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Axonal transport and axonal processing of low molecular weight proteins from the abdominal ganglion of Aplysia.

Axonal transport of proteins in nerves of the abdominal ganglion of Aplysia was observed after a 2 h incubation of the ganglion in tritiated amino acids. The transported proteins migrate as a series of discrete peaks, all apparently moving at a rate of 3 mm/h. This process is sensitive to both colchicine and vinblastine, the former agent reducing the amount of transported material without affecting the transport rate. The molecular weight distribution of the transported proteins, as revealed by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS), is basically unchanged for up to 20 h after labeling. Low molecular weight species (less than or equal to 18,000 daltons) make up 10-20% of the transported protein and appear to be enriched in leucine. These proteins undergo proteolytic cleavage during transport, eventually reaching a molecular weight of 3000 daltons or lower. It is suggested that these data reflect the axonal transport and processing of neurosecretory peptides synthesized by identifiable neurons of the ganglion.

Animals

Taurine in the developing rabbit visual system: changes in concentration and axonal transport including a comparison with axonally transported proteins.

[35S]Taurine injected intravitreally into rabbits was transported axonally to the optic nerve terminals. Considerably more [35S]taurine was transported in young rabbits than in mature rabbits. The time course of taurine transport did not parallel that of proteins labeled with [3H]proline in the same system. The concentration of taurine in all components of the visual system, except retina, was greater in young animals than in mature animals, and was especially high in optic nerve. The possible functions of the high concentrations of taurine and the greater amount of axonally transported taurine in developing mammalian CNS are discussed.

Age Factors

Continuation of fast axonal transport in regenerating axons in vitro.

A previous study by McLean and co-workers reported that regenerating axons of the rabbit vagus nerve were unable to sustain axonal transport in vitro for several months after nerve injury. In contrast, we found that sensory axons of the rat sciatic nerve were able to transport 3H-labeled protein into their regenerating portions distal to the site of injury within a week after injury when placed in vitro. Transport in vitro was not significantly less than transport in axons maintained in vivo for the same period. Transport occurred in the medium that was used by the McLean group, but was significantly reduced in calcium-free medium. When axon regeneration was delared, only small amounts of activity were present in the nerve distal to the site of injury, showing that labeled protein normally present in that part of the nerve was associated with axons and was not a result of local precursor uptake by nonneural elements in the sciatic nerve. We were not able to explain the failure of McLean and co-workers to demonstrate transport in vitro in regenerating vagus nerve, but we conclude that there is no general peculiarity of growing axons that makes them unable to sustain transport in vitro.

Animals

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.

Acetylcholine

The slow component of axonal transport. Identification of major structural polypeptides of the axon and their generality among mammalian neurons.

This study of the slow component of axonal transport was aimed at two problems: the specific identification of polypeptides transported into the axon from the cell body, and the identification of structural polypeptides of the axoplasm. The axonal transport paradigm was used to obtain radioactively labeled axonal polypeptides in the rat ventral motor neuron and the cat spinal ganglion sensory neuron. Comparison of the slow component polypeptides from these two sources using sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis revealed that they are identical. In both cases five polypeptides account for more than 75% of the total radioactivity present in the slow component. Two of these polypeptides have been tentatively identified as tubulin, the microtubule protein, on the basis of their molecular weights. The three remaining polypeptides with molecular weights of 212,000, 160,000, and 68,000 daltons are constitutive, and as such appear to be associated with a single structure which has been tentatively identified as the 10-nm neurofilament. The 212,000-dalton polypeptide was found to comigrate in SDS gels with the heavy chain of chick muscle myosin. The demonstration on SDS gels that the slow component is composed of a small number of polypeptides which have identical molecular weights in neurons from different mammalian species suggests that these polypeptides comprise fundamental structures of vertebrate neurons.

Animals

Axonal transport of taurine along neonatal and young adult rat optic axons.

Studies in this laboratory have indicated that taurine is axonally transported along goldfish optic nerves. In the present experiments the axonal transport of taurine was examined in neonatal and young adult rat optic axons. [35S]taurine was injected into the vitreous humor of right eyes of developing (1--15-day-old) or young adult (40-day-old) rats. At various times after injection ranging from 3 h to 7 days, right retinae and left and right geniculates were removed and assayed for radioactivity, left minus right lateral geniculate (L-RLG) radioactivity being used as an index of axonally transported [35S]taurine. Results indicated that taurine was rapidly transported along both neonatal and young optic axons, in contrast to other amino acids (i.e., leucine and proline) which are not axonally transported in this system. Significant developmental variations were seen in both L-RLG and right retinal [35S]taurine activity 24 h after injection. The amounts of L-RLG [35S]taurine corrected for retinal ganglion cell uptake in animals injected at 1,4,7 and 11 days after birth (prior to and during the major period of synaptogenesis in the geniculates) were 4.5, 3.1, 2.3 and 2.6 times higher, respectively, than those in the young adults. In contrast, the amount of corrected L-RLG [35S]taurine in animals injected at 15 days after birth (after synaptogenesis) were not significantly different from that in the young adult.

Age Factors

Ascending projections of the locus coeruleus in the rat. I. Axonal transport in central noradrenaline neurons.

Axonal transport of protein and metabolites of L-[3H(G)]3, 4-dihydroxyphenylalanine ([3H]DOPA) was studied in the central noradrenaline neurons of the pontine nucleus locus coeruleus and was correlated with regional alterations of noradrenaline content following destruction of the nucleus. Unilateral lesions of the locus coeruleus produce a partial depletion of noradrenaline in the ipsilateral hypothalamus and telencephalon, indicating that these neurons project widely to the ipsilateral forebrain. Twenty-four to 48 h following local injections of 50 micronCi [3H]proline, locus coeruleus neurons take up labeled material and transport it, presumably as protein, to ipsilateral structures in the midbrain, diencephalon and telencephalon including the neocortex. Similarly 8 h after injection of 25 micronCi [3H]DOPA into the locus coeruleus, transport of material including catecholamines occurs to ipsilateral diencephalon and telencephalon. Axonal transport of proteins to telencephalic structures is greatly diminished by selective lesions of catecholamine terminals with 6-hydroxydopamine (6-OHDA) and following destruction of the medial forebrain bundle. These results provide further support for the view that noradrenaline neurons of the locus coeruleus nucleus project widely within the neuraxis to ipsilateral structures of the brain stem, diencephalon and telencephalon, including all cortical areas. In addition, evidence is presented for a contralateral projection with a similar distribution. The rate of axonal transport of labeled protein and metabolites of [3H]DOPA including [3H]catecholamines in central noradrenaline neurons is estimated to be 3-4 mm/h and is accordingly similar to that reported for noradrenaline neurons of the peripheral sympathetic nervous system.

Adrenergic Fibers

The pathogenesis of reactive axonal swellings: role of axonal transport.

The role of axonal transport in the pathogenesis of the axonal swellings which develop at the severed ends of transected axons was studied by electron microscopic (EM) autoradiography. Proteins carried by fast anterograde transport in rat sciatic nerves were labeled with [3H]-leucine or [3H]-fucose; [3H]-leucine, [3H]-fucose, and [125I]-tetanus toxin were used to label components of retrograde transport. After the labeling procedure, the nerves were ligated and 2 to 24 hours later the animals were perfused with fixatives. The axonal swellings in both the proximal and distal stumps contained densely packed membranous organelles. The transported radioactivity in the swellings was strictly associated with these organelles, particularly pleomorphic vesicles and branched tubules derived from smooth endoplasmic reticulum. The endogenous (tritiated) substances had a similar association with the organelle collections in both the proximal stump (fast anterograde transport) and in the distal stump (retrograde transport). The exogenous marker of retrograde transport (125I-tetanus toxin) had the same autoradiographic localization. These results suggest that fast anterograde and retrograde transport are very similar processes carrying predominantly membranous organelles and constituting a system of bidirectional fast transport. The accumulations of organelles in reactive swellings are interpreted as the consequence of the acute focal interruption of this system. Studies of axonal transport provide a means for investigation of the origin and fate of axonal organelles in pathologic processes.

Animals

Absence of 'superfast' axonal transport in rat sciatic nerve.

Axonal transport of labelled protein was studied in rat sciatic nerve by analyzing nerve segments at intervals after injection of L-[3H]leucine into the lumbar spinal cord. Some nerves were sectioned before injection so that material in transit accumulated proximal to the section. The segments distal to the section served as controls for incorporation into the nerve of blood-borne label. An analysis of TCA-soluble and TCA-insoluble activity in cut and intact nerve segments was also made. No evidence was found for the existence of a 'superfast' component of axonal transport (velocity 2000 mm/day). Results showed that the most rapidly transported protein derived from the neuron soma had a conventional 'fast' velocity of 350-420 mm/day. There was no transport of TCA-soluble material. It is suggested that 'superfast' transport, detected in mice by other investigators, is an artefact resulting from failure to control for incorporation of circulating label into the sciatic nerve.

Animals

Kinetic properties of normal and perturbed axonal transport of serotonin in a single identified axon.

1. The axonal transport of pulses of [3H]serotonin was studied in an axon of the serotonergic giant cerebral neurone (GCN) of Aplysia californica. 2. [3H]serotonin was transported as a discrete peak which was followed by a relatively low, smooth trail. 3. The peak broadened as it moved along the axon, sometimes skewing in the proximal direction. 4. The velocity of the transport was highly dependent on temperature, but the rate of peak broadening was not. The velocity was 130 mm per day at 23 degrees C and 48 mm per day at 14 degrees C. The rate of broadening was 143 micrometer per mm transport at 23 degrees C and 156 micrometer per mm transport at 14 degrees C. 5. In another series of experiments, almost the entire length of the lip nerve, which contained the axon of GCN, was maintained at 1--3 degrees C to block transport. The GCN's cell body and the proximal few millimetres of the nerve were maintained at 23 degrees C. As a result, the amount of [3H]serotonin in the proximal segment of the nerve increased manyfold during periods of up to 4 hr. The concentrated pulse of [3H]serotonin resulting from this treatment was transported more slowly than normal after the cooling was terminated. Sometimes, a minor peak split from the major peak of radioactivity and was transported a normal velocity. 6. Incubation of the cerebral ganglion and nerves for 16 hr in the presence of anisomycin, an inhibitor of protein synthesis, reduced by nearly fourfold the amount of [3H]serotonin subsequently exported into the axon of the GCN. The transport velocity at this reduced concentration was less than half the normal value. If the concentration of [3H]serotonin in the axon was restored to normal in the presence of anisomycin, the velocity of transport was also returned to normal. 7. We conclude that the velocity of transport of serotonergic vesicles in the axon of the GCN is positively dependent on the local concentration of vesicles, except at very high concentrations, where the dependence is negative. The results are interpreted in the context of a model for transport in which the serotonergic vesicle is translocated along the axon in an intermittent fashion, alternating between moving and stationary states. The local concentration of the vesicles along the axon would control the observed velocity of transport by altering the partitioning between the two states, that is, by changing the percentage of time vesicles spend in each state.

Animals

The relation of axonal transport of mitochondria with microtubules and other axoplasmic organelles.

Axonal transport of mitochondria was studied in frog sciatic nerves incubated in agents selected for their known or alleged effect on microtubules or axonal flow. Quantitative data on mitochondria, microtubules, neurofilaments, endoplasmic reticulum, and cross-sectional area of the axon indicate that axonal transport of mitochondria is dependent on microtubules. When more than half of the microtubules are destroyed, the axonal transport of mitochondria is diminished in proportion to the destruction of microtubules. Axonal transport of mitochondria is not related to neurofilaments and endoplasmic reticulum. Changes in the cross-sectional area of axons, even upon reduction to half the normal size, do not noticeably affect mitochondrial transport. Cyanide which blocks oxidative metabolism also blocks axonal transport of mitochondria, but analysis of fine structure indicates that cyanide is destructive to microtubules as well.

Animals

Interganglionic axonal transport of neural peptides within the nervous system of Aplysia.

Neurons of the circumesophageal ganglia of Aplysia synthesize 1--2000 dalton peptides and subject them to axonal transport in large quantities in the pleuro-visceral connective and pedal nerves. Most of the protein transported in the connective nerves accumulates in the abdominal ganglion, although some passes out its peripheral nerves. Autoradiography revealed no evidence for terminations of the transporting axons in possible neurohemal areas of this ganglion. It is suggested that these data reflect the existence of a pathway mediating the "directed delivery" of neural peptides in this nervous system.

Animals

Regeneration of motor axons in the rat sciatic nerve studied by labeling with axonally transported radioactive proteins.

Labeling regenerating axons with axonally transported radioactive proteins provides information about the location of the entire range of axons from the fastest growing ones to those which are trapped in the scar. We have used this technique to study the regeneration of motor axons in the rat sciatic nerve after a crush lesion. From 2 to 14 days after the crush the lumbar spinal cord was exposed by laminectomy and multiple injections of [3H]proline were made stereotactically in the ventral horn. Twenty-four hours later the nerves were removed and the distribution of radioactivity along the nerve was measured by liquid scintillation counting. There was a peak of radioactivity in the regenerating axons distal to the crush due to an accumulation of label in the tips of these axons. After a delay of 3.2 +/- 0.2 (S.E.) days, this peak advanced down the nerve at a rate of 3.0 +/- 0.1 (S.E.) mm/day. The leading edge of this peak, which marks the location of the endings of the most rapidly growing labeled fibers, moved down the nerve at a rate of 4.4 +/- 0.2 mm/day after a delay of 2.1 +/- 0.2 days; this is the same time course as that of the most rapidly regenerating sensory axons in the rat sciatic nerve, measured by the pinch test. Another peak of radioactivity at the crush site, presumed to represent the ends of unregenerated axons or misdirected sprouts, declined rapidly during the first week, and more slowly thereafter.

Animals

Mechanism of axonal transport: a proposed role for calcium ions.

In vitro axonal transport of tritiated protein decreased 40 to 60 percent when neuronal cell bodies were incubated in calcium-free medium, but was not affected when only nerve trunks were exposed to calcium-free conditions. In addition, calcium-45 was transported along axons at a rate similar to that of rapidly transported tritiated protein. These data are interpreted to suggest that calcium ions are involved in the initiation of axonal transport and in the coupling of transported proteins to the transport system.

Animals

Secretion of axonally transported neural peptides from the nervous system of Aplysia.

The possibility that proteins reaching the abdominal ganglion of Aplysia by axonal transport from the circumesophageal ganglia might be subject to secretion in that structure was examined. Transported labeled protein was found to be released from the abdominal ganglion; such release was enhanced by exposure to a high K+ medium and by electrical stimulation of the transporting axons. Stimulation of release was inhibited by lowering the Ca2+/Mg2+ ratio of the medium. The released material is predominantly of 1--2000 daltons in molecular weight and appears to have been derived from a group of transported peptides of about the same size. The possibility is raised that these data may reflect the existence of a peptidergic second-order neurosecretory pathway in this nervous system.

Animals

[Impairment of rapid axonal transport and concomitant anomaly of smooth endoplasmic reticulum in acrylamide induced neuropathy].

The axonal transport of proteins was studied by radioautography in preganglionic axons of ciliary ganglia in Leghorn chickens treated by acrylamide. The slow axonal transport of proteins was hardly affected. In contrast, the fast axonal transport was severely impaired. Indeed, radioactive proteins accumulated focally at the periphery of several preterminal axons in regions showing a local disorganization of the smooth endoplasmic reticulum which seemed to be one of the earliest changes induced by acrylamide.

Acrylamides

Dynamic properties of axonal transport of proteins and glycoproteins: a study based on the effects of metaphase blocking drugs in the developing optic pathway of chick embryos.

Some properties of the axonal transport of proteins and glycoproteins along the optic pathway of chick embryos and newly hatched chicks were studied by labelling retinal ganglion cells with 3H-proline or 3H-fucose. A study of the effects of colchicine (COL) and vinblastine (VLB) on embryonic axonal transport was also carried out. Marked changes in the efficiency of axonal transport were found throughout development. In particular, the fraction of retinal ganglion cell proteins which is rapidly exported toward tectal terminals increases during embryonic life but steadily decreases after hatching. Glycoprotein transport behaves similarly except that its efficiency is relatively higher at stages when critical events of synaptic maturation in the tectum are reported to occur. Embryonic axonal transport is blocked by COL and VLB at very low intravitreal concentrations. Retinal protein synthesis and the morphology of ganglion cells are profoundly altered by the drugs: in general, COL and VLB effects were much more marked in embryonic than in mature neurons. An analysis of the time course of rapid transport along embryonic optic axons was carried out by reducing the efflux of labelled proteins from the eye by giving VLB intravitreally 2 h after the pulse. It revealed some peculiar features in the retino-tectal migration of glycoproteins and confirmed their progressive accumulation within terminals as previously described by radioautography. These results suggest that axonal transport of proteins during embryonic life undergoes changes in parallel with synaptic maturation. It may thus be considered as one of the factors controlling the genesis of neuronal networks.

Animals