Protein synthesis and fast axonal transport in regenerating goldfish retinal ganglion cells

Brain Research, 235 (1982) 213-223

213

Elsevier Biomedical Press

Research Reports P R O T E I N S Y N T H E S I S A N D FAST A X O N A L T R A N S P O R T I N R E G E N E R A T I N G G O L D F I S H R E T I N A L G A N G L I O N CELLS

IRVINE G. McQUARRIE* and BERNICE GRAFSTEIN Department of Physiology, Cornell University Medical College, New York, N Y 10021 (U.S.A.)

(Accepted August 13th, 1981) Key words: axonal growth - - retinal ganglion cells - - optic nerve regeneration - - axonal transport

- - nerve cell body reaction

SUMMARY To characterize the fast component of axonal transport in regenerating goldfish optic axons, the incorporation of L-2,3-[aH]proline into newly-synthesized proteins in the cell bodies of the retinal ganglion cells and the amount of transported labeled protein were determined at 2-36 days after cutting the optic tract. Both the incorporation and the amount of transported protein had doubled by 10 days after the lesion and continued to increase to about 5 times normal at 15 days, a time when a large proportion of the regenerating axon population had reached the optic tectum. Nearnormal levels were recovered by 36 days. In contralateral control neurons, the incorporation of L-2,3-[aH]proline was unchanged from normal throughout, whereas the amount of labeled transported protein entering control axons was decreased by 5 5 ~ at 2 and 10 days after the testing lesion, returning to normal by 15 days. An increase in fast transport velocity was seen in the regenerating axons beginning at 10 days after the lesion. However, a similar velocity increase was also seen in the contralateral control axons and in undamaged axons following removal of the cerebral hemispheres. Therefore, the velocity increase was not a specific consequence of axotomy.

INTRODUCTION Fast axonal transport (reviewed in ref. 15) contributes to axonal outgrowth in regenerating neurons by supplying the particulate membranous elements used in the * Present address for all correspondence: Department of Anatomy, Case Western Reserve University, School of Medicine, Cleveland, OH 44106, U.S.A. 0 0 0 6 - 8 9 9 3 / 8 2 / ~ / $ 0 2 . 7 5 © Elsevier Biomedical Press

214 longitudinal and radial growth of the axolemmaX2,13,18,~9,37. It has been reported that, in regenerating retinal ganglion cells of the goldfish, the velocity of fast transport doubles and the amount of fast-transported protein increases about 3-fold 17. The study in which these changes were observed, however, was carried out in a late period of regeneration, after the regenerating axons had reached the optic tectum and reestablished synaptic connections. In the present study, we have examined the characteristics of fast transport during the course of regeneration following transection of the optic tract. The post-operative intervals included: 2 days, before significant axonal elongation has occurred~4,25; 10 days, when some of the growing axons have begun to reach the optic tectumZ0; 15 days, when a large number of axons has invaded the tectum, and 36 days, when recovery of visual function is well underway, as demonstrated by the recovery of food localization8,17. We have found that the labeled amino acid incorporation by the retinal ganglion cells and the amount of labeled fast-transported protein appearing in the axons increased to reach a peak of about 5 times normal at 15 days. Brief summaries of these findings have appeared previously16,28. MATERIALS AND METHODS Goldfish (Carassius auratus), 9-11 cm in body length (13-15 cm from nose to tip of tail), were obtained in the fall and winter months from Ozark Fisheries (Stoutland, MO) and kept at 19-23 °C. For the testing lesion (transection of the optic tract), goldfish were anesthetized by immersion in 3 ~ (w/v) urethane until respirations had ceased. After a flap in the skull was opened, the intracranial adipose tissue and the cerebral hemispheres were removed by aspiration, and the left optic tract was cut with iridectomy scissors at its bifurcation near the optic tectum. The cranial flap was bent back into place and fastened with a single 10 mm Michel clip. All operated goldfish received an intravitreal isotope injection in each eye at 1 or 2 h before being killed at 2-36 days post-lesion; normal goldfish were similarly injected. For the injection solution, L-2,3-[3H]proline (45.7 Ci/mmol; New England Nuclear, Boston, MA) was dried to a residue and reconstituted with 0.15 M NaC1 to 0.65-0.85/~Ci/#l. Two #1 of this solution was injected by means of a 10 #1 syringe (Hamilton Co., Reno, NV). For tissue retrieval, goldfish were killed by decapitation; the cranial contents and optic nerves were immediately exposed and the head was placed into either Bouin's fixative or a trichloroacetic acid (TCA) fixative (90 ml 50 ~ ethanol, 10 m137 ~ formaldehyde, 5 g TCA), both of which are effective in removing unincorporated amino acids, so that the remaining radioactivity can be assumed to be in protein 6. After 48-72 h, tissues were decolorized by immersion in 70 ~ ethanol. The eyes were paraffin-embedded and sectioned at 8/zm for light microscopic radioautography3L The continuous length of optic nerve and tract from the optic nerve head to the optic tectum was removed from each side and embedded in paraffin. At each stage of dehydration, the specimens were straightened by applying gentle pressure with jeweler's forceps. Each specimen was serially cross-sectioned at 50/zm, and consecutive groups of 5 segments were collected into vials for liquid scintillation spectrometry ~7, so that radioactivity values were obtained for each ¼ mm segment of nerve.

215 As a measure of the amount of labeled transported radioactivity in the nerve, we chose the plateau level of radioactivity seen in the nerve at 2 h after injection of labeled proline (Fig. 2B, 2 h plot). This value was determined for the point on the nerve at 1.5 mm behind the eye by taking the average of the two values for the ¼ mm nerve segments centered at 1~ and 1-~ mm from the eye. The 1.5 mm point was far enough behind the eye to avoid contamination by glial incorporation of precursor that had diffused through the extracellular space 20, but not so far behind the eye as to become involved in the leading wave of axonally transported proteins on the proximal (eye) side of the testing lesion, which was located at 5.5 q- 0.3 mm (n ---- 6) from the eye (as determined from the length of the 2-day specimens). In unoperated nerves, in which the transport velocity was slower, there was no plateau but only a dip in the level of radioactivity behind the crest of the leading wave (Fig. 2A, 2 h plot). Levels of protein tadioactivity were expressed in disintegrations per minute per microCurie of isotope injected per segment length (dpm//zCi/250/~m). To compensate for differences in the amount of transported radioactivity in different animals, the distance that the wavefront of fast axonal transport had progressed was taken to be the point on the nerve at which the radioactivity had declined to 1/e of the value obtained at 1.5 mm behind the eye. To estimate the fast transport rate, and allow for differences in the time required for perikaryal processing of the transported proteins, the transport distances were measured at 1 and 2 h after injection of the isotope. To measure the transport distance in the 1 h group (where only the wave front had entered the nerve), the point at which radioactivity had fallen to 1/e times the mean radioactivity level at 1.5 mm behind the eye in the 2 h group was used. The fast transport rate was then calculated from the slope of the regression function of distance on time 1. In each retina, area measurements and silver grain counts were made at 400 × magnification in 7-10 retinal ganglion cells from each half-retina; 3-4 eyes (a total of 55-70 cells) were examined at each time interval post-lesion. Eyes from normal goldfish were similarly examined. Only cell profiles containing large nuclei surrounded by cytoplasm were selected for measurement. Because the incorporated radioactivity varied considerably within each retina, it was necessary to correct for this variation by relating the grain density in each retinal ganglion cell to the overall grain density in that part of the retina. Accordingly, the grain density (grains//zm z) for each retinal ganglion cell was expressed as a multiple of the grain density for a 155/tm~ area of the subjacent outer plexiform layer. At each time point post-lesion, the mean value for this corrected grain density was divided by the corresponding value in unoperated animals to yield 'fl'. The mean cell area at each time point post-lesion was then divided by the mean cell area in unoperated animals to yield 'fz'. Incorporation per cell (grains per cell at 2 h post-injection) at various times post-lesion is expressed as percent of normal by calculating: t"1 x t"2 × 100. RESULTS

Changes in cell size and protein synthesis during regeneration The increase in retinal ganglion cell size and protein synthesis during regenera-

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Fig. 1. Time course of changes in retinal ganglion cells that have been axotomized by the testing lesion (©) and control cells in the contralateral eye ( 0 ) compared to cells from unoperated goldfish (0). A : cross-sectional area of cells. B: density of silver grains (expressed as a multiple of background grain density) in radioautograms at 2 h after injection of L-2,3-[3H]proline. C: L-2,3-[aH]proline incorporation (grains per cell), expressed as percent of mean value for unoperated animals (see Materials and Methods). The testing lesion consisted of a unilateral optic tract cut. Each point represents combined data from 3 4 eyes. Numbers in parentheses denote numbers of cells examined. Vertical lines denote S.E. ; in A and B absence of vertical lines indicates S.E. less than radius of marking circle. Asterisks denote P ~< 0.05 compared to value for unoperated animals.

tion, as r e p o r t e d by M u r r a y a n d Grafstein 31, was b o r n e o u t by o u r m e a s u r e m e n t s (Fig. 1), a l t h o u g h the time course o f these changes was s o m e w h a t faster in the present study. A n 81 ~ increase in cross-sectional area was seen b y 10 days after optic t r a c t section, a n d the m a x i m u m size, 140 ~ a b o v e n o r m a l , was seen at 15 days (Fig. IA). By 36 days the cell area m e a s u r e m e n t was still 22 ~ greater t h a n n o r m a l . In the c o n t r o l cells on the o p p o s i t e side, the cell area was a b o u t 20 ~ greater t h a n n o r m a l at b o t h 10 a n d 15 days. I n c o r p o r a t i o n o f labeled a m i n o acid into the regenerating cells (Fig. 1C),

217 calculated from changes in area (Fig. 1A) and in corrected grain density (Fig. 1B), was nearly doubled by 10 days and rose to a maximum of about 5 times normal by 15 days. A 70 ~ increase was still present at 36 days. In the control cells there was no change from normal.

Changes in the amount of protein conveyed by fast axonal transport The amount of protein conveyed by the fast component of axonal transport was measured by the radioactivity at 1.5 mm behind the eye at 2 h after injection of labeled proline. The distribution of radioactivity in a typical regenerating optic nerve at 10 days (Fig. 2B), compared to that in a normal nerve (Fig. 2A), demonstrates the increase in the amount of labeled protein caused by the axotomy. When the timecourse of change was examined (Fig. 3A), it was evident that the amount of labeled protein in the regenerating axons had begun to increase by 10 days and reached a peak of about 5 times normal at 15 days. At 36 days it was still more than twice normal. In contralateral control optic axons, however, the amount of labeled protein was reduced to 55~ of normal at 2 days and 10 days but had returned to normal by 15 days.

Changes in fast transport velocity The velocity of fast transport at various times after the lesion was inferred from the axonal transport distances (see Materials and Methods) at 2 h post-injection. In typical cases from normal animals and from animals at 10 days after tract cut (Fig. 2) the transport distances were 3~ and 5~r mm, respectively. When the time-course of change in axonal transport distance was examined (Fig. 3B), nearly identical changes

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219 above normal, was also seen at 15 days but the axonal transport distance had returned to normal by 36 days, so that at this time there was a statistically significant difference between the control and regenerating nerves (P < 0.01). When the actual transport velocities were measured from the difference in the axonal transport distances at 1 and 2 h (Fig. 4), it was found that in normal nerves the velocity was 54.8 q- 8.0 mm/day, which is comparable to the velocities of 40-100 mm/day obtained by other methodsg,11,26. At 2 days after the lesion, the velocity in both control and regenerating nerves showed no significant difference from the value seen in normal nerves, but by 10 days the velocity had increased to 105 4- 7.4 mm/day in regenerating nerves and 91.0 4- 7.2 mm/day in control nerves, values which were not significantly different from each other. When the calculated regression functions which are used to obtain these velocities are extrapolated to zero distance, the time value on the X-axis indicates the duration of the latent period preceding the appearance of labeled proteins in the nerve. This latent period reflects the time required for synthesis of the transported material, for leading it into the fast transport channel 21, and for transport through the retina and choroid layers of the eye. At 2 days, although the velocity was unchanged from normal, the latent period may have been somewhat curtailed (Fig. 4B). At 10 days, the latent period was apparently somewhat longer than normal (54 min in regenerating axons vs 34 min in unoperated normal nerves, Fig. 4A, C), which may reflect the longer time required for the synthesis and processing of transported material in regenerating neurons 4°. The fact that the changes of transport velocity were nearly the same in control and regenerating nerves suggested the possibility that these changes might reflect some consequence of the surgery other than axotomy. The axonal transport distance was therefore determined following craniotomy alone or craniotomy plus removal of the cerebral hemispheres. The former operation did not increase the axonal transport distance significantly (Fig. 5B), but removal of the cerebral hemispheres (Fig. 5C)

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220 resulted in an increase in transport distance at least as great as that seen when removal of the hemispheres was accompanied by optic tract section (Fig. 5D). DISCUSSION

Changes in cell size, protein synthesis, and the amount of transported proteins During regeneration of goldfish retinal ganglion cells, the cell size increased to a maximum of about 2.5 times normal at 15 days after the optic tract was cut, as has previously been observed31, 39, whereas the labeled amino acid incorporation increased to a maximum of about 5 times normal at the same time. The disproportionate increase in incorporation may reflect the increased delivery of labeled proteins to the Golgi system, plasma membrane and nucleus that has been seen at this stage of regeneration 40. The increase in incorporation was very closely correlated in magnitude and time course with changes in fast axonally transported protein in the optic nerve. Some of the increased incorporation is presumably also directed to the increased production of slowly transported proteins17,22, 27. These changes in regenerating goldfish retinal ganglion cells would be consistent with the picture of increased protein synthesis during regeneration described by Br~ittgard et al. 3 for hypoglossal neurons. Not all regenerating neurons, however, show this picture. Bisby has found that regenerating rat dorsal root ganglion cells 2 and motor neurons 4 show a decrease in incorporation of amino acid into axonally transported protein, at least until the axons begin to reach their targets. Prior observations by Engh et al. 1° and by Kung 24 had already suggested that an overall increase in protein synthesis was not an inevitable concomitant of successful axonal regeneration, at least during the stages preceding reconnection of the axons. In some cases it has been shown that during regeneration there is a decrease in the synthesis of the proteins associated with the formation and metabolism of synaptic transmitters (reviewed in ref. 16). On the other hand, regeneration may involve a selective increase in the production of some proteins carried by fast axonal transport 85, which have been called 'growth-associated' proteins. Regeneration in some neurons may also be accompanied by perturbations in slow axonal transport (reviewed in ref. 16). The net result of these various changes as expressed in the overall level of protein synthesis is therefore likely to vary from one neuron to another. The changes in cell size, amino acid incorporation and amount of fast axonally transported protein that we observed in the goldfish retinal ganglion cells began to recede after about 15 days, when the invasion of the optic tectum by the regenerating fibers was well underway but probably not complete (R. K. Small, R. M. Alpert and B. Grafstein, unpublished observations). The mechanisms that lead to reversal of these changes are not yet clear. A significant factor may be that, as the axons reacquire their normal length, the time required for axonally transported materials to return to the cell body reverts to normal 4. Another possibility is that as the axons approach their normal target, trophic factors emanating from the target cells may resume their influence 33. Yet another hypothesis is that the time course of the changes is determined by an intrinsic 'clock' in the neurons as.

221 Changes in fast transport velocity and other changes in the contralateral control neurons The fast transport velocity in the regenerating neurons was nearly doubled by 10 days after the lesion, and was possibly increased even further by 15 days, as indicated by the increased axonal transport distance at 2 h after [aH]proline injection. However, nearly identical changes in transport velocity occurred in the contralateral control neurons. One factor that may contribute to this is that a small number of axons cross from one optic tract to the other (A. Springer, personal communication) and would therefore have been severed when the contralateral optic tract was cut. On the other hand, a similar increase in transport velocity in undamaged axons could be produced by removal of the cerebral hemispheres alone. Thus, in addition to axotomy itself, there may be other factors leading to the acceleration of fast axonal transport. Hormonal effects due, for example, to damage to input pathways to the hypothalamus may be involved. Another possibility is that removal of the cerebral hemispheres may have damaged second-order visual pathway neurons in the anterior and dorsalposterior nuclei of the thalamus that project to the cerebral hemispheresT,23, 34, leading to retraction of the terminals of the first-order neuronsS,a2, ~8. In addition to the increase in transport velocity, the contralateral control axons showed a 55 ~ decrease in the amount of labeled transported protein at 2 and 10 days post-lesion. Much, if not all, of this decrease is probably accounted for by the increased transport velocity, with the same amount of transported protein being distributed over a greater length of nerve. The size of the control cells was increased by about 20 ~ at 10 and 15 days postlesion, and a small transient increase can also be discerned in the study by Murray and Grafstein (Fig. 6 in ref. 31). The fact that this increase in cell size is unaccompanied by any increase in amino acid incorporation indicates that direct axotomy of recrossing axons is probably not the explanation for the increase in cell size. However, any of the other hypotheses considered above in explaining the increase in transport velocity could still apply. ACKNOWLEDGEMENTS This study was supported by USPHS Grant NS-09015 to Dr. Grafstein, USPHS Grant NS-14967 to Drs. Grafstein and McQuarrie, a Leopold Schepp Foundation fellowship to Dr. McQuarrie, and a grant from the Paralyzed Veterans of America to Dr. McQuarrie. We particularly thank Roberta Alpert for technical assistance, Marguarita Schmid for executing the illustrations, and Dr. Scott Brady for reviewing the manuscript. REFERENCES 1 Armitage, P., Statistical Methods in Medical Research, Wiley, New York, 1971, pp. 118-126, 150-163 and 281-284. 2 Bisby, M. A., Fast axonal transport of labeled protein in sensory axons during regeneration, Exp. Areurol., 61 (1978) 281-300. 3 Brattgard, S.-O., Edstrbm, J. E. and Hyd~n, H., The chemical changes in regenerating nerves, J. Neurochem., 1 (1957) 316-325.

222 4 Bulger, V. T. and Bisby, M. A., Reversal of axonal transport in regenerating nerves, J. Neurochem., 31 (1978) 1411-1418. 5 Cull, R. E., Role of nerve-muscle contact in maintaining synaptic connections, Exp. Brain Res., 20 0974) 307-310. 6 Droz, B. and Warshawsky, H., Reliability of the radioautographic technique for the detection of newly-synthesized protein, J. Histochem. Cytochem., 11 (1963)426-435. 7 Echteler, S. M. and Saidel, W. M., Forebrain connections in the goldfish support telencephalic homologies with land vertebrates, Science, 212 (1981) 683-685. 8 Edwards, D. L., Alpert, R. M. and Grafstein, B., Recovery of vision in regeneration of goldfish optic axons: enhancement of axonal outgrowth by a conditioning lesion, Exp. Neurok, 72 (1981) 672-686. 9 Elam, J. S. and Agranoff, B. W., Rapid transport of protein in the optic system of the goldfish, J. Neurochem., 18 (1971) 375-387. l0 Engh, C. A., Schofield, B. H., Doty, S. B. and Robinson, R. A., Perikaryal synthetic function following reversible and irreversible peripheral axon injuries as shown by radioautography, J. Comp. Neurol., 142 (1971) 465-480. 11 Forman, D. S., Grafstein, B. and McEwen, B. S., Rapid axonal transport of [aH]fucosyl glycoproteins in the goldfish optic system, Brain Research, 48 (1972) 327-342. 12 Frizell, M. and Sjostrand, J., The axonal transport of [aH]fucose labeled glycoproteins in normal and regenerating peripheral nerves, Brain Research, 78 (1974) 109-123. 13 Frizell, M. and Sjostrand, J., Transport of proteins, glycoproteins and cholinergic enzymes in regenerating hypoglossal nerves, J. Neurochem., 22 (1974) 845-850. 14 Grafstein, B., Role of slow axonal transport in nerve regeneration, Acta neurophath., Suppl. V (1971) 144-152. 15 Grafstein, B. and Forman, D. S., lntracellular transport in neurons, Physiol. Rev., 60 (1980) 1167-1283. 16 Grafstein, B. and McQuarrie, I. G., The role of the nerve cell body in axonal regeneration. In C. W. Cotman (Ed.), Neuronal Plasticity, Raven Press, New York, 1978, pp. 155-195. 17 Grafstein, B. and Murray, M., Transport of protein in goldfish optic nerve during regeneration, Exp. Neurol., 25 (1969) 494-508. 18 Griffin, J. W., Drachman, D. B. and Price, D. L., Fast axonal transport in motor nerve regeneration, J. NeurobioL, 7 (1976) 355-370. 19 Griffin, J. W., Price, D. L., Drachman, D. B. and Morris, J., Incorporation of axonally transported glycoproteins into axolemma during nerve regeneration, J. Cell Biol., 88 (1981) 205-214. 20 Haley, J. E., Wisniewski, H. M. and Ledeen, R. W., Extra-axonal diffusion in the rabbit optic system: a caution in axonal transport studies, Brain Research, 179 (1979) 69-76. 21 Hammerschlag, R. and LaVoie, P.-A., Initiation of fast axonal transport: involvement of calcium during transfer of proteins from Golgi apparatus to the transport system, Neuroscience, 4 (1979) 1195-1201. 22 Heacock, A. M. and Agranoff, B. W., Enhanced labeling of a retinal protein during regeneration of optic nerve in goldfish, Proc. nat. Acad. Sci. U.S.A., 73 (1976) 828-832. 23 lto, H. and Kishida, R., Telencephalic afferent neurons identified by the retrograde HRP method in the carp diencephalon, Brain Research, 149 (1978) 211-215. 24 Kung, S. H., Incorporation of tritiated precursors in the cytoplasm of normal and chromatolytic sensory neurons as shown by autoradiography, Brain Research, 25 (1971) 656-660. 25 Lanners, H. N. and Grafstein, B., Early stages of axonal regeneration in the goldfish optic tract: an electron microscopic study, J. Neurocytol., 9 (1980) 733-751. 26 McEwen, B.S. and Grafstein, B., Fast and slow components in axonal transport of protein, J. Cell Biol., 38 (1968) 494-508. 27 McQuarrie, I. G., .4xonal Regeneration in the Goldfish Optic System: the Role of the Nerve Cell Body, Ph.D. Thesis, Cornell University Graduate School of Medical Sciences, 1977, 219 pp. (University Microfilms International, Ann Arbor, 7801541). 28 McQuarrie, I. G. and Grafstein, B., Protein synthesis and fast axonal transport in regenerating goldfish retinal ganglion cells: effect of a conditioning lesion, Neurosci. Abstr., 4 (1978) 533. 29 McQuarrie, I. G. and Grafstein, B., Effect of a conditioning lesion on optic nerve regeneration in goldfish, Brain Research, 216 (1981) 253-264. 30 Murray, M., Regeneration of retinal axons into the goldfish optic tectum, J. comp. Neurol., 168 (1976) 175-196.

223 31 Murray, M. and Grafstein, B., Changes in the morphology and amino acid incorporation of regenerating goldfish optic neurons, Exp. Neurol., 23 (1969) 544-560. 32 Purves, D., Functional and structural changes in mammalian sympathetic neurones following interruption of their axons, J. PhysioL (Lond.), 252 (1975) 429-463. 33 Purves, D. and Lichtman, J. W., Elimination of synapses in the developing nervous system, Science, 210 (1980) 153-157. 34 Sharma, S. C., The retinal projections in the goldfish: an experimental study, Brain Research, 39 (1972) 213-223. 35 Skene, J. H. P. and Willard, M., Changes in axonaUy transported proteins during axon regeneration in toad retinal ganglion cells, J. Cell BioL, 89 (1981) 86-95. 36 Sumner, B. E. H., A quantitative analysis of the response of presynaptic boutons to postsynaptic motor neuron axotomy, Exp. Neurol., 46 (1975) 605-615. 37 Tessler, A., Autilio-Gambetti, L. and Gambetti, P., Axonal growth during regeneration - - a quantitative autoradiographic study, J. Cell Biol., 87 (1980) 197-203. 38 Watson, W. E., Observations on the nucleolar and total cell body nucleic acid of injured nerve cells, J. Physiol. (Lond.), 196 (1968) 655-676. 39 Whitnall, M. H. and Grafstein, B., The relationship between extracellular amino acids and protein synthesis is altered during axonal regeneration, Brain Research, 220 (1981) 362-366. 40 Whitnall, M. H. and Grafstein, B., Perikaryal routing of newly synthesized proteins in regenerating neurons: quantitative electron microscopic autoradiography, Brain Research, in press.