Brain Research, 112 (1976) 371-381
371
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
TRANSFER RNA MAY BE AXONALLY TRANSPORTED REGENERATION OF GOLDFISH OPTIC NERVES
DURING
NICHOLAS A. INGOGLIA and ROBERT TULISZEWSKI
Departments of Physiology and Neuroscience, New Jersey Medical School, Newark, New Jersey 07103 (U.S.A.) (Accepted January 12th, 1976)
SUMMARY
If [SH]uridine is injected into the eyes of goldfish during optic nerve regeneration, then the return of fibers to the optic rectum is accompanied by the appearance of [aH]RNA in the tectum. The amount of [aH]RNA arriving in the tectum is consistently greater than in non-regenerating controls and reaches maximum levels (more than 10 times controls) 24 days after optic nerve crush. When [laC]uridine is injected subarachnoidally 1 day prior to sacrificing, the amount of [14C]RNA in the rectum is approximately doubled throughout the regeneration period. In order to characterize the radioactive tectal RNA in these experiments, we have crushed the optic nerves of 15 fish, and 18 days later injected [SH]uridine into both eyes. Five days later [14C]uridine was injected subarachnoidally and all fish were sacrificed a day later. RNA was extracted and fractionated in 2.0 ~o polyacrylamide gels, The amounts of all- and 14C-labeled ribosomal as well as small molecular weight RNAs were increased during regeneration. Analysis of the area under the 28S, 18S and 4-7S RNA peaks indicated a small increase in ~4C radioactivity in each peak (1.2, 1.5, and 1.5 times control, respectively). On the other hand, aH radioactivity showed the greatest increase in the 4-7S fraction (8.0 times control) whereas large molecular weight ribosomal fractions were approximately 3 times control. Electrophoresis of the RNA on 10 ~o polyacrylamide gels demonstrated that all of the small molecular weight RNA was confined to the 4S (tRNA) peak. These results suggest that when optic nerves of goldfish regenerate, they may enter the teetum carrying 4S (transfer) RNA.
INTRODUCTION
We have shown that if [SH]uridine is injected into one eye of a goldfish, radioactive RNA accumulates in the contralateral optic tectum according to a definite time course 12. Further, during optic nerve regeneration and following intraocular injection
372 of [3H]uridine, large amounts of radioactive RNA (more than 10 times normal) are found in the tectum as optic nerves re-enter the denervated area 13. In these experiments, the evidence suggests that at least some of the [ZH]RNA in the tectum is synthesized in retinal ganglion cells and then transported along regenerating optic axons to the tectum. Recently, we have reported quantitative EM autoradiographic data showing that, under the same experimental conditions as described above, approximately 50 °/ooof the [3H]RNA in the tectum is contained in regenerating neurites or in areas identified as axonal growth cones. In non-regenerating nerves, on the other hand, the amount of radioactive RNA grains over axons was close to background levels 10. The present experiments were conducted in order to determine the specific type(s) of RNA in regenerating axons in the optic tectum. A maj.or problem in identifying axonal RNA is to distinguish it from 'noise' caused by glial RNA. Autoradiographic evidence on the distribution of subarachnoidally administered [3H]uridine has shown that this route of injection leads to incorporation of precursor into RNA primarily over cell perikarya xz, and not over regenerating axons as in intraocular administration of the precursor10, lz. Therefore, we have approached this problem by crushing the optic nerves in a group of goldfish and then at various stages of nerve regeneration we injected [3H]uridine into the eye (acting primarily as a label of transported radioactivity), and [14C]uridine into the brain (as a label of locally incorporated radioactivity). Fish were sacrificed and tectal RNA was isolated and fractionated by polyacrylamide gel electrophoresis. METHODS Goldfish (Carassius auratus) 4-5 in. in length were obtained from Ozark Fisheries (Stoutland, Missouri). Radioactive isotopes, [SH]uridine ([5-3H]uridine, 27.8 Ci/mmole) and [14C]uridine ([2-14C]uridine, 51.0 mCi/mmole), were obtained from New England Nuclear Corp. For surgery on the optic nerve, fish were anesthetized by immersion in ice water for 10 min. Using a stereomicroscope, the optic nerves were crushed a few millimeters behind the eye with curved jewelers forceps. Fish were kept at 20 °C throughout the experiments. Six, 12 or 18 days after crushing both optic nerves, [3H]uridine (4-8 #Ci in 4/tl sterile H20) was injected into both eyes. Five days after the injection of [ZH]uridine, [14C]uridine was injected subarachnoidally according to techniques described by Agranoff and Klinged, and all fish were sacrificed one day later.
Extraction of RNA Prior to sacrificing, groups of 10-16 fish were immersed in ice water for 10 min and their brains exposed by removal of the calvarium. Both optic tecta were rapidly excised from live but ice anesthetized fish and placed in 5 ml of a 50 m M sodium acetate buffer (pH 5.1), containing 10 m M EDTA, and 0.5 ~ sodium dodecyl sulfate (SDS). RNA was isolated by hot phenol extraction followed by purification with phenol/chloroform (4:1), 2 washes of isoamylalcohol/chloroform (99:1) and finally precipitated by
373 addition of 2.5 vol. of ethanol. Samples were stored at --10 °C for 3 days and then centrifuged at 40,000 × g at 0 °C. The supernatant was decanted and the precipitate dissolved in 1.0 ml of0.15 M Na acetate buffer (pH 5.3) with 5 mM magnesium acetate, and 0.5 ~ SDS. RNA was reprecipitated with ethanol two more times and its absorption measured between 300 and 220 nm in a dual beam spectrophotometer (Acta C III, Beckman Instruments). Purity of the RNA preparation was determined by comparing its absorption at 260 and 280 nm. This ratio was approximately 2.0 -4- 0.1 in all experiments. In order to monitor radioactivity at each step of the extraction, 100 #I aliquots were taken in triplicate, dissolved in 11 ml of Hydromix (Yorktown Research, So. Hacken sack, N.J.), and counted in a Nuclear Chicago Isocap 300 liquid scintillation counter-
Polyacrylamide gel electrophoresis 2.0 ~ and 7.5 ~ gels were prepared according to Loening19, while for 10 ~ gels the procedure of Weber and Osborn ~6 was followed. The 7.5 ~ gel was used as a plug for the 2.0 ~ running gel, and the length of each running gel was approximately 8.0 cm.
Purified RNA samples, 80-100 #1 in volume, (approximately 1/~g/#l) were added to the gels and electrophoresed at 5 mA/gel for periods varying from 90 min to 4.5 h. Gels were then scanned for absorbance at 260 nm, frozen and cut into 2 mm segments. One ml of a 10~ piperidine solution containing 1 mM EDTA was added to each gel slice and incubated overnight at 37 °C. Distilled deionized water (0.5 ml) was added to each sample, and after gentle shaking for 1 h, 11 ml of Hydromix was added tg. 3H and 14C radioactivity was measured simultaneously in two channels of the liquid scintillation counter and disint./min for aH and 14C were determined by equations derived from quench correction curves of standard solutions. RESULTS
In the first series of experiments both optic nerves were crushed in 45 fish and 6, 12, or 18 days later 4 #Ci of [3H]uridine was injected into both eyes. Five days later, [14C]uridine was injected subarachnoidally and fish were sacrificed on the next day. As outlined above, 45 control fish were injected with isotope and sacrificed according to the same time schedule. At the time of sacrifice, regeneration would have then progressed for 12 days in the first group (few if any fibers would have reinnervated the tectum), 18 days in the second group (early stages of reinnervation) and 24 days in the third group (most of the fibers would have re-entered the tectum). Extraction of purified RNA was performed and aliquots at each step of the extraction were assayed for radioactivity as described in Methods. Approximately 65 ~ of the radioactivity was found in the ethanol soluble material (presumably nucleotides and other small molecules), and 20 ~ in the ethanol precipitate, identified by its UV absorption at 260 nm and gel electrophoresis properties, as RNA. Fig. 1 shows a comparison of these fractions in regenerating and control fish at the three stages of regeneration studied. Results clearly show that radioactivity arriving from the eye (3H) is greatly increased as regenerating nerves are re-entering the tectum (18 and 24
374
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DAYS AFTER OPT'C NERVE CRUSH
Fig. 1. Both optic nerves were crushed and 6, 12, or 18 days later [SH]uridinewas injected into both eyes. [~4C]Uridinewas injected subarachnoidally 5 days later and all fish were sacrificed one day after [14C]uridineinjections. RNA was extracted as described in the Methods section. Values are the ratio of experimentalto control radioactivityin 3H (@) and 14C(©). A: RNA associatedradioactivity; B: ethanol soluble radioactivity. days after crushing the nerve), while the radioactivity which was locally applied (14C) is approximately doubled but is not significantly affected by the absence (12 days) or re-entry (18 and 24 days) of growing fibers. The fractionation of tectal RNA at these stages of regeneration is shown in Fig. 2. Gels were run at 5 mA/gel for 110 min, scanned at 260 nm, frozen and sliced into 2 mm sections. Radioactivity was determined as described above. The distribution of 14C radioactivity in non-regenerating fish was found to be somewhat different than in fish studied during the 3 regeneration periods (Fig. 2A). However, as shown in these representative gels, little change with time was noted in the distribution of 14C radioactivity in the major RNA absorption peaks (28S, 18S, 4-7S). aH radioactivity was dramatically affected by the stages of regeneration (Fig. 2B). Before the fibers have re-entered the tectum (12 days after nerve crush) the 3H radioactivity is close to background (radioactivity which is present is presumably due either to the presence of some fibers which escaped crushing, or to local incorporation of 3H nucleotides which enter the general circulation and then are incorporated into tectal RNA). As fibers reinnervate the tectum (18 and 24 days after the crush), the amount of [SH] RNA in all RNA species increases; the greatest increase occurring at the later stage of regeneration. The molecular species most affected by fiber re-entry appear to be the small molecular weight RNA (4-7S). In order to quantify the amount of radioactivity in each of the RNA peaks in control vs. experimental fish (24 days after optic nerve crush), several additional experiments were performed. Both optic nerves were crushed in 15 fish as described earlier, and 18 days later 3.2 #Ci of [3H]uridine was injected into both eyes (a total of 96/~Ci [aH]uridine). [14C]Uridine (0.21 /zCi in 10/A sterile water) was injected subarachnoidally 5 days later. So that we could get a higher level of radioactivity in control (non-regenerating) brains, stock [3H]uridine (1/~Ci//~l) was concentrated and 7.1 /zCi of [SH]uridine was injected into both eyes of 15 control fish (a total of 213 #Ci [3H]uridine). [14C]Uridine (0,21 #Ci) was injected intracranially 5 days later and all fish were sacrificed one day after the [14C]uridine injection. RNA was extracted from tectal homogenates as described above.
375
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Fig. 2. Distribution of R N A in 2.0% polyacrylamide gels run at 5 mA/gel for 110 min. Samples are from R N A fractions described in Fig. 1. A: 14C radioactivity, derived primarily from local tectal R N A synthesis. B: aH radioactivity derived principally from the contralateral eye. Symbols: • = 12 days; O = 18 days; • = 24 days; [ ] = control.
376
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Fig. 3. A: [aH]uridine (7.1/~Ci) was injected into both eyes of 15 fish and 5 days later [14C]uridine (0.21/~Ci) was injected subarachnoidally. Fish were sacrificed one day later and t h e R N A extracted. Gel electrophoresis was at 5 rnA/gel for 90 rain on 2.0% p o t y a e ~ i d ¢ gels. B: both optic nerves were crushed and 18 days later [SH]uridine (3.2/zCi) was injected intraoeularly. [laC]uridine (0.21/,Ci) was injected subaraelmoidally after 5 days and fish were sacrificed on day6. Electrophoresis was as described above. Symbols: aH = • ; 14C = (3.
377 TABLE I Comparison of total radioactivity in R N A peaks of control optic tecta and optic tecta receiving regenerating optic fibers following intraocular injection of [aH]uridine and subarachnoidal injection of [14C]uridine. Values are obtained from results of experiments shown in Fig. 3.
O.D. Peak aH (disint./min.)
28S 18S 4-7S
Regenerating
14C (disint./min.)
Regenerating
Normal
Regenerating
normal
Normal
Regenerating
normal
2073 1641 442
6,209 5,531 3,547
3.0 3.4 8.0
1070 725 194
1,304 1,113 283
1.2 1.5 1.5
Adjusted for amount of isotope injected (7.1/tCi, normal; 3.2/~Ci, regenerating).
The amount of purified RNA recovered, determined by measuring its absorption at 260 nm (1.00.D. = 40 #g RNA), varied somewhat from experiment to experiment. In the experiment shown here, 435 pg of RNA was obtained from the tecta of experimental fish, while 267 #g of RNA was obtained from controls. Seventy-three pg of control and experimental RNA preparations were layered on 2.0 ~ gels (8.0 cm in length) and run at 5 mA/gel for 90 min. The distribution of radioactivity along typical gels is shown in Fig. 3. The amount of both 3H and 14C radioactivity in the 3 RNA peaks (28S, 18S and 4-7S) is greater in regenerating preparations compared to controls, with the largest increase apparently in the 4-7S fraction. In order to quantify the differences in peak 4S
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Fig. 4. Both optic nerves were crushed in 15 fish and 18 days later 4/~Ci of [aH]uridine was injected into both eyes. Six days later fish were sacrificed and R N A was extracted from 30 pooled tecta. Electrophoresis of purified R N A was conduced on an 8 cm 10 % polyacrylamide gel column for 4.5 h, at 5 mA/gel. The labeled 4S peak indicates the extent of migration of both yeast 4S and all-labeled 4S mammalian cell marker RNA. Symbols: - - - - - - , O.D.; - - • - - , aH(disint./min.).
378 heights, the amount of radioactivity in control vs. regenerating groups was normalized for the amount of 3H radioactivity injected (7.1/~Ci/eye normal, 3.2/~Ci/eye regenerating. Determining the area under the curves of the 3 RNA peaks (using Simpson's rule for integrating successive points on a curve2a), gives the values shown in Table I. Results show a small increase in the amount of 14C radioactivity (local RNA synthesis) in all RNA peaks after regeneration. 3H radioactivity, however, is increased significantly in regenerating compared to normal fish in all 3 RNA peaks. By far the largest difference is seen in the small molecular weight RNA (8.0 times control). To determine if the aH radioactivity in the low molecular weight peak was associated specifically with the 4S, tRNA fraction, the experiments outlined in Fig. 3b were repeated except that intracranial injections of [14C]uridine were omitted. Purified RNA samples were layered on 8-9 cm long 10 ~ polyacrylamide gels. Samples were run for 4.5 h at 5 mA/gel, scanned at 260 nm, frozen, sectioned and counted as described earlier. Yeast soluble RNA (Calbiochem) and [aH]4S mammalian cell marker RNA (Schwarz-Mann) were run under the same conditions and the extent of migration of the 4S RNA was determined. Results (Fig. 4) show that although there are several optical density peaks migrating in front of and behind the 4S peak, virtually all of the [aH]RNA co-migrates with the 4S (tRNA) markers. DISCUSSION These experiments confirm our previous observations that when [3H]uridine is injected into the eye of goldfish during optic nerve regeneration, there is a large increase in the amount of tectal [3H]RNA as regenerating fibers are establishing reconnections la. The peak in the arrival of RNA (124 days) coincides approximately with the arrival of incoming fibers21, 42. Radioactivity in the ethanol fraction is also affected by the absence or re-entry of fibers and this too agrees with earlier studies involving radioactivity in a trichloroacetic acid soluble fraction 13. The significance of this finding is not known, but the transport of labeled nucleotides in non-regenerating nerves has been reported elsewhere and some speculation has been made on the possible role of axonally transported nucleotides in normal neuronal function 22. A comparison of the molecular species of RNA in normal tecta and tecta receiving regenerating fibers shows that the RNA species most affected by our experimental conditions is small molecular weight RNA. This RNA has the same mobility as yeast 4S and radioactive mammalian celt 4S RNA markers on 10~ polyacrylamide gels. Hence we have identified it as 4S RNA, probably containing tRNA, The major increase in 4S RNA is seen only in RNA derived from radioactivity injected into the eye (all) and not in RNA synthesized locally in the tectum itself (14C). These results raise several questions. First, was the [3H]RNA in the rectum synthesized in the retina and then axonally transported or rather, synthesized in the rectum itself from transported radioactive precursors? In another publication we have summarized our reasons for postulating that when optic nerves regenerate, some of the RNA found in the rectum is synthesized in the retina and is then axonally
379 transported la. This question is as yet not fully resolved. However, the present experiments show that [aH]uridine injected into the eye results in a different pattern of alllabeled tectal RNA species than [14C]uridine injected subarachnoidally. These results lend further evidence that all-labeled RNA may have been synthesized outside of the tectum and then was transported along the regenerating optic fibers. An additional argument supporting axonal RNA transport in regenerating nerves comes from recent EM findings. In these studies the experimental design was similar to that described in Fig. 4, except that tissue was prepared for quantitative EM autoradiography rather than gel electrophoresis. Results showed a proportionately larger number of RNA associated radioactive grains in regenerating axons and growth cones than in axons of non-regenerating controls10. Thus these findings are consistent with the concept of axonal transport of RNA. A second question raised by these experiments pertains to the molecular species of RNA found in regenerating axons. Our results show increases in all RNA species during regeneration (Table I) and it is possible, as has been suggested elsewherea-5, 7, 20, that all RNA molecules, including large molecular weight ribosomal RNA, are present in axons. However, it seems likely to us that only transfer RNA is axonal and that the ribosomal RNA detected in our preparation is due to local tectal RNA synthesis in glial cells surrounding the regenerating axons. We base this reasoning on several lines of evidence. First, in EM autoradiographic studies, significant amounts of [aH]RNA were observed over extra-axonal regions 10 and it is probable that this RNA is composed of ribosomal as well as small molecular weight RNA. This could account for the 3-fold increase in ribosomal RNA without suggesting an intra-axonal origin for large molecular weight RNAs. Second, Lasek et al. have demonstrated both in Myxicola and Squid giant axons, that axoplasm isolated free of RNA contamination from supporting cells, contains only 4S RNA and no ribosomal components. They have suggested that at least in these species only transfer RNA is intraaxonal is. Finally, Brink and Karnovsky have reported experiments in which a~p labeled bacterial tRNA was injected into rat eyes and radioactive tRNA was detected along the optic nerve6. Although some of this RNA, when unprotected, was degraded, a significant portion of the [a2P]tRNA appeared in fact to be axonally transported. These results lead us to interpret our data as suggesting that the large increases in all-labeled 4S RNA in the newly innervated tectum is due to fibers entering the tectum carrying labeled tRNA. The results of Brink and Karnovsky are also consistent with the position that RNA molecules can be axonally transported. This, however, does not preclude the possibility that some axonal RNA may be synthesized in glial cells in the tectum and then transferred into the axonsL Regardless of the origin of axonal RNA the question arises as to the function of the large quantities of RNA (and perhaps only tRNA) in regenerating axons. The most completely studied function of tRNA is in protein synthesis where it serves as an amino acid donor to a growing polypeptide chain (reviewed by Sirlin) 2a. However, it is difficult to postulate that this is the role of tRNA in the axons we are studying, in the light of finding no ribosomes in these growing fibers (Weis and Ingoglia, unpublished results).
380 In experiments, carried out in non-nervous tissue, it has been suggested that the supply o f available t R N A s m a y be rate limiting to protein synthesis 27, and Jacobson t'~ has shown that t R N A tyrosine can bind to a m u t a n t enzyme o f Drosophila and thus inhibit its activity. These studies indicate that t R N A molecules m a y be capable o f modifiying protein synthesis at a posttranscriptional step. There is no evidence, however, that t R N A plays such a role in nervous tissue. Results from a n u m b e r o f laboratories have shown that the a m o u n t and turnover o f t R N A s vary during development of Drosophila 27, chick embryos 25 as welt as in m a m m a l i a n brainS,9,11,15. Specific t R N A s have also been shown to be affected by behavioral training in goldfish 16,17. These experiments suggest a correlation between t R N A activity and embryonic developmental stages, as well as learning. However, the significance o f these events in either o f the functional states described is not known. In a previous publication, we suggested that R N A contained in growing axons m a y be important in neuronal recognition 1~. A l t h o u g h the present experiments offer evidence that the axonal R N A may be t R N A , the functional significance o f this R N A remains to be demonstrated. ACKNOWLEDGEMENTS The authors are indebted to Dr. J. Sirlin and Dr. R. Wilson for their advice and criticism during the course of these experiments, and Dr. Les Michelson for his help in the analysis o f the data. We thank Ms. Janet M c F a r t a n d and Mr. Paul Hartunian for their excellent technical assistance. This w o r k was supported by a Neurological Diseases and Stroke G r a n t NS11259 from N I H . Mr. Tuliszewski was supported as a Summer Research Fellow by a grant from Merck C o m p a n y Foundation.
REFERENCES 1 Agranoff, B. W. and Klinger, P. D., Puromycin effect on memory fixation in the goldfish, Science, 146 (1964) 952-953. 2 Autilio-Gambetti, L., Gambetti, P. and Shafer, B., RNA and axonal flow. Biochemical and autoradiographic study in the rabbit optic system, Brain Research, 53 (1973) 387-398. 3 Bondy, S. C., Axonal migration of various RNA species along the optic tract of the chick, J. Neurochem., 19 (1972) 1769-1776. 4 Bondy, S. C. and Purdy, J. L., Migration of ribosomes along the axons of the chick visual pathway, Biochim. biophys. Acta (Amst.), 390 (1975) 332-341. 5 Bray, J. J. and Austin, L., Flow of protein and ribonucleic acid in peripheral nerve, J. Neurochem., 15 (1968) 731-740. 6 Brink, J. J. and Karnovsky, M. L., Axonal flow of exogenous RNA in the rat optic nerve, J. Neurochern., 21 (1973) 1003-1007. 7 Casola, L., Davis, G. S. and Davis, R. E., Evidence for RNA transport in rat optic nerve, J. Neurochem., 16 (1969) 1037-1041. 8 Dellweg, H., Gerner, R. and Wacker, A., Quantitative and qualitative changes in RNAs of rat brain dependent on age and training experiments, J. Neurochem., 15 (1968) 1109-1119. 9 Frazer, J. M. and Yang, W. K,, Isoaccepting tRNA's in liver and brain of young and old BC3F1 mice, Arch. Biochem., 153 (1972) 610-618. 10 Gambetti, P., Ingoglia, N., Weis, P. and Autilio-Gambetti, L., RNA distribution in goldfish
381 optic tectum after intraocular injection of aH-uridine. An EM autoradiographic study during optic nerve regeneration, Abstr. 5th Ann. Meeting Soc. Neurosci., (1975) 797. 11 Harris, C. L. and Maas, J. W., Transfer RNA and the regulation of protein synthesis in rat cerebral cortex during neural development, J. Neurochem., 22 (1974) 741-749. 12 Ingoglia, N. A., Grafstein, B., McEwen, B. S. and McQuarrie, I. G., Axonal transport of radioactivity in the goldfish optic system following intraocular injection of labeled R N A precursors, J. Neurochem., 20 (1973) 1605-1615. 13 lngoglia, N. A., Weis, P. and Mycek, J., Axonal transport of RNA during regeneration of the optic nerves of goldfish, J. Neurobiol., 6 (1975) 549-564. 14 Jacobson, K. B., Role of an isoacceptor transfer ribonucleic acid as an enzyme inhibitor: effect on tryptophan pyrrolase of drosophila, Nature New Biol., 231 (1971) 17-19. 15 Johnson, T. C., Mathews, R. A. and Chou, L., tRNA methyltransferase activity in neonatal and mature mammalian neural tissue, J. Neurochem., 23 (1974) 489-496. 16 Kaplan, B. B., Dyer, J. C. and Sirlin, J. L., Macromolecules and behavior: Effects of behavioral training on transfer RNAs of goldfish brain, Brain Research, 56 (1973) 239-248. 17 Kaplan, B. B. and Sirlin, J. L., Macromolecules and behavior. II. Training induced alteration in leucine transfer RNA of goldfish brain, Brain Research, 83 (1975) 451-468. 18 Lasek, R., Dabrowski, C. and Nordlander, R., Analysis of axoplasmic RNA from invertebrate giant axons, Nature New BioL, 244 (1973) 162-165. 19 Loening, U. E., Fractionation of RNA by polyacrylamide gel electrophoresis, Meth. med. Res., 12 (1970) 359-368. 20 Miani, N., DiGirolamo, A. and DiGirolamo, M., Sedimentation characteristics of axonal RNA in rabbit, J. Neurochem., 13 (1966) 755-759. 21 Murray, M. and Grafstein, B., Changes in the morphology and amino acid incorporation of regenerating goldfish optic nerves, Exp. Neurol., 23 (1969) 544--560. 22 Schubert, P. and Kreutzberg, G. W., Axonal transport of adenosine and uridine derivatives and transfer to postsynaptic neurons, Brain Research, 76 (1974) 526-530. 23 Sirlin, J. L., Biology of RNA, Academic Press, New York, N.Y., 1972. 24 Sperry, R. W., Functional regeneration in the optic system. In W. F. Windle (Ed.), Regeneration in the Central Nervous System, Thomas, Springfield, I11., 1955, pp. 66-76. 25 Sundari, R. M., Cherayil, J. D. and Jacob, T. M., Changes in transfer ribonucleic acid during the development of chick embryo, Indian J. Biochem. Biophys., 11 (1974) 43-46. 26 Weber, K. and Osborn, M., The reliability of molecular weight determination by dodecyl sulfatepolyacrylamide gel electrophoresis, J. biol. Chem., 244 (1969) 4406-4412. 27 White, B. N., Tener, G. M., Holden, J. and Suzuki, D. T., Analysis of tRNA's during the development of Drosophila. Develop. BioL, 33 (1973) 185-195. 28 Wylie, C. R., Jr., Advanced Engineering Mathematics, 2nd. ed., McGraw Hill, New York, 1960, p. 160.
371
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
TRANSFER RNA MAY BE AXONALLY TRANSPORTED REGENERATION OF GOLDFISH OPTIC NERVES
DURING
NICHOLAS A. INGOGLIA and ROBERT TULISZEWSKI
Departments of Physiology and Neuroscience, New Jersey Medical School, Newark, New Jersey 07103 (U.S.A.) (Accepted January 12th, 1976)
SUMMARY
If [SH]uridine is injected into the eyes of goldfish during optic nerve regeneration, then the return of fibers to the optic rectum is accompanied by the appearance of [aH]RNA in the tectum. The amount of [aH]RNA arriving in the tectum is consistently greater than in non-regenerating controls and reaches maximum levels (more than 10 times controls) 24 days after optic nerve crush. When [laC]uridine is injected subarachnoidally 1 day prior to sacrificing, the amount of [14C]RNA in the rectum is approximately doubled throughout the regeneration period. In order to characterize the radioactive tectal RNA in these experiments, we have crushed the optic nerves of 15 fish, and 18 days later injected [SH]uridine into both eyes. Five days later [14C]uridine was injected subarachnoidally and all fish were sacrificed a day later. RNA was extracted and fractionated in 2.0 ~o polyacrylamide gels, The amounts of all- and 14C-labeled ribosomal as well as small molecular weight RNAs were increased during regeneration. Analysis of the area under the 28S, 18S and 4-7S RNA peaks indicated a small increase in ~4C radioactivity in each peak (1.2, 1.5, and 1.5 times control, respectively). On the other hand, aH radioactivity showed the greatest increase in the 4-7S fraction (8.0 times control) whereas large molecular weight ribosomal fractions were approximately 3 times control. Electrophoresis of the RNA on 10 ~o polyacrylamide gels demonstrated that all of the small molecular weight RNA was confined to the 4S (tRNA) peak. These results suggest that when optic nerves of goldfish regenerate, they may enter the teetum carrying 4S (transfer) RNA.
INTRODUCTION
We have shown that if [SH]uridine is injected into one eye of a goldfish, radioactive RNA accumulates in the contralateral optic tectum according to a definite time course 12. Further, during optic nerve regeneration and following intraocular injection
372 of [3H]uridine, large amounts of radioactive RNA (more than 10 times normal) are found in the tectum as optic nerves re-enter the denervated area 13. In these experiments, the evidence suggests that at least some of the [ZH]RNA in the tectum is synthesized in retinal ganglion cells and then transported along regenerating optic axons to the tectum. Recently, we have reported quantitative EM autoradiographic data showing that, under the same experimental conditions as described above, approximately 50 °/ooof the [3H]RNA in the tectum is contained in regenerating neurites or in areas identified as axonal growth cones. In non-regenerating nerves, on the other hand, the amount of radioactive RNA grains over axons was close to background levels 10. The present experiments were conducted in order to determine the specific type(s) of RNA in regenerating axons in the optic tectum. A maj.or problem in identifying axonal RNA is to distinguish it from 'noise' caused by glial RNA. Autoradiographic evidence on the distribution of subarachnoidally administered [3H]uridine has shown that this route of injection leads to incorporation of precursor into RNA primarily over cell perikarya xz, and not over regenerating axons as in intraocular administration of the precursor10, lz. Therefore, we have approached this problem by crushing the optic nerves in a group of goldfish and then at various stages of nerve regeneration we injected [3H]uridine into the eye (acting primarily as a label of transported radioactivity), and [14C]uridine into the brain (as a label of locally incorporated radioactivity). Fish were sacrificed and tectal RNA was isolated and fractionated by polyacrylamide gel electrophoresis. METHODS Goldfish (Carassius auratus) 4-5 in. in length were obtained from Ozark Fisheries (Stoutland, Missouri). Radioactive isotopes, [SH]uridine ([5-3H]uridine, 27.8 Ci/mmole) and [14C]uridine ([2-14C]uridine, 51.0 mCi/mmole), were obtained from New England Nuclear Corp. For surgery on the optic nerve, fish were anesthetized by immersion in ice water for 10 min. Using a stereomicroscope, the optic nerves were crushed a few millimeters behind the eye with curved jewelers forceps. Fish were kept at 20 °C throughout the experiments. Six, 12 or 18 days after crushing both optic nerves, [3H]uridine (4-8 #Ci in 4/tl sterile H20) was injected into both eyes. Five days after the injection of [ZH]uridine, [14C]uridine was injected subarachnoidally according to techniques described by Agranoff and Klinged, and all fish were sacrificed one day later.
Extraction of RNA Prior to sacrificing, groups of 10-16 fish were immersed in ice water for 10 min and their brains exposed by removal of the calvarium. Both optic tecta were rapidly excised from live but ice anesthetized fish and placed in 5 ml of a 50 m M sodium acetate buffer (pH 5.1), containing 10 m M EDTA, and 0.5 ~ sodium dodecyl sulfate (SDS). RNA was isolated by hot phenol extraction followed by purification with phenol/chloroform (4:1), 2 washes of isoamylalcohol/chloroform (99:1) and finally precipitated by
373 addition of 2.5 vol. of ethanol. Samples were stored at --10 °C for 3 days and then centrifuged at 40,000 × g at 0 °C. The supernatant was decanted and the precipitate dissolved in 1.0 ml of0.15 M Na acetate buffer (pH 5.3) with 5 mM magnesium acetate, and 0.5 ~ SDS. RNA was reprecipitated with ethanol two more times and its absorption measured between 300 and 220 nm in a dual beam spectrophotometer (Acta C III, Beckman Instruments). Purity of the RNA preparation was determined by comparing its absorption at 260 and 280 nm. This ratio was approximately 2.0 -4- 0.1 in all experiments. In order to monitor radioactivity at each step of the extraction, 100 #I aliquots were taken in triplicate, dissolved in 11 ml of Hydromix (Yorktown Research, So. Hacken sack, N.J.), and counted in a Nuclear Chicago Isocap 300 liquid scintillation counter-
Polyacrylamide gel electrophoresis 2.0 ~ and 7.5 ~ gels were prepared according to Loening19, while for 10 ~ gels the procedure of Weber and Osborn ~6 was followed. The 7.5 ~ gel was used as a plug for the 2.0 ~ running gel, and the length of each running gel was approximately 8.0 cm.
Purified RNA samples, 80-100 #1 in volume, (approximately 1/~g/#l) were added to the gels and electrophoresed at 5 mA/gel for periods varying from 90 min to 4.5 h. Gels were then scanned for absorbance at 260 nm, frozen and cut into 2 mm segments. One ml of a 10~ piperidine solution containing 1 mM EDTA was added to each gel slice and incubated overnight at 37 °C. Distilled deionized water (0.5 ml) was added to each sample, and after gentle shaking for 1 h, 11 ml of Hydromix was added tg. 3H and 14C radioactivity was measured simultaneously in two channels of the liquid scintillation counter and disint./min for aH and 14C were determined by equations derived from quench correction curves of standard solutions. RESULTS
In the first series of experiments both optic nerves were crushed in 45 fish and 6, 12, or 18 days later 4 #Ci of [3H]uridine was injected into both eyes. Five days later, [14C]uridine was injected subarachnoidally and fish were sacrificed on the next day. As outlined above, 45 control fish were injected with isotope and sacrificed according to the same time schedule. At the time of sacrifice, regeneration would have then progressed for 12 days in the first group (few if any fibers would have reinnervated the tectum), 18 days in the second group (early stages of reinnervation) and 24 days in the third group (most of the fibers would have re-entered the tectum). Extraction of purified RNA was performed and aliquots at each step of the extraction were assayed for radioactivity as described in Methods. Approximately 65 ~ of the radioactivity was found in the ethanol soluble material (presumably nucleotides and other small molecules), and 20 ~ in the ethanol precipitate, identified by its UV absorption at 260 nm and gel electrophoresis properties, as RNA. Fig. 1 shows a comparison of these fractions in regenerating and control fish at the three stages of regeneration studied. Results clearly show that radioactivity arriving from the eye (3H) is greatly increased as regenerating nerves are re-entering the tectum (18 and 24
374
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DAYS AFTER OPT'C NERVE CRUSH
Fig. 1. Both optic nerves were crushed and 6, 12, or 18 days later [SH]uridinewas injected into both eyes. [~4C]Uridinewas injected subarachnoidally 5 days later and all fish were sacrificed one day after [14C]uridineinjections. RNA was extracted as described in the Methods section. Values are the ratio of experimentalto control radioactivityin 3H (@) and 14C(©). A: RNA associatedradioactivity; B: ethanol soluble radioactivity. days after crushing the nerve), while the radioactivity which was locally applied (14C) is approximately doubled but is not significantly affected by the absence (12 days) or re-entry (18 and 24 days) of growing fibers. The fractionation of tectal RNA at these stages of regeneration is shown in Fig. 2. Gels were run at 5 mA/gel for 110 min, scanned at 260 nm, frozen and sliced into 2 mm sections. Radioactivity was determined as described above. The distribution of 14C radioactivity in non-regenerating fish was found to be somewhat different than in fish studied during the 3 regeneration periods (Fig. 2A). However, as shown in these representative gels, little change with time was noted in the distribution of 14C radioactivity in the major RNA absorption peaks (28S, 18S, 4-7S). aH radioactivity was dramatically affected by the stages of regeneration (Fig. 2B). Before the fibers have re-entered the tectum (12 days after nerve crush) the 3H radioactivity is close to background (radioactivity which is present is presumably due either to the presence of some fibers which escaped crushing, or to local incorporation of 3H nucleotides which enter the general circulation and then are incorporated into tectal RNA). As fibers reinnervate the tectum (18 and 24 days after the crush), the amount of [SH] RNA in all RNA species increases; the greatest increase occurring at the later stage of regeneration. The molecular species most affected by fiber re-entry appear to be the small molecular weight RNA (4-7S). In order to quantify the amount of radioactivity in each of the RNA peaks in control vs. experimental fish (24 days after optic nerve crush), several additional experiments were performed. Both optic nerves were crushed in 15 fish as described earlier, and 18 days later 3.2 #Ci of [3H]uridine was injected into both eyes (a total of 96/~Ci [aH]uridine). [14C]Uridine (0.21 /zCi in 10/A sterile water) was injected subarachnoidally 5 days later. So that we could get a higher level of radioactivity in control (non-regenerating) brains, stock [3H]uridine (1/~Ci//~l) was concentrated and 7.1 /zCi of [SH]uridine was injected into both eyes of 15 control fish (a total of 213 #Ci [3H]uridine). [14C]Uridine (0,21 #Ci) was injected intracranially 5 days later and all fish were sacrificed one day after the [14C]uridine injection. RNA was extracted from tectal homogenates as described above.
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Fig. 2. Distribution of R N A in 2.0% polyacrylamide gels run at 5 mA/gel for 110 min. Samples are from R N A fractions described in Fig. 1. A: 14C radioactivity, derived primarily from local tectal R N A synthesis. B: aH radioactivity derived principally from the contralateral eye. Symbols: • = 12 days; O = 18 days; • = 24 days; [ ] = control.
376
NORMAL TECTA A
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Fig. 3. A: [aH]uridine (7.1/~Ci) was injected into both eyes of 15 fish and 5 days later [14C]uridine (0.21/~Ci) was injected subarachnoidally. Fish were sacrificed one day later and t h e R N A extracted. Gel electrophoresis was at 5 rnA/gel for 90 rain on 2.0% p o t y a e ~ i d ¢ gels. B: both optic nerves were crushed and 18 days later [SH]uridine (3.2/zCi) was injected intraoeularly. [laC]uridine (0.21/,Ci) was injected subaraelmoidally after 5 days and fish were sacrificed on day6. Electrophoresis was as described above. Symbols: aH = • ; 14C = (3.
377 TABLE I Comparison of total radioactivity in R N A peaks of control optic tecta and optic tecta receiving regenerating optic fibers following intraocular injection of [aH]uridine and subarachnoidal injection of [14C]uridine. Values are obtained from results of experiments shown in Fig. 3.
O.D. Peak aH (disint./min.)
28S 18S 4-7S
Regenerating
14C (disint./min.)
Regenerating
Normal
Regenerating
normal
Normal
Regenerating
normal
2073 1641 442
6,209 5,531 3,547
3.0 3.4 8.0
1070 725 194
1,304 1,113 283
1.2 1.5 1.5
Adjusted for amount of isotope injected (7.1/tCi, normal; 3.2/~Ci, regenerating).
The amount of purified RNA recovered, determined by measuring its absorption at 260 nm (1.00.D. = 40 #g RNA), varied somewhat from experiment to experiment. In the experiment shown here, 435 pg of RNA was obtained from the tecta of experimental fish, while 267 #g of RNA was obtained from controls. Seventy-three pg of control and experimental RNA preparations were layered on 2.0 ~ gels (8.0 cm in length) and run at 5 mA/gel for 90 min. The distribution of radioactivity along typical gels is shown in Fig. 3. The amount of both 3H and 14C radioactivity in the 3 RNA peaks (28S, 18S and 4-7S) is greater in regenerating preparations compared to controls, with the largest increase apparently in the 4-7S fraction. In order to quantify the differences in peak 4S
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Fig. 4. Both optic nerves were crushed in 15 fish and 18 days later 4/~Ci of [aH]uridine was injected into both eyes. Six days later fish were sacrificed and R N A was extracted from 30 pooled tecta. Electrophoresis of purified R N A was conduced on an 8 cm 10 % polyacrylamide gel column for 4.5 h, at 5 mA/gel. The labeled 4S peak indicates the extent of migration of both yeast 4S and all-labeled 4S mammalian cell marker RNA. Symbols: - - - - - - , O.D.; - - • - - , aH(disint./min.).
378 heights, the amount of radioactivity in control vs. regenerating groups was normalized for the amount of 3H radioactivity injected (7.1/~Ci/eye normal, 3.2/~Ci/eye regenerating. Determining the area under the curves of the 3 RNA peaks (using Simpson's rule for integrating successive points on a curve2a), gives the values shown in Table I. Results show a small increase in the amount of 14C radioactivity (local RNA synthesis) in all RNA peaks after regeneration. 3H radioactivity, however, is increased significantly in regenerating compared to normal fish in all 3 RNA peaks. By far the largest difference is seen in the small molecular weight RNA (8.0 times control). To determine if the aH radioactivity in the low molecular weight peak was associated specifically with the 4S, tRNA fraction, the experiments outlined in Fig. 3b were repeated except that intracranial injections of [14C]uridine were omitted. Purified RNA samples were layered on 8-9 cm long 10 ~ polyacrylamide gels. Samples were run for 4.5 h at 5 mA/gel, scanned at 260 nm, frozen, sectioned and counted as described earlier. Yeast soluble RNA (Calbiochem) and [aH]4S mammalian cell marker RNA (Schwarz-Mann) were run under the same conditions and the extent of migration of the 4S RNA was determined. Results (Fig. 4) show that although there are several optical density peaks migrating in front of and behind the 4S peak, virtually all of the [aH]RNA co-migrates with the 4S (tRNA) markers. DISCUSSION These experiments confirm our previous observations that when [3H]uridine is injected into the eye of goldfish during optic nerve regeneration, there is a large increase in the amount of tectal [3H]RNA as regenerating fibers are establishing reconnections la. The peak in the arrival of RNA (124 days) coincides approximately with the arrival of incoming fibers21, 42. Radioactivity in the ethanol fraction is also affected by the absence or re-entry of fibers and this too agrees with earlier studies involving radioactivity in a trichloroacetic acid soluble fraction 13. The significance of this finding is not known, but the transport of labeled nucleotides in non-regenerating nerves has been reported elsewhere and some speculation has been made on the possible role of axonally transported nucleotides in normal neuronal function 22. A comparison of the molecular species of RNA in normal tecta and tecta receiving regenerating fibers shows that the RNA species most affected by our experimental conditions is small molecular weight RNA. This RNA has the same mobility as yeast 4S and radioactive mammalian celt 4S RNA markers on 10~ polyacrylamide gels. Hence we have identified it as 4S RNA, probably containing tRNA, The major increase in 4S RNA is seen only in RNA derived from radioactivity injected into the eye (all) and not in RNA synthesized locally in the tectum itself (14C). These results raise several questions. First, was the [3H]RNA in the rectum synthesized in the retina and then axonally transported or rather, synthesized in the rectum itself from transported radioactive precursors? In another publication we have summarized our reasons for postulating that when optic nerves regenerate, some of the RNA found in the rectum is synthesized in the retina and is then axonally
379 transported la. This question is as yet not fully resolved. However, the present experiments show that [aH]uridine injected into the eye results in a different pattern of alllabeled tectal RNA species than [14C]uridine injected subarachnoidally. These results lend further evidence that all-labeled RNA may have been synthesized outside of the tectum and then was transported along the regenerating optic fibers. An additional argument supporting axonal RNA transport in regenerating nerves comes from recent EM findings. In these studies the experimental design was similar to that described in Fig. 4, except that tissue was prepared for quantitative EM autoradiography rather than gel electrophoresis. Results showed a proportionately larger number of RNA associated radioactive grains in regenerating axons and growth cones than in axons of non-regenerating controls10. Thus these findings are consistent with the concept of axonal transport of RNA. A second question raised by these experiments pertains to the molecular species of RNA found in regenerating axons. Our results show increases in all RNA species during regeneration (Table I) and it is possible, as has been suggested elsewherea-5, 7, 20, that all RNA molecules, including large molecular weight ribosomal RNA, are present in axons. However, it seems likely to us that only transfer RNA is axonal and that the ribosomal RNA detected in our preparation is due to local tectal RNA synthesis in glial cells surrounding the regenerating axons. We base this reasoning on several lines of evidence. First, in EM autoradiographic studies, significant amounts of [aH]RNA were observed over extra-axonal regions 10 and it is probable that this RNA is composed of ribosomal as well as small molecular weight RNA. This could account for the 3-fold increase in ribosomal RNA without suggesting an intra-axonal origin for large molecular weight RNAs. Second, Lasek et al. have demonstrated both in Myxicola and Squid giant axons, that axoplasm isolated free of RNA contamination from supporting cells, contains only 4S RNA and no ribosomal components. They have suggested that at least in these species only transfer RNA is intraaxonal is. Finally, Brink and Karnovsky have reported experiments in which a~p labeled bacterial tRNA was injected into rat eyes and radioactive tRNA was detected along the optic nerve6. Although some of this RNA, when unprotected, was degraded, a significant portion of the [a2P]tRNA appeared in fact to be axonally transported. These results lead us to interpret our data as suggesting that the large increases in all-labeled 4S RNA in the newly innervated tectum is due to fibers entering the tectum carrying labeled tRNA. The results of Brink and Karnovsky are also consistent with the position that RNA molecules can be axonally transported. This, however, does not preclude the possibility that some axonal RNA may be synthesized in glial cells in the tectum and then transferred into the axonsL Regardless of the origin of axonal RNA the question arises as to the function of the large quantities of RNA (and perhaps only tRNA) in regenerating axons. The most completely studied function of tRNA is in protein synthesis where it serves as an amino acid donor to a growing polypeptide chain (reviewed by Sirlin) 2a. However, it is difficult to postulate that this is the role of tRNA in the axons we are studying, in the light of finding no ribosomes in these growing fibers (Weis and Ingoglia, unpublished results).
380 In experiments, carried out in non-nervous tissue, it has been suggested that the supply o f available t R N A s m a y be rate limiting to protein synthesis 27, and Jacobson t'~ has shown that t R N A tyrosine can bind to a m u t a n t enzyme o f Drosophila and thus inhibit its activity. These studies indicate that t R N A molecules m a y be capable o f modifiying protein synthesis at a posttranscriptional step. There is no evidence, however, that t R N A plays such a role in nervous tissue. Results from a n u m b e r o f laboratories have shown that the a m o u n t and turnover o f t R N A s vary during development of Drosophila 27, chick embryos 25 as welt as in m a m m a l i a n brainS,9,11,15. Specific t R N A s have also been shown to be affected by behavioral training in goldfish 16,17. These experiments suggest a correlation between t R N A activity and embryonic developmental stages, as well as learning. However, the significance o f these events in either o f the functional states described is not known. In a previous publication, we suggested that R N A contained in growing axons m a y be important in neuronal recognition 1~. A l t h o u g h the present experiments offer evidence that the axonal R N A may be t R N A , the functional significance o f this R N A remains to be demonstrated. ACKNOWLEDGEMENTS The authors are indebted to Dr. J. Sirlin and Dr. R. Wilson for their advice and criticism during the course of these experiments, and Dr. Les Michelson for his help in the analysis o f the data. We thank Ms. Janet M c F a r t a n d and Mr. Paul Hartunian for their excellent technical assistance. This w o r k was supported by a Neurological Diseases and Stroke G r a n t NS11259 from N I H . Mr. Tuliszewski was supported as a Summer Research Fellow by a grant from Merck C o m p a n y Foundation.
REFERENCES 1 Agranoff, B. W. and Klinger, P. D., Puromycin effect on memory fixation in the goldfish, Science, 146 (1964) 952-953. 2 Autilio-Gambetti, L., Gambetti, P. and Shafer, B., RNA and axonal flow. Biochemical and autoradiographic study in the rabbit optic system, Brain Research, 53 (1973) 387-398. 3 Bondy, S. C., Axonal migration of various RNA species along the optic tract of the chick, J. Neurochem., 19 (1972) 1769-1776. 4 Bondy, S. C. and Purdy, J. L., Migration of ribosomes along the axons of the chick visual pathway, Biochim. biophys. Acta (Amst.), 390 (1975) 332-341. 5 Bray, J. J. and Austin, L., Flow of protein and ribonucleic acid in peripheral nerve, J. Neurochem., 15 (1968) 731-740. 6 Brink, J. J. and Karnovsky, M. L., Axonal flow of exogenous RNA in the rat optic nerve, J. Neurochern., 21 (1973) 1003-1007. 7 Casola, L., Davis, G. S. and Davis, R. E., Evidence for RNA transport in rat optic nerve, J. Neurochem., 16 (1969) 1037-1041. 8 Dellweg, H., Gerner, R. and Wacker, A., Quantitative and qualitative changes in RNAs of rat brain dependent on age and training experiments, J. Neurochem., 15 (1968) 1109-1119. 9 Frazer, J. M. and Yang, W. K,, Isoaccepting tRNA's in liver and brain of young and old BC3F1 mice, Arch. Biochem., 153 (1972) 610-618. 10 Gambetti, P., Ingoglia, N., Weis, P. and Autilio-Gambetti, L., RNA distribution in goldfish
381 optic tectum after intraocular injection of aH-uridine. An EM autoradiographic study during optic nerve regeneration, Abstr. 5th Ann. Meeting Soc. Neurosci., (1975) 797. 11 Harris, C. L. and Maas, J. W., Transfer RNA and the regulation of protein synthesis in rat cerebral cortex during neural development, J. Neurochem., 22 (1974) 741-749. 12 Ingoglia, N. A., Grafstein, B., McEwen, B. S. and McQuarrie, I. G., Axonal transport of radioactivity in the goldfish optic system following intraocular injection of labeled R N A precursors, J. Neurochem., 20 (1973) 1605-1615. 13 lngoglia, N. A., Weis, P. and Mycek, J., Axonal transport of RNA during regeneration of the optic nerves of goldfish, J. Neurobiol., 6 (1975) 549-564. 14 Jacobson, K. B., Role of an isoacceptor transfer ribonucleic acid as an enzyme inhibitor: effect on tryptophan pyrrolase of drosophila, Nature New Biol., 231 (1971) 17-19. 15 Johnson, T. C., Mathews, R. A. and Chou, L., tRNA methyltransferase activity in neonatal and mature mammalian neural tissue, J. Neurochem., 23 (1974) 489-496. 16 Kaplan, B. B., Dyer, J. C. and Sirlin, J. L., Macromolecules and behavior: Effects of behavioral training on transfer RNAs of goldfish brain, Brain Research, 56 (1973) 239-248. 17 Kaplan, B. B. and Sirlin, J. L., Macromolecules and behavior. II. Training induced alteration in leucine transfer RNA of goldfish brain, Brain Research, 83 (1975) 451-468. 18 Lasek, R., Dabrowski, C. and Nordlander, R., Analysis of axoplasmic RNA from invertebrate giant axons, Nature New BioL, 244 (1973) 162-165. 19 Loening, U. E., Fractionation of RNA by polyacrylamide gel electrophoresis, Meth. med. Res., 12 (1970) 359-368. 20 Miani, N., DiGirolamo, A. and DiGirolamo, M., Sedimentation characteristics of axonal RNA in rabbit, J. Neurochem., 13 (1966) 755-759. 21 Murray, M. and Grafstein, B., Changes in the morphology and amino acid incorporation of regenerating goldfish optic nerves, Exp. Neurol., 23 (1969) 544--560. 22 Schubert, P. and Kreutzberg, G. W., Axonal transport of adenosine and uridine derivatives and transfer to postsynaptic neurons, Brain Research, 76 (1974) 526-530. 23 Sirlin, J. L., Biology of RNA, Academic Press, New York, N.Y., 1972. 24 Sperry, R. W., Functional regeneration in the optic system. In W. F. Windle (Ed.), Regeneration in the Central Nervous System, Thomas, Springfield, I11., 1955, pp. 66-76. 25 Sundari, R. M., Cherayil, J. D. and Jacob, T. M., Changes in transfer ribonucleic acid during the development of chick embryo, Indian J. Biochem. Biophys., 11 (1974) 43-46. 26 Weber, K. and Osborn, M., The reliability of molecular weight determination by dodecyl sulfatepolyacrylamide gel electrophoresis, J. biol. Chem., 244 (1969) 4406-4412. 27 White, B. N., Tener, G. M., Holden, J. and Suzuki, D. T., Analysis of tRNA's during the development of Drosophila. Develop. BioL, 33 (1973) 185-195. 28 Wylie, C. R., Jr., Advanced Engineering Mathematics, 2nd. ed., McGraw Hill, New York, 1960, p. 160.
