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Retinal projections in the hagfish, Eptatretus burgeri
295
Brain Research, 262 (1983) 295-298 Elsevier Biomedical Press
Retinal projections in the hagfish, Eptatretus burgeri T O Y O K A Z U K U S U N O K I and F U M I A K I AMEMIYA
Department of A natomy, Yokohama City University, School of Medicine, 2-33, Urafune-cho, Minami-ku, Yokohama 232 (Japan) (Accepted October 18th, 1982)
Key words: hagfish - retinal projections - horseradish peroxidase - degeneration
The retinal projections of hagfish were investigated by anterograde transport of HRP and the Nauta-Gygax method 9. The pathway coincided with the commissura postoptica of Jansen ~ after complete crossing within the hypothalamus. Many projections were found in the contralateral 'area pretectalis', but there were only a few projections in the tectum mesencephali, the pars ventralis thalami, and the n. tuberculi posterioris of Jansen 5.
In contrast to other cyclostomes such as the lampreys, the hagfish has subcutaneous eyes without lenses, and neither the chiasma opticum nor the tr. opticus is located outside the diencephalon, but the n. opticus decussates within the hypothalamus, as found by Jansen 5 and others. Experimental studies on retinal projections in lampreys have been done 6,v.~3, but to our knowledge, there have been no such studies on retinal projections in the hagfish brain. Therefore, in the present study we attempted to elucidate the course and termination of the optic tract in the hagfish. The fish used were Eptatretus burgeri caught in the coastal waters of Japan in April. A 20% solution of HRP (TOYOBO RZ: 3, 6) in 0.02 M Tris-HC1 buffer (pH 8.4) was injected with a microcapillary pipette into one eye in 11 animals. After 9 26 days at 18 °C in an aquarium, the animals were anesthetized and then killed by intracardial perfusion with 2% saline followed by a fixative containing 0.1 M phosphate buffer, 2.5% glutaraldehyde, and 1% paraformaldehyde. The brains were cut into 60/~m serial frontal sections in a cryostat, and then stained with the method of Adams ~. This technique gave better results than the TMB and DAB methods. The optimum survival period was 14~18 days. For anterograde degeneration, one eye each was removed from 10 animals, which were then kept 0006-8993/83/0000-0000/$03.00©1983 Elsevier Biomedical Press
for 9-30 days at 18 °C in the aquarium. After intracardial perfusion with 2% saline followed by 10% formol-saline, the brains were sectioned serially at 30/~m in a cryostat. The sections were treated with the method of Nauta and Gygax9. Two additional animals were similarly processed as controls for each method. The nomenclature of the hagfish brain follows Jansen 5. The description shown in Fig. 1 was based on the resuits of anterograde transport of HRP, because better pictures were obtained with HRP, although both methods yielded similar results. The labeled fibers, which were fine fibers with varicosities, reached the ventral surface of the hypothalamus from the eye through a slender optic nerve (Figs. IA, 2). After entering the hypothalamus, the bundle of fibers ran dorsomedially to cross with the other side of the tr. opticus. It was not clear whether any fibers or collaterals terminate in the n. postopticus of the hypothalamus, but a few labeled fine fibers were found within this nucleus. After complete crossing, the bundle of labeled fibers dispersed within the commissura postoptica, in which labeled fibers passed contralaterally to the lateral part of the pars ventralis thalami (Fig. 1B). Some of the fibers could be traced dorsally into the n. diffusus hypothalami, and other fibers or collaterals seemed to terminate on cells in the lateral part of the pars ventralis thalami, and on a small num-
296
n.ll
A
B
C
O
Fig. 1. Semi-schematic drawings of transverse sections through the hagfish brain, showing locations of anterogradely labeled fibers ( ~ ) and their destinations (: :). A: level of the chiasma opticum. B: level of the commissura postoptica. C: middle part of the diencephalon. D: transitional region between the diencephalon and mesencephalon, ap. 'area pretectalis'; aq, aqueductus mesencephali; b, area basalis; cp, commissura posterior; ctd, commissura tecti diencephali; e, eminentia thalami; ha, habenula; hi, primordium hippocampi; hy, hypothalamus; hyd, n. diffusus hypothalami; ip, n. interpeduncularis; np, n. postopticus; n. II, n. opticus; p, n. tuberculi posterioris; pc, commissura postoptica; td, pars dorsalis thalami; tv, pars ventralis thalami; v. hy, ventriculus hypothalamicus; 1, 2, 3, 4 and 5, layers 1, 2, 3, 4 and 5 of the telencephalon. Fig. 2. The n. opticus entering the hypothalamus from the ventral brain surface, anterograde labeling. Bar = 100/~m. Fig. 3. Labeled fibers with varicosities in the commissura postoptica (pc) and a terminal arbor of an optic fiber in the medial border of the n. tuberculi posterioris (p). Bar = 50/~m.
ber of cells constituting the rostromedial border of the n. tuberculi posterioris, which is located~ lateral to the pars ventralis thalami (Figs. 1C, 3). However, no fibers were found in the central region of the n. tuberculi posterioris. After the bundle gave off these terminations, the remaining labeled fibers swung dorsalward and then caudomedialward beneath the peripheral layer of the pars dorsalis thalami to approach the rostral end of the tectum mesencephali through the n. profundus of the pars dorsalis thalami. As regards the pathway of the tr. opticus, the course of the labeled fibers in the present study coincided with the postoptic commissure of JansenL
The labeled fibers terminated mainly in a cell group situated dorsolateral to the commissura posterior and just under the molecular layer of the brain surface in the medial corner of the transitional region between the diencephalon and mesencephalon (Figs. 1D, 4, 5). These were granule cells, about 5-7/~m in diameter, extending within a limit of about 250 ~m rostrocaudally, and having the same maximum width in transverse sections. These cells were somewhat more crowded than those in surrounding areas, so that the cell group was easily recognizable in Nissl preparations. Also, degenerating debris was found scattered in the same region (Fig. 6).
297
Fig. 4. Transverse section at the rostral end of the rectum mesencephali, counterstained with Neutral red. Marked HRP labeling is seen in the 'area pretectalis'. Bar indicates 500 ~tm. rm, n. reticularis mesencephali: other abbreviations as in Fig. 1. Fig. 5. Higher magnification of the 'area pretectalis' in Fig. 4. Marker bar: 100 gin. Fig. 6. Degenerating debris in the "area pretectalis', 10 days after removal of one eye, stained by the Nauta Gygax method, transverse section, d, dorsal: m, medial. Bar: 50/~m.
Nothing comparable to the structures found in the tectum opticum of other vertebrates was observed in the hagfish. Namely, there were only a few labeled fine fibers in an area in the molecular layer and in a thin superficial region of the layer of the large fiber tracts of Jansen 5 dorsal to the commissura tecti mesencephali. The lamination detectable in other animals with labeled fibers or degeneration was lacking in the tectum mesencephali here. Unfortunately it was impossible to follow the route of the fibers from the tr. opticus to the tectum in the present preparations. In one case, a recrossed fiber could be followed into the ipsilateral mesencephalic tegmentum through the commissura posterior~ but its termination was unclear.
In the medial pretectal region, the n. pretectalis in the shark and ray ~2, and the n. posterodorsalis in some snakes l°.L~ and lizards 2, which are situated dorsolateral to the commissura posterior, receive retinal projections. Also, in the lamprey retinal projections are observed in the superficial layer of the pretectum 6-7.~3. In the blind cave fish, which has degenerate eyes sunken beneath layers of connective and lipid tissues, retinal efferents are distributed both to the area pretectalis, which is located lateral to the commissura posterior, and to the medial third of the stratum opticum of the tectum opticum ~4. In consideration of these findings, it seems probable that the cell group receiving the optic projections in hagfish is topologically comparable to the pretectal region in other ani-
298
reals. Therefore we have named this cell group in the hagfish the 'area pretectalis'. It presumably corresponds to the dorsomedial optic nucleus proposed by Ebbesson 3. Since no histochemical stratification is found in the tectum opticum of hagfish 8, in contrast with the adult lamprey which has well developed eyes, and on the basis of the present observations, we think
that hagfish do not depend on visual input for feeding. We wish to thank Prof. S. Kinoshita, Director of Misaki Marine Biological Station, Faculty of Science, University of Tokyo, Japan, for the courtesy of supplying the hagfish, Dr. R. C. Goris for revising the manuscript, and Miss M. Yoshimoto for excellent technical assistance.
1 Adams, J. C. Technical considerations on the use of horseradish peroxidase as a neuronal marker, Neuroscience, 2 (1977) 141 145. 2 Butler, A. B. and Northcutt R. G., Retinal projections in Iguana iguana and A nolis carolinensis, Brain Research, 26(1971) 1 13. 3 Ebbesson, S. O. E., A proposal for a common nomenclature for some optic nuclei in vertebrates and the evidence for a common origin of two such cell groups, Brain Behav. Evol., 6 (1972) 75-91. 4 Halpern, M. and Frumin, N., Retinal projections in a snake, Thamnophis sirtaHs, J. Morph., 141 (1973)359 382. 5 Jansen, J., The brain of M),xine glutinosa, J. comp. Neurol.,49(1930)359 507. 6 Kennedy, M. C. and Rubinson, K., Retinal projections in larval, transforming and adult sea lamprey, Petromyzon marinus, J. Comp. Neurol., 171 (1977)465 480. 7 Kosareva, A. A., Retinal projections in lamprey (Lampetrafluviatilis), J. Hirnforsch., 21 (1980) 243 256. 8 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. 9 Nauta, W. J. H. and Gygax, P. A., Silver impregnation of degenerating axons in the central nervous system: a modified technic, Stain Technol., 29 (1954) 91 93. 10 Northcutt, R. G. and Butler, A. B., Retinal projections in the northern water snake Natrix sipedon sipedon (L.), J. Morph., 142(1974) 117-136. 11 Schroeder, D. M., Retinal afferents and efferents of an infrared sensitive snake, Crotalus viridis, J. comp. Neurol., 170(1981)29-42. 12 Smeets, W. J. A. J., Retinal pathways in two chondrichthyans, the shark Scyliorhinus canicula and the ray Raja clavata, J. camp. Neurol., 195 (1981) 1 11. 13 Vesselkin, N. P., Ermakova, T. V., Rep6rant, J., Kosareva, A. A. and Kenigfest, N. B., The retinofugal and retinopetal systems in Lampetrafluviatilis. An experimental study using radioautographic and HRP methods, Brain Research, 195 (1980) 453 460. 14 Voneida, T. J. and Sligar, C. M., A comparative neuroanatomic study of retinal projections in two fishes: As(vanax hubbsi (the blind cave fish), and Astvanax mexicanus, J. comp. Neurol., 165 (1976) 89 106.
Brain Research, 262 (1983) 295-298 Elsevier Biomedical Press
Retinal projections in the hagfish, Eptatretus burgeri T O Y O K A Z U K U S U N O K I and F U M I A K I AMEMIYA
Department of A natomy, Yokohama City University, School of Medicine, 2-33, Urafune-cho, Minami-ku, Yokohama 232 (Japan) (Accepted October 18th, 1982)
Key words: hagfish - retinal projections - horseradish peroxidase - degeneration
The retinal projections of hagfish were investigated by anterograde transport of HRP and the Nauta-Gygax method 9. The pathway coincided with the commissura postoptica of Jansen ~ after complete crossing within the hypothalamus. Many projections were found in the contralateral 'area pretectalis', but there were only a few projections in the tectum mesencephali, the pars ventralis thalami, and the n. tuberculi posterioris of Jansen 5.
In contrast to other cyclostomes such as the lampreys, the hagfish has subcutaneous eyes without lenses, and neither the chiasma opticum nor the tr. opticus is located outside the diencephalon, but the n. opticus decussates within the hypothalamus, as found by Jansen 5 and others. Experimental studies on retinal projections in lampreys have been done 6,v.~3, but to our knowledge, there have been no such studies on retinal projections in the hagfish brain. Therefore, in the present study we attempted to elucidate the course and termination of the optic tract in the hagfish. The fish used were Eptatretus burgeri caught in the coastal waters of Japan in April. A 20% solution of HRP (TOYOBO RZ: 3, 6) in 0.02 M Tris-HC1 buffer (pH 8.4) was injected with a microcapillary pipette into one eye in 11 animals. After 9 26 days at 18 °C in an aquarium, the animals were anesthetized and then killed by intracardial perfusion with 2% saline followed by a fixative containing 0.1 M phosphate buffer, 2.5% glutaraldehyde, and 1% paraformaldehyde. The brains were cut into 60/~m serial frontal sections in a cryostat, and then stained with the method of Adams ~. This technique gave better results than the TMB and DAB methods. The optimum survival period was 14~18 days. For anterograde degeneration, one eye each was removed from 10 animals, which were then kept 0006-8993/83/0000-0000/$03.00©1983 Elsevier Biomedical Press
for 9-30 days at 18 °C in the aquarium. After intracardial perfusion with 2% saline followed by 10% formol-saline, the brains were sectioned serially at 30/~m in a cryostat. The sections were treated with the method of Nauta and Gygax9. Two additional animals were similarly processed as controls for each method. The nomenclature of the hagfish brain follows Jansen 5. The description shown in Fig. 1 was based on the resuits of anterograde transport of HRP, because better pictures were obtained with HRP, although both methods yielded similar results. The labeled fibers, which were fine fibers with varicosities, reached the ventral surface of the hypothalamus from the eye through a slender optic nerve (Figs. IA, 2). After entering the hypothalamus, the bundle of fibers ran dorsomedially to cross with the other side of the tr. opticus. It was not clear whether any fibers or collaterals terminate in the n. postopticus of the hypothalamus, but a few labeled fine fibers were found within this nucleus. After complete crossing, the bundle of labeled fibers dispersed within the commissura postoptica, in which labeled fibers passed contralaterally to the lateral part of the pars ventralis thalami (Fig. 1B). Some of the fibers could be traced dorsally into the n. diffusus hypothalami, and other fibers or collaterals seemed to terminate on cells in the lateral part of the pars ventralis thalami, and on a small num-
296
n.ll
A
B
C
O
Fig. 1. Semi-schematic drawings of transverse sections through the hagfish brain, showing locations of anterogradely labeled fibers ( ~ ) and their destinations (: :). A: level of the chiasma opticum. B: level of the commissura postoptica. C: middle part of the diencephalon. D: transitional region between the diencephalon and mesencephalon, ap. 'area pretectalis'; aq, aqueductus mesencephali; b, area basalis; cp, commissura posterior; ctd, commissura tecti diencephali; e, eminentia thalami; ha, habenula; hi, primordium hippocampi; hy, hypothalamus; hyd, n. diffusus hypothalami; ip, n. interpeduncularis; np, n. postopticus; n. II, n. opticus; p, n. tuberculi posterioris; pc, commissura postoptica; td, pars dorsalis thalami; tv, pars ventralis thalami; v. hy, ventriculus hypothalamicus; 1, 2, 3, 4 and 5, layers 1, 2, 3, 4 and 5 of the telencephalon. Fig. 2. The n. opticus entering the hypothalamus from the ventral brain surface, anterograde labeling. Bar = 100/~m. Fig. 3. Labeled fibers with varicosities in the commissura postoptica (pc) and a terminal arbor of an optic fiber in the medial border of the n. tuberculi posterioris (p). Bar = 50/~m.
ber of cells constituting the rostromedial border of the n. tuberculi posterioris, which is located~ lateral to the pars ventralis thalami (Figs. 1C, 3). However, no fibers were found in the central region of the n. tuberculi posterioris. After the bundle gave off these terminations, the remaining labeled fibers swung dorsalward and then caudomedialward beneath the peripheral layer of the pars dorsalis thalami to approach the rostral end of the tectum mesencephali through the n. profundus of the pars dorsalis thalami. As regards the pathway of the tr. opticus, the course of the labeled fibers in the present study coincided with the postoptic commissure of JansenL
The labeled fibers terminated mainly in a cell group situated dorsolateral to the commissura posterior and just under the molecular layer of the brain surface in the medial corner of the transitional region between the diencephalon and mesencephalon (Figs. 1D, 4, 5). These were granule cells, about 5-7/~m in diameter, extending within a limit of about 250 ~m rostrocaudally, and having the same maximum width in transverse sections. These cells were somewhat more crowded than those in surrounding areas, so that the cell group was easily recognizable in Nissl preparations. Also, degenerating debris was found scattered in the same region (Fig. 6).
297
Fig. 4. Transverse section at the rostral end of the rectum mesencephali, counterstained with Neutral red. Marked HRP labeling is seen in the 'area pretectalis'. Bar indicates 500 ~tm. rm, n. reticularis mesencephali: other abbreviations as in Fig. 1. Fig. 5. Higher magnification of the 'area pretectalis' in Fig. 4. Marker bar: 100 gin. Fig. 6. Degenerating debris in the "area pretectalis', 10 days after removal of one eye, stained by the Nauta Gygax method, transverse section, d, dorsal: m, medial. Bar: 50/~m.
Nothing comparable to the structures found in the tectum opticum of other vertebrates was observed in the hagfish. Namely, there were only a few labeled fine fibers in an area in the molecular layer and in a thin superficial region of the layer of the large fiber tracts of Jansen 5 dorsal to the commissura tecti mesencephali. The lamination detectable in other animals with labeled fibers or degeneration was lacking in the tectum mesencephali here. Unfortunately it was impossible to follow the route of the fibers from the tr. opticus to the tectum in the present preparations. In one case, a recrossed fiber could be followed into the ipsilateral mesencephalic tegmentum through the commissura posterior~ but its termination was unclear.
In the medial pretectal region, the n. pretectalis in the shark and ray ~2, and the n. posterodorsalis in some snakes l°.L~ and lizards 2, which are situated dorsolateral to the commissura posterior, receive retinal projections. Also, in the lamprey retinal projections are observed in the superficial layer of the pretectum 6-7.~3. In the blind cave fish, which has degenerate eyes sunken beneath layers of connective and lipid tissues, retinal efferents are distributed both to the area pretectalis, which is located lateral to the commissura posterior, and to the medial third of the stratum opticum of the tectum opticum ~4. In consideration of these findings, it seems probable that the cell group receiving the optic projections in hagfish is topologically comparable to the pretectal region in other ani-
298
reals. Therefore we have named this cell group in the hagfish the 'area pretectalis'. It presumably corresponds to the dorsomedial optic nucleus proposed by Ebbesson 3. Since no histochemical stratification is found in the tectum opticum of hagfish 8, in contrast with the adult lamprey which has well developed eyes, and on the basis of the present observations, we think
that hagfish do not depend on visual input for feeding. We wish to thank Prof. S. Kinoshita, Director of Misaki Marine Biological Station, Faculty of Science, University of Tokyo, Japan, for the courtesy of supplying the hagfish, Dr. R. C. Goris for revising the manuscript, and Miss M. Yoshimoto for excellent technical assistance.
1 Adams, J. C. Technical considerations on the use of horseradish peroxidase as a neuronal marker, Neuroscience, 2 (1977) 141 145. 2 Butler, A. B. and Northcutt R. G., Retinal projections in Iguana iguana and A nolis carolinensis, Brain Research, 26(1971) 1 13. 3 Ebbesson, S. O. E., A proposal for a common nomenclature for some optic nuclei in vertebrates and the evidence for a common origin of two such cell groups, Brain Behav. Evol., 6 (1972) 75-91. 4 Halpern, M. and Frumin, N., Retinal projections in a snake, Thamnophis sirtaHs, J. Morph., 141 (1973)359 382. 5 Jansen, J., The brain of M),xine glutinosa, J. comp. Neurol.,49(1930)359 507. 6 Kennedy, M. C. and Rubinson, K., Retinal projections in larval, transforming and adult sea lamprey, Petromyzon marinus, J. Comp. Neurol., 171 (1977)465 480. 7 Kosareva, A. A., Retinal projections in lamprey (Lampetrafluviatilis), J. Hirnforsch., 21 (1980) 243 256. 8 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. 9 Nauta, W. J. H. and Gygax, P. A., Silver impregnation of degenerating axons in the central nervous system: a modified technic, Stain Technol., 29 (1954) 91 93. 10 Northcutt, R. G. and Butler, A. B., Retinal projections in the northern water snake Natrix sipedon sipedon (L.), J. Morph., 142(1974) 117-136. 11 Schroeder, D. M., Retinal afferents and efferents of an infrared sensitive snake, Crotalus viridis, J. comp. Neurol., 170(1981)29-42. 12 Smeets, W. J. A. J., Retinal pathways in two chondrichthyans, the shark Scyliorhinus canicula and the ray Raja clavata, J. camp. Neurol., 195 (1981) 1 11. 13 Vesselkin, N. P., Ermakova, T. V., Rep6rant, J., Kosareva, A. A. and Kenigfest, N. B., The retinofugal and retinopetal systems in Lampetrafluviatilis. An experimental study using radioautographic and HRP methods, Brain Research, 195 (1980) 453 460. 14 Voneida, T. J. and Sligar, C. M., A comparative neuroanatomic study of retinal projections in two fishes: As(vanax hubbsi (the blind cave fish), and Astvanax mexicanus, J. comp. Neurol., 165 (1976) 89 106.