AXONAL TRANSPORT OF [3H]GLUCOSE RADIOACTIVITY IN THE OPTIC SYSTEM OF SCARDINIUS ERYTHROPHTHALMUS1

Abstract— The optic system of Scardinius erythrophthalmus has been used to study the axonal translocation of radioactivity from [³H]glucose. Intraocularly injected precursors were transported intra-axonally along the optic nerve towards the contralateral optic tectum. In comparison with the well known properties of axonal protein transport there were remarkable differences in the proximo-distal translocation of [³H]glucose. These were: (1) a delay in the labelling of the structures investigated, after tracer application; (2) only a rapid phase of transport; and (3) no accumulation of radioactivity in the region of nerve terminals in the optic tectum connected with the injected eye. The transported material was almost exclusively in the form of TCA-soluble compounds and was mainly glucose itself or its low molecular derivatives, but not glycogen. The rate of transport was decreased by lowered temperatures and was not immediately dependent on retinal protein synthesis. Colchicine blocked the axonal transport of glucose by up to 60–70 per cent.     

Axonal transport of RNA during regeneration of the optic nerves of goldfish

The distribution of radioactive RNA and RNA precursors in the goldfish optic tecta following intraocular injection of ³H-uridine has been studied during various stages of optic nerve regeneration. ³H-uridine was injected into the posterior chamber of the right eye 17, 30, or 60 days after both optic nerves were crushed. Fish were sacrificed at time intervals ranging from 0.5 to 21 days after injection. One day prior to sacrificing, ¹⁴C-proline was also injected into the right eye as a marker of fast axonal protein transport. Seventeen to 23 days after crushing, the approximate time of nerve reconnection, the amount of radioactive RNA appearing in the left optic tectum was increased by more than ten times control values. Approximately 30 days after crushing the nerve, when the reconnected nerve is maturing, RNA values were still elevated, but significantly decreased from the earlier stage. By 60 days after crushing the optic nerve, the amounts of RNA in the left tectum was close to normal. Evidence suggesting that, at least, some of the radioactive RNA in the tectum originated from RNA transported along optic axons rather than from RNA synthesized locally in the tectum was provided by autoradiographic experiments. Autoradiograms of paraffin sections taken from the goldfish optic tecta after the intraocular injection of ³H-uridine showed a distribution of grains in a linear pattern, suggesting a distribution over the incoming fibers during the reconnection stage of regeneration. Electron microscopic autoradiography of glutaraldehyde fixed epoxy sections confirmed that a significant number of grains (shown to be ³H-RNA) were, in fact, over regenerating optic axons. Intracranial injection of ³H-uridine, during the same stage of regeneration, on the other hand, resulted in a distribution of grains, specifically over cell perikarya. These experiments suggest that during the reconnection phase of nerve regeneration, large amounts of RNA may be carried within regenerating optic axons as they enter the optic tectum.     

THE EFFECT OF INTRAOCULAR INJECTION OF CORDYCEPIN ON RETINAL RNA SYNTHESIS AND ON RNA AXONALLY TRANSPORTED DURING REGENERATION OF THE OPTIC NERVES OF GOLDFISH

Abstract— The presence of relatively large amounts of RNA has been demonstrated in regenerating axons of the goldfish optic nerve. Previous experiments have suggested that this R NA may be composed of only small molecular weight 4S RNA. The present experiments were performed in order to see if inhibiting RNA transport by intraocular injections of cordycepin causes a selective depletion of 4S RNA arriving in the contralateral optic tectum, and thus add further evidence that 4S RNA is axonally transported. Optic nerves were crushed in a group of goldfish and 18 days later 10.0 /tg of cordycepin was injected into the right eye followed 3 h later by injections of [³H]uridine into the same eye. Six days later the amount of axonally transported [³H]RNA was decreased by 89% compared with non-cordycepin treated controls. The effect of cordycepin on retinal RNA synthesis was shown by autoradiography to be primarily on retinal ganglion cell RNA synthesis with lesser effects on other cellular elements of the retina. SDS polyacrylamide gel electrophoresis at both 1 and 6 days after intraocular injections of cordycepin and [³H]uridine, showed that cordycepin blocks the retinal synthesis of ribosomal RNAs but appeared to have little effect on the synthesis of 4S RNA. When transported RNA in the tectum was fractionated by gel electrophoresis 6 days after injection, it was found that the amount of ribosomal RNA was decreased by approx 70% as a result of cordycepin pretreatment. This correlated well with the effect of cordycepin on the transport of available RNA precursors (also decreased by approx 70%) and is consistent with the contention that in these experiments ribosomal RNA is synthesized in the tectum itself and is not axonal. The amount of [³H] 4S RNA arriving in the tectum, however, was decreased by greater than 90% suggesting that its presence in the tectum was not entirely dependent on the availability of ³H precursors for local synthesis in the tectum. These results are consistent with data suggesting that 4S RNA is the predominant, if not the only, RNA species axonally transported during regeneration of goldfish optic nerves.     

Protein phosphorylation: Localization in regenerating optic axons

A number of axonal proteins display changes in phosphorylation during goldfish optic nerve regeneration (Larrivee and Grafstein, 1989). (1) To determine whether the phosphorylation of these proteins was closely linked to their synthesis in the retinal ganglion cell body, cycloheximide was injected intraocularly into goldfish whose optic nerves had been regenerating for 3 weeks. Cycloheximide reduced the incorporation of [³H]proline and³²P orthophosphate into total nerve protein by 84% and 46%, respectively. Of the 20 individual proteins examined, 17 contained less than 15% of the [³H]proline label measured in corresponding controls, whereas 18 proteins contained 50% or more of the³²P label, suggesting that phosphorylation was largely independent of synthesis. (2) To deterine whether the proteins were phosphorylated in the ganglion cell axons, axonal transport of proteins was blocked by intraocular injection of vincristine. Vincristine reduced [³H]proline labeling of total protein by 88% and³²P labeling by 49%. Among the individual proteins [³H]proline labeling was reduced by 90% or more in 18 cases but³²P labeling was reduced only by 50% or less. (3) When³²P was injected into the cranial cavity near the ends of the optic axons, all of the phosphoproteins were labeled more intensely in the optic tract than in the optic nerve. These results suggest that most of the major phosphoproteins that undergo changes in phosphorylation in the course of regeneration are phosphorylated in the optic axons.Abbreviations SDS sodium lauryl sulfate - GAP growth associated protein - TCA trichloracetic acid - kD kilodalton     

Phosphorylation of Proteins in Normal and Regenerating Goldfish Optic Nerve

Within 6 h after radiolabeled phosphate was injected into the eye of goldfish, labeled acid-soluble and acid-precipitable material began to appear in the optic nerve and subsequently also in the lobe of the optic tectum, to which the optic axons project. From the rate of appearance of the acid-precipitable material, a maximal velocity of axonal transport of 13-21 mm/day could be calculated, consistent with fast axonal transport group II. Examination of individual proteins by two-dimensional gel electrophoresis revealed that approximately 20 proteins were phosphorylated in normal and regenerating nerves. These ranged in molecular weight from approximately 18,000 to 180,000 and in pI from 4.4 to 6.9. Among them were several fast transported proteins, including protein 4, which is the equivalent of the growth-associated protein GAP-43. In addition, there was phosphorylation of some recognizable constituents of slow axonal transport, including alpha-tubulin, a neurofilament constituent (NF), and another intermediate filament protein characteristic of goldfish optic axons (ON2). At least some axonal proteins, therefore, may become phosphorylated as a result of the axonal transport of a phosphate carrier. Some of the proteins labeled by intraocular injection of 32P showed changes in phosphorylation during regeneration of the optic axons. By 3-4 weeks after an optic tract lesion, five proteins, including protein 4, showed a significant increase in labeling in the intact segment of nerve between the eye and the lesion, whereas at least four others (including ON2) showed a significant decrease. When local incorporation of radiolabeled phosphate into the nerve was examined by incubating nerve segments in 32P-containing medium, there was little or no labeling of the proteins that showed changes in phosphorylation during regeneration. Segments of either normal or regenerating nerves showed strong labeling of several other proteins, particularly a group ranging in molecular weight from 46,000 to 58,000 and in pI from 4.9 to 6.4. These proteins were presumably primarily of nonneuronal origin. Nevertheless, if degeneration of the axons had been caused by removal of the eye 1 week earlier, most of the labeling of these proteins was abolished. This suggests that phosphorylation of these proteins depends on the integrity of the optic axons.     

Axonal transport of lipid in goldfish optic axons

After injection of labeled glycerol, choline, or serine into the eye of goldfish, labeled lipids were axonally transported along the optic nerve to the optic tectum. although the different precursors were presumably incorporated into somewhat different lipid populations, all three were approximately equally effective in labeling the lipids transported to the tectum, but the amount of transported material remaining in the nerve was different, being highest with choline and lowest with serine. The labeled lipids appeared in the tectum within 6 hr of the injection, indicating a fast rate of transport, but continued to accumulate over a period of 1–2 weeks, which presumably reflects the time course of their release from the cell body. Since there was a gradual increase in the proportion of labeled lipid in the tectum during this period, some other process in addition to fast axonal transport may have affected the distribution of the lipids along the optic axons. When [³H]choline was used as precursor, the transported material included a small amount of TCA-soluble material, which was probably mainly phosphorylcholine, with labeled acetylcholine appearing in only insignificant amounts. With serine, which gave rise to a large amount of axonally transported protein in addition to lipid, a late increase in the amount of labeled lipid in the tectum was seen, accompanied by a decrease in labeling of the protein fraction.     

Association of axonally transported heparan sulfate with isolated synaptic plasma membrane

Studies on isolated synaptic plasma membranes (SPM) have detected little if any heparan sulfate or other glycosaminoglycans (GAGs), while more recent studies employing proteoglycan antibodies have localized heparan sulfate proteoglycan in presynaptic plasma membrane of intact tissue. To further address the issue of proteoglycans in synaptic plasma membrane of intact tissue. To further address the issue of proteoglycans in synaptic plasma membrane, we have investigated the possible presence of axonally transported GAGs in SPM isolated from the goldfish optic tectum. SPMs isolated from tecta following rapid axonal transport of³⁵SO₄ labeled molecules down the optic nerve, showed specific radioactivity approximately two-fold higher than the starting homogenate. Treatment of the transport labeled SPM with the enzyme heparitinase liberated 21% of the radioactivity, indicating the presence of a significant fraction of trnasported label in heparan sulfate. In a separate series of experiments a GAG fraction was isolated from transport labeled SPM and was found to consist of heparan sulfate containing 28% of transported radioactivity. Chondroitin (4 or 6) sulfate, which undergoes axonal transport in the goldfish optic system, was not found associated with SPM. Taken together the results support immunological evidence for the presence of heparan sulfate proteoglycans in presynaptic plasma membrane.To whom to address reprint request..     

Axonal transport of glycerophospholipids following intracerebral injection of glycerol into substantia nigra or lateral geniculate body

[2-³H]Glycerol was injected into one substantia nigra of adult rats. Incorporation of radioactivity into lipids at the injection site was maximal by 2 hr, after which it declined. Rapidly transported³H-labeled lipids were just beginning to accumulate in the primary projection site, the ipsilateral corpus striatum by 2 hr, as evidenced by 20-fold higher levels of lipid radioactivity in the projection site relative to control regions. However, the bulk of labeled lipid arrived between 6 hr and 3 days postinjection, suggesting either a prolonged period of release of rapidly transported lipids from the nerve cell bodies or a slow rate of transport for the later arriving lipids. Colchicine applied locally to the fibers of this tract blocked the axonal transport of lipids to the striatum almost completely. Choline and ethanolamine phosphoglycerides were the major transported lipids, accounting for approximately 60% and 25%, respectively, of the total. Similar results were obtained in studies of [2-³H]glycerol-labeled lipids synthesized in the lateral geniculate body and transported to the visual cortex. The rapid axonal transport of lipids labeled with [³²P]phosphate (injected simultaneously with [2-³H]glycerol) could also be demonstrated in both tracts. However, in contrast to [2-³H]glycerol, considerable amounts of³²P soluble label were present in the projection sites, and colchicine only partially blocked the accumulation of³²P-labeled lipid. These results demonstrate the relative utility of [2-³H]glycerol as a lipid precursor for examination of axonal transport in intrabrain tracts. Characteristics of lipid axonal transport in these two intrabrain tracts are similar to each other and are also similar to those previously described for retinal ganglion cells, indicating a common requirement for the axonal transport of these membrane constituents to axons and nerve endings in widely divergent CNS tracts.Presented in part at the 11th meeting of the American Society for Neurochemistry, Houston, Texas, March 1980.     

Plasticin, a novel type III neurofilament protein from goldfish retina: increased expression during optic nerve regeneration.

The goldfish visual pathway displays a remarkable capacity for continued development and plasticity. The intermediate filament proteins in this pathway are unexpected and atypical, suggesting these proteins provide a structure that supports growth and plasticity. Using a goldfish retina lambda gt10 library, we have isolated a full-length cDNA clone that encodes a novel type III intermediate filament protein. The mRNA for this protein is located in retinal ganglion cells, and its level dramatically increases during optic nerve regeneration. The protein is transported into the optic nerve within the slow phase of axonal transport. We have named this protein plasticin because it was isolated from a neuronal pathway well known for its plasticity.     

AXONAL TRANSPORT OF RADIOACTIVITY IN THE GOLDFISH OPTIC SYSTEM FOLLOWING INTRAOCULAR INJECTION OF LABELLED RNA PRECURSORS

After injection of the tritiated RNA precursors [³H]guanosine, [³H]uridine or [³H]orotic acid into the eye of goldfish, labelled TCA-soluble material and RNA appeared to be axonally transported to the contralateral optic tectum. From the time courses of arrival in the tectum,‘average’rates of transport of 6 mm/day for the soluble material and 1·7 mm/day for the RNA were calculated. If the optic nerve was cut after the transported material had arrived in the tectum, about 60 per cent of the TCA-soluble material disappeared by 7 days after the cut, but almost none of the RNA. After a further 8- to 13-day period, the TCA-soluble material had declined by a further 50 per cent from the 7-day value, but the RNA by only 20 per cent. Thus, relatively little RNA was lost when the optic axons degenerated, an observation which suggested that the RNA might be extra-axonal. However, if the optic nerve was crushed before the arrival of the transported material, RNA did not appear in the tectum until the regenerating optic nerve fibres arrived. Therefore, the presence of RNA must be dependent on intact nerve fibres. Moreover, in the earliest stages of regeneration the proportion of transported RNA to TCA-soluble material was considerably higher than normal, suggesting that the regenerating fibres arrived in the tectum already carrying RNA. This implies that the RNA itself was transported in the optic fibres.     

58,000 Dalton Intermediate Filament Proteins of Neuronal and Nonneuronal Origin in the Goldfish Visual Pathway

A group of proteins in the goldfish optic nerve with a molecular weight of 58K daltons was analyzed by two-dimensional gel electrophoresis. Results show that the proteins are differentially phosphorylated and found exclusively in a cytoskeletal-enriched fraction. The proteins from this fraction can be reconstituted into typical intermediate filament structures, as shown by electron microscopy. Two components which are of neuronal origin are transported within the slow phase of transport. The 58K proteins are the most abundant proteins in the optic nerve, and they are distinct from actin and tubulin. It was concluded that they are intermediate filament proteins. Cytoskeletal preparations of rat spinal cord, rat optic nerve, and goldfish optic nerve were compared by one-dimensional gel electrophoresis. The rat spinal cord contains glial fibrillary acidic protein (GFAP), and the rat optic nerve contains vimentin and GFAP, in addition to the neurofilament triplet. A typical mammalian neurofilament triplet is not detected in the goldfish optic nerve, while the major cytoskeletal constituent is a 58K band which coelectrophoreses with vimentin in the rat optic nerve by one-dimensional gel electrophoresis.     

THE EFFECT OF CORDYCEPIN ON THE APPEARANCE OF [3H]RNA IN THE GOLDFISH OPTIC TECTUM FOLLOWING INTRAOCULAR INJECTION OF [3H]URIDINE

Abstract— Although biochemical and electron microscopic evidence has shown that RNA molecules may be found within axons, the origin of this RNA is not known. In order to determine if the RNA found in axons is synthesized in the nerve cell body and axonally transported, we have studied the effect of the RNA inhibitor cordycepin (3′-deoxyadenosine) on the retinal synthesis and axonal migration of radioactive RNA. Ten μg of cordycepin was injected into the right eye of 11 fish and 3 h later [³H]uridine was injected into the same eye. Twelve control fish were injected with [³H]uridine only and all fish were sacrificed 6 days later. Results of RNA extraction of retina and tecta showed that cordycepin decreased retinal RNA synthesis by approx 24%, while inhibiting the amount of [³H]RNA appearing in the contralateral tectum by 74%. Since the transport of RNA precursors was depressed by only 50%, (significantly different from the effect on RNA, P < 0.01) it seems unlikely that the action of cordycepin in decreasing tectal [³H]RNA levels was due solely to a decrease in the availability of labeled precursors for tectal RNA synthesis. For the purpose of blocking tectal RNA synthesis, 200 μg of cordycepin was injected intracranially several days after the intraocular injection of [³H]uridine. This route of cordycepin administration failed to significantly block the appearance of [³H]RNA in the tectum, suggesting that at least some of the [³H]RNA in the tectum was synthesized before arrival in the tectum itself. To be sure that cordycepin itself was not being transported, we injected cordycepin into the right eye of fish and 5 days later, injected fish intracranially with [³H]uridine. Autoradiograms were prepared and grains were counted over the fiber layers of left (experimental) and right (control) tecta. No significant difference was observed in the number of grains of left vs right tecta indicating that cordycepin itself is not axonally transported. These experiments support earlier findings from our laboratory which suggest that RNA may be axonally transported in goldfish optic fibers.     

A COMPARISON OF THE AXONAL TRANSPORT OF TAURINE AND PROTEINS IN THE GOLDFISH VISUAL SYSTEM

Abstract— Radioactive cystathionine, a metabolic precursor of taurine, was injected into the right eye of goldfish. At various times after injection the retina and both optic tecta were extracted with trichloroacetic acid (TCA) and the amount and nature of the radioactivity was determined. Radioactive taurine and inorganic sulfate were present in the TCA-soluble extract of retina and radioactive taurine and a small amount of inorganic sulfate was found in the contralateral optic tectum. That taurine is migrating intraaxonally and is not diffusing in extraaxonal spaces is suggested from experiments in which the migration of taurine was compared with that of [¹⁴C]mannitol, used here as a marker of extracellular diffusion. In the time studied (up to 15 h) mannitol did not migrate to the tectum, whereas taurine was detectable in the tectum as early as 8 h after injection. Since intra-axonal diffusion of amino acids and other small molecules in this system has been ruled out, it is likely that taurine is being transported axonally. The axonal transport of taurine was found to be similar to the fast component of protein transport because: (1) their rates of transport are similar, (2) the transport of both is blocked by the protein synthesis inhibitor cycloheximide, (3) vinblastine, which disrupts neurotubules, appears to have similar effects on both protein and taurine transport, and (4) both rapidly transported proteins and taurine remain mostly intra-axonal once they have been transported to the tectum. Taurine and proteins differ in that rapidly transported proteins are primarily paniculate in nature and localized to a large extent in nerve endings, while taurine is primarily in a soluble fraction and is present in nerve endings only in trace amounts. We suggest that taurine may be loosely linked to a newly synthesized protein in the soma and is then transported along with that protein on a similar conveying mechanism in the axoplasm.     

Axonal transport of RNA during regeneration of the optic nerves of goldfish.

The distribution of radioactive RNA and RNA precursors in the goldfish optic tecta following intraocular injection of 3H-uridine has been studied during various stages of optic nerve regeneration. 3H-uridine was injected into the posterior chamber of the right eye 17, 30, or 60 days after both optic nerves were crushed. Five were sacrificed at time intervals ranging from 0.5 to 21 days after injection. One day prior to sacrificing, 14C-proline was also injected into the right eye as a marked of fast axonal protein transport. Seventeen to 23 days after crushing, the approximate time of nerve reconnection, the amount of radioactive RNA appearing in the left optic tectum was increased by more than ten times control values. Approximately 30 days after crushing the nerve, when the reconnected nerve is maturing, RNA values were still elevated, but significantly decreased from the earlier stage. By 60 days after crushing the optic nerve, the amounts of RNA in the left tectum was close to normal. Evidence suggesting that, at least, some of the radioactive RNA in the tectum originated from RNA transported along optic axons rather than from RNA synthesized locally in the tectum was provided by autoradiographic experiments. Autoradiograms of paraffin sections taken from the goldfish optic tecta after the intraocular injection of 3H-uridine showed a distribution of grains in a linear pattern, suggesting a distribution over the incoming fibers during the reconnection stage of regeneration. Electron microsocpic autoradiography of glutaraldehyde fixed epoxy sections confirmed that a significant number of grains (shown to be 3H-RNA) were, in fact, over regenerating optic axons. Intracranial injection of 3H-uridine, during the same stage of regeneration, on the other hand, resulted in a distribution of grains, specifically over cell perikaprya. These experiments suggest that during the reconnection phase of nerve regeneration, large amounts of RNA may be carried within regenerating optic axons as they enter the optic tectum.     

AXOPLASMIC TRANSPORT IN THE OPTIC NERVE AND TRACT OF THE RABBIT

The axonal transport of labelled proteins was studied in the optic system of adult rabbits after an intraocular injection of [³H]Ieucine. It was demonstrated that the precursor was incorporated into protein, which was transported along the axons of the retinal ganglion cells. Intraocularly injected puromycin inhibited protein synthesis in the retina and markedly inhibited the appearance of labelled protein in the optic nerve and tract. It was further demonstrated by intracisternal injection of [³H]leucine that an intraocular injection of puromycin did not affect the local protein synthesis in the optic nerve and tract. Cell fractionation studies of the optic nerve and tract showed that the rapidly migrating component, previously described as moving at an average rate of 110-150 mm/day, was largely associated with the microsomal fraction. About 40 per cent of the total protein-bound radioactivity in this component was found in the microsomal fraction and about 15 per cent was recovered in the soluble protein fraction. Most of the labelled material moving at a rate of 1-5-2 mm/day was soluble protein. The specific radioactivity of this component was about ten times greater than that of the fast one. In the slow component about 50 per cent of the radioactivity was found in the soluble protein fraction and about 10 per cent of the radioactivity was recovered in the microsomal fraction. Radioautography demonstrated incorporated label in the neuropil structures in the lateral geniculate body as early as 4-8 hr after intraocular injection. The labelling of the neuropil increased markedly during the first week, and could be observed after 3 weeks.     

RAPIDLY LABELLING AND EXPORTED NEURONAL PROTEIN; [3H]FUCOSE AS A PRECURSOR, AND EFFECTS OF CYCLOHEXIMIDE AND COLCHICINE

Abstract— The characteristics of a rapidly labelled and rapidly transported neuronal perikaryal protein fraction (Rose & Sinha . 1974a) were investigated in three experiments. (1) The kinetics of labelling of neuronal cell body and neuropil fractions from [³H]fucose were followed and shown to be similar to those from [³H]lysine, the label first appearing in the neuronal fraction and then being exported. The neuronal/neuropil incorporation ratio fell from 1.37 at 1 h to 0.77 at 4 h. (2) When cycloheximide (5 mg/kg) was injected intraperitoneally 15 min after [³H]lysine, incorporation into neuronal protein was inhibited to a greater extent (85%) than into neuropil (60%). (3) Colchicine was injected at a dose (40 μg/kg) sufficient to prevent accumulation of radioactively labelled protein into synaptosomes but insufficient to affect total incorporation of precursor into protein. [³H]Lysine was injected 1 h after colchicine and neurons and neuropil fractions made 1 h and 4 h later; colchicine inhibited the export of labelled protein from the neuronal perikaryon and its accumulation in the neuropil. We conclude that the rapidly labelled neuronal protein is partially glycoprotein in character and may be normally transported from the cell body by way of the axonal/(dendritic?) flow mechanism.     

Axonal migration of various ribonucleic acid species along the optic tract of the chick

—The avian visual system has been used to study the axonal transport of RNA and protein. After monocular injection of radioactive uridine into 1-day-old chicks, a considerable amount of labelled RNA migrated along the optic tract to the optic tectum contralateral to the injected eye. This RNA was largely ribosomal, although it was contained in several subcellular fractions. The migration of RNA appeared to be a slow process. However, following monocular injection of radioactive proline, the migration of ribosomal protein was rapid. This discrepancy was resolved by examination of the kinetics of labelling of RNA and protein within the retina after intraocular injection of a mixture of labelled uridine and proline. Cytoplasmic RNA was labelled much more slowly than cytoplasmic protein. This lag in labelling of RNA could account for the delayed arrival of RNA at the contralateral optic lobe and suggests that ribosomes may travel rapidly along the axon. In other experiments, eyes were removed 4 days after the injection of labelled precursors. After a further 14 days, the remaining radioactivity in RNA and protein of contralateral optic lobes was 5–15% of that attributable to migration along the axon in control, unenucleated birds. Thus, the survival of the bulk of migrating macromolecules depends on the integrity of synaptic terminals. This observation suggests that both RNA and protein migrate within the axon rather than extra-axonally, and that they remain largely within the nerve cells along the axons of which they are transported.     

Protein synthesis and transport in the regenerating goldfish visual system

The nature of the proteins synthesized in the goldfish retina and axonally transported to the tectum during optic nerve regeneration has been examined. Electrophoretic analysis of labeled soluble retinal proteins by fluorography verified our previous observation of a greatly enhanced synthesis of the microtubule subunits. In addition, labeling of a tubulin-like protein in the retinal particulate fraction was also increased during regeneration. Like soluble tubulin, the particulate material had an apparent MW of 53–55K and could be tyrosylated in the presence of cycloheximide and [³H]tyrosine. Comparison of post-crush and normal retinal proteins by two-dimensional gel electrophoresis also revealed a marked enhancement in the labeling of two acidic 68–70K proteins. Analysis of proteins slowly transported to the optic tectum revealed changes following nerve crush similar to those observed in the retina, with enhanced labeling of both soluble and particulate tubulin and of 68–70K polypeptides. The most striking change in the profile of rapidly transported protein was the appearance of a labeled 45K protein which was barely detectable in control fish.     

Axonal transport of the mitochondria-specific lipid, diphosphatidylglycerol, in the rat visual system

Rats 24 d old were injected intraocularly with [2-3H]glycerol and [35S]methionine and killed 1 h-60 d later. 35S label in protein and 3H label in total phospholipid and a mitochondria-specific lipid, diphosphatidylglycerol(DPG), were determined in optic pathway structures (retinas, optic nerves, optic tracts, lateral geniculate bodies, and superior colliculi). Incorporation of label into retinal protein and phospholipid was nearly maximal 1 h postinjection, after which the label appeared in successive optic pathway structures. Based on the time difference between the arrival of label in the optic tract and superior colliculus, it was calculated that protein and phospholipid were transported at a rate of about 400 mm/d, and DPG at about half this rate. Transported labeled phospholipid and DPG, which initially comprised 3-5% of the lipid label, continued to accumulate in the visual structures for 6-8 d postinjection. The distribution of transported material among the optic pathway structures as a function of time differed markedly for different labeled macromolecules. Rapidly transported proteins distributed preferentially to the nerve endings (superior colliculus and lateral geniculate). Total phospholipid quickly established a pattern of comparable labeling of axon (optic nerve and tract) and nerve endings. In contrast, the distribution of transported labeled DPG gradually shifted toward the nerve ending and stabilized by 2-4 d. A model is proposed in which apparent "transport" of mitochondria is actually the result of random bidirectional saltatory movements of individual mitochondria which equilibrate them among cell body, axon, and nerve ending pools.     

THE EXTENT OF AXOPLASMIC TRANSPORT DURING DEVELOPMENT, DETERMINED BY MIGRATION OF VARIOUS RADIOACTIVELY-LABELLED MATERIALS

Abstract— Several isotopic precursors have been monocularly injected into chick embryos and into day-old or 15-day-old chicks. After various intervals, the incorporation of various isotopes into acid insoluble material within the retina of the injected eye and within the optic lobes, was determined. Radioactive proline and fucose were used as precursors of protein and glycoprotein respectively while uridine was used as an RNA precursor. The proportion of rapidly migrating proteins and glycoproteins was reduced during maturation. The extent of RNA migrating also appeared to decline during development. The proportion of synthesized protein that was transported was relatively constant and independent of the amino acid used. Around 30 per cent of retinally synthesized glycoprotein migrated distally and this migrating material appeared to contain very few sialic acid residues. A considerable amount of retinally synthesized gangliosides also appeared rapidly in the distal regions of the optic nerve.