Two rates of fast axonal transport of [3H]glycoprotein in an identified invertebrate neuron

Brain Research, 229 (1981) 445~55 Elsevier/North-Holland Biomedical Press

445

T W O R A T E S OF FAST A X O N A L T R A N S P O R T OF [ Z H ] G L Y C O P R O T E I N I N AN IDENTIFIED INVERTEBRATE NEURON

DANIEL J. GOLDBERG and RICHARD T. AMBRON Departments of Pharmacology, Anatomy and Neurology and Center for Neurobiology and Behavior, Columbia University College of Physicians and Surgeons, New York, N Y 10032 (U.S.A.)

(Accepted May 21st, 1981) Key words: axonal transport - - glycoprotein - - Aplysia - - identified neuron

SUMMARY Recently, the kinetics of fast axonal transport of a single type of organelle, the serotonergic storage vesicle, was described in an identified axon of Aplysia using a pulsing technique combined with intracellular injection of [3H]serotonin. Here we extend the single axon studies by analyzing the movement of pulses of [aH]glycoprotein, following injection into the giant Aplysia cell, R2, of the amino sugar [aH]Nacetylgalactosamine. This glycoprotein precursor has been shown to label several organelles in this neuron. [aH]Glycoprotein is found to move in the axon of R2 at 2 rates of fast transport, 174 and 105 m m per day at room temperature. We suggest that the 2 rates reflect movements of 2 different types of organelle.

INTRODUCTION The macroscopic properties of fast axonal transport, such as velocities and temperature dependence, have been studied extensively. Material moving by fast transport is predominantly particulate and, in a given nerve, usually has been described as migrating at only one fast velocity, typically several hundred m m per day at, or when adjusted to, 38 °Cla,15,19, 20. In addition to the fast rate, some nerves also seem to move material at an intermediate rate, 5-10 times slower than the fast velocity but much faster than the slow axoplasmic flow by which cytoplasm and cytoskeletal structures move18, 22. Until recently, virtually all of these studies were done on whole nerves, containing thousands or millions of axons. Within the last several years, Schwartz and his colleagues have demonstrated fast transport of neurotransmittersl~, ~2,~6, glycoproteins 1,3 and glycolipids 21 in single identified neurons of the marine mollusc, Aplysia. In 0006-8993/81/0000-0000/$02.75 © Elsevier/North-Holland Biomedical Press

446 these studies, labeled material was exported from the cell body into the axon throughout the experiment, yielding a moving front of radioactivity in the nerve that was sometimes broad and ill-defined. Recently, Goldberg et al. analyzed the movement of discrete pulses of [3H]serotonin in the axon of the serotonergic giant cerebral neuron (GCN) 1°. Because [3H]serotonin is transported in this cell exclusively in a characteristic compound vesicle, these experiments allowed a detailed study of kinetic parameters of fast transport for a single type of organelle in a single axon. We now extend these studies of kinetic parameters of fast transport in single axons by describing the movement of pulses of radioactivity generated with a precursor that labels several types of organelle, the amino sugar N-acetylgalactosamine (NAGA). When injected into R2, the neuron used in the present studies, [ZH]NAGA is incorporated into glycoprotein components of compound vesicles, lucent vesicles and smooth endoplasmic reticulum 4, in contrast to [3H]serotonin in GCN, which labels only compound vesicles. We report here the co-existence of 2 fast rates of transport in the axon of R2. MATERIALS AND METHODS

Aplysia californiea (Pacific Bio-Marine, Venice, CA) weighing 100-250 g were kept until use at 15 °C in aquaria containing aerated Instant Ocean (Aquarium Systems, Eastlake, OH). Animals were fed periodically with seaweed. Intraeellular injection Methods for intrasomatic injection of radioactive material into R2 have been described previously 6. Briefly, the abdominal ganglion, with attached nerves and pleuropedal ganglia, was removed from the animal and pinned in a dish containing an artificial sea water supplemented with amino acids, vitamins and glucose. The cell body of R2, the largest in the abdominal ganglion, was impaled with a double-barreled microelectrode, and 0.2-1.0 nl of solution containing about 20,000-100,000 cpm of [3H]NAGA (17.5 Ci/mmol, New England Nuclear, Boston, MA) was rapidly ( 1-5 min) injected by application of pressure. The cell was used only if its resting potential was at least --42 mV and an antidromic action potential could be elicited by stimulating the right pleuroabdominal connective, which contains the major axon of R2. Injection was done at room temperature (22-23 °C). The usual procedure for creating a pulse of labeled material in the axon was to ligate the right connective with 6-0 surgical silk at its origin at the abdominal ganglion 110-115 rain after [3H]NAGA injection. The nervous system was then left at room temperature for 1, 2, 4 or 7 h, when the ganglion and the connective, pulled until taut, were pinned to an aluminum block and rapidly frozen with solid CO2. In a few experiments, 18 #M anisomycin was added to the supplemented artificial sea water 5 h after intracellular injection, and the nervous system maintained an additional 15 h at 15 °CL After re-impalement of the cell to ascertain that the resting and action potentials were still satisfactory, the ganglion and nerves were pinned and frozen on an aluminum block as described above.

447

Distribution of [3H]glycoproteins along the axon The frozen connective was cut into sequential 1 mm sections using a Mickle gel slicer. Each section was homogenized at 0 °C in a ground glass grinder containing 10 trichloroacetic acid (TCA)-I ~o phosphotungstic acid (PTA), and the precipitated proteins collected on glass fiber pads (Whatman GF/C). Lipid was removed by washing with ethanol-ether (1:2) and ether and the radioactivity on the pads was counted by liquid scintillation in Omnifluor-toluenel,L In some experiments, portions of the frozen connective expected to contain peaks of [3H]glycoprotein were homogenized in 50 mM Tris-HC1 (pH 7.6) and centrifuged at 105,000 × g. [3H]Glycoprotein in the supernatant was precipitated with TCA-PTA and counted. The membrane pellet was extracted sequentially with chloroform-methanol mixtures to remove lipid 21 and with sodium dodecyl sulfate (SDS) and formic acid to recover [3H]glycoprotein. Radioactivity in the SDS and formic acid extracts is defined as the particulate glycoprotein content of the cell1, 4.

Chemical analyses of transported [3H]glycoproteins Extraction from pads. Glass fiber pads were removed from the scintillation vials and washed with toluene, ethanol-ether (1:2) and ether. Dried pads containing a peak of [3H]glycoprotein were combined, cut into small pieces, and extracted at 70 °C with 3 ~ SDS, 1 ~ 2-mercaptoethanol (2-MSH) for 20 min. After centrifugation, extracted [3H]glycoproteins were isolated by acetone precipitation 1. SDS polyacrylarnide gel electrophoresis. Acetone-precipitated [3H]glycoproteins were treated for 20 min with 2 ~ SDS-5 ~ 2-MSH in 0.063 M Tris-HC1 (pH 7.1) at 70 °C. The extract was electrophoresed in SDS on a 3 ~ stacking gel-7.5 ~ running gel according to the method of Laemmli 17. Dansylated beef serum albumin was included in each sample as an internal standard for estimating molecular weight 1. The gels were frozen on a plate with solid COz and cut into sequential 1 mm segments using a Mickle gel slicer. Each slice was dissolved in a glass scintillation vial by heating with 30 hydrogen peroxide at 55 °C and counted after addition of scintillation fluid containing Triton X-100. Pronase digestion. [3H]Glycoproteins were digested with pronase (Calbiochem, San Diego, CA; 45 U/mg) for 72 h at 37 °C in a volume of 0.5 ml containing approximately 0.2 mg substrate protein, 0.5 mg enzyme, and a drop of toluene1, 4. Digests were examined by gel filtration on a column (0.9 × 104 cm) of Sephadex G-50 (Pharmacia, Piscataway, N J) in 0.1 M pyridine-acetate (.pH 4.7). RESULTS R2 is a giant neuron whose cell body is just beneath the dorsal surface of the abdominal ganglion. As with other invertebrate central neurons, only one axon arises from the cell body. This axon bifurcates within the ganglionic neuropil about 1 mm from the cell body. A very thin branch exits the ganglion in the branchial nerve; the majol branch is in the right pleuroabdominal connective, extending unbranched about 40-45 mm to the right pleural ganglion s. We used the right connective for all of our studies.

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DISTANCE ALONG CONNECTIVE (mm) Fig. 1. Distribution of [~H]glycoprotein along right connective. Separate nerves, l , 2, 4 and 7 h after proximal ligation of connective. Each vial was counted to ± 5 ~ accuracy, with background of 18 cpm already subtracted.

Movement of pulse of [3H]glycoprotein in axon [SH]NAGA injected into the cell body of R2 is incorporated into membraneassociated [3H]glycoproteins, some of which are exported into the axon where they move by fast axonal transport. Macromolecular radioactivity that appears in the right connective is restricted to the axon of R21,3. We first examined the time required for export and found that significant amounts of [SH]glycoprotein began to appear in the right connective about 100 min after injection of [3H]NAGA. To create as narrow a pulse of [3H]glycoprotein as possible, the right connective was ligated at its origin 110115 min after injection. The initial pulse of [ZH]glycoprotein typically split into a few peaks during migration along the axon. The distribution of radioactivity along the right connective at various times after ligation is shown in the representative profiles in Fig. 1. Because we wanted to create as narrow a pulse as possible, radioactivity was allowed to appear in the connective for only 10-15 min before ligation. Consequently, the peaks of [3H]glycoprotein subsequently observed were small. By analyzing 8-10 nerves at each

449 of the 4 post-ligation times, however, we f o u n d that some peaks appeared at each time with reasonable consistency (Fig. 2). T w o sets o f these peaks can be fit closely by straight lines, one representing a c o n s t a n t velocity of m o v e m e n t of 174 m m per day, a n d the other a c o n s t a n t velocity of 105 m m per day. We designate as Peak C the set o f peaks fit by the 174 m m per day line, Peak B the set of peaks fit by the other line, a n d Peak A the proximal group of peaks n o t fit b y a straight line.

Biochemical analysis of [3H]glycoprotein in peaks We wished to k n o w whether the peaks of radioactivity in the a x o n actually represented organellar material rather t h a n soluble glycoprotein, some of the latter having previously been f o u n d to appear in the connective after i n t r a s o m a t i c injection 1. We thus allowed 3 connectives to r e m a i n 4 h after ligation a n d then analyzed the radioactivity in regions of the nerves that would be expected to c o n t a i n the 3 peaks of radioactivity appearing at that time. A l m o s t all of the [3H]glycoprotein in the regions c o n t a i n i n g the 2 more distal peaks was particulate (Table I). Somewhat less, b u t still the p r e p o n d e r a n t portion, of the [3H]glycoprotein in the region c o n t a i n i n g the most

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Fig. 2. Mean locations of consistent peaks of [aH]glycoprotein in right connective at several times after injection of [SH]NAGA into R2 cell body - - 2.8 h ( = 1 h after ligation), 3.8 h ( = 2 h after ligation), 5.8 ( = 4 h after ligation) and 8.8 h ( = 7 h after ligation). In each nerve, an accumulation of [SH]glycoprotein was defined as a peak if it contained at least 5 % of the total [~H]glycoprotein in the whole nerve (less the 1 mm segment next to the ligation). Peaks that were located within a few mm of one another from one nerve to the next were considered to be manifestations of the same peak. For each of the 4 times, only those peaks that appeared in a majority of the nerves studied are plotted on this graph. The location of each peak is the mean (4- S.E.M.) of its location in individual nerves. Number of nerves studied at 2.8 h = 10, at 3.8 h = 10, at 5.8 h = 9, and at 8.8 h = 8. In all nerves, the cell body is assumed to be 1 mm from the ligation, because of the intraganglionicaxon segment. Points are fit by linear regression.

450 TABLE I

Distribution o f macromolecular radioactivity in peaks moving along R2's axon Three R2's were injected with [3H]NAGA. 110 min later each right connective was ligated and axonal transport was allowed to proceed for 4 h. The tissue was then frozen and the connective sectioned into mm segments. Segments containing peaks of radioactivity (A, 0-5 mm; B, 12-15 ram; C, 27-32 mm; see Fig. 2) were combined, added to unlabeled carrier tissue and fractionated. Radioactivity in membrane and soluble glycoprotein and in glycolipid was determined as described in Materials and Methods. The remaining radioactivity in each peak was acid-soluble.

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Fig. 3. Gel filtration of [3H]glycopeptides resulting from pronase digestion of labeled glycoproteins in Peaks A, B and C from R2's axon. Glass fiber pads from 30 individual experiments and containing [3H]glycoproteins from each of the 3 peaks, were combined and extracted with hot SDS. The glycoproteins were recovered from the extract by acetone precipitation and were digested with pronase for 72 h. The digest was then analyzed by gel filtration on a column (0.9 × 104 cm) of Sephadex G-50. The excluded volume was determined by blue dextran (BD).

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Fig. 4. SDS polyacrylamide gel electrophoresis of rapidly transported [aH]glycoproteins in R2's axon. R2 was injected with [aH]NAGA and 5 h later the tissue was placed in anisomycin to block export. After 15 h at 15 °C, the tissue was frozen, the connective sectioned, and [att]glycoproteins in each segment collected on a glass fiber pad after addition of TCA-PTA. Pads containing the distal-most peaks of labeled glycoprotein (corresponding to Peaks B and C above) were combined, and the glycoprotein was removed and electrophoresed as described in Materials and Methods. Roman numerals denote identified glycoproteinslm, a.

proximal peak was also particulate. All 3 regions also contained some [3H]glyeolipid which, however, was removed during the analyses of the distribution of radioactivity along the axon whose results are shown in Fig. 1 (see Materials and Methods). [3H]NAGA, when injected into the cell body of R2, labels a few types of organelles that subsequently appear in the axon, specifically, compound vesicles, lucent vesicles and smooth endoplasmic reticulum3, 4. However, we did not know how many of these 3 types were represented in the very brief pulse of [aH]glycoprotein we studied or, consequently, whether Peak A, B and C each represented a different type of organelle. The least ambiguous way of finding out would have been to analyze the peaks using quantitative electron microscopic autoradiography, but there was far too little radioactivity to allow such analysis. We thus tried to obtain an indirect answer by biochemically analyzing the [aH]glycoprotein composition of the peaks. Here, also, the paucity of radioactivity prevented use of the best method, separation of the [aH]glycoproteins by SDS gel electrophoresis; NAGA-labeled [aH]glycoproteins in Aplysia have been extensively characterized with this tectmiquel,4, 5. Instead, we digested the [3H]glycoproteins from the peaks with pronase, and analyzed the digests by gel filtration on a column of Sephadex G-50. The elution patterns for the digests from Peak B and Peak C were similar, indicating that the sugar portions of their glycoproteins, at least, were indistinguishable by size (Fig. 3). The elution pattern for Peak A was different from the other 2 patterns. The explanation for the similarity of the chromatographic elution profiles of the pronase digests of Peak B and Peak C is perhaps provided by an experiment we did using anisomycin. This inhibitor of protein synthesis blocks export of [aH]glycoprotein from cell body to axon within 30 min of its addition to the sea water bathing a

452 ganglionL We exposed an abdominal ganglion to l 8/~M anisomycin 5 h after injection of [3H]NAGA into the cell body of R2, the preparation being kept at 15 °C instead of room temperature. By preventing further export, the drug created a crude pulse of radioactivity in the right connective, considerably broader than the usual pulses caused by ligation, but also much richer in radioactivity. After 15 h more at 15 °C, the connective was frozen, and the [aH]glycoprotein that had reached the end of the nerve analyzed by SDS gel electrophoresis. This radioactivity presumably corresponds to material found in Peak B and Peak C in our usual pulse protocol. The gel electropherogram showed that the radioactivity consisted primarily of just one of the 5 major glycoproteins that are labeled by [aH]NAGA (Fig. 4), the one that has been designated component V (mol. wt. 90,000 daltons) 1. Previous results also suggested that component V is the glycoprotein transported most rapidly along R2's axon a. Thus, the similarity in elution profiles for the pronase digests from Peak B and Peak C may have resulted from both peaks consisting predominantly of one and the same [3H]glycoprotein. DISCUSSION

Origin of f3H]glycoprotein peaks in axon These experiments provide evidence that [3H]glycoproteins move in the axon of R2 by more than one rate of fast axonal transport. At each of the 4 post-ligation times we studied, there was more than one peak of [aH]glycoprotein in the axon. When the position of these peaks is plotted as a function of time after ligation, we find that 2 sets of the peaks can be closely fit by straight lines (Fig. 2). These lines intersect the abscissa at the same point, indicating that the 2 sets of [aH]glycoprotein exit from the cell body simultaneously. Also, this time of exit, 102 min post-injection, is appropriate; radioactive material would reach the right connective just priol to ligation, needing about 10 min to traverse the 1 mm intraganglionic axonal segment. For these reasons, we think the 2 sets of peaks each represent [3H]glycoprotein moving in the axon at a fast constant rate, that is, by fast axonal transport. So, we conclude that there co-exist in R2 two rates of fast transport, one of about 174 mm per day (Peak C) and the other of about 105 mm per day (Peak B) at room temperature. There is a considerable amount of [3H]glycoprotein that scarcely moves from the ligation, represented by the 4 proximal peaks in Fig. 2 that we have collectively tel med Peak A. Often, it comprised the largest fraction of [3H]glycoprotein in the axon. Clearly, this material does not move by fast transport after ligation. If, however, it left the cell body together with the materials in Peak B and Peak C, it must have moved through the intraganglionic axonal segment by fast t~ ansport so as to reach the right connective before ligation. There are two likely alternative explanations for the behavior of this material. Firstly, the material may have been partly or completely immobilized by the local nerve trauma caused by ligation. In support of this idea is the fact that a large amount of apparently immobilized radioactivity usually remained at the ligature in experiments in which the transport of pulses of [aH]serotonin in GCN was studied 1°. On the other hand, the proximal radioactivity may represent [aH]glyco-

453 protein-containing membranes that were incorporated into normally stationary axonal organelles, such as smooth endoplasmic reticulum. It is not clear, however, why the material should be deposited as a large peak proximally, rather than uniformly along the axon, unless the proximal area is specialized, perhaps as a processing or sorting area for membrane components. Although we find that some [3H]NAGA diffuses into the axon before ligation, it is very unlikely that substantial amounts of either the proximal stationary [3H]glycoprotein or the more distal Peaks B and C resulted from intra-axonal incorporation of sugar into membranes, because it has been found that only about 5 ~o of [3H]NAGA injected directly into the axon is incorporated into [3H]glycoprotein in the time span of our experiments 5.

Comparison with previous work Two such fast rates of transport in a nerve, or nerve bundle, have been repolted previously only in the adrenergic fibers running from the locus coeruleus to the hypothalamus in rat brain. In these axons, [3H]glycoprotein was found to move at rates of 48 and 96 mm per day is. In mammalian optic nerve ~2 2 rates for fast transport of protein have been demonstrated, but one is several times slower than the other, as opposed to the less than 2-fold difference seen in our experiments. In studies of the movement of pulses of [3H]protein in garfish olfactory nerve 15 and cat sciatic nerve 19, which are long nerve bundles in which the longitudinal distribution of radioactivity can be accurately measured, the [3H]protein apparently migrates as a single peak. We cannot explain why only one migrating peak was observed in these other pulse studies while we observe 2 peaks in our study, other than to point out differences in experimental design. Firstly, we used a single identified axon, whereas the sciatic nerve contains thousands of sensory axons ([3H]leucine is injected into the dorsal root ganglion) and the olfactory nerve is composed of millions of axons. Secondly, we allowed radioactivity to enter the axon for only 10-15 min, while radioactivity was most often allowed to enter the sciatic nerve for about 90 min (assuming a 30 min delay between ganglionic injection and appearance of radioactivity in the nerve) and the olfactory nerve for about 260 min. Thirdly, we analyzed the transport of [3H]glycoprotein, whereas [ZH]protein was the marker used in the other studies. What these differences have in common is that they would narrow the 'window' through which transported material is observed in our experiments; however, this does not necessarily mean that we would more easily detect multiple rates. Finally, there is the obvious phyletic difference, although fast transport seems to have the same basic properties in vertebrates and invertebrates. Mechanism for generation oJ'2 fast transport rates in a single axon Although it has been found that [3H]NAGA injected into the soma of R2 labels compound vesicles, lucent vesicles and smooth endoplasmic reticulum in the axon 3, we do not know if labeled members of all of these types of organelle were present in the axons we analyzed, because the labeling periods were so brief. However, because pulsed [ZH]serotonin, which labels only the compound vesicle in GCN's axon, moves

454 as a single peak, it is reasonable to suggest that the 2 [aH]glycoprotein peaks in R2's axon are generated by 2 labeled types of organelle that move at different velocities, rather t h a n a single type with 2 velocities. It has been proposed by G o l d b e r g et al.9, TM a n d by Gross la that organelles move in the anterograde direction in a stop-and-go fashion, as observed by light microscopy for retrograde t r a n s p o r t 7. If so, one organelle type could have a greater net velocity t h a n a n o t h e r by m o v i n g more frequently rather t h a n more rapidly, that is, by spending less time stationary in the axoplasm. This is p r o b a b l y true of retrograde transport, where mitochondria, which have a m u c h slower net velocity t h a n the fastest transported material, can be seen to move rapidly but rarely 7. P r o o f for this idea awaits the identification of the organelles moving at the 2 rates in R2. ACKNOWLEDGEMENTS We t h a n k Mr. D a v i d Weissman for technical assistance. This work was supported by a n I r m a T. Hirschl Career Scientist Award, a n Alfred P. Sloan Research Fellowship a n d N I H Research G r a n t NS 14711 to D.J.G., a n d by N I H Research Career Development A w a r d NS 00350 a n d N I H Research G r a n t NS 14555 to R.T.A.

REFERENCES 1 Ambron, R. T., Goldman, J. E. and Schwartz, J. 14., Axonal transport of newly synthesized glycoproteins in a single identified neuron of Aplysia californica, J. Cell Biol., 61 (1974) 665-675. 2 Ambron, R. T., Goldman, J. E. and Schwartz, J. H., Effect of inhibiting protein synthesis on axonal transport of membrane glycoproteins in an identified neuron of Aplysia, Brain Research, 94 (1975) 307-323. 3 Ambron, R. T. and Schwartz, J. H., Regional aspects of neuronal glycoprotein and glycolipid synthesis. In R. U. Margolis and R. K. Margolis (Eds.), Complex Carbohydrates of Nervous Tissue, Plenum, New York, 1979, pp. 269-289. 4 Ambron, R. T., Sherbany, A. A., Shkolnik, L. J. and Schwartz, J. H., Distribution of membrane glycoproteins among the organelles of a single identified neuron of Aplysia, I., Brain Research, 207 (1981) 17-32. 5 Ambron, R. T. and Treistman, S. N., Glycoproteins are modified in the axon of R2, the giant neuron of Aplysia californica, after intra-axonal injection of [aH]N-acetylgalactosamine, Brain Research, 121 (1977) 287-309. 6 Eisenstadt, M., Goldman, J. E., Kandel, E. R., Koike, H., Koester, J. and Schwartz, J. 14, Intrasomatic injection of radioactive precursors for studying transmitter synthesis in identified neurons of Aplysia californica, Proc. nat. Acad. Sci. (Wash.), 70 (1973) 3371-3375. 7 Forman, D. S., Padjen, A. L. and Siggins, G. R., Axonal transport of organelles visualized by light microscopy: cinemicrographic and computer analysis, Brain Research, 136 (1977) 197-213. 8 Gillette, R. and Pomeranz, B., A study of neuron morphology in Aplysia californica using Procion yellow dye, Comp. Biochem. PhysioL, 44A (1973) 1257-1259. 9 Goldberg, D. J., Goldman, J. E. and Schwartz, J. H., Alterations in amounts and rates of serotonin transported in an axon of the giant cerebral neurone of Aplysia californica, J. Physiol. (Lond.), 259 (1976) 473-490. 10 Goldberg, D. J., Schwartz, J. H. and Sherbany, A. A., Kinetic properties of normal and perturbed axonal transport of serotonin in a single identified axon, J. Physiol. (Lond.), 281 (1978) 559-579. 11 Goldman, J. E., Kim, K. S. and Schwartz, J. H., Axonal transport of [aH]serotonin in an identified neuron of Aplysia californica, J. Cell BioL, 70 (1976) 304-318.

455 12 Gotoh, H. and Schwartz, J. H., Specific axonal transport of [all]histamine after intrasomatic injection of C2, an identified Aplysia neuron, Neurosci. Abstr., 6 (1980) 502. 13 Gross, G. W., The effect of temperature on the rapid axoplasmic transport in C-fibers, Brain Research, 56 (1973) 359-363. 14 Gross, G. W., The microstream concept of axoplasmic and dendritic transport, Advanc. Neurok, 12 (1975) 283-296. 15 Gross, G. W. and Beidler, L. M., Fast axonal transport in the C-fibers of garfish olfactory nerve, J. Neurobiol., 6 (1975) 213-232. 16 Koike, H., Eisenstadt, M. and Schwartz, J. H., Axonal transport of newly synthesized acetylcholine in an identified neuron of Aplysia, Brain Research, 37 (1972) 152-159. 17 Laemmli, V. K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature New Biol., 227 (1970) 680-685. 18 Levin, B. E., Axonal transport of [3H]fucosyl glycoproteins in noradrenergic neurons in the rat brain, Brain Research, 130 (1977) 421-432. 19 Ochs, S., Retention and redistribution of proteins in mammalian nerve fibers by axoplasmic transport, J. Physiol. (Lond.), 253 (1975) 459-475. 20 Schwartz, J. H., Axonal transport: components, mechanisms, and specificity, Ann. Rev. Neurosci., 2 (1979) 467-504. 21 Sherbany, A. A., Ambron, R. T. and Schwartz, J. H., Membrane glycolipids: regional synthesis and axonal transport in a single identified neuron ofAplysia californica, Science, 203 (1979) 78-81. 22 Willard, M., Cowan, W. M. and Vagelos, P. R., The polypeptide composition of intra-axonally transported proteins: evidence for four transport velocities, Proc. nat. Acad. Sci. (Wash.), 71 (1974) 2183-2187.