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Aberrant regeneration in goldfish after crushing one optic nerve
214
Brahl Research, 199 (1980) 2 1 4 2 I ~ ) E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
Aberrant regeneration in goldfish after crushing one optic nerve
ALAN
D. S P R I N G E R
Department of Anatomy, New York Medical College, Basic Science Building, Valhalla, N.Y. 10595
(u.s.A.) ( A c c e p t e d J u n e 12th, 1980)
Key word~: g o l d f i s h - - o p t i c n e r v e --- r e g e n e r a t i o n - - a u t o r a d i o g r a p h y
Optic nerve regeneration in mature, lower vertebrates has been examined in numerous studies attempting to elucidate the principles that determine the orderly development of the visual system. Neuroanatomical and electrophysiological techniques have demonstrated that each optic nerve of goldfish innervates the contralateral optic tectum (COT)L Following regeneration, the retinotopic projection that existed prior to nerve section is re-established a and normal vision is restoredl,le, 14. In some situations, an optic nerve will spontaneously regenerate to the ipsilateral optic tectum (IOT), even when the COT is intact 15. When the optic nerve of one eye was crushed at the same time as the other eye was enucleated, most fibers of the remaining nerve regenerated back to the COT. However, a small number of fibers grew back to the denervated IOT and deployed themselves retinotopically. These fibers were evident in the lOT at the longest survival time examined (50 days). One explanation for this spontaneous IOT projection is that removing one eye eliminated nerve fibers from the ipsilateral optic tract (IOTr). The presence of the opposite nerve at the chiasm would have mechanically blocked the regenerating fibers from entering the erroneous IOTr and tectum. To evaluate this hypothesis, the present experiment sought to determine whether optic nerve fibers would regenerate to the lOT when only one nerve was crushed and when the opposite eye was left intact. Common goldfish 5-6 cm in body length were anesthetized and the left optic nerve was crushed behind the eye. They were kept in aerated tanks at 30 °C and separate groups of 2-3 fish were injected intraocularly (left eye) with 25 /zCi of [3H]proline at 3, 7, 15 and 31 days following surgery 15. Additional fish had the right eye removed and the left optic nerve crushed. These fish were injected with [3H]proline into the remaining eye 31 days following surgery. The fish were killed after 24 h of incorporation and their brains were fixed in alcohol-formalin-acetic acid. Paraffin sections (10 /zm) were dipped in Kodak NTB2 emulsion, exposed for 11-28 days, developed in Dektol and stained with hematoxylin-eosin or cresyl violet tS. Silver grain densities above background levels were interpreted as representing the presence of regenerating nerve fibers.
215 Regenerating optic nerve fibers reached the chiasm within 4 days following the crush of one optic nerve. Fibers were not evident in the COT at this time, indicating that the nerve crush was effective. By 8 days post-crush, the medial and lateral portions of the COT were densely labeled 14. Surprisingly, appreciable label was present in the IOTr, as well as throughout the lOT (Fig. 1). Compact rows of silver grains were found in the stratum opticum and in the other tectal laminae known to receive retinal fibersS, 11 (Fig. 2). The label was apparent throughout the rostro-caudal extent of the lOT. Labeling of the lOT was appreciably reduced in the fish examined at 16 days post-crush. Silver grains, possibly reflecting individual fascicles of nerve fibers, were only found in the stratum opticum of the rostral IOT of both fish (Figs. 3 and 4). Fibers were not detected in the lOT or IOTr at 32 days post-crush. In contrast, the lOT was extensively labeled 32 days post-crush (Figs. 5 and 6) in fish that had a right eye enucleation at the time of left optic nerve crush. (Several anomalous contralateral areas were also transiently labeled: nucleus rotundus, pituitary, cerebellum and nucleus isthmi.) In a previous report 15, regeneration of fibers to the lOT following left nerve crush and right eye enucleation was attributed to the enucleation. It was assumed that the state of degeneration was identical in both optic tracts at the time the nerve reached the chiasm, and therefore fibers were misguided into the IOTr and IOT. The present results suggest that nerve fibers will regenerate to the lOT even when an eye is not removed, making it unlikely that degeneration products profoundly affect the entrance of fibers into the IOTr. When one eye is removed, the regenerating fibers (of the opposite eye) that reach the lOT appear to remain there indefinitely, while when the eye is left intact, their presence in the lOT appears transitory. One explanation for the apparent absence of the anomalous lOT fibers at 32 days post-crush is that they are dispersed within the lOT and are not readily detectable with autoradiography. This interpretation seems unlikely since fibers should have been seen in the IOTr, where they would not be expected to disperse. Alternatively, since the ipsilateral fibers are few in number 15, a progressive diminution in transported labeled protein may render them difficult to visualize. Regenerating fibers transport 8-10 times more labeled protein than do either normal intact fibers or fibers that have completed regenerating 6. A more intriguing explanation for these results is that termination sites were not available in the lOT and, therefore, the aberrant fibers degenerated or retracted. The anomalous fibers remain in the lOT in fish that had one eye removed and the opposite nerve crushed because the enucleation denervated the lOT and numerous terminations were available. Evidence for retraction or degeneration is based on two observations: (a) at 16 days post-crush fibers were only seen in the stratum opticum of the rostral lOT and IOTr, while at 8 days post-crush they were evident throughout the lOT and IOTr, and (b) at 32 days post-crush fibers could not be readily detected in the IOTr. Retraction of optic nerve fibers in frogs is reported to occur when they spontaneously regenerate to the opposite retina following unilateral nerve crush 2. The disappearance of these fibers was also thought to occur because of the unavailability of appropriate sites. It is unlikely that maintaining fish at 30 °C rather than 19 °C caused the
216 anomalous projection since a similar result was mentioned in passing by Meyer ~°. As yet, high temperatures appear to only result in faster regeneration and not in different patterns o f regeneration. Retraction of fibers when termination sites are unavailable is a tenable hypothesis. However, such a p h e n o m e n o n would be inconsistent with several results obtained in manipulations o f the goldfish visual system. F o r example: (a) following unilateral tectal ablation, the interrupted optic nerve fibers innervate the remaining ipsilateral rectum, even when the native optic nerve fibers are left intact 9 and (b) the ipsilateral retinotectal projection displaces significant portions of the existing C O T projection 13. Foreign fibers apparently capture zones that were occupied by the native fibers. Thus it appears that foreign fibers will not necessarily retract f r o m an IOT. It remains to be determined why foreign fibers successfully compete with native fibers in some instances and not in others. Foreign fibers that innervate the I O T following a tectal ablation are likely to be the primary axons o f the retinal ganglion cells. Those fibers that reach the I O T following the crush o f one optic nerve may be collateral branches o f primary axons that project to the COT. Therefore, it is possible that primary axons are more capable of competing for termination zones than are collateral branches. This hypothesis assumes that competition between primary axons and collateral branches will lead to retraction o f the collaterals if the primary axons are already in place. In developing rodents, an ipsilateral projection to the superior colliculus initially occupies several laminae that are also occupied by primary axons f r o m the contralateral eyeL Ipsilaterally projecting retinal fibers that terminate in the superior cotliculus o f rodents appear to be collateral branches 3. Subsequently, the ipsilateral fibers retract and come to occupy a restricted area. The hypothesis also assumes that primary axons f r o m two different origins will compete with one another and fiber segregation, not retraction, will occur4,16. The present results on goldfish optic nerve regeneration are comparable to the observations in the m a m m a l i a n developmental studies, with the i m p o r t a n t difference
Fig. 1. Dark-field photomicrograph of a cross-section through the lOT just caudal to the rostral portion of the valvula cerebelli (V). The fish was killed 8 days following unilateral optic nerve crush. High silver grain densities are most evident at the medial (M) and lateral (L) edges of the tectum. Fig. 2. Enlargement of region shown in Fig. 1. Silver grains are evident in the stratum opticum (SO), stratum fibrosum et griseum superficiale (SFGS), stratum griseum centrale (SGC) and stratum album centrale (SAC). Scale as in Fig. 6. Fig. 3. Cross-section through rostral IOT of a fish killed 16 days after unilateral nerve crush. Silver grain densities were only seen in the stratum opticum (arrow) and optic tract (not shown). Fig. 4. Enlargement of silver grains shown in Fig. 3. This compact row of grains appears to be a fascicle of nerve fibers and could be traced through several sections. Scale same as in Fig. 6. Fig. 5. Cross-section through lOT more caudal to that shown in Fig. 1. The fish received a nerve crush and unilateral enucleation and was killed 32 days after surgery. Silver grains are located throughout the IOT. Fig. 6. Enlargement of region indicated in Fig. 5. Silver grains are densest in several tectal lamina. See Fig. 2 for notations.
I
218 that the results in m a m m a l s indicate retraction from the i n a p p r o p r i a t e l a m i n a of the correct structure, while in fish retraction is observed to occur in fibers that grow to the w r o n g side of the brain. Retraction of nerve fibers from i n a p p r o p r i a t e t e r m i n a t i o n zones could be a general m e c h a n i s m that functions d u r i n g both regeneration and development. I t h a n k Dr. S. S h a r m a for valuable criticism. This research was supported by BNS-7922257 from N S F .
1 Arora, H.L. and Sperry, R.W., Color discrimination after optic nerve regeneration in the fish Astronatus ocellatus, Develop. Biol., 7 (1963) 234-243. 2 Bohn, R.C. and Stelzner, D.J. Aberrant retino-retinal pathway during early stages of regeneration in adult Rana pipiens, Brain Research, 160 (1979) 139-144. 3 Cunningham, T.J., Early eye removal produces excessive bilateral branching in the rat: application of cobalt filling method, Science, 194 (1976) 857-859. 4 Constantine-Paton, M. and Law, M.I., Eye-specific termination bands in tecta of three-eyed frogs, Science, 202 (1978) 639-641. 5 Gaze, R.M., The Formation of Nerve Connections, Academic Press, London, 1970. 6 Grafstein, B. and Murray, M., Transport of protein in goldfish optic nerve during regeneration, Exp. Neurol., 25 (1969) 494-508. 7 Land, P.W. and Lund, R.D., Development of the rat's uncrossed retinotectal pathway and its relation to plasticity studies, Science, 205 (1979) 695-700. 8 Landreth, G.E., Neale, E.A., Neale, J.H., Duff, R.S., Braford, M.R., Northcutt, R.G. and Agranoff, B.W. Evaluation of [aH]proline for radioautographic tracing of axonal projections in the teleost visual system, Brain Research, 91 (1975) 25-42. 9 Levine, R.L. and Jacobson, M., Discontinuous mapping of retina onto tectum innervated by both eyes, Brain Research, 98 (1975) 172-176. 10 Meyer, R.L., Mapping the normal and regenerating retinotectal projection of goldfish with autoradiographic methods, J. comp. Neurol., 189 (1980) 273-289. 11 Neale, J.H., Neale, E.A. and Agranoff, B.W., Radioautography of the optic tectum of the goldfish after intraocular injection of [aH[proline, Science, 176 (1972) 407-410. 12 Sperry, R.W., Restoration of vision after crossing of optic nerves and after contralateral transplantation of eye, J. Neurophysiol., 8 (1945) 15-28. 13 Springer, A.D., Autoradiographic and visual analyses of two types of ipsilateral retinotectal projections of goldfish, Neurosci. Abstr., 5 (1979) 684. 14 Springer, A.D. and Agranoff, B.W., Effect of temperature on rate of goldfish optic nerve regeneration: a radioautographic and behavioral study, Brain Research, 128 (1977) 405-415. 15 Springer, A.D., Heacock, A.M., Schmidt, J.T. and Agranoff, B.W., Bilateral tectal innervation by regenerating optic nerve fibers in goldfish: a radioautographic, electrophysiological and behavioral study, Brain Research, 128 (1977) 417-427. 16 Wiesel, T.N., Hubel, D.H. and Lam, D.M.K., Autoradiographic demonstration of ocular-dominance columns in the monkey striate cortex by means of transneuronal transport, Brain Research, 79 (1974) 273-279.
Brahl Research, 199 (1980) 2 1 4 2 I ~ ) E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
Aberrant regeneration in goldfish after crushing one optic nerve
ALAN
D. S P R I N G E R
Department of Anatomy, New York Medical College, Basic Science Building, Valhalla, N.Y. 10595
(u.s.A.) ( A c c e p t e d J u n e 12th, 1980)
Key word~: g o l d f i s h - - o p t i c n e r v e --- r e g e n e r a t i o n - - a u t o r a d i o g r a p h y
Optic nerve regeneration in mature, lower vertebrates has been examined in numerous studies attempting to elucidate the principles that determine the orderly development of the visual system. Neuroanatomical and electrophysiological techniques have demonstrated that each optic nerve of goldfish innervates the contralateral optic tectum (COT)L Following regeneration, the retinotopic projection that existed prior to nerve section is re-established a and normal vision is restoredl,le, 14. In some situations, an optic nerve will spontaneously regenerate to the ipsilateral optic tectum (IOT), even when the COT is intact 15. When the optic nerve of one eye was crushed at the same time as the other eye was enucleated, most fibers of the remaining nerve regenerated back to the COT. However, a small number of fibers grew back to the denervated IOT and deployed themselves retinotopically. These fibers were evident in the lOT at the longest survival time examined (50 days). One explanation for this spontaneous IOT projection is that removing one eye eliminated nerve fibers from the ipsilateral optic tract (IOTr). The presence of the opposite nerve at the chiasm would have mechanically blocked the regenerating fibers from entering the erroneous IOTr and tectum. To evaluate this hypothesis, the present experiment sought to determine whether optic nerve fibers would regenerate to the lOT when only one nerve was crushed and when the opposite eye was left intact. Common goldfish 5-6 cm in body length were anesthetized and the left optic nerve was crushed behind the eye. They were kept in aerated tanks at 30 °C and separate groups of 2-3 fish were injected intraocularly (left eye) with 25 /zCi of [3H]proline at 3, 7, 15 and 31 days following surgery 15. Additional fish had the right eye removed and the left optic nerve crushed. These fish were injected with [3H]proline into the remaining eye 31 days following surgery. The fish were killed after 24 h of incorporation and their brains were fixed in alcohol-formalin-acetic acid. Paraffin sections (10 /zm) were dipped in Kodak NTB2 emulsion, exposed for 11-28 days, developed in Dektol and stained with hematoxylin-eosin or cresyl violet tS. Silver grain densities above background levels were interpreted as representing the presence of regenerating nerve fibers.
215 Regenerating optic nerve fibers reached the chiasm within 4 days following the crush of one optic nerve. Fibers were not evident in the COT at this time, indicating that the nerve crush was effective. By 8 days post-crush, the medial and lateral portions of the COT were densely labeled 14. Surprisingly, appreciable label was present in the IOTr, as well as throughout the lOT (Fig. 1). Compact rows of silver grains were found in the stratum opticum and in the other tectal laminae known to receive retinal fibersS, 11 (Fig. 2). The label was apparent throughout the rostro-caudal extent of the lOT. Labeling of the lOT was appreciably reduced in the fish examined at 16 days post-crush. Silver grains, possibly reflecting individual fascicles of nerve fibers, were only found in the stratum opticum of the rostral IOT of both fish (Figs. 3 and 4). Fibers were not detected in the lOT or IOTr at 32 days post-crush. In contrast, the lOT was extensively labeled 32 days post-crush (Figs. 5 and 6) in fish that had a right eye enucleation at the time of left optic nerve crush. (Several anomalous contralateral areas were also transiently labeled: nucleus rotundus, pituitary, cerebellum and nucleus isthmi.) In a previous report 15, regeneration of fibers to the lOT following left nerve crush and right eye enucleation was attributed to the enucleation. It was assumed that the state of degeneration was identical in both optic tracts at the time the nerve reached the chiasm, and therefore fibers were misguided into the IOTr and IOT. The present results suggest that nerve fibers will regenerate to the lOT even when an eye is not removed, making it unlikely that degeneration products profoundly affect the entrance of fibers into the IOTr. When one eye is removed, the regenerating fibers (of the opposite eye) that reach the lOT appear to remain there indefinitely, while when the eye is left intact, their presence in the lOT appears transitory. One explanation for the apparent absence of the anomalous lOT fibers at 32 days post-crush is that they are dispersed within the lOT and are not readily detectable with autoradiography. This interpretation seems unlikely since fibers should have been seen in the IOTr, where they would not be expected to disperse. Alternatively, since the ipsilateral fibers are few in number 15, a progressive diminution in transported labeled protein may render them difficult to visualize. Regenerating fibers transport 8-10 times more labeled protein than do either normal intact fibers or fibers that have completed regenerating 6. A more intriguing explanation for these results is that termination sites were not available in the lOT and, therefore, the aberrant fibers degenerated or retracted. The anomalous fibers remain in the lOT in fish that had one eye removed and the opposite nerve crushed because the enucleation denervated the lOT and numerous terminations were available. Evidence for retraction or degeneration is based on two observations: (a) at 16 days post-crush fibers were only seen in the stratum opticum of the rostral lOT and IOTr, while at 8 days post-crush they were evident throughout the lOT and IOTr, and (b) at 32 days post-crush fibers could not be readily detected in the IOTr. Retraction of optic nerve fibers in frogs is reported to occur when they spontaneously regenerate to the opposite retina following unilateral nerve crush 2. The disappearance of these fibers was also thought to occur because of the unavailability of appropriate sites. It is unlikely that maintaining fish at 30 °C rather than 19 °C caused the
216 anomalous projection since a similar result was mentioned in passing by Meyer ~°. As yet, high temperatures appear to only result in faster regeneration and not in different patterns o f regeneration. Retraction of fibers when termination sites are unavailable is a tenable hypothesis. However, such a p h e n o m e n o n would be inconsistent with several results obtained in manipulations o f the goldfish visual system. F o r example: (a) following unilateral tectal ablation, the interrupted optic nerve fibers innervate the remaining ipsilateral rectum, even when the native optic nerve fibers are left intact 9 and (b) the ipsilateral retinotectal projection displaces significant portions of the existing C O T projection 13. Foreign fibers apparently capture zones that were occupied by the native fibers. Thus it appears that foreign fibers will not necessarily retract f r o m an IOT. It remains to be determined why foreign fibers successfully compete with native fibers in some instances and not in others. Foreign fibers that innervate the I O T following a tectal ablation are likely to be the primary axons o f the retinal ganglion cells. Those fibers that reach the I O T following the crush o f one optic nerve may be collateral branches o f primary axons that project to the COT. Therefore, it is possible that primary axons are more capable of competing for termination zones than are collateral branches. This hypothesis assumes that competition between primary axons and collateral branches will lead to retraction o f the collaterals if the primary axons are already in place. In developing rodents, an ipsilateral projection to the superior colliculus initially occupies several laminae that are also occupied by primary axons f r o m the contralateral eyeL Ipsilaterally projecting retinal fibers that terminate in the superior cotliculus o f rodents appear to be collateral branches 3. Subsequently, the ipsilateral fibers retract and come to occupy a restricted area. The hypothesis also assumes that primary axons f r o m two different origins will compete with one another and fiber segregation, not retraction, will occur4,16. The present results on goldfish optic nerve regeneration are comparable to the observations in the m a m m a l i a n developmental studies, with the i m p o r t a n t difference
Fig. 1. Dark-field photomicrograph of a cross-section through the lOT just caudal to the rostral portion of the valvula cerebelli (V). The fish was killed 8 days following unilateral optic nerve crush. High silver grain densities are most evident at the medial (M) and lateral (L) edges of the tectum. Fig. 2. Enlargement of region shown in Fig. 1. Silver grains are evident in the stratum opticum (SO), stratum fibrosum et griseum superficiale (SFGS), stratum griseum centrale (SGC) and stratum album centrale (SAC). Scale as in Fig. 6. Fig. 3. Cross-section through rostral IOT of a fish killed 16 days after unilateral nerve crush. Silver grain densities were only seen in the stratum opticum (arrow) and optic tract (not shown). Fig. 4. Enlargement of silver grains shown in Fig. 3. This compact row of grains appears to be a fascicle of nerve fibers and could be traced through several sections. Scale same as in Fig. 6. Fig. 5. Cross-section through lOT more caudal to that shown in Fig. 1. The fish received a nerve crush and unilateral enucleation and was killed 32 days after surgery. Silver grains are located throughout the IOT. Fig. 6. Enlargement of region indicated in Fig. 5. Silver grains are densest in several tectal lamina. See Fig. 2 for notations.
I
218 that the results in m a m m a l s indicate retraction from the i n a p p r o p r i a t e l a m i n a of the correct structure, while in fish retraction is observed to occur in fibers that grow to the w r o n g side of the brain. Retraction of nerve fibers from i n a p p r o p r i a t e t e r m i n a t i o n zones could be a general m e c h a n i s m that functions d u r i n g both regeneration and development. I t h a n k Dr. S. S h a r m a for valuable criticism. This research was supported by BNS-7922257 from N S F .
1 Arora, H.L. and Sperry, R.W., Color discrimination after optic nerve regeneration in the fish Astronatus ocellatus, Develop. Biol., 7 (1963) 234-243. 2 Bohn, R.C. and Stelzner, D.J. Aberrant retino-retinal pathway during early stages of regeneration in adult Rana pipiens, Brain Research, 160 (1979) 139-144. 3 Cunningham, T.J., Early eye removal produces excessive bilateral branching in the rat: application of cobalt filling method, Science, 194 (1976) 857-859. 4 Constantine-Paton, M. and Law, M.I., Eye-specific termination bands in tecta of three-eyed frogs, Science, 202 (1978) 639-641. 5 Gaze, R.M., The Formation of Nerve Connections, Academic Press, London, 1970. 6 Grafstein, B. and Murray, M., Transport of protein in goldfish optic nerve during regeneration, Exp. Neurol., 25 (1969) 494-508. 7 Land, P.W. and Lund, R.D., Development of the rat's uncrossed retinotectal pathway and its relation to plasticity studies, Science, 205 (1979) 695-700. 8 Landreth, G.E., Neale, E.A., Neale, J.H., Duff, R.S., Braford, M.R., Northcutt, R.G. and Agranoff, B.W. Evaluation of [aH]proline for radioautographic tracing of axonal projections in the teleost visual system, Brain Research, 91 (1975) 25-42. 9 Levine, R.L. and Jacobson, M., Discontinuous mapping of retina onto tectum innervated by both eyes, Brain Research, 98 (1975) 172-176. 10 Meyer, R.L., Mapping the normal and regenerating retinotectal projection of goldfish with autoradiographic methods, J. comp. Neurol., 189 (1980) 273-289. 11 Neale, J.H., Neale, E.A. and Agranoff, B.W., Radioautography of the optic tectum of the goldfish after intraocular injection of [aH[proline, Science, 176 (1972) 407-410. 12 Sperry, R.W., Restoration of vision after crossing of optic nerves and after contralateral transplantation of eye, J. Neurophysiol., 8 (1945) 15-28. 13 Springer, A.D., Autoradiographic and visual analyses of two types of ipsilateral retinotectal projections of goldfish, Neurosci. Abstr., 5 (1979) 684. 14 Springer, A.D. and Agranoff, B.W., Effect of temperature on rate of goldfish optic nerve regeneration: a radioautographic and behavioral study, Brain Research, 128 (1977) 405-415. 15 Springer, A.D., Heacock, A.M., Schmidt, J.T. and Agranoff, B.W., Bilateral tectal innervation by regenerating optic nerve fibers in goldfish: a radioautographic, electrophysiological and behavioral study, Brain Research, 128 (1977) 417-427. 16 Wiesel, T.N., Hubel, D.H. and Lam, D.M.K., Autoradiographic demonstration of ocular-dominance columns in the monkey striate cortex by means of transneuronal transport, Brain Research, 79 (1974) 273-279.