves, and their fiber connections. The efferent facial nucleus, J. Comp. Neurol., 65 (1936) 503-602. 6 Black, D., 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 (1917) 467-484. 7 Brown, M.C., Jansen, J.K.S. and Van Essen, D., Polyneuronal innervation of skeletal muscle in new-born rats and its elimination during maturation, J. Physiol. (London), 261 (1976) 387-422. 8 Dawson, J.A., The oral cavity, the 'jaws' and the horny teeth of Myxine glutinosa. In A. Brodal and R. F~inge (Eds.), The Biology of Myxine, Universitetsforslaget, Oslo, 1963, pp. 231-255. 9 Fuller, P.M., Afferent and efferent components of the facial nerve in the bullfrog (Rana catesbeiana), J. Morphol., 159 (1979) 245-252. 10 Homma, S., Organization of the trigeminal motor nucleus before and after metamorphosis in lampreys, Brain Research, 140 (1978) 33-42. 11 Jansen, J,, The brain of Myxine glutinosa, J. Comp. Neurol., 49 (1930) 359-507. 12 Jansen, J.K.S. and Andersen, P., Anatomy and physiology of the skeletal muscles. In A. Brodal and R. Fringe (Eds.), The Biology of Myxine, Universitetsforslaget, Oslo, 1963, pp. 161-194.
13 Kappers, C.U.A., Huber, G.C. and Crosby, E.C., The
Comparative Anatomy of the Nervous System of Vertebrates Including Man, Hafner, New York, 1967, pp. 516-644. 14 Koyama, H., Kishida, R. and Kusunoki, T., Organization of sensory and motor nuclei of the trigeminal nerve in lampreys, Neurosci. Res., in press (abstract). 15 Kusunoki, T., Kadota, T. and Kishida, R., Chemoarchitectonics of the brain stem of the hagfish, Eptatretus burgeri, with special reference to the primordial cerebellum, J. Hirnforsch., 23 (1982) 109-119. 16 Luiten, P.G.M., A somatotopic and functional representation of the respiratory muscles in the trigeminal and facial motor nuclei of the carp (Cyprinus carpio L.), J. Comp. NeuroL, 166 (1976) 191-200. 17 Matesz, C. and Szekely, G., The motor column and sensory projections of the branchial cranial nerves in the frog, J. Comp. Neurol., 178 (1978) 157-176. 18 Mizuno, N., Konishi, A. and Sato, M., Localization of masticatory motoneurons in the cat and rat by means of retrograde axonal transport of horseradish peroxidase, J. Comp. Neurol., 164 (1975) 105-116. 19 Moody, S.A. and Meszler, R.M., Subnuclear organization of the ophidian trigeminal motor nucleus. I. Localization of neurons and synaptic bouton distribution, J. Comp. Neurol., 190 (1980) 463-486. 20 Moody, S.A. and Meszler, R.M., Subnuclear organization of the ophidian trigeminal motor nucleus. II. Ultrastructural measurements on motoneurons innervating antagonistic muscles, J. Comp. NeuroL, 190 (1980) 487-500. 21 Peters, A., The peripheral nervous system. In A. Brodal and R. Fringe (Eds.), The Biology of Myxine, Universitetsforslaget, Oslo, 1963, pp. 92-123. 22 Rovainen, C.M., Neurobiology of lampreys, Physiol. Rev., 59 (1979) 1007-1077. 23 Wild, J.M. and Zeigler, H.P., Central representation and somatotopic organization of the jaw muscles within the facial and trigeminal nuclei of the pigeon (Columba livia), J. Comp. Neurol., 192 (1980) 175-20l. 24 Woodburne, R.T., A phylogenetic consideration of the primary and secondary centers and connections of the trigemihal complex in a series of vertebrates, J. Comp. Neurol., 65 (1936) 403-501.
263
Elsevier BRE 12081
Organization of the Trigeminal and Facial Motor Nuclei in the Hagfish, Eptatretus burgeri: a Retrograde HRP Study REIJI KISHIDA, HIDEKI ONISH1, HIDEO NISHIZAWA, TETSUO KADOTA, RICHARD C. GORIS and TOYOKAZU KUSUNOKI
Department of Anatomy, Yokohama City University, School of Medicine, 2-33 Urafune-cho, Minami-ku, Yokohama (Japan) (Accepted 18 March 1986)
Key words: Trigeminal nerve - - Facial nerve - - Motor nucleus - - Jaw muscle - - Hagfish - Retrograde horseradish peroxidase transport
We studied the trigeminal and facial motor nuclei of the hagfish by the retrograde HRP method. We distinguished 4 components in a single column of the motor nuclei of the trigeminal nerve and the facial nerve, viz., the pars magnocellularis of the trigeminal motor nucleus (mVm), the anterior part of the pars parvocellularis of the trigeminal motor nucleus (mVpl), the posterior part of the pars parvocellularis of the trigeminal motor nucleus (mVp2) and the facial motor nucleus (mVII). Although in Nissl preparations only the mVm could be distinguished from the rest of the nucleus, the boundaries of the other 3 components were clearly demarcated in HRP preparations. Intramuscular injections into two representative antagonistic jaw muscles revealed that there was no apparent topological organization of the neurons pertaining to the opening and closing muscles in the mVm and mVpl, but both antagonistic muscles were innervated bilaterally. Although the hagfish does possess a cartilaginous jaw, the organization pattern of the motor nuclei of the jaw muscles seems to be the most primitive of all living vertebrates.
INTRODUCTION In gnathostomes (jawed vertebrates), the jaw muscles are innervated by the motor n e u r o n s of the nV and the nVII (in teleosts 16, reptiles 19'2°, birds 23, and mammalslS). Despite their classification as agnathans, hagfishes do possess a cartilaginous jaw, which differs in shape and direction of m o v e m e n t from the jaw of gnathostomes and is capable of bitings. In the brain of hagfishes, the motor nuclei of nV and n V I l are found in a single column. Attempts have been made to distinguish the respective nuclei in this column on the basis of cytoarchitecture, but with only moderate success. A t the rostral end of the column a region of large cells belonging to the m V m has been distinguished and behind it a region of smaller cells, which has been described as the combined m V - V l l p 6'11. Further, an extra group of cells lateral to the m V - V I I p t5 has been described whose affiliation with these motor nuclei is not clear.
In the present work we have used retrograde H R P transport in an effort to identify with certainty the nuclei of the two motor nerves and to compare the organization pattern of the jaw muscles innervating motor nuclei in an agnathan with the organization patterns of those in gnathostomes, as described in the literature. MATERIALS AND METHODS A total of 21 adult hagfish, Eptatretus burgeri were used, 11 for H R P application to the nerves, 6 for H R P intramuscular injection and 4 for controls. Each fish operated on was first anesthetized with 1% ethyl carbamate (Urethane) in artificial seawater. It was then wrapped in a polyvinyl sheet and buried in shaved ice, with only the head protruding. For application to the nerves, an incision was made in the skin of the head and a nerve rma-re (n = 3) of the nV the rmp (n = 4) of the nV or the n V l I , (n = 4), was exposed (Fig. 1). The nerve was cut just distal to the
Correspondence: R. Kishida, Department of Anatomy, Yokohama City University School of Medicine, 2-33 Urafune-cho, Minamiku, Yokohama, 232 Japan. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
264 rma
were injected per muscle. A f t e r 7 - 1 3 days of survival, the fish were perfused and sections m a d e as above. As controls, p a r a f f i n - e m b e d d e d brains from the remaining 4 animals were used. These were sectioned at 15tim, 3 frontally and 1 horizontally, and all were stained with Cresyl violet. The sections were used to obtain a normal picture of the trigeminal and facial m o t o r nuclei and the other cell groups a r o u n d it. RESULTS
Fig. 1. Sketch of a dorsal view of the trigeminal and facial nuclei and nerves of Eptatretus burgeri reconstructed from standard preparations. Triangles indicate the location of cell bodies seen in Nissl preparations. Dotted portions indicate sites of HRP application. Scale, 500/~m.
brain capsule and the short end protruding from the brain capsule was sucked into a short length of plastic catheter. The proximal end of the catheter was first sealed with quick-drying glue. Then the catheter was filled with 50% H R P ( T o y o b o , Japan) in distilled water containing 2.5% L-a-lysophosphatidylcholine (Sigma). The distal end of the catheter was then sealed with the same glue, the wound was sutured and the fish were allowed to recover. F i v e - 7 days after the operation the fish were perfused and the brain with its enveloping structures removed. F r o n t a l frozen serial sections were cut at 40 tim and m o u n t e d alternately on two series of slide glasses. The sections were reacted for H R P with a modified D A B m e t h o d ~ and alternate sections were counterstained with Cresyl violet. Other details of the p r o c e d u r e were as described by A m e m i y a et ai. 3. For intramuscular injection, the anesthetized fish was turned belly up and a midline incision was m a d e in the skin from the mouth to the gills, exposing the underlying musculature. H R P was injected unilaterally into either of two muscles controlling jaw movement, viz., the m. protractor dentium profundus and one of its antagonists, the m. tubulatus. T h r e e fish
In normal, Nissl-stained sections, the m V - V l l p a p p e a r e d as sketched in Fig. 1. A t the rostral end, there was the pars magnocellularis of the mVm. Under this and extending caudally was the pars parvocellularis of the m V - V l l p . Lateral to the m V - V l l p was a separate cell group, nucleus A of Kusunoki 15. W h e n H R P was applied to the nerves, the axons, cell bodies and even fine details of dendrites were stained heavily and clearly. H R P t r a n s p o r t e d retrogradely from the rmp was found only in the m o t o r nucleus of this branch, but H R P from the rma-re and
Jl ',.2..,/ Fig. 2. Sketch of a dorsal view showing the borders of the trigeminal and facial nuclei. Broken lines with numbers indicate the borders of the dendritic fields of (1) the mVm, (2) the mVpl and mVp2 and (3) the mVII. Black triangles indicate the areas which contained HRP-labeled neurons in the present work. The areas containing white triangles, in contrast, were not labeled. Scale, 500/~m.
265 from nVII was found in both m o t o r and sensory nuclei. No neurons outside the m o t o r and sensory nuclei were stained. The m o t o r and sensory nuclei could be clearly distinguished from one a n o t h e r , a s could the pathways to each from the nerve root. F o r this reason, we have chosen to ignore the sensory systems in this work, and to confine our descriptions to the m o t o r nuclei and pathways. In the animals that received intramuscular injection of H R P , only the p e r i k a r y o n of neurons in the area of distribution of the r m p (see below) was labeled and the labeling was light. No axons or dendrites were labeled.
H R P application to the ramus muscularis posterior When H R P was applied to the rmp, neurons in the
region of large cells of the V nucleus at the rostral end of the column were labeled. This is the pars magnocellularis of Jansen 11 (mVm). In the m V - V I I p immediately caudal to the m V m , a few neurons were labeled in the rostral one-third, which we have n a m e d the anterior part of the pars parvocellularis of nV ( m V p l , Fig. 2). The rostral end of the m V p l is located medially to the m V m (Figs. 2 and 3C, D). There was no labeling in the caudal two-thirds of the m V - V I I p , or in the trigeminal and utricular ganglia. Also, among the labeled neurons, there a p p e a r e d to be no sensory neurons, for none had the characteristically r o u n d e d shape of sensory cells. L a b e l e d neurons of the m V m were large and multipolar, with relatively few and short dendrites (Figs. 2 and 3). Some of the dendrites had varicosities, mostly
Fig. 3. Retrograde HRP labeling in the brainstem after application of the enzyme to the rmp of the nV. Frontal sections. A: rostral part of the mVm. ×30. Only the ventral part of the rV is labeled; the same holds for C below. B: magnified view of A. x75. C: caudal part of the mVm. x30. The mVpl appears at this point medially to the mVm. D: magnified view of C. x75. Small triangles, the small neurons of the mVpl. Arrows, axonal loops of the mVm.
266
Fig. 4. HRP labeling after simultaneous application to the rma and the re (a sensory branch). Cresyl violet counterstaining. A" section through the center of the mVm. x 30. B: magnified view of the same section. Triangles, labeled mVpl neurons, x 112.5. Asterisks, unlabeled mVm neurons.
Fig. 5. The same HRP application as in Fig. 4. A: center of the mVp2. x30. Dorsally to the mVp2, part of the sV has been labeled anterogradely. Nucleus A of Kusunoki (a) is not labeled. B - D : magnified views of the section adjacent to A. B: some of the dorsal dendrites extend to the sV. Dots indicate the border of the sV. x 87.5. C: the ventral dendrites are highly developed at the ventral surface of the brain, x 87.5. D: magnified view of the mVp2. × 75. White triangles indicate the cell sketched in Fig. 6. Black triangles in C and D indicate the ventral surface of the brain.
267 along their distal parts. The m V m borders on the sV, and some of its dendrites entered this nucleus (Fig. 3B, D). Most of the dendrites extended fanwise into the medial reticular formation. Some of the axons of this nucleus first ran mediodorsally and then reversed course in a hairpin turn before exiting from the brain. Of the axons showing this reverse of course, some were thick and some were thin (Fig. 3C, D). H R P application to the rma-re
Although H R P was applied to the rma-re as a single unit, the two fiber tracts could be distinguished clearly in the sections. Large numbers of labeled neurons were found throughout the m V - V I I p , i.e., in both the m V p l and mVp2 parts of the mVp (Fig. 2), except the caudal one-fifth (mVII), but there was no labeling in the mVm. Labeled neurons were apparent also along the ventral border of the mVm (Fig. 4), and in both the trigeminal and utricular ganglia (Fig. 5A). It could be clearly seen that the neurons in these two ganglia appertained to the sensory fibers of the re. The m V p l and mVp2 neurons were small and multipolar, with long and profusely branched dendrites. Many dendrites extended outside the complex dorsally, medially and ventrally; laterally, only a few dendrites extended outside the complex (Figs. 2 and 5A, D). Two types of dendrites could be distinguished: one type gradually grew slender as it branched repeatedly in its course from the cell body; the other type became abruptly and extremely slender, to the extent that it could almost be taken for an axon. Of these slender dendrites, again, two types could be' distinguished. One type remained inside the complex and was without varicosities. The other type extended well outside the complex and had many varicosities. Fig. 6 shows a sketch of a typical neuron from the complex. Dorsally, some of the dendrites entered the sV (Fig. 5B). Medially, some of them reached the region of the Miiller cells in the reticular formation (Fig. 7). Ventrally, there was a dense plexus of the slender dendrites just beneath the ventral surface of the brain (Fig. 5C). As was seen in the mVm, some of the axons of the m V p l first ran mediodorsally and then reversed course in a hairpin turn before exiting from the brain. The nucleus A of Kusunoki, situated laterally to the m V p l and mVp2, was not labeled either by applica-
tion to the rma or to the ramus muscularis posterior (Fig. 5A). H R P application to the n V I I
Labeled neurons were situated in the caudal fifth of the m V - V I I p , the area labeled mVII in Fig. 2; i.e., in the area which was not labeled by application of H R P to the rma. There were also a few labeled neurons in the utricular ganglion. Although in the dorsal view shown in Fig. 2 the m V I I seems to be overlapping a part of the mVp2 and in Nissl staining seems to be continuous with the mVp2, in frontal sections of H R P labeled specimens, the boundary between the two is clearly demarcated (Fig. 8). The dendritic field of the mVII was much smaller than that of the m V p l and mVp2 (Fig. 2), and the dendrites ran dorsomedially and ventromedially, mostly outside the mVp2 (Fig. 8B). H R P injection into the muscles
There was only a light scattering of H R P granules in the perikaryon of neurons (Fig. 9). Labeled perikarya were found only in the m V m and the m V p l , but bilaterally, in spite of the unilateral injection, although there was a tendency for the labeling to be slightly heavier on the injected side. The distribution of labeling was the same for both the m. protractor dentium profundus and its antagonist, the m. tubulatus, in spite of the fact that they were injected separately in different animals. In other words, there was no apparent topological organization of the neurons pertaining to either muscle. But there was a significant difference between the two muscles in the number of neurons labeled (Table I).
TABLE I Percentage of labeled neurons after i.m. injection of HRP
Best case of 3 shown for each of the two muscles. Muscle
Labeled nucleus mVm injected side
m. pro. dent. prof. 18.6% m. tubulatus 72.7%
mVpl contralateral side
injected contraside lateral side
17.0% 71.4%
13.8% 40.0%
10.1% 35.7%
268
~
D
~/_ /.
•
•
•
•
,$
A
/ ,P
. IP
Fig. 6. Sketch of a single HRP-labeled neuron from the rma. Squares indicate the border of the mVp2. Fine arrows indicate slender dendrites with varicosities. H e a v y arrow indicates one of these running toward the ventral surface. Triangles indicate slender dendrites without varicosities. Scale, 200 ~ m .
269
Fig. 7. A: frontal section through the center of the mVp2 labeled with HRP applied to the rma and counterstained with Cresyl violet. x 75. B: magnifiedview of the square in A. x 225. Dendrites of labeled neurons from the mVp2 (arrows) extend into the area occupied by Miiller cells in the reticular formation.
DISCUSSION We have clearly identified the border between the motor nuclei of nV and nVII and we also found subdivisions in the mVp, namely, the rostral mVpl and the caudal mVp2. The mVp and mVIIm have been treated together in some previous papers 6'11, because, on the basis of cytoarchitecture, only two components could be distinguished in the motor V - V I I nuclei column: the mVm and the m V - V I I p . We have distinguished 4 components: the mVm, mVpl, mVp2 and mVII. In his study on Bdellostoma and Myxine, Addens 2 proposed that there are two nVII roots, the rVIIml and rVIIm2, and that the rVIIml fuses with the nV roots in adult animals. However, judging from his Figs. 73 and 74, the rVIIml of Addens corresponds to the rma of our study, which is clearly a branch of nV on the basis of its peripheral location. Addens examined the nerves only at their intracranial roots, where the rma is indeed located slightly posterior to the rmp of nV (see Fig. 1). Addens also proposed that there are 3 nuclei in the motor V - V I I nuclei column: the nucl.Vm.lat (our mVm), the nucl.Vm.med (our mVpl), and the nucl.VIIm (our mVp2 and mVII). The fact that he was able to distinguish the mVpl on the sole basis of the normal preparations which he used in his study was a notable accomplishment. He was not, however, able to distinguish the mVp2 from the mVII,
which we have done very clearly in this HRP study. Further, Addens was of the opinion that the rma pertained only to the neurons of the mVp2, but we have shown that this nerve contains axons from the entire m V p l - m V p 2 complex. The hairpinlike reversals of direction seen in some of the axons of the mVm and the mVpl have been reported by Jansen 11 for Myxine, but they have not been reported from the trigeminal system of any other animal. In higher animals axons of the nVI1 have been reported to make a hairpin turn around the nucleus of the nVI before exiting from the brain 5, but the axons of the nV all exit directly, except in some teleosts 13,24. In the hagfish nVI is lacking, and the axons of nVII all exit directly. We have no idea what significance these hairpin turns in the hagfish trigeminal axons might have. The 'turns' observed in the nVII and nV in higher animals all seem to be detouring around some other brain structure, such as the nucleus of the nVI, but in the hagfish there is no apparent reason for the sharp reversal of direction, and there is no set location for the turning point of the individual axons (Fig. 3C, D). Our HRP study has also elucidated the precise configurations of the motor neurons of nV and nVII, because the HRP filled not only the somas but also almost all of the dendrites, as in Golgi preparations. This excellent labeling could be due to a reduced rate of metabolism in the hagfishes as compared with higher vertebrates, permitting the HRP to remain
270 unchanged in the nervous system for a longer time. H o w e v e r , even taking into account the better-thanusual labeling, our H R P - s t a i n e d m o t o r neurons seem to possess a m o r e d e v e l o p e d dendritic system than most j a w e d vertebrates in the literature that have been studied with H R P methods (teleosts 16, amphibians 9, reptiles 4'2°, birds 23, mammalslS). This suggests
Fig. 8. Sections through the border region of the mVp2 and the mVII. Cresyl violet counterstaining. ×75. A: when HRP was applied to the rma, the neurons of the mVp2 (medial side of the section) were labeled, but not the neurons of the mVII (lateral side). B: when HRP was applied to the nVII, rVII and the mVII were labeled, but not the mVp2. The dendrites of the mVII extend medially, but do not pass through the mVp2.
two functional aspects of this animal. First, the m o t o r reflex arch of nV and n V I I in hagfishes may be simpler than in higher vertebrates. In fact, the dorsal dendrites extend into the sensory nucleus of nV, and there is a strong possibility that they m a k e direct m o n o s y n a p t i c connections with the p r i m a r y afferents from the skin of the head. F u r t h e r , the medial and v e n t r o m e d i a l dendritic fields c o r r e s p o n d to the reticular terminal areas of the direct spinal afferent fibers (unpublished data) and this indicates the existence of m o n o s y n a p t i c or bisynaptic m o t o r reflex arches with the primary afferents from the skin of the body. H i g h e r vertebrates have one or m o r e interneurons in these reflex arches and the associated neurons have only a few short dendrites. Second, the m V p l and mVp2 may have some mechanism for synchronization with the spinal cord or the o t h e r brainstem areas, because some of the axon-like dendrites with varicosities from cells in the m V p l and m V p 2 seem to have direct contacts with some Mtiller cell bodies and with ascending and descending axons in the flm (Fig. 7). On the other hand, the axon-like dendrites without varicosities that remain within the nucleus may function as mediators of synchronization with other neurons in the nucleus. Both in H R P application through the r m p and in
Fig. 9. HRP labeling after injection into the m. tubulatus. Light Cresyl violet counterstaining. A: frontal section through the caudal part of the mVm. ×75. B: magnified view of the mVpl in A. ×225. C: magnified view of the mVm in A. ×225. Arrows indicate cell bodies containing HRP granules.
271 that done through intramuscular injection, only motor neurons were labeled. This indicates a probable lack of even a primitive mesencephalic trigeminal nucleus or other neurons mediating proprioception in the jaw muscles or associated structures. This is in accord with the lack of any reference to muscle spindles in the literature. In our preparations, we could distinguish clearly two types of cells: large multipolar cells with relatively few and short dendrites in the mVm, and small multipolar cells with long and profusely branched dendrites in the m V p l , mVp2. This is true of cells in the motor nucleus of nV in most jawed vertebrates (teleosts 16, amphibians 17, reptiles 19'2°, birds 23 and mammalslS). In other cyclostomes (i.e., the lampreysl°'22), the existence in this nucleus of large and small cells had not been reported until recently, when it was noted by Koyama et alJ 4. Regarding reptiles 2°, birds 23 and mammals TM, it has been speculated that the large cells are alpha motoneurons and that at least some of the small cells are gamma motoneurons innervating the jaw closing muscles. We do not believe that the small cells in our preparations are gamma cells, mainly because there are no muscle spindles in either the opening or closing muscles, there is no evidence of any other kind of proprioception involved and both the opening and closing muscles are innervated by both large and small cells, in contrast to the vertebrates cited above, which have alpha neurons only in the opening muscles. However, Jansen and Andersen 12 have reported fast muscle fibers innervated by large diameter axons and slow muscle fibers innervated by small diameter axons in another species of hagfish. We therefore propose that our large cells are neurons controlling the fast muscle fibers and the small cells neurons controlling the slow muscle fibers of the jaw muscles of Eptatretus burge-
ity of the muscles. The location of this branching can be inferred from the fact that H R P application to the rmp labels only ipsilateral neurons, indicating that the branching must take place distally to the point of application. Again referring to Table I, the number of neurons labeled in the mVm is about double those labeled in the m V p l . This is because the m V m sends all of its axons to the rmp, whereas the m V p l sends a good proportion of its axons also to the rma, as shown by the results of the H R P application to the rma, where neurons are labeled in both the m V p l and the mVp2. The proposal that individual neurons branch to innervate simultaneously several muscles is underscored well by the total amount of neurons labeled in the mVm (Table I). Although the two injected muscles, the m. protractor dentium profundus and the m. tubulatus, make up less than 50% of the total mass of the jaw muscles (there are 5 muscles altogether 8, all of which are innervated by the rmp21), ca. 90% of the neurons in the mVm were labeled by the injection. This means that at least a part of these neurons branch to innervate the other >50% of the jaw muscles not injected. A similar case of polyneuronal innervation of skeletal muscles has been reported by Brown et al. 7 in newborn rats, a presumably primitive condition which disappears as the pups grow, whereas it persists even in adult hagfish, which are the most primitive of vertebrates. In conclusion, the jaw mechanism of the hagfish is a very simple one, characterized by several muscles (two openers and 3 closers) whose individual components are incapable of independent movement, but function as a single unit under the control of bilateral neurons in both opening and closing movements.
ri.
ACKNOWLEDGEMENTS
As shown in Table I, the number of neurons labeled by unilateral intramuscular injection was nearly equal on both the injected side and the contralateral side. This indicates that each neuron on whichever side controls both ipsilateral and contralateral muscles by appropriate branching in the immediate vicin-
We wish to thank Prof. S. Kinoshita, Director of Misaki Marine Biological Station, Faculty of Science, University of Tokyo, for the courtesy of supplying the hagfish. We also thank Dr. F. Amemiya and Dr. H. Koyama for discussing our results.
272 ABBREVIATIONS
trigeminal motor nucleus posterior part of the pars parvocellularis of the trigeminal motor nucleus mV-VIIp pars parvocellularis of the trigeminal and facial motor nucleus mVII facial motor nucleus nV trigeminal nerve nVII facial nerve ot optic tectum re ramus externus of nV rma ramus muscularis anterior of nV rmp ramus muscularis posterior of nV rs ramus sensorius of nV rV root of nV rVII root of nVII sV trigeminal sensory nucleus V ventral vent reduced ventricles mVp2
A a
aal com.v D d DAB fai tim gu gV HRP M mVm mVpl
axon nucleus A of Kusunoki area acoustico-lateralis commissura ventralis dorsal diencephalon diaminobenzidine fibrae arcuatae internae fasciculus longitudinalis medialis ganglion utriculare ganglion of nV horseradish peroxidase Miiller cell pars magnocellularis of the trigeminal motor nucleus anterior part of the pars parvocellularis of the
REFERENCES 1 Adams, J.C., Heavy metal intensification of DAB-based HRP reaction product, J. Histochem. Cytochem., 29 (1981) 775. 2 Addens, J.L., The motor nuclei and roots of the cranial and first spinal nerves of vertebrates. Part 1. Introduction. Cyclostomes, Z. Anat. Entwickl.-Gesch., 101 (1933) 307-410. 3 Amemiya, F., Kishida, R., Goris, R.C., Onishi, H. and Kusunoki, T., Primary vestibular projections in the hagfish, Eptatretus burgeri, Brain Research, 337 (1985) 73-79. 4 Barbas-Henry, H.A., The motor nuclei and primary projections of the facial nerve in the monitor lizard Varanus exanthematicus, J. Comp. Neurol., 207 (1982) 105-113. 5 Barnard, J.W., A phylogenetic study of the visceral afferent areas associated with the facial, glossopharyngeal, and vagus nerves, and their fiber connections. The efferent facial nucleus, J. Comp. Neurol., 65 (1936) 503-602. 6 Black, D., 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 (1917) 467-484. 7 Brown, M.C., Jansen, J.K.S. and Van Essen, D., Polyneuronal innervation of skeletal muscle in new-born rats and its elimination during maturation, J. Physiol. (London), 261 (1976) 387-422. 8 Dawson, J.A., The oral cavity, the 'jaws' and the horny teeth of Myxine glutinosa. In A. Brodal and R. F~inge (Eds.), The Biology of Myxine, Universitetsforslaget, Oslo, 1963, pp. 231-255. 9 Fuller, P.M., Afferent and efferent components of the facial nerve in the bullfrog (Rana catesbeiana), J. Morphol., 159 (1979) 245-252. 10 Homma, S., Organization of the trigeminal motor nucleus before and after metamorphosis in lampreys, Brain Research, 140 (1978) 33-42. 11 Jansen, J,, The brain of Myxine glutinosa, J. Comp. Neurol., 49 (1930) 359-507. 12 Jansen, J.K.S. and Andersen, P., Anatomy and physiology of the skeletal muscles. In A. Brodal and R. Fringe (Eds.), The Biology of Myxine, Universitetsforslaget, Oslo, 1963, pp. 161-194.
13 Kappers, C.U.A., Huber, G.C. and Crosby, E.C., The
Comparative Anatomy of the Nervous System of Vertebrates Including Man, Hafner, New York, 1967, pp. 516-644. 14 Koyama, H., Kishida, R. and Kusunoki, T., Organization of sensory and motor nuclei of the trigeminal nerve in lampreys, Neurosci. Res., in press (abstract). 15 Kusunoki, T., Kadota, T. and Kishida, R., Chemoarchitectonics of the brain stem of the hagfish, Eptatretus burgeri, with special reference to the primordial cerebellum, J. Hirnforsch., 23 (1982) 109-119. 16 Luiten, P.G.M., A somatotopic and functional representation of the respiratory muscles in the trigeminal and facial motor nuclei of the carp (Cyprinus carpio L.), J. Comp. NeuroL, 166 (1976) 191-200. 17 Matesz, C. and Szekely, G., The motor column and sensory projections of the branchial cranial nerves in the frog, J. Comp. Neurol., 178 (1978) 157-176. 18 Mizuno, N., Konishi, A. and Sato, M., Localization of masticatory motoneurons in the cat and rat by means of retrograde axonal transport of horseradish peroxidase, J. Comp. Neurol., 164 (1975) 105-116. 19 Moody, S.A. and Meszler, R.M., Subnuclear organization of the ophidian trigeminal motor nucleus. I. Localization of neurons and synaptic bouton distribution, J. Comp. Neurol., 190 (1980) 463-486. 20 Moody, S.A. and Meszler, R.M., Subnuclear organization of the ophidian trigeminal motor nucleus. II. Ultrastructural measurements on motoneurons innervating antagonistic muscles, J. Comp. NeuroL, 190 (1980) 487-500. 21 Peters, A., The peripheral nervous system. In A. Brodal and R. Fringe (Eds.), The Biology of Myxine, Universitetsforslaget, Oslo, 1963, pp. 92-123. 22 Rovainen, C.M., Neurobiology of lampreys, Physiol. Rev., 59 (1979) 1007-1077. 23 Wild, J.M. and Zeigler, H.P., Central representation and somatotopic organization of the jaw muscles within the facial and trigeminal nuclei of the pigeon (Columba livia), J. Comp. Neurol., 192 (1980) 175-20l. 24 Woodburne, R.T., A phylogenetic consideration of the primary and secondary centers and connections of the trigemihal complex in a series of vertebrates, J. Comp. Neurol., 65 (1936) 403-501.