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The effect of inhibition of axonal RNA transport on the restoration of retinotectal projections in regenerating optic nerves of goldfish
Brain Research, 156 (1978) 141-145 © Elsevier/North-Holland Biomedical Press
141
The effect of inhibition of axonal RNA transport on the restoration of retinotectal projections in regenerating optic nerves of goldfish
NICHOLAS A. INGOGLIA and S. C. SHARMA Departments of Physiology and Neuroscience, New Jersey Medical School, Newark, N.J. 07103 and Department of Ophthahnology, New York Medical College, New York, N. Y. 10029 (U.S.A.)
(Accepted June 22nd, 1978)
When the optic nerve of a goldfish is crushed or cut, the portion of the axon distal to the crush initially degenerates 14, but is replaced within a month by an axon sprout regenerating from the proximal stump 13,16. The regenerating fibers face two distinct problems if they are to establish functional reconnections with appropriate postsynaptic cells in the optic tectum. The first of these is physical elongation or growth; the second is to find the correct tectal target cell so that the order of visual information processing is retained. Although neurons in the central nervous system of mammals do not regenerate, the retinal ganglion cells of the goldfish are capable of complete functional regeneration with remarkable specificity in their reconnections, i.e. the best evidence to date suggests that these growing fibers return to the tectum to reinnervate the same tectal areas that they had innervated prior to optic nerve transectionZ,~L The way in which the axons grow as well as the signals used by the axon and/or the target cells to achieve specificity is poorly understood. The best evidence for axonal growth indicates that materials necessary for establishing axonal membranes and organelles are synthesized in the neuronal cell body and then exported from the soma into the stump of the axon to be conveyed by axonal transport into the growing axon (reviewed by Grafstein, 1975) 6. Since there is little evidence of protein synthesis in growing axons, and some recent studies suggest that glia surrounding the axon do not contribute proteins to the growing axon 1~, it is likely that axonal transport of proteins is the sole source of constituent proteins for the growing axon. The mechanisms underlying axonal guidance are poorly understood. However, it is likely that axonal transport of materials from the soma into the growing tips of the axon plays an important role in this phenomenon. Since a mature axon has little 'need' for guidance factors, it also seems likely that materials present in a regenerating axon, but not present, or present in reduced amounts, in mature axons would be prime candidates for playing a role in growth or guidance. There are several substances which have been shown to be distributed in this manner. Glycoproteins, for example, are axonally transported in preference to enzymes involved in transmitter synthesis in regenerating rat hypoglossal nerves, whereas this preference is reversed in fully
142 connected functioning axons 1. This finding is consistent with the hypothesis that axonally transported proteins are the prime source of the rebuilding proteins of the axon. The axonal transport of the diamine, putrescine, has been demonstrated in regenerating but not in mature optic axons of goldfish% Furthermore, during optic nerve regeneration in goldfish, some RNA molecules appear to be synthesized in retinal ganglion cells and are then transported into the growing axon 1°,11. Recent EM autoradiographic evidence has shown the presence of [3H]RNA within regenerating axons and axonal growth cones in the optic tectum. This RNA was apparently synthesized in the retina and axonally transported to the tectum as regenerating fibers were forming connections with tectal cells 2. Further evidence suggests that the intraaxonal RNA may be only 4S RNA, i.e. ribosomal RNA appears to be excluded from the axon and is probably not transported axonally in this system 7,l°. Since proteins are probably not synthesized in regenerating goldfish optic axons, the role of transported RNA in regenerating axons is in question. The aim of this study was to determine the effect of inhibition of axonal RNA transport during nerve regeneration on the restoration of appropriate retino-tectal (RT) connections. In order to monitor successful and appropriate reconnections we have employed standard electrophysiologic recording techniques 5. These techniques do not record from tectal fibers 'en passage' but only from terminal arborizations and, therefore, in spite of the fact that the electrical potentials recorded are presynaptic, they are only recorded from fibers which have made or are about to make synaptic contacts 5. Studies have shown that following nerve transection the ability to record a normal R-T map closely coincides with the return of visual function (unpublished data). Goldfish (Carassius auratus, 10-l 2 cm in length, obtained from Ozark Fisheries, Stoutland, Mo.) were kept at approximately 22 °C for the duration of the experiment. Following anaesthesia by ice-water immersion, both optic nerves were crushed a few millimeters behind the eye using curved jewelers forceps. At various times after crushing the nerves the RNA synthesis inhibitor cordycepin (3'-deoxyadenosine; Sigma) was injected into the right eye only, and fish were prepared for electrophysiologic recording at various times after these injections. Cordycepin was chosen because it has been shown that at the doses used (t0 #g/eye) it is an effective blocker of RNA transport 8, appearing to block the transport of 4S RNA by greater than 90 ~7. Further, when injected into the eye, its effect appears to be primarily on retinal ganglion cells while sparing cells in other retinal layers (see Fig. 2 of ref. 7). Since both optic nerves were crushed, but only the right eye was injected with cordycepin, the left eye-right tectum visual pathway served as a control for the effect of environmental factors on nerve regeneration. Thus, the R-T map was first monitored in the right tectum at various times after crushing the nerve; if this map was complete or at least partially restored we assumed that successful regeneration was occurring. Following this procedure we studied the right eye (cordycepin-injected)-left tectum pathway and recorded the visual map from the left tectum.
143 TABLE I The effect of intraoculur injections of cordycepin on the restoration of the retino-tectal map during regeneration o['goldfish optic' axons Time of injection after crush (days)
Dose of cordycepin (ltg)
Electrophysiologic recording aJter nerve crush (days)
(a) (b) (c) (d)
0.0 10.0 10.0 10.0
24 24 28 34
1 5 2 I
48 41
l 2
34 8 days after injection 1 day after injection 6daysafterinjection I h after injection
1 1 1 1 l
18 18 18 18
(e) 16, 19, 22 (f) 26, 29, 32 (g) (h) (i) (j) (k)
26, 29, 32 Normal (not regenerating) Normal(not regenerating) Normal (not regenerating) Normal (not regenerating)
0.5/lnj. 0.5/lnj. 10.0/tnj. 10.0 10.0 10.0 20.0
Number Description O[" R-T rnap*
Normal No response No response Faint response rostral/no response caudal No response Some normal response rostral No response Normal Normal Normal Normal
* A normal R-T map is defined as one in which responses are similar to those recorded, (1) in an intact retino-tectal system, or (2) at the same stage of regeneration but without drug treatment. In h-k the optic nerve was not crushed In all cases studied, cordycepin either blocked or significantly delayed the reestablishment of the R-T map (Table I, b-g), suggesting that blocking axonal R N A transport interferes with some aspect of the regeneration of retinal fibers. However, cordycepin not only blocks R N A synthesis but also interferes secondarily with protein synthesis. Thus, it seems possible that cordycepin might be blocking retinal protein synthesis, and that the inability to record a map following cordycepin administration might be due to inhibition of protein synthesis thereby affecting retinal morphology, optic axon integrity, or both. In other words, cordycepin might be causing degeneration of retinal cells and/or the optic nerve, and in this way was preventing the restoration of a map. To test this possibility we studied the effect of intraocular injections of similar doses of cordycepin on normal (non-regenerating) retino-tectal projections. Results showed that in the doses used and at the times studied cordycepin had no effect on the integrity of the R-T map (Table I, h-k). Thus, it appears that the effect of cordycepin on the re-establishment of the R-T map during regeneration is not due to alterations of any of the properties of the mature visual pathways, but is confined to effects on regenerating nerves. Since cordycepin also affects protein synthesis and transport, it was necessary to determine if the effects of cordycepin on the restoration of the R-T m a p could be due to an effect on proteins rather than R N A . To determine whether inhibition of retinal protein synthesis and transport could also block the restoration of the R-T map, experiments were undertaken in which retinal protein synthesis was inhibited by intraocular injections of the protein synthesis inhibitor cycloheximide (Sigma). In both non-regenerating (Table II, a and b) and regenerating preparations,
144 TABLE I!
The effect of intraocular injections of cvcloheximide on the restoration of the retino-tectal ;nap during regeneration of goldfish optic axons Time of injection after crush (days)
(a) (b) (c) (d) (e) (f) (g) (h) (i) (j) (k)
Normal (not regenerating) Normal (not regenerating) 13 18 21 24 27 30 30 45 18, 20, 22
Number Description ~1" R-T map*
(/~g)
Electrophysiologic recording after nerve crush (days)
10.0 10.0 10.0 1.0 1.0 10.0 10.0 10.0 10.0 10.0 10.0/Inj.
1 after injection 6 after injection 19 19 22 25 31 31 60 69 24
1 1 1 2 l 1 1 I I 1 3
Dose of eycloheximide
Normal Normal Mostly normal Normal Normal Normal Normal Normal Normal Normal No response
* A normal R-T map is defined'as one in which responses are similar to those recorded; (1) in an intact retino-tectal system, or (2) at the same stage of regeneration but without drug treatment: In a and b the optic nerve was not crushed. single injections of 10.0 #g of cycloheximide, a dose which blocks axonal protein transport by 90 ~ one day after injection (unpublished data), had no effect on the restoration of the R-T map at any time points studied (Table tl, c-j). However, the effect of cycloheximide on protein synthesis is readily reversible. In these experiments the inhibition of protein synthesis did not last for more than 18-24 h. In the cordycepin experiments the inhibition was of longer duration, the effect of cordycepin on R N A and protein synthesis still being apparent 12 days after a single injection (unpublished data). Thus, in an attempt to mimic the cordycepin block of protein transport by cycloheximide, it was necessary to give repeated injections of cycloheximide. When cycloheximide was given in doses of 10.0 #g/injection 18, 20 and 22 days after crushing the optic nerves, no responses were recorded from the optic rectum (Table II, k). These results show that a 'momentary' interruption (18 h) in the delivery of proteins to the optic tectum has no effect on the restoration of the R-T map, but a prolonged block (over a 6-day period) does prevent the m a p from formin& Therefore, these experiments do not distinguish between the roles that axonally transported R N A and protein might be playing in nerve regeneration. These experiments show that inhibiting R N A synthesis and transport with cordycepin blocks the successful regeneration of retinal fibers to the optic tectum. However, this effect could be unrelated to axonal R N A transport, i.e., it is possible that the drug prevented the growth of the axons and thus the optic fibers never reached the tectum. Another possibility is that cordycepin inhibited the synthesis and transport of a protein important in growth or guidance. We have been unable to rule out these possibilities. However, it is clear that R N A molecules, derived from retinal ganglion cells, are
145 conveyed to the optic tectum along regenerating but not mature optic axonsL Further, this RNA appears to be only 4S 7,1°, and is clearly within regenerating axons as well as axonal growth cones. While this study shows that interrupting the arrival of RNA in the tectum also interrupts the re-establishment of the R-T map, the specific role of 4S RNA transport, during regeneration of goldfish visual fibers, is not yet clear. Supported by Grants N.S. 11259 and N.E.I. 01426 from N.I.H. and Career Development Award EYI0010I to S.C.S.
1 Frizell, M. and Sj6strand, J., Transport of proteins, glycoproteins and cholinergic enzymes in regenerating hypoglossal neurons, J. Neurochem., 22 (1974) 845-850. 2 Gambetti, P., Ingoglia, N. A., Autilio-Gambetti, L. and Weis, P., Distribution of [3H]RNA in goldfish optic tectum following intraocular or intracranial injection of [3Hluridine. Evidence of axonal migration of RNA in regenerating optic fibers, Brain Research, in press. 3 Gaze, R. M., The Formation of Nerve Connections: A Consideration of Neural Specificity, Modulation and Comparable Phenomena, Academic Press, London, 1970. 4 Gaze, R. M. and Jacobson, M., A study ofretinotectal projections during regeneration of the optic nerve, Proc. roy. Soc. B, 157 (1963) 420-448. 5 Gaze, M. and Sharma, S. C., Axial differences in the reinnervation of the goldfish optic rectum by regenerating optic nerve fibers, Exp. Brain Res., 10 (1970) 171 179. 6 Grafstein, B., The nerve cell body response to axotomy, Exp. Neurol., 48 (1975) 32 51. 7 Ingoglia, N. A., The effect ofintraocular injection of cordycepin on retinal RNA synthesis and on RNA axonally transported during regeneration of the optic nerves of goldfish, J. Neurochem. (1978) in press. 8 lngoglia, N. A., Sellin, L. C. and Lindquist, T. D., The effect of cordycepin on the appearance of [3H]RNA in the goldfish optic rectum following intraocular injection of [3H ]uridine, J. Neurochem., 27 (1976) 179 184. 9 lngoglia, N. A., Sturman, J. A. and Eisner, R. A., Axonal transport of putrescine, spermidine and spermine in normal and regenerating goldfish optic nerves, Brain Research, 130 (1977) 433-445. 10 lngoglia, N. A. and Tuliszewski, R., Transfer RNA may be axonally transported during regeneration of goldfish optic nerves, Brain Research, 112 (1976) 371-381. 11 Ingoglia, N., Weis, P. and Mycek, J., Axonal transport of RNA during regeneration of the optic nerves of goldfish, J. Neurobiol., 6 (1975) 549-563. 12 Jacobson, M., Neuronal recognition in the retinotectal system. In S. H. Barondes (Ed.), Neuronal Recognition, Plenum Press, New York, 1976. 13 Jacobson, M. and Gaze, R. M., Selection of appropriate tectal connections by regenerating optic nerve fibers in adult goldfish, Exp. Neurol., 13 (1965) 418-430. 14 Murray, M., Regeneration of retinal axons into the goldfish optic tectum, J. comp. Neurol., 168 (1976) 175 196. 15 Peck, K., Gambetti, P. and Autilio-Gambetti, L., Glio-axonal transfer of protein in regenerating goldfish optic system, Amer. Assoc. Neuropathol., Abstr. 53rd Ann. Meet. (1977) 622. 16 Sperry, R. W., Patterning of central synapses in regeneration of the optic nerve in teleosts, Physiol. Zool., 28 (1948) 351-361.
141
The effect of inhibition of axonal RNA transport on the restoration of retinotectal projections in regenerating optic nerves of goldfish
NICHOLAS A. INGOGLIA and S. C. SHARMA Departments of Physiology and Neuroscience, New Jersey Medical School, Newark, N.J. 07103 and Department of Ophthahnology, New York Medical College, New York, N. Y. 10029 (U.S.A.)
(Accepted June 22nd, 1978)
When the optic nerve of a goldfish is crushed or cut, the portion of the axon distal to the crush initially degenerates 14, but is replaced within a month by an axon sprout regenerating from the proximal stump 13,16. The regenerating fibers face two distinct problems if they are to establish functional reconnections with appropriate postsynaptic cells in the optic tectum. The first of these is physical elongation or growth; the second is to find the correct tectal target cell so that the order of visual information processing is retained. Although neurons in the central nervous system of mammals do not regenerate, the retinal ganglion cells of the goldfish are capable of complete functional regeneration with remarkable specificity in their reconnections, i.e. the best evidence to date suggests that these growing fibers return to the tectum to reinnervate the same tectal areas that they had innervated prior to optic nerve transectionZ,~L The way in which the axons grow as well as the signals used by the axon and/or the target cells to achieve specificity is poorly understood. The best evidence for axonal growth indicates that materials necessary for establishing axonal membranes and organelles are synthesized in the neuronal cell body and then exported from the soma into the stump of the axon to be conveyed by axonal transport into the growing axon (reviewed by Grafstein, 1975) 6. Since there is little evidence of protein synthesis in growing axons, and some recent studies suggest that glia surrounding the axon do not contribute proteins to the growing axon 1~, it is likely that axonal transport of proteins is the sole source of constituent proteins for the growing axon. The mechanisms underlying axonal guidance are poorly understood. However, it is likely that axonal transport of materials from the soma into the growing tips of the axon plays an important role in this phenomenon. Since a mature axon has little 'need' for guidance factors, it also seems likely that materials present in a regenerating axon, but not present, or present in reduced amounts, in mature axons would be prime candidates for playing a role in growth or guidance. There are several substances which have been shown to be distributed in this manner. Glycoproteins, for example, are axonally transported in preference to enzymes involved in transmitter synthesis in regenerating rat hypoglossal nerves, whereas this preference is reversed in fully
142 connected functioning axons 1. This finding is consistent with the hypothesis that axonally transported proteins are the prime source of the rebuilding proteins of the axon. The axonal transport of the diamine, putrescine, has been demonstrated in regenerating but not in mature optic axons of goldfish% Furthermore, during optic nerve regeneration in goldfish, some RNA molecules appear to be synthesized in retinal ganglion cells and are then transported into the growing axon 1°,11. Recent EM autoradiographic evidence has shown the presence of [3H]RNA within regenerating axons and axonal growth cones in the optic tectum. This RNA was apparently synthesized in the retina and axonally transported to the tectum as regenerating fibers were forming connections with tectal cells 2. Further evidence suggests that the intraaxonal RNA may be only 4S RNA, i.e. ribosomal RNA appears to be excluded from the axon and is probably not transported axonally in this system 7,l°. Since proteins are probably not synthesized in regenerating goldfish optic axons, the role of transported RNA in regenerating axons is in question. The aim of this study was to determine the effect of inhibition of axonal RNA transport during nerve regeneration on the restoration of appropriate retino-tectal (RT) connections. In order to monitor successful and appropriate reconnections we have employed standard electrophysiologic recording techniques 5. These techniques do not record from tectal fibers 'en passage' but only from terminal arborizations and, therefore, in spite of the fact that the electrical potentials recorded are presynaptic, they are only recorded from fibers which have made or are about to make synaptic contacts 5. Studies have shown that following nerve transection the ability to record a normal R-T map closely coincides with the return of visual function (unpublished data). Goldfish (Carassius auratus, 10-l 2 cm in length, obtained from Ozark Fisheries, Stoutland, Mo.) were kept at approximately 22 °C for the duration of the experiment. Following anaesthesia by ice-water immersion, both optic nerves were crushed a few millimeters behind the eye using curved jewelers forceps. At various times after crushing the nerves the RNA synthesis inhibitor cordycepin (3'-deoxyadenosine; Sigma) was injected into the right eye only, and fish were prepared for electrophysiologic recording at various times after these injections. Cordycepin was chosen because it has been shown that at the doses used (t0 #g/eye) it is an effective blocker of RNA transport 8, appearing to block the transport of 4S RNA by greater than 90 ~7. Further, when injected into the eye, its effect appears to be primarily on retinal ganglion cells while sparing cells in other retinal layers (see Fig. 2 of ref. 7). Since both optic nerves were crushed, but only the right eye was injected with cordycepin, the left eye-right tectum visual pathway served as a control for the effect of environmental factors on nerve regeneration. Thus, the R-T map was first monitored in the right tectum at various times after crushing the nerve; if this map was complete or at least partially restored we assumed that successful regeneration was occurring. Following this procedure we studied the right eye (cordycepin-injected)-left tectum pathway and recorded the visual map from the left tectum.
143 TABLE I The effect of intraoculur injections of cordycepin on the restoration of the retino-tectal map during regeneration o['goldfish optic' axons Time of injection after crush (days)
Dose of cordycepin (ltg)
Electrophysiologic recording aJter nerve crush (days)
(a) (b) (c) (d)
0.0 10.0 10.0 10.0
24 24 28 34
1 5 2 I
48 41
l 2
34 8 days after injection 1 day after injection 6daysafterinjection I h after injection
1 1 1 1 l
18 18 18 18
(e) 16, 19, 22 (f) 26, 29, 32 (g) (h) (i) (j) (k)
26, 29, 32 Normal (not regenerating) Normal(not regenerating) Normal (not regenerating) Normal (not regenerating)
0.5/lnj. 0.5/lnj. 10.0/tnj. 10.0 10.0 10.0 20.0
Number Description O[" R-T rnap*
Normal No response No response Faint response rostral/no response caudal No response Some normal response rostral No response Normal Normal Normal Normal
* A normal R-T map is defined as one in which responses are similar to those recorded, (1) in an intact retino-tectal system, or (2) at the same stage of regeneration but without drug treatment. In h-k the optic nerve was not crushed In all cases studied, cordycepin either blocked or significantly delayed the reestablishment of the R-T map (Table I, b-g), suggesting that blocking axonal R N A transport interferes with some aspect of the regeneration of retinal fibers. However, cordycepin not only blocks R N A synthesis but also interferes secondarily with protein synthesis. Thus, it seems possible that cordycepin might be blocking retinal protein synthesis, and that the inability to record a map following cordycepin administration might be due to inhibition of protein synthesis thereby affecting retinal morphology, optic axon integrity, or both. In other words, cordycepin might be causing degeneration of retinal cells and/or the optic nerve, and in this way was preventing the restoration of a map. To test this possibility we studied the effect of intraocular injections of similar doses of cordycepin on normal (non-regenerating) retino-tectal projections. Results showed that in the doses used and at the times studied cordycepin had no effect on the integrity of the R-T map (Table I, h-k). Thus, it appears that the effect of cordycepin on the re-establishment of the R-T map during regeneration is not due to alterations of any of the properties of the mature visual pathways, but is confined to effects on regenerating nerves. Since cordycepin also affects protein synthesis and transport, it was necessary to determine if the effects of cordycepin on the restoration of the R-T m a p could be due to an effect on proteins rather than R N A . To determine whether inhibition of retinal protein synthesis and transport could also block the restoration of the R-T map, experiments were undertaken in which retinal protein synthesis was inhibited by intraocular injections of the protein synthesis inhibitor cycloheximide (Sigma). In both non-regenerating (Table II, a and b) and regenerating preparations,
144 TABLE I!
The effect of intraocular injections of cvcloheximide on the restoration of the retino-tectal ;nap during regeneration of goldfish optic axons Time of injection after crush (days)
(a) (b) (c) (d) (e) (f) (g) (h) (i) (j) (k)
Normal (not regenerating) Normal (not regenerating) 13 18 21 24 27 30 30 45 18, 20, 22
Number Description ~1" R-T map*
(/~g)
Electrophysiologic recording after nerve crush (days)
10.0 10.0 10.0 1.0 1.0 10.0 10.0 10.0 10.0 10.0 10.0/Inj.
1 after injection 6 after injection 19 19 22 25 31 31 60 69 24
1 1 1 2 l 1 1 I I 1 3
Dose of eycloheximide
Normal Normal Mostly normal Normal Normal Normal Normal Normal Normal Normal No response
* A normal R-T map is defined'as one in which responses are similar to those recorded; (1) in an intact retino-tectal system, or (2) at the same stage of regeneration but without drug treatment: In a and b the optic nerve was not crushed. single injections of 10.0 #g of cycloheximide, a dose which blocks axonal protein transport by 90 ~ one day after injection (unpublished data), had no effect on the restoration of the R-T map at any time points studied (Table tl, c-j). However, the effect of cycloheximide on protein synthesis is readily reversible. In these experiments the inhibition of protein synthesis did not last for more than 18-24 h. In the cordycepin experiments the inhibition was of longer duration, the effect of cordycepin on R N A and protein synthesis still being apparent 12 days after a single injection (unpublished data). Thus, in an attempt to mimic the cordycepin block of protein transport by cycloheximide, it was necessary to give repeated injections of cycloheximide. When cycloheximide was given in doses of 10.0 #g/injection 18, 20 and 22 days after crushing the optic nerves, no responses were recorded from the optic rectum (Table II, k). These results show that a 'momentary' interruption (18 h) in the delivery of proteins to the optic tectum has no effect on the restoration of the R-T map, but a prolonged block (over a 6-day period) does prevent the m a p from formin& Therefore, these experiments do not distinguish between the roles that axonally transported R N A and protein might be playing in nerve regeneration. These experiments show that inhibiting R N A synthesis and transport with cordycepin blocks the successful regeneration of retinal fibers to the optic tectum. However, this effect could be unrelated to axonal R N A transport, i.e., it is possible that the drug prevented the growth of the axons and thus the optic fibers never reached the tectum. Another possibility is that cordycepin inhibited the synthesis and transport of a protein important in growth or guidance. We have been unable to rule out these possibilities. However, it is clear that R N A molecules, derived from retinal ganglion cells, are
145 conveyed to the optic tectum along regenerating but not mature optic axonsL Further, this RNA appears to be only 4S 7,1°, and is clearly within regenerating axons as well as axonal growth cones. While this study shows that interrupting the arrival of RNA in the tectum also interrupts the re-establishment of the R-T map, the specific role of 4S RNA transport, during regeneration of goldfish visual fibers, is not yet clear. Supported by Grants N.S. 11259 and N.E.I. 01426 from N.I.H. and Career Development Award EYI0010I to S.C.S.
1 Frizell, M. and Sj6strand, J., Transport of proteins, glycoproteins and cholinergic enzymes in regenerating hypoglossal neurons, J. Neurochem., 22 (1974) 845-850. 2 Gambetti, P., Ingoglia, N. A., Autilio-Gambetti, L. and Weis, P., Distribution of [3H]RNA in goldfish optic tectum following intraocular or intracranial injection of [3Hluridine. Evidence of axonal migration of RNA in regenerating optic fibers, Brain Research, in press. 3 Gaze, R. M., The Formation of Nerve Connections: A Consideration of Neural Specificity, Modulation and Comparable Phenomena, Academic Press, London, 1970. 4 Gaze, R. M. and Jacobson, M., A study ofretinotectal projections during regeneration of the optic nerve, Proc. roy. Soc. B, 157 (1963) 420-448. 5 Gaze, M. and Sharma, S. C., Axial differences in the reinnervation of the goldfish optic rectum by regenerating optic nerve fibers, Exp. Brain Res., 10 (1970) 171 179. 6 Grafstein, B., The nerve cell body response to axotomy, Exp. Neurol., 48 (1975) 32 51. 7 Ingoglia, N. A., The effect ofintraocular injection of cordycepin on retinal RNA synthesis and on RNA axonally transported during regeneration of the optic nerves of goldfish, J. Neurochem. (1978) in press. 8 lngoglia, N. A., Sellin, L. C. and Lindquist, T. D., The effect of cordycepin on the appearance of [3H]RNA in the goldfish optic rectum following intraocular injection of [3H ]uridine, J. Neurochem., 27 (1976) 179 184. 9 lngoglia, N. A., Sturman, J. A. and Eisner, R. A., Axonal transport of putrescine, spermidine and spermine in normal and regenerating goldfish optic nerves, Brain Research, 130 (1977) 433-445. 10 lngoglia, N. A. and Tuliszewski, R., Transfer RNA may be axonally transported during regeneration of goldfish optic nerves, Brain Research, 112 (1976) 371-381. 11 Ingoglia, N., Weis, P. and Mycek, J., Axonal transport of RNA during regeneration of the optic nerves of goldfish, J. Neurobiol., 6 (1975) 549-563. 12 Jacobson, M., Neuronal recognition in the retinotectal system. In S. H. Barondes (Ed.), Neuronal Recognition, Plenum Press, New York, 1976. 13 Jacobson, M. and Gaze, R. M., Selection of appropriate tectal connections by regenerating optic nerve fibers in adult goldfish, Exp. Neurol., 13 (1965) 418-430. 14 Murray, M., Regeneration of retinal axons into the goldfish optic tectum, J. comp. Neurol., 168 (1976) 175 196. 15 Peck, K., Gambetti, P. and Autilio-Gambetti, L., Glio-axonal transfer of protein in regenerating goldfish optic system, Amer. Assoc. Neuropathol., Abstr. 53rd Ann. Meet. (1977) 622. 16 Sperry, R. W., Patterning of central synapses in regeneration of the optic nerve in teleosts, Physiol. Zool., 28 (1948) 351-361.