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Primary vestibular projections in the Hagfish,Eptatretus burgeri
Brain Research, 337 (1985) 73-79
73
Elsevier BRE 10802
Primary Vestibular Projections in the Hagfish, Eptatretus burgeri FUMIAKI AMEMIYA, REIJI KISHIDA, RICHARD C. GORIS, HIDEKI ONISHI and TOYOKAZU KUSUNOKI
Department of Anatomy, Yokohama City University, 2-33 Urafune-cho, Minami-ku, Yokohama, 232 (Japan) (Accepted September 11th, 1984)
Key words: vestibular nerve - - ganglion cells - - acoustico-lateral area - - brainstem - - hagfish - transganglionic horseradish peroxidase (HRP) transport
The VIIIth cranial nerve projections in the hagfish, which has only one circular canal in the ear, were studied by transganglionic HRP transport. This nerve has two branches, the nervus utricularis (N. utr.) and the nervus saccularis (N. sac.), each with its own ganglion, the ganglion utriculare (G. utr.) and the ganglion sacculare (G. sac.), respectively. Although the G. sac. has uniformly small cells, the G. utr. consists of two separate cell masses, a ventral mass of large cells and a dorsal mass of small cells. The small cells were labeled in both ganglia after horseradish peroxidase (HRP) injection into the endolymphatic space. The greater part of the terminal areas of these two branches overlapped in the ventral nucleus of the area acoustico-lateralis, but the terminals of the N. sac. extended slightly further in a caudal direction. No projections to the primordial cerebellum and no retrogradely labeled cells in the brain were found. The large cells in the ventral part of the G. utr. seem to be general cutaneous neurons, and the dorsal part of the area acousticolateralis seems to receive lateral line input. IIqTRODUCTION The labyrinthine system of the cyclostomes has the most primitive form of all living vertebrates4,19. Although the primitive e a r of l a m p r e y s has two semicircular canals ~3, the ear of the hagfishes has only a single canal in the form of a thick ring, which in Myxine is o r i e n t a t e d about 55 ° to the vertical plane14,18. It has been found by d e g e n e r a t i o n studies that the vestibular nerve fibers in l a m p r e y s p r o j e c t to the ventral nucleus of the octavolateralis area and to the cerebellum16. H o w e v e r , no e x p e r i m e n t a l data from such active tracing m e t h o d s have been r e p o r t e d regarding the central p r o j e c t i o n s of the vestibular nerve in hagfishes. Recently, the horseradish peroxidase ( H R P ) m e t h o d has been used effectively in the hagfishes for tracing tracts both a n t e r o g r a d e l y and r e t r o g r a d e l y within the brain and optic nerves2,11. This bidirectional labeling provides m o r e information a b o u t the fiber connections in the nervous system than the degeneration methods. T h e r e f o r e , we a p p l i e d the H R P m e t h o d to the labyrinth of the hagfish to elucidate
the afferent and efferent connections of the vestibular nerve to the brainstem. MATERIALS AND METHODS
H R P method Twelve adult (body length 4 0 - 5 0 cm) hagfish, Eptatretus burgeri, were anesthetized with 0.02% tricaine m e t h a n e s u l f o n a t e (MS-222) and placed in a holder. During the o p e r a t i o n the animals were respired by circulating artificial sea water containing 0.02% MS-222 from m o u t h to gills. A longitudinal p a r a m e d i a n skin incision was m a d e over the cartilaginous otic capsule. A f t e r removing the roof of the capsule, the m e m b r a n o u s circular canal was exposed and lifted up gently with a fine thread. The tip of a glass micropipette (o.d. 4 0 - 6 0 / ~ m ) was inserted into the m e m b r a n o u s duct, and 50% H R P in distilled water was injected into the e n d o l y m p h a t i c space by air pressure. A f t e r the injection, the micropipette was pulled out and the hole was i m m e d i a t e l y closed with glue. F o u r to 21 days after the injection (a 4 - 1 0 day sur-
Correspondence: F. Amemiya, Department of Anatomy, Yokohama City University, School of Medicine, 2-33, Urafune-cho, Minami-ku, Yokohama, 232 Japan. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
74 vival period proved best), the animals were perfused intracardially with 2% saline containing 5 I U heparin, then with 0.15 M phosphate buffer, p H 7.3, and finally with fixative containing 1% paraformaldehyde, 5% glutaraldehyde, and 4% sucrose in 0.15 M phosphate buffer, pH 7.3. The heads were severed from the trunks, and the skin, mouth and teeth were removed. The remaining head structures were then stored for 24-48 h in 0.1 M phosphate buffer, p H 7.3, containing 30% sucrose. Frontal frozen serial sections were cut at 40 ktm and mounted alternately on two series of slide glasses which had previously been dipped into a chrom-alum gelatin solution and dried. The sections were dried at room temperature with a fan, and treated with a modified diaminobenzidine ( D A B ) method 1. Alternate slides were counterstained with cresyl violet or neutral red.
Bodian, Nissl, Klaver-Barrera and hematoxylin-eosin methods An additional three animals were prepared for the standard staining methods. Two brains were embedded in paraffin, sectioned frontally at 15 ~m, and stained with the Bodian and Nissl methods. One whole head without the teeth was embedded in celloidin. Frontal serial sections of this head were cut at 30 ~m and stained alternately with the Klfiver-Barrera and hematoxylin-eosin methods. A side view of the brain and peripheral organs was then reconstructed from these preparations (Fig. 1). The nomenclature of the brain follows Jansen 9.
al side of the circular canal at the level of the end of the medulla, ran dorsad, penetrated the dense connective tissue surrounding the brain, extended rostrally, and ended near the brain surface at the caudal end of the G. sac. as a blind tube (Fig. 1). H R P labeling was successful in 10 of the 12 experimental animals. In 5 cases, the H R P was confined to the rostral half of the endolymphatic space; here there was labeling of the N. utr. and the G. utr., but not of the N. sac. or the G. sac. In 4 cases the H R P was confined to the caudal half of the endolymphatic space, and here the N. sac. and G. sac. were labeled, but not the N. utr. or the G. ut.r. In the one remaining case, the H R P had filled the entire endolymphatic space, and both nerves with their respective ganglia were labeled. In the 5 cases where only the N. and G. utr. were stained, labeled cells were found only in the dorsolateral portion of the G. utr., and were small bipolar neurons (12-24 ~m). In Bodian preparations, the G. utr. consisted of two separate cell masses, a ventral mass of large pseudounipolar neurons (20-40/am) and a dorsal mass of small bipolar cells (12-24 ~tm) (Fig. 2a). The axons of labeled cells ran dorsally, entered the medulla via several rootlets, and continued toward the ventral part of the area acoustico-lateralis (Figs. 3a and 4A-2). Near the entrance to the brain the axons had a small number of varicosities, which
1 2345 I II I I
RESULTS The vestibular nerve of Eptatretus had two components, one anterior and the other posterior, which for convenience we shall call here, following Ayers and Worthington3 and Peters ~7, the nervus utricularis (N. utr.) and nervus saccularis (N. sac.). Both of these could be seen to contain a number of myelinated fibers in the Kli.iver-Barrera preparations, and each had its own ganglion, the ganglion utriculare (G. utr.) and ganglion sacculare (G. sac.), respectively. The G. sac. was situated caudodorsally to the G. utr. The root of the N. facialis passed through the G. utr. The orientation of the circular canals is such that the upper part is inclined outward 45 ° to the vertical plane. The endolymphatic duct arose from the medi-
5mm
Fig. 1. Side view of the brain and peripheral organs reconstructed from frontal serial sections stained with the KlfiverBarrera and hematoxylin-eosin methods. Numbers (1-6) indicate the levels of the frontal sections in Fig. 4. Abbreviations in this and subsequent figures: AAL, area acoustico-lateralis; BO, bulbus olfactorius; CC, circular canal; CrA, crista anterior; CrP, crista posterior; D, diencephalon; DE, ductus endolymphaticus; G. sac., ganglion sacculare; G. utr., ganglion utriculare; lob.v, lobus vagi; m, nucleus acoustico-lateralis magnocellularis; MC, macula communis; MS, medulla spinalis; N. sac., nervus saccularis; N. utr., nervus utricularis; N.v, ventral nucleus of the area acoustico-lateralis; N.V, nervus trigeminus; N.VII, nervus facialis; T, telencephalon; TO, tectum opticum; Vs, nucleus sensorius nervi trigemini; IX-Xm, nucleus motorius IX-X; Xm, nucleus motorius X.
75
Fig. 2. Frontal sections of G. utr. and G. sac.; Bodian staining, x 160; a: G. utr., showing the ventral mass of large cells (L, inside dotted line) and the dorsal mass of small cells (S). In HRP preparations (inset, x 60) only the mass of small cells was stained, b: G. sac., made up entirely of small cells (S). In HRP preparations (inset, x 60) the entire ganglion was stained.
were more n u m e r o u s after the axon e n t e r e d the brain (Fig. 3c). In the terminal area the axons ramified with many varicosities (Fig. 3d); there were both ascending and descending rami (Fig. 4A). The terminal area of N. utr. was situated medially in the horn of the m e d u l l a (Figs. 1, 3a, and 4 A ) , a position corresponding to the ventral part of the area acoustico-lateralis of Myxine 9 and Bdellostoma 3. In the 4 cases where only the N. and G. sac. were labeled, all labeled cells in the ganglion were small (12-24/~m) and bipolar; in B o d i a n p r e p a r a t i o n s , the G. sac. consisted entirely of small bipolar cells (Fig. 2b). The rootlets of the N. sac. e n t e r e d the medulla just caudal to the entrance of the rootlets of N. utr. (Figs. 3b and 4B-3). A x o n a l varicosities and m a n n e r of termination were the same as in N. utr. The greater part of the terminal area of N. sac. overlapped the terminal area of N. utr., but the terminal area of N. sac. e x t e n d e d slightly further in a caudal
direction (Fig. 4). In the single case where both nerves and both ganglia were labeled, the results were identical to those of the above two paragraphs; there was no additional labeling. In Bodian and K l f i v e r - B a r r e r a p r e p a r a t i o n s , a ventral and a dorsal portion could be distinguished in the area acoustico-lateralis. The ventral portion corr e s p o n d e d to the terminal area of the H R P study, and we here name it the ventral nucleus of the area acoustico-lateralis. In our material its rostral pole reached the anterior end of the m e d u l l a r y horn, and the caudal pole was at almost the same level as the rostral end of the nucleus motorius I X - X (Fig. 4). The rostrocaudal length of the ventral nucleus was 1360/~m; and in frontal sections it was 560 x 280 # m at its largest portion (Fig. 4). The caudalmost part of the ventral nucleus contained the nucleus acousticolateralis magnoceltularis of Jansen 9 (Figs. 1 and 4).
76
iii~,iiii Ill
I Fig. 3. a: frontal section of HRP preparation of medulla oblongata at the point where N. utr. enters. The caudal end of the tectum opticum (TO) is also seen. x 30. b: frontal section as above, at the point where N. sac. enters. The caudal end of the tectum opticum can no longer be seen. x 30. c: high-power view of HRP-labeled fibers of N. utr. in a section adjacent to that of a. Dotted line indicates the brain surface (outside is to the right). Arrows indicate varicosities, which are seen both inside and outside the brain, x 310. d: terminal area of the fibers in c. The fibers branch profusely and have numerous varicosities (arrows). x 310.
DISCUSSION
Sarnat and Netskyl9 stated that cyclostomes have an open lymphatic duct communicating between the labyrinth and the outside. However, in Eptatretus there was no such duct; the endolymphatic duct was a blind tube, which made it easy to confine the H R P in our experiments to the endolymphatic space. The endolymphatic canal of Myxine also does not c o m m u n i cate with the outside 4A4,i8. O n the basis of traditional staining methods, Ayers and W o r t h i n g t o n 3 reported that in Bdellostoma, G. utr. contains two sizes of cells, large (46-66 # m ) cells which are the ganglion cells of general cutaneous fibers, and small (16-26 p m ) cells which are the ganglion cells of the acoustic fibers. They also stated that the cells of the G. sac. were similar in size and character to the acoustic cells of the G. utr. Although the
cells in our
Eptatretus were a little smaller than the
cells in Bdellostoma, our results confirmed the existence of the smaller cells in both G. utr. and G. sac. and their vestibular nature. The nature of the larger cells in G. utr. is not clear from our work, but their pseudounipolar shape and the course of N. facialis seem to indicate that they are indeed general cutaneous cells of N. facialis. Peters 17 reported that there are no myelinated fibers in cyclostomes, but we recognized a n u m b e r of myelinated fibers in both the N. utr. and N. sac. of our preparations. We also found myelinated fibers in N. II, V, VII, IX and X, and in parts of the main central tract, such as the fasciculus longitudinalis medialis (Amemiya, unpublished). This could be an adaptation to life in the shallow coastal waters that Eptatretus burgeri inhabits, where it could be subject to more stimulation and be required to react with great-
77
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Fig. 4. Schematic representation of frontal sections at the levels indicated in Fig. 1. Large dots indicate cell bodies. A r e a s indicated with small dots are terminal areas. D a s h e d lines are H R P - s t a i n e d fibers. Note that N. sac. enters further caudad than N. utr., and its terminal area also extends further caudad than that of N. utr.
78 er alacrity than other hagfishes. In our material, anterogradely labeled postganglionic axons had varicosities not only in the brain, but also in the peripheral roots. In previous anterograde and retrograde H R P studies using Eptatretus2.11, we reported similar varicosities in retinal fibers. Such varicosities in peripheral nerves outside the brain may serve to mediate axon-axon interactions in the peripheral nervous system. In previous reports about the area acoustico-lateralis of hagfishes3,9,12, there has been no mention of clear subdivisions except that of the caudally situated nucleus acoustico-lateralis magnocellularis. However, we have clearly shown that the octavius terminal field is restricted to the ventral nucleus in Eptatretus. The octavius terminal fields are separate from those of the lateral line in the lampreyS6, dogfish 5, bony fishes 15 and frogs8. Here we have identified the same situation in the hagfish. It appears, therefore, that in all anamniotic vertebrates, at least at the level of first order neurons, the octavolateralis area has two separate components: the ventral octavius area (our 'ventral nucleus') and the dorsal lateral line area. Although the octavius tract has two separate peripheral courses, the N. utr. and N. sac., their terminal areas in the ventral nucleus showed considerable overlap and intermingling, without evidence of topic organization. However, the caudal magnocellular acoustico-lateral nucleus seemed to receive more information from the N. sac. than from the N. utr. In the lamprey the primary octavius projection areas are the cerebellum and the ventral nucleus of
the octavolateralis area, which contains three distinct aggregations of large neurons, the anterior, intermediate and posterior octavomotor nuclei ~6. In our Eptatretus, the primary octavius projection area was the ventral nucleus of the area acoustico-lateralis, which showed no topic organization except for the caudalmost magnocellular nucleus; there were no projections to the primordial cerebellum10.12. This indicates that the primary octavius nerve projection area in the brain of the hagfish is more primitive than that of the lamprey, which has a more advanced labyrinthine system than the hagfish4,19. Vestibular efferent neurons have been reported in a number of different vertebrates (e.g. the goldfish 20, the frog 21, the pigeon6, the cat 22 and the squirrel monkeyT). A m o n g the cyclostomes, the lamprey also has efferent nerve endings in the labyrinthS3; but no efferent endings have been noted in the neuroepithelia of the hagfish4, TM. Therefore, the fact that there were no retrogradely labeled cells in the brain of Eptatretus in our H R P study was not due to technical failure, but to the lack of efferent neurons in this species.
REFERENCES
6 Eden, A. R. and Correia, M. J., Identification of multiple groups of efferent vestibular neurons in the adult pigeon using horseradish peroxidase and DAPI, Brain Research, 248 (1982) 201-208. 7 Goldberg, J. M. and Fermlndez, C., Efferent vestibular system in the squirrel monkey: anatomical location and influence on afferent activity, J. Neurophysiol., 43 (1980) 986-1025. 8 Jacoby, J. and Rubinson, K., The acoustic and lateral line nuclei are distinct in the premetamorphic frog, Rana catesbeiana. J. comp. Neurol., 216 (1983) 152-161. 9 Jansen, J., The brain of Myxine glutinosa, J. cornp. Neurol., 49 (1930) 359-507. 10 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.
1 Adams, J. C., Heavy metal intensification of DAB-based HRP reaction product, J. Histochem. Cytochem., 29 (1981) 775. 2 Amemiya, F., Afferent connections to the tectum mesencephali in the hagfish, Eptatretus burgeri: an HRP study, J. Hirnforsch., 24 (1983) 225-236. 3 Ayers, H. and Worthington, J., The finer anatomy of the brain of Bdellostoma dombeyi. 1. The acoustico-lateral system, Amer. J. Anat., 8 (1908) 1-33. 4 Baird, I. L., Some aspects of the comparative anatomy and evolution of the inner ear in sub-mammalian vertebrates, Brain Behav. Evol., 10 (1974) 11-36. 5 Boord, R. L. and Roberts, B. L., Medullary and cerebellar projections of the statoacoustic nerve of the dogfish, Scyliorhinus canicula, J. comp. Neurol., 193 (1980) 57-68.
ACKNOWLEDGEMENTS 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 Mr. H. Shugyo, Mr. S. Takahashi and Mr. H. Nishizawa for technical help.
79 11 Kusunoki, T. and Amemiya, F., Retinal projections in the hagfish, Eptatretus burgeri, Brain Research, 262 (1983) 295-298. 12 Larsell, O., Myxinidae. In J. Jansen (Ed.), The Compara-
tive Anatomy and Histology of the Cerebellum from Myxinoids Through Birds, The University of Minnesota Press, 13
14
15
16 17
Minneapolis, 1967, pp. 8-13. Lowenstein, O., Osborne, M. P. and Thornhill, R. A., The anatomy and ultrastructure of the labyrinth of the lamprey (Lampetra fluviatilis L.) Proc. roy. Soc. B., 170 (1968) 113-134. Lowenstein, O. and Thornhill, R. A., The labyrinth of Myxine: anatomy, ultrastructure and electrophysiology, Proc. roy. Soc. B., 176 (1970) 21-42. Maler, L., The acousticolateral area of bony fishes and its cerebellar relations, Brain Behav. Evol., 10 (1974) 130-145. Northcutt, R. C., Central projections of the eighth cranial nerve in lampreys, Brain Research, 167 (1979) 163-167. Peters, A., The peripheral nervous system. In A. Brodal
18
19 20
21
22
and R. F/inge (Eds.), The Biology ofMyxine, Universitetsforslaget, Oslo, 1963, pp. 92-123. Ross, D. M., The sense organs ofMyxineglutinosa L. In A. Brodal and R. F~inge (Eds.), The Biology of Myxine, Universitetsforlaget, Oslo, 1963, pp. 150-160. Sarnat, H. B. and Netsky, M. G., Evolution of the Nervous System, Oxford University Press, New York, 1974, 318 pp. Strutz, J., Schmidt, C. L. and St/irmer, C., Origin of efferent fibers of the vestibular apparatus in goldfish. A horseradish peroxidase study, Neurosci. Lett., 18 (1980) 5-9. Strutz, J., Spatz, W. B., Schmidt, S. C. and Stiimer, C., Origin of centrifugal fibers to the labyrinth in the frog (Rana esculenta). A study with the fluorescent retrograde neuronal tracer 'fast blue', Brain Research, 215 (1980) 323-328. Warr, W. B., Olivocochlear and vestibular efferent neurons of the feline brain stem: their location, morphology and number determined by retrograde axonal transport and acetylcholinesterase histochemistry, J. comp. Neurol., 161 (1975) 159-182.
73
Elsevier BRE 10802
Primary Vestibular Projections in the Hagfish, Eptatretus burgeri FUMIAKI AMEMIYA, REIJI KISHIDA, RICHARD C. GORIS, HIDEKI ONISHI and TOYOKAZU KUSUNOKI
Department of Anatomy, Yokohama City University, 2-33 Urafune-cho, Minami-ku, Yokohama, 232 (Japan) (Accepted September 11th, 1984)
Key words: vestibular nerve - - ganglion cells - - acoustico-lateral area - - brainstem - - hagfish - transganglionic horseradish peroxidase (HRP) transport
The VIIIth cranial nerve projections in the hagfish, which has only one circular canal in the ear, were studied by transganglionic HRP transport. This nerve has two branches, the nervus utricularis (N. utr.) and the nervus saccularis (N. sac.), each with its own ganglion, the ganglion utriculare (G. utr.) and the ganglion sacculare (G. sac.), respectively. Although the G. sac. has uniformly small cells, the G. utr. consists of two separate cell masses, a ventral mass of large cells and a dorsal mass of small cells. The small cells were labeled in both ganglia after horseradish peroxidase (HRP) injection into the endolymphatic space. The greater part of the terminal areas of these two branches overlapped in the ventral nucleus of the area acoustico-lateralis, but the terminals of the N. sac. extended slightly further in a caudal direction. No projections to the primordial cerebellum and no retrogradely labeled cells in the brain were found. The large cells in the ventral part of the G. utr. seem to be general cutaneous neurons, and the dorsal part of the area acousticolateralis seems to receive lateral line input. IIqTRODUCTION The labyrinthine system of the cyclostomes has the most primitive form of all living vertebrates4,19. Although the primitive e a r of l a m p r e y s has two semicircular canals ~3, the ear of the hagfishes has only a single canal in the form of a thick ring, which in Myxine is o r i e n t a t e d about 55 ° to the vertical plane14,18. It has been found by d e g e n e r a t i o n studies that the vestibular nerve fibers in l a m p r e y s p r o j e c t to the ventral nucleus of the octavolateralis area and to the cerebellum16. H o w e v e r , no e x p e r i m e n t a l data from such active tracing m e t h o d s have been r e p o r t e d regarding the central p r o j e c t i o n s of the vestibular nerve in hagfishes. Recently, the horseradish peroxidase ( H R P ) m e t h o d has been used effectively in the hagfishes for tracing tracts both a n t e r o g r a d e l y and r e t r o g r a d e l y within the brain and optic nerves2,11. This bidirectional labeling provides m o r e information a b o u t the fiber connections in the nervous system than the degeneration methods. T h e r e f o r e , we a p p l i e d the H R P m e t h o d to the labyrinth of the hagfish to elucidate
the afferent and efferent connections of the vestibular nerve to the brainstem. MATERIALS AND METHODS
H R P method Twelve adult (body length 4 0 - 5 0 cm) hagfish, Eptatretus burgeri, were anesthetized with 0.02% tricaine m e t h a n e s u l f o n a t e (MS-222) and placed in a holder. During the o p e r a t i o n the animals were respired by circulating artificial sea water containing 0.02% MS-222 from m o u t h to gills. A longitudinal p a r a m e d i a n skin incision was m a d e over the cartilaginous otic capsule. A f t e r removing the roof of the capsule, the m e m b r a n o u s circular canal was exposed and lifted up gently with a fine thread. The tip of a glass micropipette (o.d. 4 0 - 6 0 / ~ m ) was inserted into the m e m b r a n o u s duct, and 50% H R P in distilled water was injected into the e n d o l y m p h a t i c space by air pressure. A f t e r the injection, the micropipette was pulled out and the hole was i m m e d i a t e l y closed with glue. F o u r to 21 days after the injection (a 4 - 1 0 day sur-
Correspondence: F. Amemiya, Department of Anatomy, Yokohama City University, School of Medicine, 2-33, Urafune-cho, Minami-ku, Yokohama, 232 Japan. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
74 vival period proved best), the animals were perfused intracardially with 2% saline containing 5 I U heparin, then with 0.15 M phosphate buffer, p H 7.3, and finally with fixative containing 1% paraformaldehyde, 5% glutaraldehyde, and 4% sucrose in 0.15 M phosphate buffer, pH 7.3. The heads were severed from the trunks, and the skin, mouth and teeth were removed. The remaining head structures were then stored for 24-48 h in 0.1 M phosphate buffer, p H 7.3, containing 30% sucrose. Frontal frozen serial sections were cut at 40 ktm and mounted alternately on two series of slide glasses which had previously been dipped into a chrom-alum gelatin solution and dried. The sections were dried at room temperature with a fan, and treated with a modified diaminobenzidine ( D A B ) method 1. Alternate slides were counterstained with cresyl violet or neutral red.
Bodian, Nissl, Klaver-Barrera and hematoxylin-eosin methods An additional three animals were prepared for the standard staining methods. Two brains were embedded in paraffin, sectioned frontally at 15 ~m, and stained with the Bodian and Nissl methods. One whole head without the teeth was embedded in celloidin. Frontal serial sections of this head were cut at 30 ~m and stained alternately with the Klfiver-Barrera and hematoxylin-eosin methods. A side view of the brain and peripheral organs was then reconstructed from these preparations (Fig. 1). The nomenclature of the brain follows Jansen 9.
al side of the circular canal at the level of the end of the medulla, ran dorsad, penetrated the dense connective tissue surrounding the brain, extended rostrally, and ended near the brain surface at the caudal end of the G. sac. as a blind tube (Fig. 1). H R P labeling was successful in 10 of the 12 experimental animals. In 5 cases, the H R P was confined to the rostral half of the endolymphatic space; here there was labeling of the N. utr. and the G. utr., but not of the N. sac. or the G. sac. In 4 cases the H R P was confined to the caudal half of the endolymphatic space, and here the N. sac. and G. sac. were labeled, but not the N. utr. or the G. ut.r. In the one remaining case, the H R P had filled the entire endolymphatic space, and both nerves with their respective ganglia were labeled. In the 5 cases where only the N. and G. utr. were stained, labeled cells were found only in the dorsolateral portion of the G. utr., and were small bipolar neurons (12-24 ~m). In Bodian preparations, the G. utr. consisted of two separate cell masses, a ventral mass of large pseudounipolar neurons (20-40/am) and a dorsal mass of small bipolar cells (12-24 ~tm) (Fig. 2a). The axons of labeled cells ran dorsally, entered the medulla via several rootlets, and continued toward the ventral part of the area acoustico-lateralis (Figs. 3a and 4A-2). Near the entrance to the brain the axons had a small number of varicosities, which
1 2345 I II I I
RESULTS The vestibular nerve of Eptatretus had two components, one anterior and the other posterior, which for convenience we shall call here, following Ayers and Worthington3 and Peters ~7, the nervus utricularis (N. utr.) and nervus saccularis (N. sac.). Both of these could be seen to contain a number of myelinated fibers in the Kli.iver-Barrera preparations, and each had its own ganglion, the ganglion utriculare (G. utr.) and ganglion sacculare (G. sac.), respectively. The G. sac. was situated caudodorsally to the G. utr. The root of the N. facialis passed through the G. utr. The orientation of the circular canals is such that the upper part is inclined outward 45 ° to the vertical plane. The endolymphatic duct arose from the medi-
5mm
Fig. 1. Side view of the brain and peripheral organs reconstructed from frontal serial sections stained with the KlfiverBarrera and hematoxylin-eosin methods. Numbers (1-6) indicate the levels of the frontal sections in Fig. 4. Abbreviations in this and subsequent figures: AAL, area acoustico-lateralis; BO, bulbus olfactorius; CC, circular canal; CrA, crista anterior; CrP, crista posterior; D, diencephalon; DE, ductus endolymphaticus; G. sac., ganglion sacculare; G. utr., ganglion utriculare; lob.v, lobus vagi; m, nucleus acoustico-lateralis magnocellularis; MC, macula communis; MS, medulla spinalis; N. sac., nervus saccularis; N. utr., nervus utricularis; N.v, ventral nucleus of the area acoustico-lateralis; N.V, nervus trigeminus; N.VII, nervus facialis; T, telencephalon; TO, tectum opticum; Vs, nucleus sensorius nervi trigemini; IX-Xm, nucleus motorius IX-X; Xm, nucleus motorius X.
75
Fig. 2. Frontal sections of G. utr. and G. sac.; Bodian staining, x 160; a: G. utr., showing the ventral mass of large cells (L, inside dotted line) and the dorsal mass of small cells (S). In HRP preparations (inset, x 60) only the mass of small cells was stained, b: G. sac., made up entirely of small cells (S). In HRP preparations (inset, x 60) the entire ganglion was stained.
were more n u m e r o u s after the axon e n t e r e d the brain (Fig. 3c). In the terminal area the axons ramified with many varicosities (Fig. 3d); there were both ascending and descending rami (Fig. 4A). The terminal area of N. utr. was situated medially in the horn of the m e d u l l a (Figs. 1, 3a, and 4 A ) , a position corresponding to the ventral part of the area acoustico-lateralis of Myxine 9 and Bdellostoma 3. In the 4 cases where only the N. and G. sac. were labeled, all labeled cells in the ganglion were small (12-24/~m) and bipolar; in B o d i a n p r e p a r a t i o n s , the G. sac. consisted entirely of small bipolar cells (Fig. 2b). The rootlets of the N. sac. e n t e r e d the medulla just caudal to the entrance of the rootlets of N. utr. (Figs. 3b and 4B-3). A x o n a l varicosities and m a n n e r of termination were the same as in N. utr. The greater part of the terminal area of N. sac. overlapped the terminal area of N. utr., but the terminal area of N. sac. e x t e n d e d slightly further in a caudal
direction (Fig. 4). In the single case where both nerves and both ganglia were labeled, the results were identical to those of the above two paragraphs; there was no additional labeling. In Bodian and K l f i v e r - B a r r e r a p r e p a r a t i o n s , a ventral and a dorsal portion could be distinguished in the area acoustico-lateralis. The ventral portion corr e s p o n d e d to the terminal area of the H R P study, and we here name it the ventral nucleus of the area acoustico-lateralis. In our material its rostral pole reached the anterior end of the m e d u l l a r y horn, and the caudal pole was at almost the same level as the rostral end of the nucleus motorius I X - X (Fig. 4). The rostrocaudal length of the ventral nucleus was 1360/~m; and in frontal sections it was 560 x 280 # m at its largest portion (Fig. 4). The caudalmost part of the ventral nucleus contained the nucleus acousticolateralis magnoceltularis of Jansen 9 (Figs. 1 and 4).
76
iii~,iiii Ill
I Fig. 3. a: frontal section of HRP preparation of medulla oblongata at the point where N. utr. enters. The caudal end of the tectum opticum (TO) is also seen. x 30. b: frontal section as above, at the point where N. sac. enters. The caudal end of the tectum opticum can no longer be seen. x 30. c: high-power view of HRP-labeled fibers of N. utr. in a section adjacent to that of a. Dotted line indicates the brain surface (outside is to the right). Arrows indicate varicosities, which are seen both inside and outside the brain, x 310. d: terminal area of the fibers in c. The fibers branch profusely and have numerous varicosities (arrows). x 310.
DISCUSSION
Sarnat and Netskyl9 stated that cyclostomes have an open lymphatic duct communicating between the labyrinth and the outside. However, in Eptatretus there was no such duct; the endolymphatic duct was a blind tube, which made it easy to confine the H R P in our experiments to the endolymphatic space. The endolymphatic canal of Myxine also does not c o m m u n i cate with the outside 4A4,i8. O n the basis of traditional staining methods, Ayers and W o r t h i n g t o n 3 reported that in Bdellostoma, G. utr. contains two sizes of cells, large (46-66 # m ) cells which are the ganglion cells of general cutaneous fibers, and small (16-26 p m ) cells which are the ganglion cells of the acoustic fibers. They also stated that the cells of the G. sac. were similar in size and character to the acoustic cells of the G. utr. Although the
cells in our
Eptatretus were a little smaller than the
cells in Bdellostoma, our results confirmed the existence of the smaller cells in both G. utr. and G. sac. and their vestibular nature. The nature of the larger cells in G. utr. is not clear from our work, but their pseudounipolar shape and the course of N. facialis seem to indicate that they are indeed general cutaneous cells of N. facialis. Peters 17 reported that there are no myelinated fibers in cyclostomes, but we recognized a n u m b e r of myelinated fibers in both the N. utr. and N. sac. of our preparations. We also found myelinated fibers in N. II, V, VII, IX and X, and in parts of the main central tract, such as the fasciculus longitudinalis medialis (Amemiya, unpublished). This could be an adaptation to life in the shallow coastal waters that Eptatretus burgeri inhabits, where it could be subject to more stimulation and be required to react with great-
77
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Fig. 4. Schematic representation of frontal sections at the levels indicated in Fig. 1. Large dots indicate cell bodies. A r e a s indicated with small dots are terminal areas. D a s h e d lines are H R P - s t a i n e d fibers. Note that N. sac. enters further caudad than N. utr., and its terminal area also extends further caudad than that of N. utr.
78 er alacrity than other hagfishes. In our material, anterogradely labeled postganglionic axons had varicosities not only in the brain, but also in the peripheral roots. In previous anterograde and retrograde H R P studies using Eptatretus2.11, we reported similar varicosities in retinal fibers. Such varicosities in peripheral nerves outside the brain may serve to mediate axon-axon interactions in the peripheral nervous system. In previous reports about the area acoustico-lateralis of hagfishes3,9,12, there has been no mention of clear subdivisions except that of the caudally situated nucleus acoustico-lateralis magnocellularis. However, we have clearly shown that the octavius terminal field is restricted to the ventral nucleus in Eptatretus. The octavius terminal fields are separate from those of the lateral line in the lampreyS6, dogfish 5, bony fishes 15 and frogs8. Here we have identified the same situation in the hagfish. It appears, therefore, that in all anamniotic vertebrates, at least at the level of first order neurons, the octavolateralis area has two separate components: the ventral octavius area (our 'ventral nucleus') and the dorsal lateral line area. Although the octavius tract has two separate peripheral courses, the N. utr. and N. sac., their terminal areas in the ventral nucleus showed considerable overlap and intermingling, without evidence of topic organization. However, the caudal magnocellular acoustico-lateral nucleus seemed to receive more information from the N. sac. than from the N. utr. In the lamprey the primary octavius projection areas are the cerebellum and the ventral nucleus of
the octavolateralis area, which contains three distinct aggregations of large neurons, the anterior, intermediate and posterior octavomotor nuclei ~6. In our Eptatretus, the primary octavius projection area was the ventral nucleus of the area acoustico-lateralis, which showed no topic organization except for the caudalmost magnocellular nucleus; there were no projections to the primordial cerebellum10.12. This indicates that the primary octavius nerve projection area in the brain of the hagfish is more primitive than that of the lamprey, which has a more advanced labyrinthine system than the hagfish4,19. Vestibular efferent neurons have been reported in a number of different vertebrates (e.g. the goldfish 20, the frog 21, the pigeon6, the cat 22 and the squirrel monkeyT). A m o n g the cyclostomes, the lamprey also has efferent nerve endings in the labyrinthS3; but no efferent endings have been noted in the neuroepithelia of the hagfish4, TM. Therefore, the fact that there were no retrogradely labeled cells in the brain of Eptatretus in our H R P study was not due to technical failure, but to the lack of efferent neurons in this species.
REFERENCES
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ACKNOWLEDGEMENTS 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 Mr. H. Shugyo, Mr. S. Takahashi and Mr. H. Nishizawa for technical help.
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