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Aberrant retinotectal pathways induced by larval unilateral optic nerve section in Xenopus
Neuroscience Letters, 18 (1980) 137-142 © Elsevier/North-Holland Scientific Publishers Ltd.
137
ABERRANT RETINOTECTAL PATHWAYS INDUCED BY LARVAL UNILATERAL OPTIC NERVE SECTION IN XENOPUS
DAVID TAY and CHARLES STRAZNICI(Y Centre for Neuroscience and Department of Human Morphology, School of Medicine, Flinders University of South Australia, Bedford Park, S.A. 5042 (Australia) (Received March 19th, 1980) (Accepted March 24th, 1980)
SUMMARY
Following unilateral optic nerve section in Xenopus tadpoles and toadlets, the reformation of visual pathways and retinotectal projections was analyzed using [3H]proline autoradiography. Bilateral retinotectal projections were found in 61°70 of the animals operated on between stages 42 and 58. In these animals, the projections were established through multiple aberrant pathways which included the oculomotor nerve, the trigeminal nerve and the posterior commissure. In contrast, in animals with optic nerve section at stage 62 or after metamorphosis, the regenerated optic fibres arrived at the contralateral and ipsilateral tecta through normal visual pathways.
In normal frogs the retina projects topographically to the contralateral optic tectum and to both the contralateral and the ipsilateral diencephalic visual centres [4, 11, 14]. Following unilateral optic nerve section in post-metamorphic Xenopus, regeneration occurs and the contralateral retinotectal projection is restored [6]. Recent observations on the optic nerve regeneration in post-metamorphic Xenopus have shown both morphologically and electrophysiologically, that regenerated optic fibres not only re-established the contralateral retinotectal projection but also regularly innervated the ipsilateral tectum [7, 15, 16]. In contrast, optic nerve regeneration studies in mid- and late-larval Xenopus tadpoles failed to demonstrate bilateral retinotectal projections [5]. In order to resolve the apparent disparity between the results of larval and metamorphic optic nerve regeneration, a time series experiment was carried out in animals ranging from early larval stages to after metamorphosis. It is shown that, following optic nerve section, regular bilateral tectal projections via normal visual pathways can be induced only from the time of metamorphic climax.
138 Xenopus tadpoles at various stages of development ranging from stage 42 [12] to metamorphosis and juvenile toads were used. Operations were performed under MS222 (tricaine methane sulphonate, Sandoz) anaesthesia. In young tadpoles the right optic nerve was approached from the dorsal surface of the head, exposed and cut with a sharp tungsten needle about 1-2 mm behind the eye ball. In stage 62 tadpoles and post-metamorphic Xenopus the optic nerve was reached through the pharynx and sectioned proximal to the optic chiasma as described in previous studies [7, 15]. Tadpoles were reared to metamorphosis and beyond while the postmetamorphic toadlets were kept for 20-180 days after the surgery. Twenty-four hours prior to the sacrifice, 5 ~Ci k-[5-3H]proline (specific activity 23 Ci/mmol, Amersham) was injected into the right eye. The brain was fixed in Bouins and embedded in paraffin. Transverse serial sections were cut at 10 tzm and processed for autoradiography [13]. Deparaffinized sections were coated with Ilford K2 nuclear emulsion, exposed for 14 days in light-tight boxes at 4°C, developed in Kodak D19 and counterstained with Harris's haematoxylin. The pathways taken by the regenerating optic fibres and their tectal terminations were reconstructed by camera lucida drawings of every eighth section of the autoradiographs. Forty-six animals with successful optic nerve regeneration were included in this report (Table I). Nine animals were discarded owing to poor regeneration or to lack of optic nerve formation. In 14 animals (30°7o) regenerated optic fibres grew back to the contralateral tectum, while in the remaining animals (7007o) optic nerve regeneration included both the contralateral and the ipsilateral tecta. In the former the contralateral retinotectal pathway was normal except that regenerated fibres were confined to the margin of the optic tract in the diencephalon, confirming the results of previous observations [5]. In contrast, bilateral retinotectal pathways to TABLE I T H E RESULTS OF O P T I C FIBRE R E G E N E R A T I O N IN A N I M A L S W I T H R I G H T O P T I C NERVE SECTION A T VARIOUS D E V E L O P M E N T A L STAGES W A M and M A M denote weeks and months after metamorphosis, respectively. Retinotectal projection Time of optic nerve section Unilateral
Bilateral
Stage 42 Stage 50 Stage 52
2 3
l 2 15
Stage 55 Stage 58
1 6
Stage 62 2 WAM 3 MAM
2
2 2 9
139 both the contralateral and the ipsilateral tecta were formed through a variety of abnormal fibre tracts. Between stages 42 and 52, bilateral optic nerve regeneration occurred in the majority of animals (Table I). Regenerated optic fibres in these animals entered the brain through the oculomotor (Fig. 1C) and trigeminal nerve roots (Fig. ID). In some animals the bulk of the regenerated optic fibres entered the brain via the optic nerve as well as the oculomotor or trigeminal nerve roots. Detailed analysis of the aberrant visual pathways revealed that the majority of the regenerated optic fibres crossed over the midline in the diencephalon or in the
D Fig. 1. Bright-field photographs of transverse brain sections from autoradiographs. Right is on the left and dorsal is at the top. Bars represent 500 #m. A: section across the mid-diencephalon in an animal with post-metamorphic optic nerve section. Arrows point at the courses of regenerating optic fibre bundles along the lateral margin of the diencephalon. Arrowhead points at the optic nerve through which the regenerating optic fibres enter the brain. B: section at the level of posterior commissure in an animal with stage 50 optic nerve section. Arrow indicates the position of recrossing optic fibres to the ipsilateral side. Note that the ipsilateral tectum is not evenly innervated. C: section across the mid-tectum in an animal with stage 50 optic nerve section. The a n o m a l o u s entry of regenerated optic fibres through the oculomotor nerve root (arrowhead) and their rostromedial course (arrow) are indicated. D: section at the level of the posterior tectal poles in an animal with stage 52 optic nerve section. Arrowhead indicates the entry of regenerated optic fibres to the brain via the trigeminal nerve root.
140 tegmentum to innervate the contralateral tectum, while a small bundle of such fibres reached the tectum ipsilaterally (Fig. 1B, C). In all cases, the optic fibres coursed dorsally towards the contralateral and ipsilateral pretectal areas and entered the tectum through the dorsomedial and ventrolateral brachia of the optic tract (Fig. 2B, C). Six animals of this group showed, in addition to the multiple entries to the brain, that a portion of the regenerated optic fibres arrived from the contralateral side to the ipsilateral tectum through the posterior commissure (Figs. 1B and 2A). Following optic nerve section at mid-larval stages (stages 55-58), only one out of eight animals formed bilateral retinotectal projections. In this animal, a portion of the regenerated optic fibres were guided from the optic chiasma to the ipsilateral tectum by the retinal fibres from the other eye. In the other 7 animals, the restored contralateral retinotectal projection involved normal optic fibre trajectory. In 13 out of the 15 animals where the optic nerve was sectioned at stage 62 or later (Table I), both contralateral and ipsilateral retinotectal projections developed. In the two remaining animals only contralateral regeneration was found. Irrespective of unilateral or bilateral tectal innervations in these animals, the regenerated optic fibres followed the course of normal visual pathways; traversing from the optic chiasma alongside the lateral margin of the diencephalon to the rostral pole of the tectum (Fig. 1A). It has been demonstrated that the ipsilateral retinothalamic projection is formed at or around stage 58 in Xenopus, well after the establishment of the contralateral retinotectal and retinothalamic projections [3, 10]. One might assume that
Fig. 2. Graphical reconstruction of aberrant pathways of regenerated optic fibres (dotted lines) following early larval optic nerve section. Arrowhead points rostrally. Course of regenerated optic nerve fibres: A, via optic nerve (II), in the chiasma, optic tracts and posterior commissure (arrow) to the rectum; B, via oculomotor nerve (IlI), in the tegmentum and pretectal area to the tectum; and C, via trigeminal nerve (V), in the hindbrain, tegmentum and pretectal area to the rectum. Small arrowheads indicate entry points o f optic fibres to the rectum.
141 regenerating fibres follow degenerated axonic debris of their predecessors to reform previous connections, in which case no ipsilateral fibre growth can be expected before stage 58. Indeed, the present observations show that wherever ipsilateral retinotectal growth was found before stage 62 optic nerve section, it involved anomalous pathways and entry points of regenerated fibres. In contrast, bilateral fibre regeneration, without the involvement of abnormal optic fibre tracts, was induced only at or after stage 62 optic nerve section. It is conceivable that optic nerve section at early larval stages involves the damage to adjacent trunks of the oculomotor and/or trigeminal nerves being in close proximity to the optic nerve in the retro-orbital tissue. Regenerating optic fibres may possibly join the trunks of the optic, oculomotor and/or trigeminal nerves. Contact, mechanical guidance along these nerves, assures the entry of regenerating optic fibres to the brain through one or more of the above nerve roots. Regardless of the point of entry to the brain, optic fibres regrew to the tectal surface after having coursed through entirely foreign territories. It would appear from these observations that mechanical guidance cues alone are not sufficient to ensure the eventual arrival of optic fibres from the abnormal entry point to the contralateral and ipsilateral tecta. ACKNOWLEDGEMENTS
We wish to thank Mrs. Theresa Clark for her skilled assistance in preparing the autoradiographic materials. This study was supported by a research grant from the Australian Research Grant Committee.
REFERENCES 1 Attardi, D.G. and Sperry, R.W., Preferential selection of central pathways by regenerating optic fibres, Exp. Neurol., 7 (1963) 46-64. 2 Beazley, L.D. and Lamb, A.H., Rerouted axons in Xenopus tadpoles form normal visuotectal projection, Brain Res., 179 (1979) 373-378. 3 Currie, J. and Cowan, W.M., Evidence of the late development of the uncrossed retinothalamic projection in the frog Rana pipiens, Brain Res., 71 (1974) 133-139. 4 Gaze, R.M., The representation of the retina on the optic lobe of the frog, Quart. J. exp. Physiol., 43 (1958) 209-214. 5 Gaze, R.M. and Grant, P., The diencephalic course of regenerating retinotectal fibres in Xenopus tadpoles, J. Embryol. exp. Morph., 44 (1978) 201-216. 6 Gaze, R.M. and Jacobson, M., A study of the retinotectal projection during regeneration of the optic nerve in the frog, Proc. roy. Soc. B, 157 (1963) 420-448. 7 Glastonbury, J. and Straznicky, K., Aberrant ipsilateral retinotectal projection following optic nerve section in Xenopus, Neurosci. Lett., 7 (1978) 67-72. 8 Grant, P. and Ma, P.M., Effect of eye rotation in the development of optic fibre pathway in Xenopus laevis, Neurosci. Abstr., 5 (1979) 516.
142 9 I0 11 12 13 14 15
16
Hibbard, E., Visual recovery following regeneration of the optic nerve through the oculomotor nerve root in Xenopus, Exp. Neurol., 19 (1967) 350-356. Khalil, S.H. and Szekely, G., The development of the ipsilateral retinothalamic projections in the Xenopus toad, Acta biol. Acad. Sci. hung., 27 (1976) 253-260. Lazar, G., The projection of the retinal quadrants on the optic centres in the frog, Acta morph. Acta Sci. hung., 19 (1971) 325-334. Nieuwkoop, P.D. and Faber, .1., Normal Table of Xenopus laevis (Daudin), North-Holland, Amsterdam, 1956. Rogers, A.W., Techniques of Autoradiography, 3rd edn. Elsevier/North-Holland, Amsterdam, 1979. Scalia, F. and Fite, K., A retinotopic analysis of the central connections of the optic nerve in the frog, J. comp. Neurol., 158 (1974) 455-478. Straznicky, C., Tay, D. and Glastonbury, .1., Regeneration of an abnormal ipsilateral visuotectal projection in Xenopus is delayed by the presence of optic fibres from the other eye, J. Embryol. exp. Morph., in press. Tay, D. and Straznicky, K., The pattern of early optic nerve regeneration from compound eyes to the tectum in Xenopus, Proc. Aust. physiol, pharmacol. Soc., 9 (1978) 65.
137
ABERRANT RETINOTECTAL PATHWAYS INDUCED BY LARVAL UNILATERAL OPTIC NERVE SECTION IN XENOPUS
DAVID TAY and CHARLES STRAZNICI(Y Centre for Neuroscience and Department of Human Morphology, School of Medicine, Flinders University of South Australia, Bedford Park, S.A. 5042 (Australia) (Received March 19th, 1980) (Accepted March 24th, 1980)
SUMMARY
Following unilateral optic nerve section in Xenopus tadpoles and toadlets, the reformation of visual pathways and retinotectal projections was analyzed using [3H]proline autoradiography. Bilateral retinotectal projections were found in 61°70 of the animals operated on between stages 42 and 58. In these animals, the projections were established through multiple aberrant pathways which included the oculomotor nerve, the trigeminal nerve and the posterior commissure. In contrast, in animals with optic nerve section at stage 62 or after metamorphosis, the regenerated optic fibres arrived at the contralateral and ipsilateral tecta through normal visual pathways.
In normal frogs the retina projects topographically to the contralateral optic tectum and to both the contralateral and the ipsilateral diencephalic visual centres [4, 11, 14]. Following unilateral optic nerve section in post-metamorphic Xenopus, regeneration occurs and the contralateral retinotectal projection is restored [6]. Recent observations on the optic nerve regeneration in post-metamorphic Xenopus have shown both morphologically and electrophysiologically, that regenerated optic fibres not only re-established the contralateral retinotectal projection but also regularly innervated the ipsilateral tectum [7, 15, 16]. In contrast, optic nerve regeneration studies in mid- and late-larval Xenopus tadpoles failed to demonstrate bilateral retinotectal projections [5]. In order to resolve the apparent disparity between the results of larval and metamorphic optic nerve regeneration, a time series experiment was carried out in animals ranging from early larval stages to after metamorphosis. It is shown that, following optic nerve section, regular bilateral tectal projections via normal visual pathways can be induced only from the time of metamorphic climax.
138 Xenopus tadpoles at various stages of development ranging from stage 42 [12] to metamorphosis and juvenile toads were used. Operations were performed under MS222 (tricaine methane sulphonate, Sandoz) anaesthesia. In young tadpoles the right optic nerve was approached from the dorsal surface of the head, exposed and cut with a sharp tungsten needle about 1-2 mm behind the eye ball. In stage 62 tadpoles and post-metamorphic Xenopus the optic nerve was reached through the pharynx and sectioned proximal to the optic chiasma as described in previous studies [7, 15]. Tadpoles were reared to metamorphosis and beyond while the postmetamorphic toadlets were kept for 20-180 days after the surgery. Twenty-four hours prior to the sacrifice, 5 ~Ci k-[5-3H]proline (specific activity 23 Ci/mmol, Amersham) was injected into the right eye. The brain was fixed in Bouins and embedded in paraffin. Transverse serial sections were cut at 10 tzm and processed for autoradiography [13]. Deparaffinized sections were coated with Ilford K2 nuclear emulsion, exposed for 14 days in light-tight boxes at 4°C, developed in Kodak D19 and counterstained with Harris's haematoxylin. The pathways taken by the regenerating optic fibres and their tectal terminations were reconstructed by camera lucida drawings of every eighth section of the autoradiographs. Forty-six animals with successful optic nerve regeneration were included in this report (Table I). Nine animals were discarded owing to poor regeneration or to lack of optic nerve formation. In 14 animals (30°7o) regenerated optic fibres grew back to the contralateral tectum, while in the remaining animals (7007o) optic nerve regeneration included both the contralateral and the ipsilateral tecta. In the former the contralateral retinotectal pathway was normal except that regenerated fibres were confined to the margin of the optic tract in the diencephalon, confirming the results of previous observations [5]. In contrast, bilateral retinotectal pathways to TABLE I T H E RESULTS OF O P T I C FIBRE R E G E N E R A T I O N IN A N I M A L S W I T H R I G H T O P T I C NERVE SECTION A T VARIOUS D E V E L O P M E N T A L STAGES W A M and M A M denote weeks and months after metamorphosis, respectively. Retinotectal projection Time of optic nerve section Unilateral
Bilateral
Stage 42 Stage 50 Stage 52
2 3
l 2 15
Stage 55 Stage 58
1 6
Stage 62 2 WAM 3 MAM
2
2 2 9
139 both the contralateral and the ipsilateral tecta were formed through a variety of abnormal fibre tracts. Between stages 42 and 52, bilateral optic nerve regeneration occurred in the majority of animals (Table I). Regenerated optic fibres in these animals entered the brain through the oculomotor (Fig. 1C) and trigeminal nerve roots (Fig. ID). In some animals the bulk of the regenerated optic fibres entered the brain via the optic nerve as well as the oculomotor or trigeminal nerve roots. Detailed analysis of the aberrant visual pathways revealed that the majority of the regenerated optic fibres crossed over the midline in the diencephalon or in the
D Fig. 1. Bright-field photographs of transverse brain sections from autoradiographs. Right is on the left and dorsal is at the top. Bars represent 500 #m. A: section across the mid-diencephalon in an animal with post-metamorphic optic nerve section. Arrows point at the courses of regenerating optic fibre bundles along the lateral margin of the diencephalon. Arrowhead points at the optic nerve through which the regenerating optic fibres enter the brain. B: section at the level of posterior commissure in an animal with stage 50 optic nerve section. Arrow indicates the position of recrossing optic fibres to the ipsilateral side. Note that the ipsilateral tectum is not evenly innervated. C: section across the mid-tectum in an animal with stage 50 optic nerve section. The a n o m a l o u s entry of regenerated optic fibres through the oculomotor nerve root (arrowhead) and their rostromedial course (arrow) are indicated. D: section at the level of the posterior tectal poles in an animal with stage 52 optic nerve section. Arrowhead indicates the entry of regenerated optic fibres to the brain via the trigeminal nerve root.
140 tegmentum to innervate the contralateral tectum, while a small bundle of such fibres reached the tectum ipsilaterally (Fig. 1B, C). In all cases, the optic fibres coursed dorsally towards the contralateral and ipsilateral pretectal areas and entered the tectum through the dorsomedial and ventrolateral brachia of the optic tract (Fig. 2B, C). Six animals of this group showed, in addition to the multiple entries to the brain, that a portion of the regenerated optic fibres arrived from the contralateral side to the ipsilateral tectum through the posterior commissure (Figs. 1B and 2A). Following optic nerve section at mid-larval stages (stages 55-58), only one out of eight animals formed bilateral retinotectal projections. In this animal, a portion of the regenerated optic fibres were guided from the optic chiasma to the ipsilateral tectum by the retinal fibres from the other eye. In the other 7 animals, the restored contralateral retinotectal projection involved normal optic fibre trajectory. In 13 out of the 15 animals where the optic nerve was sectioned at stage 62 or later (Table I), both contralateral and ipsilateral retinotectal projections developed. In the two remaining animals only contralateral regeneration was found. Irrespective of unilateral or bilateral tectal innervations in these animals, the regenerated optic fibres followed the course of normal visual pathways; traversing from the optic chiasma alongside the lateral margin of the diencephalon to the rostral pole of the tectum (Fig. 1A). It has been demonstrated that the ipsilateral retinothalamic projection is formed at or around stage 58 in Xenopus, well after the establishment of the contralateral retinotectal and retinothalamic projections [3, 10]. One might assume that
Fig. 2. Graphical reconstruction of aberrant pathways of regenerated optic fibres (dotted lines) following early larval optic nerve section. Arrowhead points rostrally. Course of regenerated optic nerve fibres: A, via optic nerve (II), in the chiasma, optic tracts and posterior commissure (arrow) to the rectum; B, via oculomotor nerve (IlI), in the tegmentum and pretectal area to the tectum; and C, via trigeminal nerve (V), in the hindbrain, tegmentum and pretectal area to the rectum. Small arrowheads indicate entry points o f optic fibres to the rectum.
141 regenerating fibres follow degenerated axonic debris of their predecessors to reform previous connections, in which case no ipsilateral fibre growth can be expected before stage 58. Indeed, the present observations show that wherever ipsilateral retinotectal growth was found before stage 62 optic nerve section, it involved anomalous pathways and entry points of regenerated fibres. In contrast, bilateral fibre regeneration, without the involvement of abnormal optic fibre tracts, was induced only at or after stage 62 optic nerve section. It is conceivable that optic nerve section at early larval stages involves the damage to adjacent trunks of the oculomotor and/or trigeminal nerves being in close proximity to the optic nerve in the retro-orbital tissue. Regenerating optic fibres may possibly join the trunks of the optic, oculomotor and/or trigeminal nerves. Contact, mechanical guidance along these nerves, assures the entry of regenerating optic fibres to the brain through one or more of the above nerve roots. Regardless of the point of entry to the brain, optic fibres regrew to the tectal surface after having coursed through entirely foreign territories. It would appear from these observations that mechanical guidance cues alone are not sufficient to ensure the eventual arrival of optic fibres from the abnormal entry point to the contralateral and ipsilateral tecta. ACKNOWLEDGEMENTS
We wish to thank Mrs. Theresa Clark for her skilled assistance in preparing the autoradiographic materials. This study was supported by a research grant from the Australian Research Grant Committee.
REFERENCES 1 Attardi, D.G. and Sperry, R.W., Preferential selection of central pathways by regenerating optic fibres, Exp. Neurol., 7 (1963) 46-64. 2 Beazley, L.D. and Lamb, A.H., Rerouted axons in Xenopus tadpoles form normal visuotectal projection, Brain Res., 179 (1979) 373-378. 3 Currie, J. and Cowan, W.M., Evidence of the late development of the uncrossed retinothalamic projection in the frog Rana pipiens, Brain Res., 71 (1974) 133-139. 4 Gaze, R.M., The representation of the retina on the optic lobe of the frog, Quart. J. exp. Physiol., 43 (1958) 209-214. 5 Gaze, R.M. and Grant, P., The diencephalic course of regenerating retinotectal fibres in Xenopus tadpoles, J. Embryol. exp. Morph., 44 (1978) 201-216. 6 Gaze, R.M. and Jacobson, M., A study of the retinotectal projection during regeneration of the optic nerve in the frog, Proc. roy. Soc. B, 157 (1963) 420-448. 7 Glastonbury, J. and Straznicky, K., Aberrant ipsilateral retinotectal projection following optic nerve section in Xenopus, Neurosci. Lett., 7 (1978) 67-72. 8 Grant, P. and Ma, P.M., Effect of eye rotation in the development of optic fibre pathway in Xenopus laevis, Neurosci. Abstr., 5 (1979) 516.
142 9 I0 11 12 13 14 15
16
Hibbard, E., Visual recovery following regeneration of the optic nerve through the oculomotor nerve root in Xenopus, Exp. Neurol., 19 (1967) 350-356. Khalil, S.H. and Szekely, G., The development of the ipsilateral retinothalamic projections in the Xenopus toad, Acta biol. Acad. Sci. hung., 27 (1976) 253-260. Lazar, G., The projection of the retinal quadrants on the optic centres in the frog, Acta morph. Acta Sci. hung., 19 (1971) 325-334. Nieuwkoop, P.D. and Faber, .1., Normal Table of Xenopus laevis (Daudin), North-Holland, Amsterdam, 1956. Rogers, A.W., Techniques of Autoradiography, 3rd edn. Elsevier/North-Holland, Amsterdam, 1979. Scalia, F. and Fite, K., A retinotopic analysis of the central connections of the optic nerve in the frog, J. comp. Neurol., 158 (1974) 455-478. Straznicky, C., Tay, D. and Glastonbury, .1., Regeneration of an abnormal ipsilateral visuotectal projection in Xenopus is delayed by the presence of optic fibres from the other eye, J. Embryol. exp. Morph., in press. Tay, D. and Straznicky, K., The pattern of early optic nerve regeneration from compound eyes to the tectum in Xenopus, Proc. Aust. physiol, pharmacol. Soc., 9 (1978) 65.