Deposition and transfer of axonally transported phospholipids in rat sciatic nerve

Radioactive glycerol, ethanolamine, or choline injected into the vicinity of the cell bodies of rat sciatic nerve sensory fibers is incorporated into phospholipid. Some newly synthesized ethanolamine and choline phosphoglycerides are subsequently committed to transport down the sciatic nerve axons at a rate of several hundred millimeters per day. Most labeled choline phosphoglycerides move uniformly down the axons; in contrast, the crest of moving ethanolamine phosphoglycerides is continually attenuated. These data, as well as differences in the clearance of these phospholipids distal to a nerve ligature, suggest that various classes of labeled phospholipids are differentially unloaded from the transport vector (possibly by exchange with unlabeled lipid in stationary axonal structures) during movement down the axons. The extent of unloading appears to be defined by the base moiety; both diacyl and plasmalogen species of ethanolamine phosphoglycerides exchange extensively with stationary axonal lipids, while most choline phosphoglycerides continue down the axons. Autoradiographic studies with 3H-choline and 3H-ethanolamine demonstrated that most unloaded phospholipid is initially deposited in axonal structures; some of this unloaded lipid is subsequently transferred to the axon/myelin interface (axolemma?) and then to myelin. Although transported ethanolamine phosphoglycerides exchange more extensively with lipids in stationary axonal structures than do choline phosphoglycerides, at early times more label from 3H-choline is found in myelin. A model to resolve this seeming discrepancy is proposed, wherein a differential topographic localization of phospholipid classes in the membrane of the transport vector allows for a preferential extensive exchange of transported ethanolamine phosphoglycerides with lipids in stationary axonal structures, while choline phosphoglycerides become available for rapid transfer to myelin by a process involving vesicle fusion with axolemma.     

Ultrastructural localization of slow retrograde axonal transport: an autoradiographic study

We used [3H]-N-succinimidylpropionate ([3H]-N-SP) to covalently label endogenous intra-axonal proteins within the nerve in order to study their bidirectional transport. At the time of injection virtually all of the labeled proteins are found at the injection site. At later times specific patterns of labeled proteins are found within the nerve both proximal to and distal from the injection site, as a result of retrograde and anterograde axonal transport, respectively. We undertook the current study to determine the ultrastructural distribution of the [3H]-N-SP-labeled transported proteins in the nerve. One microliter of [3H]-N-SP was injected subepineurially in sciatic nerve, and 5 days later the nerves were processed either for light and electron microscopic autoradiography or for gel electrophoresis and fluorography. At the injection site the labeled proteins are predominantly myelin proteins. Distally, a pattern similar to that described for slow anterograde transport is seen. Proximal to the injection site a constellation dominated by the 68-kilodalton protein is seen. Light microscopic autoradiography shows diffuse labeling both in axons and in myelin at the injection site, with predominant axonal labeling distant from the injection site. Electron microscopic autoradiography of segments distal to the injection site show silver grains which are distributed within the axoplasm without apparent relationship to organelles. In contrast, segments proximal to the injection site show silver grains which seem closely related to membrane-bound organelles, predominantly mitochondria. These results suggest that slow retrograde transport has a unique subcellular distribution that is distinct from that of slow anterograde transport.     

Axonal transport of enzymes and labeled proteins in experimental axonopathy induced by p-bromophenylacetylurea

Axonal transport was studied by several techniques in the sciatic nerves of adult male Sprague-Dawley rats with neuropathy induced by treatment with p-bromophenylacetylurea (BPAU) in dimethylsulfoxide solution. Control rats were treated with solvent alone. BPAU, 200 mg/kg, induced severe muscle weakness in the hindlimbs, beginning after a latent period of 1 week and progressing to near total paralysis by 2 weeks. Axonal transport of the endogenous transmitter enzymes, acetylcholinesterase, dopamine-beta-hydroxylase and choline acetyltransferase, was normal at both 2 and 15 days after administration of BPAU, as judged by the accumulation of enzyme activity above and below a set of double ligatures on the sciatic nerve. The velocity of fast anterograde transport of [35S] methionine labeled protein was also unaffected by BPAU. However, 4 abnormalities of transport were detected in BPAU- treated rats: (1) doubling of the time for initiation of fast anterograde transport after precursor injection in the dorsal root ganglion, (2) 25% fall in the velocity of slow axonal transport of [3H] leucine labeled protein, (3) 30% reduction in the proximal accumulation of fast transported labeled protein in ligated nerve, 8-30 h after injection of precursor, and (4) 50-60% reduction in distal accumulation of "early arriving" labeled protein, 8-14 h after precursor injection. The last abnormality, suggesting an impaired turnaround from anterograde to retrograde transport, was detected as soon as 2 days after BPAU administration. The turnaround abnormality was correlated with the severity of neuropathy as estimated by independent clinical scoring in the group of rats treated with 200 mg/kg of drug. However, further studies showed that turnaround was delayed even in rats treated with doses as low as 50 mg/kg, which never led to clinically evident neuropathy. Nevertheless it is proposed that the abnormalities of transport play a role, as yet undefined, in the distal axonopathy caused by BPAU.     

Anterograde to retrograde reversal of fast axonal transport within cold blocked and rewarmed intact axons

The possibility that anterograde to retrograde reversal of axonal transport might take place in mid axon at a site distant from any nerve termination was investigated in sciatic nerve preparations from Xenopus laevis. The nerve, containing a pulse of anterogradely transported protein labeled with [³⁵S]methionine, was kept in a two-compartment temperature controlled chamber. One compartment containing the proximal nerve was maintained at room temperature throughout the duration of an experiment while the second compartment containing the distal nerve, and separated from the first by a thermal barrier, was initially cooled to 3–4°C and later warmed to room temperature. Transport of labeled proteins in the nerve was detected with a position-sensitive detector of ionizing radiation. With the distal portion of the nerve cold, the pulse of labeled protein transported up to the thermal barrier and stopped. When the distal part of the nerve was warmed to room temperature, retrograde and anterograde pulses of label propagated away from the thermal barrier with no time delay. The retrograde pulse could be collected on the distal side of a proximally placed tie and could be eliminated by treatment of the proximal nerve with vinblastine or dinitrophenol. Functional and structural evidence indicated that the cold block and thermal barrier were not destructive to the axons. Electron microscopy showed that the numerical density of axonal microtubules distal to the cold block was decreased about seven fold during the cold treatment and that this decrease could be prevented by 10 μmol/1 taxol. Taxol also prevented anterograde to retrograde reversal at the thermal barrier when the distal nerve was warmed, but did not prevent continued anterograde transport. We conclude that anterograde to retrograde transport reversal can occur in mid axon and that altered microtubule structure can result in transport reversal.     

Fast axonal transport of labeled protein in sensory axons during regeneration.

Fast axonal transport of labeled protein was studied in sensory axons of the rat sciatic nerve at various intervals after crushing the sciatic nerve, and at various times after injection of precursor [³H]leucine into the L5 dorsal root ganglia. The velocity of transport was normal in both intact and regenerated portions of the injured nerve. In response to axotomy there was a short-term decrease in the proportion of ganglion-synthesized labeled protein that was transported, but this returned to normal values as regeneration proceeded. Transported protein accumulated proximal to the site of injury, and regenerated axons distal to the injury became heavily labeled with transported protein. The pattern of labeling of the injured nerve gradually returned to normal during a period of 30 days. Molecular weights were assigned to 23 polypeptides which comprise the major fast-transported protein of normal axons. There were few changes in the relative amounts of the transported polypeptides in regenerating axons. It was concluded that regeneration of sensory axons can be sustained without major changes in those parameters of fast axonal transport which were examined.     

Observations on labeling of neuronal cell bodies, axons, and terminals after injection of horseradish peroxidase into rat brain.

We observed with light microscopy the evolution of neuronal labeling phenomena in rats with two hours to eight days survival following injection of horseradish peroxidase (HRP) into the neostriatum. We compared the results of neostriatal injection with those injection of the overlying neocortex. The following conclusions were reached. The zone of diffuse background HRP around the injection site began to shrink after 18 hours. The extent of the early zone of diffusion was marked from 18 hours to two days by a universal granular labeling of neurons. This granular cell labeling appears to be the result of earlier diffuse cell labeling rather than retrograde axonal transport. Retrograde labeling of cells takes place from terminals and from damaged axons. Anterograde labeling of what appear to be terminals and fine preterminal axons takes place only from cells in the injection area, and not from either intact or damaged axons of passage. Usually the axons engaged in such retrograde or anterograde transport are not visibly labeled. Labeling of cells and terminals is generally optimal at 12 to 24 hours, but corticostriatal cells in layer V of the neocortex are seen best at 24 to 36 hours. The area from which retrograde labeling of cells takes place is larger than that from which anterograde labeling of terminals occurs. Massive intra-axonal movement of HRP away from the injection site occurs within a few hours in axons of passage as well as those originating there. This axonal labeling fades in most axons by 24 hours, but certain axons remain darkly labeled. The latter are probably damaged, since labeled axons which appear to be degenerating are later (3–5 days) seen in the same place, and Fink-Heimer staining of adjacent sections also shows degenerating axons in these regions. The early massive labeling of axons can lead to short-lived labeling of complete axonal arborizations within the range of this intra-axonal movement of HRP. But it seems to have no relation to the longer-lived labeling of distant cells and terminals, which presumably results from active axonal transport. This suggests that HRP entering axons of passage is not captured by the axonal transport mechanisms.     

Bidirectional axonal transport of glycoproteins in goldfish optic nerve

The goldfish visual system was used to study the relationships between anterograde and retrograde transport of axonal glycoproteins. After intraocular injection of radioactive glucosamine, determinations were made of the normal time course of appearance of labeled glycoproteins in the optic nerve and tectum and of their time course of accumulation on both sides of an optic nerve crush. The labeled glycoproteins, transported at a maximum velocity of about 80 mm/day, continued to pass through the optic nerve in significant amounts for as long as 24 h after the injection, with a maximum at about 6 to 14 h. Retrograde transport of labeled materials back from the optic tectum to the same point in the nerve began about 5 h later, indicating a minimum possible retrograde velocity of about 36 mm/day and a maximum possible lag time in the axon terminals (with the assumption of equal retrograde and anterograde velocities) of 1 to 2 h. When delivery of glycoproteins from the retina to the optic tectum was interrupted by a nerve crush 8 h after injection, a component with rapid turnover in the tectum was revealed having a half-life of not more than 6 h. At least 40% of this turnover could be attributed to retrograde transport. The amount of labeled glycoprotein transported in a retrograde direction 8 to 10 h after injection was greatly elevated in regenerating axons 2 weeks after the optic tract was cut.     

Toxic neurofilamentous axonopathies and fast anterograde axonal transport. I. The effects of single doses of acrylamide on the rate and capacity of transport

Acrylamide (ACR) produces a neuropathy in the central and peripheral nervous systems characterized by neurofilament-containing swellings in the distal nerve and eventual dying-back degeneration of axons. The effects of a single exposure to ACR on the rate and quantity of protein transported in the rat sciatic nerve has been measured to determine whether fast axonal transport is compromised by this toxicant. Using the segmental analysis of radioactive label of proteins following 3H-leucine injections into the DRG, ACR (50-100 mg/kg) significantly reduced the rate of fast anterograde transport by 9.3 to 20.8% but, more importantly, reduced the quantity of transported protein by 42.4 to 51.3%. The non-neurotoxic analogue methylene bis-acrylamide did not significantly change either parameter. The reductions in transport were not due to general effects of the toxicant upon protein synthesis. Therefore, fast anterograde transport was significantly affected by a single exposure to ACR in the same magnitude as retrograde transport. Discovery of these dramatic changes was due to differences from previous studies in the time frame of study of transport in relation to toxicant injection and to measurements of the quantity of protein transported rather than only the rate. These changes may be significant in terms of the pathogenesis of distal nerve degeneration.     

4S RNA is transported axonally in normal and regenerating axons of the sciatic nerves of rats

Experiments were designed to determine if following injection of [³H]uridine into the lumbar spinal cord of the rat, [³H]RNA could be demonstrated within axons of the sciatic nerve, and if 4S RNA is the predominant RNA species present in these axons.

In one experiment the left sciatic nerve of a rat was crushed. Two days later 170 μCi of [³H]uridine was injected into the vicinity of the lumbar ventral horn cells. Ten days after injection, rats were sacrificed and sciatic nerves were prepared for autoradiography. Photomicrographs were taken of labeled areas of intact and regenerating nerves and grains were counted over Schwann cells, myelin, axons and other unspecified areas. In both intact and regenerating sciatic nerves more than 20% of the silver grains were associated with motor axons and approximately 40% were found over cytoplasm of Schwann cells surrounding these axons. These data indicate an intra-axonal localization of RNA in sciatic nerve axons, as well as an active transfer of RNA precursors from axons to their surrounding Schwann cells.

In separate studies, the left sciatic nerve was crushed and 10 days later [³H]-uridine was bilaterally injected intraspinally into 6 rats. Four control rats were sacrificed at 14 or 20 days after injection. In the remaining 2 rats the sciatic nerve was cut 14 days after injection and the distal part of the nerve was allowed to degenerate for 6 days before sacrificing the rat. Thus, the distal portion of the nerve contained Schwann cells labeled by axonal transport but lacked intact axons. RNA was isolated from experimental and control nerve segments by hot phenol extraction and ethanol precipitation. RNA species (28S, 18S and 4S) were separated by polyacrylamide gel electrophoresis and radioactivity was measured in a liquid scintillation counter. Control groups had RNA profiles similar to those already described²⁰, with greater than 30% of the radioactivity present as 4S RNA. The proximal portions of nerve taken from the group in which nerves were cut, had a similar amount of radioactivity present as 4S RNA. However, in the distal segments of these nerves (in which the axons had degenerated thus creating an ‘axon-less’ nerve) the amount of radioactivity in the 4S peak decreased to approximately 15% of the total RNA, suggesting that 4S RNA is the predominant if not the only RNA present in these axons. These results strongly indicate that both intact and regenerating sciatic nerves of rats selectively transport 4S RNA along their motor axons.     

Vacor neuropathy: ultrastructural and axonal transport studies

Distal axonal degeneration has been correlated with abnormalities of fast axonal transport in several toxic neuropathies. We have investigated axonal transport in experimental PNU (N-3-pyridylmethyl-N'-p-nitrophenylurea; Vacor) neuropathy, because of the rapid and synchronous degeneration of many terminal axons after a single dose of PNU. Almost all axon terminals at neuromuscular junctions in hindfoot muscles degenerated by 24 hours after the administration of PNU. Fewer affected axons were found in intramuscular nerve twigs, and fewer still in the posterior tibial nerves. No abnormal myelinated axons were found in the sciatic nerve in the thigh. Fast axonal transport in the sciatic nerve remained normal to the mid-thigh, but a reduced amount of labeled transported material reached the posterior tibial nerve at the ankle (27% reduction). Autoradiography showed that nearly no transported material reached the intramuscular nerves and neuromuscular junctions of the hindfeet. These results suggest that toxic impairment of fast anterograde axonal transport may contribute to the axonal degeneration produced by PNU.     

Calcitonin gene-related peptide (CGRP) in the regenerating rat sciatic nerve

Calcitonin-gene related peptide (CGRP) is a neuromodulatory peptide present in motoneurons and a subpopulation of sensory neurons of the adult peripheral nervous system. Here we have investigated the changes in axonal transport of CGRP and CGRP receptor expression in the injured and regenerating rat sciatic nerve using CGRP-immunocytochemistry, radioimmunoassay and quantitative in situ receptor autoradiography techniques. Axotomy led to a gradual and prolonged, 2.5- to 3.5-fold increase in specific CGRP binding to the distal part of the crushed sciatic nerve, beginning 4-6 days after axotomy. An even stronger, up to 30-fold increase was observed after 30-42 day denervation in the distal part of the transected sciatic nerve, where neurite reinnervation was prevented by retroversion and ligation of the proximal nerve stump. Reconnection of the proximal and distal nerve stumps 21 days after transection did not lead to a major reduction in specific CGRP binding but prevented a further increase that occurred between 21 and 42 days after transection without reconnection. In contrast, the anterograde axonal transport of CGRP decreased after axotomy to 40-50% of the control values 6-8 days after nerve crush but recovered towards normal levels during successful regeneration. Interestingly, the retrograde axonal transport of CGRP appeared to amount to only 10-20% of the anterograde transport, suggesting that the peptide may be released by the regenerating neurites into the endoneurium of the injured peripheral nerve. In view of the persistent upregulation in endoneural CGRP binding after axotomy these data indicate that axonal CGRP could play a regulatory role in mediating axonal-endoneural cell interaction during peripheral nerve regeneration.     

Peripheral nerve autografts to the rat spinal cord: Studies with axonal tracing methods

In young adult female rats, autologous sciatic nerve segments were transplanted to the thoracic region of the spinal cord. The grafts became well innervated but led to no obvious functional improvement. The origin and termination of axons in the grafts was studied by retrograde neuronal labeling with horseradish peroxidase (HRP) and radioautographic axonal tracing. Studies with HRP indicated that some axons in the grafts originated from intrinsic CNS neurons with their cell bodies in nearby segments of the spinal cord and that others arose from dorsal root ganglia at the level of the grafts and at least 7 segments distal to them. After tritiated amino acids were injected into lumbar dorsal root ganglia, labeled axons could be followed into the grafts but not into the rostral spinal cord stumps. Together with other experimental observations, these results demonstrate a correlation between success or failure of elongation of dorsal root fibers and peripheral or central ensheathment at the axonal tip. The corticospinal tract was studied both with radioautography and retrograde axonal transport of HRP but no extension of its axons into peripheral nerve grafts was detected under these experimental conditions. The findings implicate both neuroglial and axonal factors in the feeble regenerative response usually seen after injury to the spinal cord.     

Axonal transport of phospholipid in goldfish optic system.

After injection of [³H] glycerol into the eye of goldfish, labeled lipid was conveyed by axonal transport in the optic axons to the optic tectum. The transported material was found to consist almost entirely of phosphoglycerides, including both zwitterionic and acidic species. The time course of appearance of the labeled phospholipid in the optic tectum suggested that it might be axonally transported at a rate intermediate between the rates of the fast and slow components in the axonal transport of protein. This possibility, however, was contradicted by the following evidence: a) the rate of transport of the phospholipid that first appeared in the tectum was the same as that of the fast protein component; b) during the first few days after the precursor injection the phase difference between the rates of bulid-up of labeled phospholipid in the optic nerve and tectum such as would be expected with an intermediate rate of transport was not seen; c) the accumulation of phospholipid in the tectum was rapidly terminated after the optic axons were separated from their cell bodies, as would be expected from a fast rather than intermediate transport rate in the isolated axons. The results suggest that the axonal transport of most of the phospholipid had the same rate as the fast component of protein transport, and that the prolonged period of accumulation of the phospholipid in the tectum reflected a prolonged period of release from the cell body into the axon. Only a small proportion of the phospholipid might have been transported at a slower rate. Inhibition of protein synthesis in the retina reduced the amount of transported phospholipid appearing in the tectum almost as much as the transported protein. It is probable, therefore, that the transport of phospholipid occurs in association with protein, possibly in the form of an assembled membrane structure. Whereas the protein that is transported is newly synthesized, the transported lipid need not be.     

Axonal transport and localization of B-50/GAP-43-like immunoreactivity in regenerating sciatic and facial nerves of the rat

Neurons that can regenerate their axons following axotomy increase their synthesis and axonal transport of a growth-associated protein, called GAP-43, which has been shown to be identical to the synaptic phosphoprotein B-50. The function of B-50/GAP-43 to the process of regeneration is unknown. We used a polyclonal, affinity-purified antibody against B-50 to study the axonal transport and localization of B-50/GAP-43-like immunoreactivity (B50LI) in the regenerating sciatic and facial nerves of adult rats. Quantitative data were obtained by densitometry of the B-50 band in immunoblots of nerve segments, which had been run on SDS-polyacrylamide gels. In the regenerating sciatic nerve, anterograde accumulation at a collection ligature was 3.0 times higher than retrograde accumulation. The mobile fraction of B50LI was only 0.28 of total B50LI and traveled with a mean anterograde velocity of 5.3 mm/hr. B50LI distribution in the newly regenerated portion of the nerve revealed maximal B50LI levels midway between the position of the crush and the fastest-growing axons. Immunocytochemistry of this portion of the nerve demonstrated B50LI to be associated with regenerating axons but also to a large extent with extra-axonal structures outlining the Schwann cell bands of Büngner. This zone of B50LI-positive Schwann cell bands was found to extend more distally in nerves in which regeneration had processed longer, e.g., up to 5 mm distal to the crush after 3 d and 8 mm after 4 d. Further distal to this zone, many fine regenerating axonal profiles could be detected with B-50 antibody, but were neurofilament negative. These findings raise the possibility of an extra-axonal function of B-50/GAP-43, as this protein might be secreted from regenerating axons and might play a role in axon-Schwann cell interactions during axonal maturation.     

Axonal transport in the motor neurons of rats with neuropathy induced by p-bromophenylacetylurea

Axonal transport was studied in sciatic motor neurons of rats with neuropathy induced by p-bromophenylacetylurea (BPAU) in dimethylsulfoxide solution. Control rats were treated with the vehicle alone. To label rapidly transported proteins, the rats received an injection of 35S-methionine into the ventral horn of the spinal cord at the L1 vertebral level. Radiolabeled protein was collected at ligatures applied on the sciatic nerve at intervals thereafter. In animals with severe motor weakness owing to treatment with BPAU, 400 mg/kg, there was evidence of increased delivery of labeled protein into the axon during the early period after isotope injection, but reduced delivery later. A dose-dependent decrease in the amount of labeled protein recirculated by retrograde axonal transport was also noted. A significant reduction in the amount of protein transported retrogradely was also detected during the latent subclinical phase of the neuropathy. The velocity of rapid anterograde transport, examined in unligated sciatic nerves, was unaffected by BPAU treatment. However, the lag time between precursor injection and the onset of transport was shorter in BPAU-treated rats than in controls. This effect was not explainable on the basis of fluctuations in core body temperature. The results are consistent with the view that disturbances of rapid anterograde and retrograde transport play a role in the peripheral neurotoxicity of BPAU. Attention is directed to the possibility that the transport disturbances and the subsequent neuropathy are related to alterations in the processing of rapidly transported membrane-limited organelles in the nerve cell bodies.     

Retrograde axonal transport in rat sciatic nerve after nerve crush injury

We investigated the quantitative alterations in retrograde transport of proteins following a nerve crush injury using the 3H N-succinimidyl propionate (3H NSP) method in rat sciatic nerve. After subepineurial injection of 3H NSP into the nerve the amount of radioactively labeled proteins accumulating in the cell bodies of the motor and sensory neurons was determined 1 day or 7 days later in nerves which had been crushed distal to the injection site 1, 3, 5, 7, or 33 days prior to 3H NSP labeling. One day accumulation in the DRG and spinal cord was not altered by nerve crush. Seven day accumulation in the DRG was initially slightly increased, then fell to 73% of control by 7 days, remaining reduced 33 days after crush. Seven day accumulation in the spinal cord was reduced to 25% of control 1 day after crush and remained at that low level except for 5 days post-crush when a normal amount of labeled protein was transported to the spinal cord. The time course of these changes suggests that quantitative alterations in retrograde transport may be involved in the long-term trophic interactions between the cell body and periphery, but are too slow to account for the earliest perikaryal responses to injury. In addition, the difference between the alterations of retrograde transport in motor and sensory neurons may reflect fundamental differences in the composition of retrograde transport in those different systems.     

Movement of labeled macromolecules to the goldfish optic tectum following intraocular injection of 125I-labelled tetanus toxin.

This study demonstrates that radioiodinated tetanus toxin, whose retrograde axonal transport has been established, is taken up by the retinal ganglion cell and transported in the anterograde direction. The time course of arrival of labeled macromolecules in the optic tectum following intraocular injection of ¹²⁵I-labeled toxin is contrasted with the axonal transport of [³H]proline-labeled protein and [³H]fucose-labeled glycoproteins in this system. The rapid axonal transport of tritium-labeled proteins is complete within 24 hr, whereas the movement of ¹²⁵I-labeled macromolecules into the optic tectum contralateral to the injected eye is detected after 12 hr and continues for several days. The small amount of toxin-related radioactivity in the optic nerve 5 days after injection indicates that, similar to glycoprotein transport, little, if any, toxin moves at the rate of slowly transported proline-labeled protein. The appearance of ¹²⁵I-labeled macromolecules in the optic tectum is blocked by exposure of the toxin to antitetanus antibody prior to injection into the eye. The anterograde axonal transport of radioiodinated bovine serum albumin is not detected in this system. The data suggest that following intraocular injection, labeled tetanus toxin is either transported at a variety of rapid and intermediate rates following a brief uptake interval or moves along the optic nerve at a minimum rate of 14 mm/day after its slow and continuous uptake into the retinal ganglion cell.     

Neurofilament redistribution in transected nerves: evidence for bidirectional transport of neurofilaments

Nerve fibers of the C57BL/6/Ola mouse exhibit very slow Wallerian degeneration following axotomy, thus allowing prolonged observation of mammalian axons separated from their cell bodies. The present study utilized teased-fiber preparations, silver histochemistry, immunocytochemistry, and electron microscopy to examine the distribution of axonal components in the distal stumps of axotomized sciatic nerves in C57BL/6/Ola mice. In examining nerve segments at varying intervals after nerve transection, we found no evidence of proximal-to-distal "emptying out" of the cytoskeleton, as would be predicted if the cytoskeleton in these transected nerves were undergoing anterograde transport as an assembled structure. Instead, we observed a gradual redistribution of cytoskeletal constituents over time, dominated by the progressive accumulation of neurofilaments at the severed ends of axons. In particular, there were massive accumulations at the proximal ends of the distal stumps. These results strongly suggest that, at least in transected nerve fibers, neurofilaments can be transported bidirectionally.     

Retrograde and anterograde axonal transport demonstrated by intracerebral injection of a labeled protein-acylating agent

Following injection of the tritium-labeled protein-acylating agent, N-succinimidyl propionate, into the neostriatum in rats, retrograde labeling of nigrostriatal cells in the pars compacta of the substantia nigra and anterograde labeling of the striatonigral terminal field in the nigral pars reticulata were demonstrated by autoradiography, at 18 hr survival. Labeling of the corticopontine terminal field and of corticothalamic cell bodies in cortical layer 6 were also seen, deriving from corticofugal fibers of passage through the striatal injection site. In comparison, neostriatal injection of horseradish peroxidase was found to yield nigral cell and terminal field labeling, but the corticopontine terminal field was not labeled, and very few cortical cells in layer 6 were labeled. These results show that the retrograde and anterograde axonal transport of endogenous proteins can be profitably studied by this in vivo protein-acylating technique, and suggest that the method may prove useful as a connection-tracing technique, especially because of the novel attribute of labeling terminal fields by anterograde axonal transport following injections into white matter axonal bundles, with concomitant labeling of the cell bodies of origin of these axons.     

Retrograde Axonal Transport of the α-Subunit of the GTP-binding Protein Gz in Mouse Sciatic Nerve: a Potential Pathway for Signal Transduction In Neurons

We have utilized antibodies against the α subunit of G_z in fluorescence immunohistochemistry to determine whether this GTP-binding protein can translocate along nerves by intra-axonal transport. After ligation of the mouse sciatic nerve we found an increase in G_z-like immunoreactivity on the proximal and distal side with time, suggesting that the α subunit undergoes orthograde axonal transport and also returns to the cell body by retrograde axonal transport in the sciatic nerve. Unlike the retrograde transport of G_iα, shown in a previous study to be present in most sciatic axons, G_zα only accumulated in a subpopulation of axons, suggesting that different G-proteins could convey information specific to neuronal subtypes. These results support our proposal that G_z may play a second messenger role in communicating information from the terminals back to cell bodies. G_iαand G_zα may be representative of relatively stable signalling molecules by which the signal from some neurotrophic molecules can be translocated from the neuronal periphery to the cell body without the need for the retrograde transport of the neurotrophic factor itself.