Intraocular tetrodotoxin in goldfish hinders optic nerve regeneration

Brain Research, 269 (1983) 1-14

1

Elsevier

Research Reports Intraocular Tetrodotoxin in Goldfish Hinders Optic Nerve Regeneration D. L O U I S E E D W A R D S * a n d B E R N I C E G R A F S T E I N * *

Department of Physiology, Cornell University Medical College, New York, N Y 10021 (U. S.A.) (Accepted N o v e m b e r 16th, 1982)

Key words: nerve regeneration - visual p a t h w a y - axonal t r a n s p o r t - vision - axon - goldfish optic nerve - tetrodotoxin

Repeated intraocular injection of tetrodotoxin (TTX) was used to produce a maintained block of neural activity in goldfish optic axons which were regenerating following a crush of the optic nerve. The recovery of visual function was delayed in the TTXtreated fish, as demonstrated by delays in the return of the startle reaction to sudden illumination, the dorsal light reflex and food pellet localization. A single injection of T r x at the time of optic nerve crush delayed recovery of the startle reaction but not of food localization. There was no loss of ganglion cells in the TTX-treated animals, but the number of regenerated axons detectable by light microscopy was reduced. Also, axonal transport of radioactively labeled protein in some of the regenerating axons showed a deficit at 21--28 days after the lesion, i.e., during innervation of the tectum. Incorporation of labeled amino acid into protein in the retinal ganglion cells was depressed during the same period, but both the transport and incorporation had returned to normal by 36 days after the lesion. These results, together with the results of the accompanying electrophysiological analysis of the e flqe c ts of TTX58 .59, su ggest that TTX treatment interferes with two separate events in regeneration, one occurring soon after the nerve lesion and the other during innervation of the tectum. During at least the first of these events the effect of TTX treatment may be to reduce the number or size of the regenerating axon branches. We propose that the TTX effect may be mediated by a reduction in the axonal transport of certain materials, including gangliosides, nucleosides, or proteins specifically involved in regeneration. INTRODUCTION

Does the physiological activity of nerve cells affect their growth? This is a question with important theoretical and practical implications in relation to problems of development and regeneration in the nervous system. That nervous system development could proceed in the absence of activity was indicated by the classical work of Matthews and Detweiler in 19263s, showing that normal reflexes developed in Ambystoma embryos raised in anesthetic solution. More recently, studies in tissue culture have shown that nerve terminals forming in culture solutions containing a local anesthetic functioned normallys,53. Experiments on the visual system in vivo have also suggested that activity has little effect on development of the optic axons. For exam-

pie, in an ingenious experiment, Harris grafted an embryonic axolotl eye on a developing newt embryo possessing endogenous tetrodotoxin (TTX), a toxin that blocks conduction of action potentials: the axolotl retina developed normally and the axons of the retinal ganglion cells grew to the appropriate tectal layers. In developing rabbit retina blocked with TTX the resulting intraretinal organization appeared normal and the retinal ganglion cells made correct connections with the lateral geniculate body22. These studies support the view that neuronal development, at least through the stages of axonal outgrowth and initial synapse formation, does not require physiological activity. Nevertheless, there have now been many demonstrations that nervous system structure and function may be significantly disturbed as a

* Present address: D e p a r t m e n t o f Neurobiology, H a r v a r d Medical School, Boston, M A 02115, U.S.A. ** To w h o m c o r r e s p o n d e n c e should be addressed. 0006-8993/83/$03.00 © 1983 Elsevier Science Publishers B.V.

result of modified physiological activity during development. Altered afferent connectivity in the mammalian visual cortex resulting from monocular visual deprivation z6 is perhaps the best known example. In lower vertebrates, changes in lighting conditions during development or during lesion-induced sprouting of the optic axons can affect the organization of the visual pathway 7,31.7t,73. Also, mammalian neurons in dissociated cell culture that normally exhibit electrical activity under control conditions show impaired maturation and probably a lower survival rate when they are cultured in TTX-containing medium 4. In the present study, we have used the regenerating goldfish optic nerve to determine whether axonal regeneration is affected when neural activity in the optic nerve has been blocked with intraocular TTX. Regeneration in this system is now well characterized in terms of axonal outgrowth 2,33~4°,47,establishment of retinotectal connectionsT M , and recovery of visual function2,1°,17.64. Also, there have been detailed descriptions of the accompanying changes in the retinal ganglion cell bodies, including changes in morphology49,5°,7° and in RNA and protein synthesis6,23,4°,45,5°.7° as well as alterations in axonal transport of various materials ~7,27,28,4°.In this paper, we have assayed the effect of TTX on regeneration in terms of the recovery of various visually mediated behaviors and the time course of arrival of radioactively labeled regenerating axons in the optic tectum, as well as histological changes in the optic axons and in the retinal ganglion cell bodies. In the two subsequent papers~8,59 an electrophysiological analysis of the TTX effects on regeneration is presented.

MATERIALS AND METHODS

Surgical technique Goldfish 7.5-10 cm in body length (Ozark Fisheries, Stoutland, MO) were used. They were normally kept at 20 °C on a 12 h/12 h light-dark cycle. For surgery they were anesthetized in 400 mg/liter tricaine methanesulfonate (Finquel, Ayerst Laboratories, New York, NY). Under a dissecting microscope, the connective tissue

around the dorsal surface of the right eye was cut with iridectomy scissors. The eye was gently rotated forward and the nerve crushed for 3 s close to the back of the orbit with a pair of jewellers' forceps with angled tips. Fish were returned to their tank and recovered within 5 min.

Intraocular injections As soon as fish resumed swimming after surgery, they were injected in the operated eye with 0.07 ~g (0.7 #1) TTX (Sigma) in 2.6 mM citrate buffer (pH 5) or 0.7/LI of 2.6 mM citrate buffer (Sigma). The dose of TTX used usually abolished visual activity for 2.5-3 days. When a prolonged block of activity was required, TTX or citrate was reinjected every 60-65 h, except in the experiments in which fish were tested for visual behavior, in which case the injections of TTX or citrate were repeated at the end of every fourth day, so that the TTX would have worn off by the time of testing.

Behavioral testing for return of visualfunction Testing for recovery of the startle reaction, dorsal light reflex and food localization was carried out as described by Edwards et aL t°. During testing, the unoperated eye was temporarily blinded with xylocaine. The testing in most experiments was done at 4-day intervals, when the TTX had worn off, and in appropriate cases the fish were reinjected with TTX or citrate immediately after testing.

A xonal transport ofprotein to the optic tectum Fish with the right optic nerve crushed were injected with TTX or citrate as described. At selected post-crush intervals, the right eye in each fish was injected with 5-6 ~tCi of L-[3,43H(N)]proline (32.5 Ci/mmol, New England Nuclear) and the fish were killed by decapitation 24 h later. The brains were exposed and the heads immediately immersed in Bodian's fixative. Forty-eight hours later, the optic tecta were removed, dissolved in Soluene-100 (Packard Instruments, Downers Grove, IL), and counted in a liquid scintillation counter (Beckman Instruments LS 7500). Data were expressed as dpm//~Ci injected per tectum. To avoid any di-

rect effects of TTX on labeling of protein each [3H]proline injection was made at least 3 days after the last TTX injection.

Histology of nerves After the heads had been fixed in Bouin's solution, the right optic nerve with the contiguous optic tract was dissected out and embedded in paraffin. Longitudinal sections were cut at 15 #m and processed by the Bodian silver technique. The n u m b e r of axons at either 1 m m (10 days) or 1.5 m m (22 days) from the site of initiation of outgrowth was counted at 400 x magnification on every fourth section and the counts were s u m m e d and multiplied by 4 to obtain the total n u m b e r of axons in the nerve. (The site of initiation of outgrowth was defined as the level along the nerve at which the parallel orientation of normal axons extending from the back of the eye changed into an irregular plexiform arrangement 39.)

Retinal ganglion cell measurements The eyes were dissected from the fixed fish heads and paraffin sections were cut at 15/zm and stained with toluidine blue. Ganglion cell profiles containing the entire nucleus and having a clear cell outline were drawn with the aid of a camera lucida, using the 100 x oil immersion objective. Sixty cells per retina were selected at points representative of the whole retina and the cell areas were measured using an electronic digitizer (Numonics, Lansdale, PA). To determine the relative numbers of ganglion cells, cells were selected using the same criteria as for cell size. For 22 day retinas, counts were made at 160 x magnification in 3 separate regions each 625 ~m in length (as measured with an eyepiece grid) in each of 6 sections per retina. In the 36 day retinas, counts were made at 400 × in 3 lengths of 250/~m each in each of 6 sections per retina.

Incorporation of [3H]proline into retinal ganglion cells TTX- and citrate-treated fish were injected in the right eye with 5-6/~Ci of L-[3,4-3H(N)]pro line (32.5 Ci/mmol). Twenty-four hours later,

the fish were killed, and after the heads had been fixed in Bouin's solution, the eyes were dissected out. Paraffin sections (8 tan) of eyes were dipped in NTB2 emulsion (Eastman Kodak, Rochester, NY), exposed for 18 h, developed with D-19, and stained with toluidine blue. Grain counts on 60 retinal ganglion cells per retina were done at 400 × magnification. Only cells with a detectable nucleus and surrounding cytoplasm were selected. The mean n u m b e r of grains per cell was calculated from each fish and the mean of those means was calculated for each group of 3-4 fish.

A utoradiography of the regenerated tectalprojection At 40 days after optic nerve crush, selected fish that had been treated with TTX or citrate for 31 days after the nerve crush were injected in the regenerating eye with 10/~Ci of [3H]proline. Fish were allowed to survive for 2-4 days before being killed by decapitation. After fixation in Bodian's solution, the brains were embedded in paraffin and serial sections cut in either the sagittal or transverse planes were processed for autoradiography as described above for the retinas, but with exposure times of about 2 weeks.

Incorporation of [3H]2-deoxyglucose into the optic tectum An intraperitoneal injection of 6-7/~Ci D-[ 1,23H]2-deoxyglucose (37.3 Ci/mmol, New England Nuclear) in 20 btl saline was made into the ventral midline of the fish. In experiments with TTX, the TTX injections were made 12-18 h before the 2-deoxyglucose injection, in order to allow any acute effects of TTX on movement or equilibrium to wear off. After 60-120 min, the fish were decapitated and the optic tecta rapidly removed. Each tectum was dissolved in Soluene100 and prepared for liquid scintillation counting. Flash stimulation used during the course of some of these experiments was provided by a Grass photic stimulator.

RESULTS

Blocking visual activity with TTX The efficacy of intraocular injection of TTX in blocking neural activity in the visual system of normal goldfish was assayed by observing the presence of the dorsal light reflex t° after unilateral TTX injection. We found that after an injection of 0.05-0.10 ttg of TTX the dorsal light reflex appeared within 10-15 min. It disappeared after about an hour but then could be consistently evoked over a period of 2.5-3 days by briefly dark adapting the fish ~°, which indicates the persistence of the TTX block for this period. This duration of the TTX effect was about equal to that determined by abolition of electrophysiological responses 58. The long duration of the block, which has also been observed upon intraocular injection of TTX into cats 3°, is probably due to sequestering of TTX in the vitreous. As confirmation that the TTX was blocking activity in the optic axons, we found that for a period of at least 18 h following a single TTX injection the incorporation of [3H]2-deoxyglucose (2-DG) in the optic tectum connected to the normal eye was 21-76% higher than in the tectum connected to the TTX-injected eye (Table I, 'Control group'). The magnitude of the difference in incorporation on the two sides of the tectum was approximately equal to that caused by excision of one eye or intraocular injection of xylocaine in one eye 1. The difference in [3H]2-DG

incorporation between the tecta was still seen when, instead of a single TTX injection, a series of injections was given at 2-3 day intervals so that activity had been continuously blocked for 8 days at the time that [3H]2-DG incorporation was tested (Table I). This shows that the effect of TTX in blocking activity did not wear off with repeated injections of the drug. If anything, the effect in the 8-day treated animals appeared to be somewhat greater than in the control group which had received only a single TTX injection. We are confident therefore, that a reasonably constant block of optic nerve activity was produced with repeated TTX injections over prolonged periods of time. The difference in tectal incorporation of [3H]2-DG produced by unilateral TTX injection was the same in animals kept in normal laboratory lighting during the 1-2 h period of [3H]2D G incorporation as in those kept in the dark, or those subjected to flashing light at various rates (Table I). These changes in lighting conditions would therefore not have been effective in producing the sustained changes in optic nerve activity that would be required for exploring the relationship between neural activity and regeneration, although they may produce some changes over relatively short periods of time I .

Regeneration of optic axons during TTX block Recovery of visual function. Nerve regeneration during a continuous block of visual activity

TABLE I

Effects of various conditions of illumination or chronic TTX injection on [3H]2-DG incorporation into optic tecta connected to uninjected and TTX-injected eyes The control group in each case consisted of animals that were kept in normal laboratory lighting during the [3H]2-DG incorporation. Flash stimulation was carried out with a Grass photic stimulator. Fish were injected with [3H]2-DG 18 h after the last injection of TI'X. The n u m b e r offish per group appears in parentheses.

Experimental treatment 8 daysTTX Dark Flash I/s Flash 2/s Flash 10/s Flash 20/s

[3H]2-DG incorp. period 60 min 120min* 60 min 120 min 120min* 120 min

* Experiment carried out at 25 °C.

Mean ratio ± S.E.M. (Tectum of uninjected eye/ tectum of TTX-injected eye) Experimental group

Control group

1.41 1.46 1.62 1.48 1.44 1.70

1.21 1.40 1.52 1.27 1.40 1.76

__. 0.08 (8) _ 0.11 (9) ± 0.19 (8) ± 0.13 (8) ± 0.12 (9) ± 0.13 (8)

__. 0.05 (6) ± 0.13(8) ± 0.15 (8) ± 0.09 (8) ± 0.13 (8) ___0.15 (8)

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Fig. 1. Effect of multiple intraocular injections of TTX on recovery of the startle reaction following an optic nerve crush. A: TI'X or citrate buffer (control) was injected into the R eye within 5 min after crush of the R optic nerve and every 4 days thereafter. Fish were tested for startle reaction beginning at day 8 and every 4 days thereafter just prior to reinjection. B: fish were treated as above, except that TTX was allowed to wear off completely during the period indicated by the double line by omitting the injection on day 16.

by intraocular TTX was initially evaluated by testing for recovery of visual function, as indicated by the reappearance of the startle reaction to sudden illumination, the dorsal light reflex and food pellet localization. The results obtained for recovery of the startle reaction are shown in Fig. 1A. In the control fish (injected with citrate buffer), the initial responses occurred at 8 days after optic nerve crush and 100% recovery for the group was reached by 20 days. The first responses of the TTX-injected fish were also seen at 8 days, but most of these fish lagged behind the citrate controls and complete recovery for the TTX group did not occur until 35 days. The normal mean recovery time of this r e a c t i o n is 12 14 d a y s 1°,19,72. B e c a u s e o f the l o n g intervals b e t w e e n test periods, w e h a v e n o t a t t e m p t e d to q u a n t i f y the c h a n g e in the r e c o v e r y time, b u t i n s p e c t i o n o f Fig. 1A indicates a n increase o f a b o u t 5 d a y s in the m o d a l value. I n o r d e r to e n s u r e t h a t the d e l a y e d r e c o v e r y o f

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Fig. 2. Effect of repeated qq'X injections on recovery of visual responses after an optic nerve crush. A: recovery of dorsal light reflex. B: recovery of food pellet localization. Schedule of injections as in Fig. 1A, with last injection day 28.

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shown in Fig JA, which had received TTX evely 4 days without interruption. Recovery of the dorsal light reflex (DLR) and food pellet localization were also delayed in TTX-treated fish (Fig. 2A, B). For the DLR, the appearance of the initial responses was about 10-15 days later in TTX-treated fish (Fig. 2A). For the food pellet localization, which is believed to depend on connections from the retina to the tectum 65, recovery began by 32 days in the citrate-injected fish, but the first positive responses for the TTX-treated fish did not occur until day 41, even though no TTX injections were made after day 28 (Fig. 2B). Complete recovery of food localization was observed in the citrate group before the first TTX-treated fish responded and the modal value for recovery in the TTX group was greater by about 8 days. In a separate set of experiments, we investigated the effect of a single injection of TTX administered at the time of nerve crush. The single injection would have produced an activity block lasting only about 2.5 3 days, but, as can be seen

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Fig. 4. Effect of single TTX injection immediately following optic nerve crush on recovery of food pellet localization.

in Fig. 3, the recovery of the startle reaction was delayed to about the same extent as when TTX was given throughout the recovery period (Fig. IA). In contrast, there was no delay in the recovery of food localization when only the single injection of TTX had been given (Fig. 4). Since the testing interval for this response was only 2 days, we would not have had any difficulty in detecting an increase in recovery time of the magnitude of that seen for the startle reaction with a single injection. Number of retinal ganglion cells. To confirm that the delayed recovery of the behavioral responses was not due to a toxic effect of TTX on the retinal ganglion cells (RGCs), measurements of R G C numbers were made from regenerating retinas at 22 days and 36 days postcrush, with TTX or citrate having been injected every 3 days. The results of such counts (Table II) show that TTX did not reduce the incidence of retinal ganglion cells in the retina after 36 days of regeneration. Thus, there was no evidence that the long-term application of TTX was toxic to regenerating RGCs.

TABLE II Incidence of retinal ganglion cells in retinas treated with T T X or citrate every 3 days following optic nerve crush Number of retinas in each group indicated in parentheses. Time after nerve crush

Mean No. of cells per 1000 lun length of retina* +_ S.E.M. TTX-treated

Citrate controls

T T X / citrate

22 days 36 days

63.4 ___ 1.3 (4) 85.6 _ 4.9 (3)

57.3 __. 3.3 (4) 82.7 _ 3.3 (4)

1.11 1.04

* Total length measured per retina = 11.25 mm for 22 day retinas, 4.5 mm for 36 day retinas.

TABLE III Number of axons detectable in silver-stained sections of optic nerves treated with T T X or citrate following an optic nerve crush The number of nerves in each group appears in parentheses. Time after crush

10days 22days

Injections of T T X *

multiple or single multiple

Counting site (mm from

Mean No. of axons in nerve + S.E.M.

initiation of outgrowth)

TTX-treated

Citrate control

T T X / citrate

1.0 1.5

590 _ 164(5) 2,489 __+ 199(7)

806 - 40 (5) 3,191 + 617(3)

0.73 0.78

* Fish which received multiple injections of TTX were injected every 3.5-4 days; those which received a single injection were injected only at the time of crushing.

Number o f regenerating axons. The effect of intraocular TTX on the numbers of regenerated axons at 10 and 22 days after nerve crush (i.e. times before and after the axons had reached the optic tectum) was determined in silver-stained longitudinal sections of optic nerves. We have found (B. Grafstein and R. M. Alpert, unpublished results) that the number of axons detectable by this method in the normal optic nerve represents about 10% of the total number calculated from electron micrographs 47. Probably only axons above a certain size and separation are distinguishable, although it is not clear whether all the axons above the critical size would be included. However, we assume that these constraints would apply equally to TTXtreated and citrate-treated preparations. The group of 10-day regenerating nerves examined (Table III) included nerves from both fish receiving multiple injections of TTX and those that had received TTX only at the time of the nerve crush, since both treatments produced similar lengthening of recovery times for the startle reaction. The average number of axons per nerve in the citrate-treated fish was close to the mean value previously obtained in this laboratory for nerves after 10 days of regeneration following a nerve crush 72. In fish treated with TTX, the average number of axons per nerve was 73% of the average number in citrate controls. A similar relationship between the TTX and citrate groups was seen at 22 days postcrush, the average number of axons in the TTX treated group being 78% of citrate controls. These results would be consistent with either a decrease in the number of regenerating axons in the nerve (e.g. fewer branches produced by each

parent axon), a reduction in the diameter of the regenerating axons, or a slower rate of axonal outgrowth. Time course o f arrival of axons at the tectum. The amount of axonally transported labeled protein arriving at the optic tectum after intraocular injection of [3H]proline was determined for TTX- or citrate-treated fish at various intervals after optic nerve crush, as an indication of the time course of arrival of RGC axons at the tectum 39. As can be seen in Fig. 5, hardly any transported radioactivity had reached the tectum in either group of fish by 15 days after the lesion, but the amount appearing in the tectum in both groups of fish increased thereafter, reaching its peak in the citrate-treated fish at about 28 days, when the regenerating axons would have covered the entire tectum (ref. 59; R. K. Small, unpublished observations). Initially, the amount of transported radioactivity appearing in the tecta of the TTX-treated ARRIVAL OF REGENERATING AXONS tN OPTIC TECTUM

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Fig. 5. Time course of arrival of axonally transported radioactive protein in optic tectum following optic nerve crush. Fish were injected in the R eye with TFX or citrate buffer within 5 min after R nerve crush and every 3 days thereafter, Amount of labeled transported material is determined from the difference between the L and R tectum 24 h after injection of[aH]proline into the R eye. P ( 0.05, (2-tailed t test) for values at 24 days.

TABLE IV

Incorporation of [3H]proline into retinal ganglion cells of retinas treated with T T X or citrate every 3 days following an optic nerve crush The number of retinas per group appears in parentheses. 60 cells were examined per retina. N.B. Proline given at end of 3rd day after TTX.

Time after crush

Mean No. of grainsper cell +_ S.E.M. TTX

Citrate

T T X / citrate

24days 36days

13.8 ___ 1.1 (3) 14.0 ___ 1.2(3)

18.3 ___0.5 (3) 13.7 ___ 1.2(3)

0.75 1.00

fish was not different from that in the controls, indicating that the time of arrival of the most rapidly regenerating axons was identical. At 21 28 days, however, a significant deficit was observed in the TTX-treated group: the maximum difference, nearly 60% less than in the citrate-injected group, was seen at 24 days. There was no difference at 36 days, and none at 45 days when the amount transported in both groups had declined in the course of recovery from the elevation of protein synthesis that occurs during regeneration in these neurons4°,5°. It is likely, therefore, that the difference in transported material reaching the tectum does not represent merely a delay in the time of arrival of the regenerating axons: rather, a fraction of the transported radioactivity fails to appear. Cell body reaction. Normally, protein synthesis in goldfish RGCs increases dramatically in response to axotomy, as indicated by increased incorporation of labeled amino aci& °,5°. Measurements on autoradiograms of retinas from fish that had been injected with [3H]proline 24 h before sacrifice showed that at 24 days postcrush there was 25% less [3H]proline incorporated into protein in RGCs of the TTX-treated fish than in citrate-treated controls (Table IV). This difference in incorporation is probably the

basis for the difference in the amount of axonally transported protein seen in the tectum at 24 days (Fig. 5), since the retinas were taken from the same fish used for the time course. The magnitude of the difference in incorporation appears to be somewhat less than the maximum change in transport because it represents the average difference in the whole population of RGCs, some of which (the most rapidly regenerating) show little or no change as indicated by their contribution of axonally transported protein to the tectum. At 36 days after the crush, the mean number of grains over retinal ganglion cells for the two groups was the same, indicating that at this time the utilization of [3H]proline for protein synthesis was not affected by TTX, consistent with the absence of any difference in axonal transport at this time. Retinas were also examined to see if the increase in cell size that is normally part of the cell body response to axotomy5° was altered by TTX treatment. At 22 days after nerve crush the mean area of RGCs in TTX-treated fish was slightly larger than for citrate controls (Table V), but by 36 days after nerve crush this difference had disappeared.

A utoradiography of the regenerated retinotectal projection. The retinotectal projections of

TABLE V

Retinal ganglion cell areas from retinas treated with T T X or citrate for 22 or 36 days following an optic nerve crush The number o f retinas in each group appears in parentheses.

Time after CrUsh

Mean RGC area (pro2) +_ S.E.M. TTX

Citrate

TTX/citrate

22days 36days

66.6 ___ 2.0(4) 65.1 ___0.4(3)

61.5 ± 1.1 (4) 64.8 ___ 1.3(3)

1.08 1.00

TTX- and citrate-treated fish (last intraocular injection on day 28) were examined at 42-44 days after nerve crush using [3H]proline autoradiography. No obvious difference was seen. In each case, the tectum contralateral to the crush showed heavy labeling in the stratum opticum (SO) and stratum fibrosum et griseum superficialis (SFGS), the primary terminal zones of the retinotectal projection. In the medial half of the tectum label was also present in stratum griseum centrale (SGC) in both groups, but very little labeling occurred in stratum album centrale (SAC) in either group. In both groups there was also light labeling in the SFGS of the ipsilateral tectum. These projections are like those that have previously been described in the optic tectum in regenerating preparations~2. DISCUSSION

The results of this study show that when the neural activity of goldfish retinal ganglion cells had been blocked by intraocular injection of TTX their axons could regenerate after an optic nerve crush and form effective synapses in the optic tectum. However, regeneration was hindered by the TTX treatment, as indicated by the delayed recovery of visually mediated behavior, by reduced numbers of axons in regenerating optic nerves, and by reductions in protein synthesis and fast axonal transport of protein. In addition, the TTX-treated animals showed enlarged multi-unit receptive fields in the regenerated retinotectal projection, even at long times after the TTX treatment had been stopped 58. Also, during the early stages of reconnection to the tectum the latency of the responses was prolonged 59. The general implication of our findings, that blocking of neural activity hindered regeneration of the goldfish optic axons, suggests that enhanced activity might promote regeneration. Some important support for this idea has recently been obtained by Diamond and co-workers 52, in their observations that impulse activity can stimulate collateral sprouting of sensory nerves in rat skin. Also, it has been shown that in peripheral nerves electrical stimulation can in-

crease sprouting of intact axons into denervated areas near their targets 24,37.From our present experiments, we can make some progress in identifying some features of the regenerative process that are likely to be affected by the modulation of neural activity, and we can suggest some hypotheses about the mechanisms involved.

Characteristics of regeneration with visual activity blocked Recovery of visual behavior The effect of TTX in hindering recovery was evident for each of the 3 kinds of visual behavior examined - startle reaction, dorsal light reflex and food pellet localization - although they are probably quite different with respect to the brain centers, mechanisms of neuronal interaction and retinal ganglion cell populations that are involved j°. For example, retinotopically patterned connections in the optic tectum and a reasonable degree of visual activity are probably important for food localization, whereas the startle reaction may depend only on achieving a critical degree of excitation in an as-yet-unidentified non-tectal center. Delayed recovery of food localization has been reported to occur when goldfish are kept in the dark during optic nerve regeneration 35. However, we have found no evidence from studies of[3H]2-DG incorporation into the tectum of normal fish that there is any maintained difference in visual activity between animals kept continuously in darkness and those kept in the light. This would be consistent with previous electrophysiological evidence that there is relatively little difference in sustained optic nerve activity in light and darkness (reviewed by Grafstein et al?8). It remains to be established, therefore, whether the impaired regenerative ability in the dark is attributable to decreased visual activity or to a more generalized defect in nervous system arousal, locomotor activity or hormonal status. Such considerations do not apply in our present study, in which the TTX-injected fish retained normal vision in the untreated eye throughout the experiment (except for a few hours during behavioral testing when activity in

10 the untreated eye was eliminated with xylocaine). The efficacy of a single TTX injection in delaying the recovery of the startle reaction suggests that much of the effect of TTX on this reaction occurs at a very early stage of regeneration. For food localization recovery, on the other hand, we found no detectable effect from a single injection of TTX at the time of the lesion. Moreover, continuous TTX treatment during the first 14 days after nerve crush did not elicit one of the characteristic electrophysiological abnormalities that can be produced by the TTX treatment, namely enlarged receptive fields in the optic tectum 58. Thus we believe that the TTX treatment may be producing its effects by interfering with two separate events, one occurring during the first few days of regeneration and affecting the rate of recovery of the startle reaction, the other occurring after 2 weeks of regeneration and affecting the rate of recovery of food localization, as well as the size of receptive fields. In the discussion that follows, we present our reasons for believing that at least the first of these events may be the production of regenerating axon branches in the optic nerve.

Outgrowth characteristics of regenerating axons Our axon counts in silver-stained nerve sections as well as the time course of appearance of transported radioactivity in the tectum indicate that in the TTX-treated animals there is some structural defect in the regenerated optic axons, manifesting itself as early as 10 days after the nerve crush, and persisting for up to 28 days. This defect might be related to an impairment in the time or rate of outgrowth of some of the regenerating axons, or to a reduction in the number or size of the regenerating axon branches Rate of outgrowth. Intraocular injection of TTX does not interfere with the rate of elongation or time of outgrowth of the fastest-growing axons in goldfish optic nerve. This may be adduced from our finding that TTX had no effect on the time of initial arrival of regenerating axons at the optic tectum as shown by the appearance of axonally transported protein in the tecturn at 15-21 days after the nerve crush. In addi-

tion, the maximum length of axonal outgrowth in the optic nerve at 10 days after nerve crush (measured by filling the regenerating axons with labeled axonally transported protein 39) does not appear to be affected by intraocular TTX injection (J. R. Sparrow, unpublished results). These observations do not eliminate the possibility that the TTX treatment might have selectively reduced the outgrowth rate of a non-leading population of axons. Selective sensitivity of various axon populations to the level of physiological activity was seen in the collateral sprouting of sensory nerves in rat skin 52, in that highthreshold sensory axons were found to increase their sprouting when they were stimulated, whereas low-threshold (possibly larger diameter?) axons did not. However, our observation that the time course of arrival of transported material in the tectum showed an absolute deficit at 21-28 days and not just a shift in timing is difficult to explain by retardation of outgrowth of some axons. Number and size of regenerating axons. During the normal course of regeneration of goldfish optic axons each parent axon appears to produce an average of 4 regenerating branches from points near the site of the injury 47. TTX treatment may reduce the number or the diameter of these branches. Either of these circumstances would be consistent with our finding of about 25% fewer silver-stained axons in the nerves from TTX-treated retinas compared to citrate-treated controls, if it is assumed that only axons greater than a certain size were visualized by the stain. The long latency of evoked responses at early regeneration times 59 is probably easier to explain in terms of smaller size of the axons; the delayed recovery of the behavioral responses is easier to explain in terms of the production of fewer branches (so that a longer regeneration time would be required before a critical degree of innervation was established in the respective target areas). Other arguments could be adduced, e.g. based on requirements for temporal summation, to support one or the other of these hypotheses, but it would be reasonable to assume that a reduced number o f a x o n branches and a reduced size of branches could occur to-

11 gether, since both would be effective in reducing the volume of the regenerating axons. Since a reduced number of axons in the nerve was seen as early as 10 days after the lesion and was probably produced even with a single injection of TTX, a reduction in axon branching in the optic nerve could account for the delay in the recovery of the startle reaction. However, it could not by itself account for the effects of TTX treatment in inhibiting recovery of food localization and preventing refinement of the retinotectal map, since early TTX treatment did not elicit these effects. We can suggest one possible explanation, based on that fact that, in addition to branching near the site of the lesion, the regenerating axons apparently branch again en route to or within the optic tectum46,48. Continued TTX treatment may impair the later stages of axonal branching - possibly terminal branching within the tectum itself may be inhibited, since the TTX-treatment period effective for preventing the refinement of the retinotectal map corresponds to the time when the axons would be invading the tectum. In spite of any axonal branching defects that may result from the TTX treatment, the total number of synaptic contacts eventually produced is probably not significantly reduced, as indicated by the normalization of the amount of axonally transported label reaching the tectum at 36 days and later, and by the nearly normal amplitude of evoked responses in the tectum 59. Thus, the process of synaptogenesis appears to be quantitatively regulated by tectal factors, as Murray et al. 5~ have also concluded from other evidence. However, it is possible that reduction of the number of intra-tectal axon branches as a result of TTX-treatment might be sufficient to produce a decrease in accuracy of the connections. With fewer branches, the competition by which incorrect or asynchronously active inputs are eliminated 25.56 might be reduced, leading to preservation of anomalous connections. This view receives some support from the reduced elimination of neuromuscular connections that was seen when the number of axons innervating a developing muscle had been reduced 5.

Mechanisms o f the T T X effect on axonal outgrowth in the optic nerve Intraocular injection of TTX rapidly abolishes orthodromic action potentials in R G C axons44,59, presumably because it blocks Na ÷ channels in RGC axons within the eye. Activity in efferent axons to the eye57 and antidromically conducted activity in the R G C axons (originating from ectopic foci at the injury site68) would also be blocked under these conditions. Moreover, TTX may act directly on the R G C soma (as it does on the soma of other axotomized neurons12), to block Na ÷ channels in the electrogenic patches of soma-dendrite membrane that develop in response to axotomy32. (Also, if TTX diffuses into other retinal layers, it would block action potential-mediated input onto the RGCs 43, but this may not make much difference, since axotomy alone may cause retraction of some presynaptic boutons from the perikaryon and dendrites67.) In view of these multiple ways in which TTX may be acting, it is not yet clear whether the impairment of regeneration is due to interference with membrane changes in the cell body or at the lesion site. If it acts on the cell body, we might expect that activity may have an influence on the synthetic capability of the neuron and hence on the supply of axonally transported material to the growing axon; if it acts at the lesion site we might anticipate that activity could either influence structural changes at the growth cone directly, or could alter retrograde axonal transport from the growth cone (with alterations in the cell body then occurring secondarily as a result of modified chemical feedback). Whatever the primary site of TTX action may be, we can envisage a number of mechanisms by which it might exert its effect on regeneration. (1) Changes in Na ÷ fluxes in neuronal structures deprived of their normal action potential activity may lead to alterations of Na÷-linked processes, such as phosphorylation of proteins or membrane transport of organic solutes60. (2) Blocking action potentials may interfere with Ca 2÷ entry into the growth cone. As has been found in developing as well as regenerating neurons 2°.41,63,66, action potentials in growing ax-

12 ons may involve inward Ca 2÷ current, particularly at the growth cone. Entry of Ca 2÷ might promote axonal outgrowth by stimulating the addition of new m e m b r a n e in the growth c o n e ~6, or by increasing the rate of elongation of the cytoskeleton 34,54. This postulated role of Ca 2÷ would be consistent with our recent observations that local application of the calcium ionophore A-23187 to the lesion site in a crushed goldfish optic nerve is effective in enhancing the rate of visual recovery 19. (3) Block of action potentials may interfere with entry of Ca 2÷ into the cell body, since Ca 2÷ currents may contribute to the cell body action potential in developing neurons 63,69 and presumably also in regenerating neurons, which develop a similar soma-dendrite-spike mechanism 12,32.The change in Ca 2÷ availability might influence the availability of materials for axonal transport, particularly materials processed in the Golgi apparatus such as glycoproteins21. The possibility that the impairment of regeneration by TTX might involve a defect in axonal transport is consistent with our recent finding that intraocular TTX treatment reduced the axonal transport and turnover of glucosaminecontaining glycoproteins in normal RGCs 9. Even more dramatic was the effect on glucosamine-containing glycolipids9, an effect which is all the more interesting because of recent observations on the ability of exogenous gangliosides to stimulate axonal branching during regeneration13,62.

Another effect of TTX that we have observed in normal RGCs is a reduction in the axonal transport of nucleosides and in their subsequent incorporation into R N A in the postsynaptic tectal neurons 9,15,16.Since axonal transport of nucleosides and 4S R N A is greatly increased during regeneration of goldfish optic axons 27,28the supply of these materials may have an influence on REFERENCES ! Aitenau, L. and Agranoff, B. W., Visual stimulation increases regional cerebral metabolism and blood flow in the goldfish, Brain Research 161 (1979) 55-61. 2 Attardi, D. G. and Sperry, R. W., Preferential selection of central pathways by regenerating optic fibers, Exp.

regeneration, although the nature of their contribution is still a matter for speculation 28. By contrast with the activity-sensitive axonal transport of glucosamine-containing compounds and nucleosides, neither the fast nor slow axonal transport of proteins labeled with various radioactive amino acids was affected even by long-term TTX treatment in normal fish9,14,16. These findings demonstrate that TTX treatment does not produce a non-specific depression of protein synthesis or axonal transport, and this is confirmed by our finding in the present study that TTX treatment did not impair amino acid incorporation or axonal transport at 36 days and later after optic nerve crush. Nevertheless, we found that amino acid incorporation was depressed by about 25% at 24 days, and in other recent experiments we have found a deficit in the amount of axonally transported labeled protein entering the optic axons even as early as 8 days after the crush (J. R. Sparrow, unpublished results). It is possible, therefore, that in regenerating neurons synthesis of transported materials is especially sensitive to block of neural activity; for example, the reduction of activity might interfere specifically with the synthesis of certain transported proteins that may be essential for axonal growth in regeneration and development 3,H,55,61. This may explain why variations in physiological activity would have a profound effect on the structure and function of the nervous system in young animals but not in adults. ACKNOWLEDGEMENTS

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