382
Brain Research, 168 (1979) 382-387 (~ Elsevier/North-HollandBiomedical Press
Evidence of collateral sprouting in the frog visual system
DENNIS J. STELZNER Department of Anatomy, Upstate Medical Center, Syracuse, N. Y. 13210 (U.S.A.)
(Accepted January 25th, 1979)
Regeneration of injured axons within the central nervous system (CNS) of mammals is usually abortive. It has been proposed that axonal regeneration fails because non-specific connections are made by regenerating axons on denervated synaptic sites in the zone of injury (regenerative sprouting hypothesis). These abnormal connections interfere with further regenerative attempts 2. A second but related hypothesis is that this non-specificity is a general property of the mammalian CNS and, after injury, denervated synaptic sites are filled abnormally by nearby intact axons. Since they occupy the previously denervated sites, these non-specific collateral sprouts could also interfere with regeneration by shutting offthe stimulus to regenerate (collateral sprouting hypothesis)11,z2. We have been testing these hypotheses in the amphibian visual system. Under most circumstances, amphibian optic axons regenerate along their original pathways and reconnect with their normal targets in the same topographical order that existed prior to optic nerve damage5,7,s,17,29. The first question we asked was whether regenerating optic axons would innervate inappropriate regions that lie in close proximity to normal projection sites of the optic tract if these non-optic zones were previously deafferented. If this occurred and regeneration to normal targets was impaired, the regenerative sprouting hypothesis cited above would be supported. However, we found no evidence of abnormal reinnervation in this experiment and regeneration was succesful 4. This is further evidence for the remarkable specificity of regenerating frog optic axons for their normal targets. In the present experiment we tested whether collateral sprouting of uninjured optic tract axons from one eye would take place within optic tract projection zones if the other eye was enucleated. We did find evidence of collateral sprouting. However, it was delayed past the time when optic axons would have regenerated back to their appropriate targets if, instead of enucleation, the optic nerve had been crushed. The right eye of 17 adult northern variety Rana pipiens was removed after severing the extraocular muscles and cutting the optic nerve in the orbit. The left eye of each frog was injected with 4 #1 of [aH]proline (10 #Ci/#l) at times varying from 1 week to 6 months after the other eye had been enucleated. Nine unoperated frogs also received intravitreal injections of proline and served as controls. These animals were
;i ,~ i. 4f; !~ii ~
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•
,
~ ~i ~i~ ~ ~}{!~i'i,~
384 killed either 24 h (n = 21) or 7 days (n = 5) after injection by vascular perfusion of 0 . 6 ~ saline followed by 10~o neutral buffered formal-saline. Brains were removed from the cranium, embedded in egg yolk and frozen sectioned coronally at 20 #m. Four series of serial sections 80 #m apart were coated with Kodak NTB-2 emulsion, exposed for 2, 4, 6 and 8 weeks at 4 °C, developed in D-19 at 12 °C and stained with cresyl violet. The distribution of labelled optic tract axons and endings in contralateral visual targets of control frogs was similar to that described previously 24. In all control animals ipsilateral projections to diencephalic targets overlapped the contralateral projection zones but did not fill the entire extent of the area to which the contralateral retina projected. For instance, in the anterior third of the corpus geniculatum thalamicum (CGT) patches of label were found only in a dorsomedial region of the neuropil and in a zone along its base (compare Figs. 4 and 5). More posteriorly in CGT the ipsilateral projection was restricted to the medial edge of the neuropil. A direct projection was found going to the rostral pole and lateral wall of the ipsilateral optic tectum but no labelled axons were found in the medial portion of the ipsilateral rectum (Fig. 1). This direct ipsilateral retinotectal projection was limited to a band in the superficial third of the stratum griseum centrale. Little difference from the normal distribution of labelled optic tract axons was found in the optic tectum during the first 3 weeks after eye removal when the intact eye of enucleated frogs was injected with [aH]proline (n = 5). By one month post-enucleation there was evidence of abnormal labelling in the ipsilateral optic tectum (n ~ 2) which increased in distribution and density with increasing time after enucleation (n 10). By 6 months postenucleation labelled axons could be traced across the tectal midline for 200-250/~m and filled the superficial layers of the ipsilateral optic tectum (Fig. 2). This abnormal projection was found only where the tectal hemispheres were joined anatomically at the midline; no abnormal projection was found in the anterior pole of the tectum where the two hemispheres were separated by other areas of the brain. This suggests that retinal axons reached the ipsilateral tectum by sprouting across the midline. This idea is further supported by our finding no increase in the amount of label in the ipsilateral optic tract to correspond with the abnormal ipsilateral tectal projection. We also found evidence suggesting that sprouting of the normal ipsilateral projection had occurred. The band of label found in the anterior pole and lateral wall of the ipsilateral tectum in normal frogs was increased in density and it spread dorsomedially in enucleated animals. Collateral sprouting was also found within the thalamus of chronic enucleated frogs (3-6 months postenucleation). For instance, the intact retina projected ipsilaterally to fill the entire CGT with the exception of its lateral edge (Fig. 3). In these animals the CGT was noticeably smaller on the side of the brain deafferented of contralateral retinal input. This raises the possibility that the change in distribution of label was due to shrinkage of the CGT. However, most of the shrinkage in the CGT had already occurred one month postenucleation before labelled axons were found throughout the CGT. Thus, the spread of label in chronic enucleated frogs must be due to intact retinal axons sprouting throughout the extent of the deafferented CGT.
385 These results indicate that frog retinal axons will sprout into adjacent deafferented regions within the ipsilateral optic tectum and corpus geniculatum thalamicum. The sprouted retinal axons remain within normal optic tract projection zones but they extend outside their normal zones of projection in each region. Previous attempts to find abnormal growth of remaining retinal axons after eye removal in amphibians have been negative. No abnormal projections were found in Xenopus laevis after enucleation at pre- or postmetamorphic stages of development using electrophysiologicalI or degeneration methods (Lund and Gaze, unpublished observations). The difference in developmental age and species compared with the present experiment may be partly responsible for these negative results. However, the use of anterograde transport of [SH]proline coupled with autoradiography appears to give greater resolution for tracing regenerated or sprouted neural connections than either electrophysiology or the degeneration methods. In previous work we were unable to detect the abnormal connections from the remaining eye of chronic enucleated frogs found in the present experiment using a modified Fink-Heimer method (Kicliter and Stelzner, unpublished observations). In addition, abnormal ipsilateral retinotectal connections are found after optic nerve regeneration in Xenopus laevis 10 or Ranapipiens30, al using the anterograde transport method even though these connections are difficult to find electrophysiologically10. The abnormal growth found in the present experiment begins much later after neural injury than does axonal sprouting in the CNS of mammals16,19,20,23,a2. In addition it does not begin until after the time at which regenerating optic tract axons in the frog reach their targetsa,7,s,17,29 even under conditions where the age of the animal, lighting conditions and water temperature were identical to the present experimental. The slow time course of sprouting may be one of the factors allowing regeneration to take place in this species. Norden et al. 21 presented evidence supporting the idea that collateral sprouting does not interfere with regeneration in the frog visual system. These investigators found no evidence of collateral sprouting of intrinsic connections within the amphibian optic rectum after enucleation. Previous studies of optic nerve regeneration in the normal frog and in frogs with unilateral tectal removal show that regenerating axons will make abnormal connections ipsilaterally in the optic tectum. These ipsilateral connections overlap or displace a portion of the normal contralateral connections from the other retina which already fill the retinal synaptic siteslO,l~,14,1~As,30. These results raise the possibility that, besides the slow time course and limited amount of collateral sprouting, optic axons regenerate successfully because they are able to either induce the formation of new synaptic sites or share space with normal intact endings. A number of reports have shown that abnormally compressed or expanded retinal connections will form in the optic tectum as a result of partial retinal or tectal ablations in fish and amphibiansS,9,13,eB,27,z3,35,a6. However, these abnormal connections only form gradually; often the normal projection is first re-establishedn,e,26,27,z4. In certain instances there is immediate compression after a partial tectal lesion if reinnervation of the tectum is delayed2S,a4. One explanation of these results suggests a change in the properties of retinal or tectal neurons which does not take place until
386 some time after portions of the retina or tectum have been removed 2~. A second possibility is that there is no compression until degenerating retinal axons remaining from the initial lesion are removed ~s. Since the remaining regenerating and intact optic tract axons appear to interact with one another in forming these compressed and expanded projectionsS,n,13, 3a, a final possibility is that these axonal interactions are not immediate and occur over a long time course. The results of the present experiment indicate a slow expansion of remaining retinal connections in the normal optic tract projection zones of the frog brain which does not begin until one month after deafferentation of retinal axons from the other eye. The similarities in the time course between the result of this experiment and the results of experiments in which portions of the tectum or retina were ablated suggests that a similar mechanism may be operating in both situations to cause or to allow retinal axons to slowly spread or to be compressed within projection zones of the optic tract. Previous work shows that damaged optic tract axons do not sprout to abnormal denervated sites during optic nerve regeneration in the frog (regenerative sprouting hypothesis) 4. The present experiment shows that abnormal growth of intact axons to sites denervated of optic tract axons does not take place over a time course that is likely to interfere with optic nerve regeneration (collateral sprouting hypothesis). In fact, because of the slow time course of axonal sprouting in the frog, the factors which induce sprouting may be quite different in mammals and amphibians. Since both regenerative and collateral sprouting occur in the mammalian CNS where axonal regeneration is abortive, it is possible that the lack of axonal sprouting found during optic nerve regeneration in the frog is related to the success of axonai regeneration. I would like to thank Dr. J. A. Horel, Dr. E. D. Weber, Mr. R. C. Bohn and Ms. D. R. Bernstein for critical reading of this manuscript, Ms. Judith A. Strauss for expert technical and photographic assistance and Ms. Nancy Wood for preparation of the manuscript. Supported by N I H Grant NS 14096.
1 Beazley, L. D., Factors determining decussation at the optic chiasma by developing retinotectal fibres in Xenopus, Exp. Brain Res., 23 (1975) 491-504. 2 Bernstein, J. J. and Bernstein, M. E., Neuronal alteration and reinnervation following axonal regeneration and sprouting in mammalian spinal cord, Brain Behav. EvoL, 8 (1973) 135-161. 3 Bohn, R. C. and Stelzner, D. J., Aberrant retinoretinal pathway during early stages of regeneration in adult Rana pipiens, Brain Research, 160 (1979) 139-144. 4 Bohn, R. C. and Stelzner, D. J., Retention of specificity for appropriate synaptic sites by regenerating frog optic axons, Neurosci. Abstr., 4 (1978) 529. 5 Cook, J. E. and Horder, T. J., The multiple factors determining retinotopic order in the growth of optic fibres into the optic tectum, Phil. Trans. B., 278 (1977) 261-276. 6 Cook, J. E. and Horder, T. J., Interactions between optic fibres in their regeneration to specific sites in the goldfish tectum, J. PhysioL (Lond.), 241 (1974) 89-90P. 7 Gaze, R. M. and Jacobson, M., A study of the retino-tectal projection during regeneration of the optic nerve in the frog, Proc. roy. Soc. B, 157 (1963) 420-448. 8 Gaze, R. M. and Keating, M. J., Further studies on the restoration of the contralateral retinotectal projection following regeneration of the optic nerve in the frog, Brain Research, 21 (1970) 183-195.
387 9 Gaze, R. M. and Sharma, S. C., Axial differences in the reinnervation of the goldfish optic tectum by regenerating optic nerve fibers, Exp. Brain Res., l0 0970) 171-181. 10 Glastonbury, J. and Straznicky, K., Aberrant ipsilateral retinotectal projection following optic nerve section in Xenopus, Neurosci. Lett., 7 (1978) 67-72. l l Guth, L. and Windle, W. F., The enigma of central nervous system regeneration, Exp. Neurol., Suppl., 5 (1970) 1-43. 12 Ingle, D., Two visual systems in the frog, Science, 181 (1973) 1053-1055. 13 Ingle, D. and Dudek, A., Aberrant retinotectal projections in the frog, Exp. NeuroL, 55 0977) 567-582. 14 Kicliter, E., Misantone, L. J. and Stelzner, D. J., Neuronal specificity and plasticity in frog visual system: anatomical correlates, Brain Research, 82 (1974) 293-297. 15 Law, M. I. and Constantine-Paton, M., Alternating retinal ganglion cell termination bands in doubly innervated frog optic tecta, Neurosci. Abstr., 4 (1978) 118. 16 Matthews, D. A., Cotman, C. W. and Lynch, G. S., An electron microscopic study of lesioninduced synaptogenesis in the dentate gyrus of the adult rat. II. Reapl~earance of morphologically normal synaptic contacts, Brain Research, I 15 (1976) 23-41. 17 Maturana, H. R., Lettvin, J. Y., McCulloch, W. S. and Pitts, W. H., Evidence that cut optic nerve fibers in a frog regenerate to their proper places in the tectum, Science, 130 (1959) 1709-1710. 18 Misantone, L. J. and Stelzner, D. J., Behavioral manifestations of competition of retinal endings for sites in doubly innervated frog optic tectum, Exp. Neurol., 45 (1974) 364-376. 19 Murray, M. and Goldberger, M. E., Restitution of function and collateral sprouting in the cat spinal cord: the partially hemisected animal, J. comp. Neurol., 158 (1974) 19-36. 20 Nadler, J. V., Cotman, C. W. and Lynch, G. S., Histochemical evidence of altered development of cholinergic fibers in the rat following lesions. I. Time course after complete unilateral entorhinal lesion at various ages, J. comp. Neurol., 171 (1977) 561-588. 21 Norden, J. J., Ostberg, A.-J. C. and Freeman, J. A., Descriptive and quantitative EM studies of the optic tectum of Xenopus following enucleation, NeuroscL Abstr., 4 (1978) 639. 22 Raisman, G., Neuronal plasticity in the septal nuclei of the adult rat, Brain Research, 14 0969) 25-48. 23 Raisman, G. and Field, P. M., A quantitative investigation of the development of collateral reinnervation after partial deafferentation of the septal nuclei, Brain Research, 50 (1973) 241-264. 24 Scalia, F., The optic pathway of the frog: nuclear organization and connections. In R. Llin~lsand W. Precht (Eds.), Frog Neurobiology, Springer-Verlag, New York, 1976, pp. 386-404. 25 Schmidt, J. T., Cicerone, C. M. and Easter, S. S., Expansion of the half retina projection to the tectum in goldfish: an electrophysiological and anatomical study, J. comp. NeuroL, 177 (1978) 257-278. 26 Sharma, S. C., Redistribution of visual projection in altered optic tectum of goldfish, Proc. nat. Acad. Sci. (Wash.), 69 (1972) 2637-2639. 27 Sharma, S. C., Visual projection in surgically created 'compound' tectum in adult goldfish, Brain Research, 93 (1975) 497-501. 28 Sharma, S. C. and Romeskie, M., Immediate 'compression' of the goldfish retinal projection to a tectum devoid of degenerating debris, Brain Research, 133 (1977) 367-370. 29 Sperry, R. W., Optic nerve regeneration with return of vision in Anurans, J. NeurophysioL, 7 (1944) 57-70. 30 Stelzner, D. J., An autoradiographic analysis of ipsilateral retinal projections of the intact or regenerated optic nerve of Rana pipiens and the effect of enucleation of the opposite eye, Neurosci. Abstr., 2 (1976) 817. 31 Stelzner, D. J. and Bohn, R. C.,Time course in the formation of abnormal ipsilateral projections by regenerating optic axons in Rana pipiens, Anat. Rec., in press. 32 Steward, O., Cotman, C. W. and Lynch, G. S., A quantitative autoradiographic and electrophysiological study of the reinnervation of the dentate gyrus by the contralateral entorhinal cortex following ipsilateral entorhinal lesions, Brain Research, 114 (1976) 181-200. 33 Udin, S. B., Rearrangements of the retinotectal projection in Rana pipiens after unilateral caudal half-tectum ablation, J. comp. NeuroL, 173 (1977) 561-583. 34 Yoon, M., Progress of topographic regulation of the visual projection in the halved optic tectum of adult goldfish, J. Physiol. (Lond.), 257 (1976) 621-643. 35 Yoon, M., Reorganization of the retinotectal projections following surgical operations on the optic tectum in goldfish, Exp. NeuroL, 33 (1971) 395-411. 36 Yoon, M., Reversibility of the reorganization of retinotectal projection in goldfish, Exp. NeuroL, 35 (1972) 565-577.
Brain Research, 168 (1979) 382-387 (~ Elsevier/North-HollandBiomedical Press
Evidence of collateral sprouting in the frog visual system
DENNIS J. STELZNER Department of Anatomy, Upstate Medical Center, Syracuse, N. Y. 13210 (U.S.A.)
(Accepted January 25th, 1979)
Regeneration of injured axons within the central nervous system (CNS) of mammals is usually abortive. It has been proposed that axonal regeneration fails because non-specific connections are made by regenerating axons on denervated synaptic sites in the zone of injury (regenerative sprouting hypothesis). These abnormal connections interfere with further regenerative attempts 2. A second but related hypothesis is that this non-specificity is a general property of the mammalian CNS and, after injury, denervated synaptic sites are filled abnormally by nearby intact axons. Since they occupy the previously denervated sites, these non-specific collateral sprouts could also interfere with regeneration by shutting offthe stimulus to regenerate (collateral sprouting hypothesis)11,z2. We have been testing these hypotheses in the amphibian visual system. Under most circumstances, amphibian optic axons regenerate along their original pathways and reconnect with their normal targets in the same topographical order that existed prior to optic nerve damage5,7,s,17,29. The first question we asked was whether regenerating optic axons would innervate inappropriate regions that lie in close proximity to normal projection sites of the optic tract if these non-optic zones were previously deafferented. If this occurred and regeneration to normal targets was impaired, the regenerative sprouting hypothesis cited above would be supported. However, we found no evidence of abnormal reinnervation in this experiment and regeneration was succesful 4. This is further evidence for the remarkable specificity of regenerating frog optic axons for their normal targets. In the present experiment we tested whether collateral sprouting of uninjured optic tract axons from one eye would take place within optic tract projection zones if the other eye was enucleated. We did find evidence of collateral sprouting. However, it was delayed past the time when optic axons would have regenerated back to their appropriate targets if, instead of enucleation, the optic nerve had been crushed. The right eye of 17 adult northern variety Rana pipiens was removed after severing the extraocular muscles and cutting the optic nerve in the orbit. The left eye of each frog was injected with 4 #1 of [aH]proline (10 #Ci/#l) at times varying from 1 week to 6 months after the other eye had been enucleated. Nine unoperated frogs also received intravitreal injections of proline and served as controls. These animals were
;i ,~ i. 4f; !~ii ~
,i ~ ~ t~ ~'¸ ~
•
,
~ ~i ~i~ ~ ~}{!~i'i,~
384 killed either 24 h (n = 21) or 7 days (n = 5) after injection by vascular perfusion of 0 . 6 ~ saline followed by 10~o neutral buffered formal-saline. Brains were removed from the cranium, embedded in egg yolk and frozen sectioned coronally at 20 #m. Four series of serial sections 80 #m apart were coated with Kodak NTB-2 emulsion, exposed for 2, 4, 6 and 8 weeks at 4 °C, developed in D-19 at 12 °C and stained with cresyl violet. The distribution of labelled optic tract axons and endings in contralateral visual targets of control frogs was similar to that described previously 24. In all control animals ipsilateral projections to diencephalic targets overlapped the contralateral projection zones but did not fill the entire extent of the area to which the contralateral retina projected. For instance, in the anterior third of the corpus geniculatum thalamicum (CGT) patches of label were found only in a dorsomedial region of the neuropil and in a zone along its base (compare Figs. 4 and 5). More posteriorly in CGT the ipsilateral projection was restricted to the medial edge of the neuropil. A direct projection was found going to the rostral pole and lateral wall of the ipsilateral optic tectum but no labelled axons were found in the medial portion of the ipsilateral rectum (Fig. 1). This direct ipsilateral retinotectal projection was limited to a band in the superficial third of the stratum griseum centrale. Little difference from the normal distribution of labelled optic tract axons was found in the optic tectum during the first 3 weeks after eye removal when the intact eye of enucleated frogs was injected with [aH]proline (n = 5). By one month post-enucleation there was evidence of abnormal labelling in the ipsilateral optic tectum (n ~ 2) which increased in distribution and density with increasing time after enucleation (n 10). By 6 months postenucleation labelled axons could be traced across the tectal midline for 200-250/~m and filled the superficial layers of the ipsilateral optic tectum (Fig. 2). This abnormal projection was found only where the tectal hemispheres were joined anatomically at the midline; no abnormal projection was found in the anterior pole of the tectum where the two hemispheres were separated by other areas of the brain. This suggests that retinal axons reached the ipsilateral tectum by sprouting across the midline. This idea is further supported by our finding no increase in the amount of label in the ipsilateral optic tract to correspond with the abnormal ipsilateral tectal projection. We also found evidence suggesting that sprouting of the normal ipsilateral projection had occurred. The band of label found in the anterior pole and lateral wall of the ipsilateral tectum in normal frogs was increased in density and it spread dorsomedially in enucleated animals. Collateral sprouting was also found within the thalamus of chronic enucleated frogs (3-6 months postenucleation). For instance, the intact retina projected ipsilaterally to fill the entire CGT with the exception of its lateral edge (Fig. 3). In these animals the CGT was noticeably smaller on the side of the brain deafferented of contralateral retinal input. This raises the possibility that the change in distribution of label was due to shrinkage of the CGT. However, most of the shrinkage in the CGT had already occurred one month postenucleation before labelled axons were found throughout the CGT. Thus, the spread of label in chronic enucleated frogs must be due to intact retinal axons sprouting throughout the extent of the deafferented CGT.
385 These results indicate that frog retinal axons will sprout into adjacent deafferented regions within the ipsilateral optic tectum and corpus geniculatum thalamicum. The sprouted retinal axons remain within normal optic tract projection zones but they extend outside their normal zones of projection in each region. Previous attempts to find abnormal growth of remaining retinal axons after eye removal in amphibians have been negative. No abnormal projections were found in Xenopus laevis after enucleation at pre- or postmetamorphic stages of development using electrophysiologicalI or degeneration methods (Lund and Gaze, unpublished observations). The difference in developmental age and species compared with the present experiment may be partly responsible for these negative results. However, the use of anterograde transport of [SH]proline coupled with autoradiography appears to give greater resolution for tracing regenerated or sprouted neural connections than either electrophysiology or the degeneration methods. In previous work we were unable to detect the abnormal connections from the remaining eye of chronic enucleated frogs found in the present experiment using a modified Fink-Heimer method (Kicliter and Stelzner, unpublished observations). In addition, abnormal ipsilateral retinotectal connections are found after optic nerve regeneration in Xenopus laevis 10 or Ranapipiens30, al using the anterograde transport method even though these connections are difficult to find electrophysiologically10. The abnormal growth found in the present experiment begins much later after neural injury than does axonal sprouting in the CNS of mammals16,19,20,23,a2. In addition it does not begin until after the time at which regenerating optic tract axons in the frog reach their targetsa,7,s,17,29 even under conditions where the age of the animal, lighting conditions and water temperature were identical to the present experimental. The slow time course of sprouting may be one of the factors allowing regeneration to take place in this species. Norden et al. 21 presented evidence supporting the idea that collateral sprouting does not interfere with regeneration in the frog visual system. These investigators found no evidence of collateral sprouting of intrinsic connections within the amphibian optic rectum after enucleation. Previous studies of optic nerve regeneration in the normal frog and in frogs with unilateral tectal removal show that regenerating axons will make abnormal connections ipsilaterally in the optic tectum. These ipsilateral connections overlap or displace a portion of the normal contralateral connections from the other retina which already fill the retinal synaptic siteslO,l~,14,1~As,30. These results raise the possibility that, besides the slow time course and limited amount of collateral sprouting, optic axons regenerate successfully because they are able to either induce the formation of new synaptic sites or share space with normal intact endings. A number of reports have shown that abnormally compressed or expanded retinal connections will form in the optic tectum as a result of partial retinal or tectal ablations in fish and amphibiansS,9,13,eB,27,z3,35,a6. However, these abnormal connections only form gradually; often the normal projection is first re-establishedn,e,26,27,z4. In certain instances there is immediate compression after a partial tectal lesion if reinnervation of the tectum is delayed2S,a4. One explanation of these results suggests a change in the properties of retinal or tectal neurons which does not take place until
386 some time after portions of the retina or tectum have been removed 2~. A second possibility is that there is no compression until degenerating retinal axons remaining from the initial lesion are removed ~s. Since the remaining regenerating and intact optic tract axons appear to interact with one another in forming these compressed and expanded projectionsS,n,13, 3a, a final possibility is that these axonal interactions are not immediate and occur over a long time course. The results of the present experiment indicate a slow expansion of remaining retinal connections in the normal optic tract projection zones of the frog brain which does not begin until one month after deafferentation of retinal axons from the other eye. The similarities in the time course between the result of this experiment and the results of experiments in which portions of the tectum or retina were ablated suggests that a similar mechanism may be operating in both situations to cause or to allow retinal axons to slowly spread or to be compressed within projection zones of the optic tract. Previous work shows that damaged optic tract axons do not sprout to abnormal denervated sites during optic nerve regeneration in the frog (regenerative sprouting hypothesis) 4. The present experiment shows that abnormal growth of intact axons to sites denervated of optic tract axons does not take place over a time course that is likely to interfere with optic nerve regeneration (collateral sprouting hypothesis). In fact, because of the slow time course of axonal sprouting in the frog, the factors which induce sprouting may be quite different in mammals and amphibians. Since both regenerative and collateral sprouting occur in the mammalian CNS where axonal regeneration is abortive, it is possible that the lack of axonal sprouting found during optic nerve regeneration in the frog is related to the success of axonai regeneration. I would like to thank Dr. J. A. Horel, Dr. E. D. Weber, Mr. R. C. Bohn and Ms. D. R. Bernstein for critical reading of this manuscript, Ms. Judith A. Strauss for expert technical and photographic assistance and Ms. Nancy Wood for preparation of the manuscript. Supported by N I H Grant NS 14096.
1 Beazley, L. D., Factors determining decussation at the optic chiasma by developing retinotectal fibres in Xenopus, Exp. Brain Res., 23 (1975) 491-504. 2 Bernstein, J. J. and Bernstein, M. E., Neuronal alteration and reinnervation following axonal regeneration and sprouting in mammalian spinal cord, Brain Behav. EvoL, 8 (1973) 135-161. 3 Bohn, R. C. and Stelzner, D. J., Aberrant retinoretinal pathway during early stages of regeneration in adult Rana pipiens, Brain Research, 160 (1979) 139-144. 4 Bohn, R. C. and Stelzner, D. J., Retention of specificity for appropriate synaptic sites by regenerating frog optic axons, Neurosci. Abstr., 4 (1978) 529. 5 Cook, J. E. and Horder, T. J., The multiple factors determining retinotopic order in the growth of optic fibres into the optic tectum, Phil. Trans. B., 278 (1977) 261-276. 6 Cook, J. E. and Horder, T. J., Interactions between optic fibres in their regeneration to specific sites in the goldfish tectum, J. PhysioL (Lond.), 241 (1974) 89-90P. 7 Gaze, R. M. and Jacobson, M., A study of the retino-tectal projection during regeneration of the optic nerve in the frog, Proc. roy. Soc. B, 157 (1963) 420-448. 8 Gaze, R. M. and Keating, M. J., Further studies on the restoration of the contralateral retinotectal projection following regeneration of the optic nerve in the frog, Brain Research, 21 (1970) 183-195.
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