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PubMed1986

The use of whole-mount preparations of nerves labelled with axonally transported radioactive proteins to study regeneration.

A method for studying the process of regeneration in the frog sciatic nerve is presented. The outgrowth of sensory axons was determined by assaying the distribution of axonally transported radioactive proteins by fluorography of whole-mount nerve preparations. After labelling, the nerves were fixed at their in vivo lengths, impregnated with a fluorographic reagent and whole-mounted on a glass-slide before exposure to an X-ray film. In this way the rate of axon elongation after nerve crush lesions could be accurately determined. Nerve regeneration preceded by a conditioning lesion will be described. After the outgrowth has been measured the method enables subsequent analysis of, for instance, the protein composition in selected parts of the same nerve. The technique should be broadly applicable for studying nerve regeneration.

Animals↗
PubMed1980

The short term accumulation of axonally transported organelles in the region of localized lesions of single myelinated axons.

Myelinated axons were isolated from the sciatic nerve of Xenopus laevis and were subjected to localized (less than 30 microns wide) lesions. In axons which were bathed in a 0.12 M potassium glutamate solution there was very little local reaction to the lesion and optically-detectable particles undergoing axoplasmic transport accumulated immediately adjacent to, and mostly distal to, the lesion. Preparations fixed for electron microscopy at times up to 3 h following the lesion showed that the axoplasmic changes about the lesion were asymmetrical. Large organelles predominated on the distal side of the lesion; these were mostly dense lamellar bodies (DLB) with mean dimensions, as determined from thin sections, of 0.48 by 0.19 microns. Multivesicular bodies, mitochondria, and a variety of smaller membrane bounded bodies also appeared in the particle accumulation distal to the lesion. Analysis of these results led to the conclusion that DLB were transported up to the lesion and represent the majority of the optically detectable particles which are transported in the retrograde direction. Small vesicles and tubules were the commonest structures which accumulated proximal to the lesion. The time course of this accumulation was consistent with the hypothesis that these structures are particulate bodies which move in the orthograde direction at about 1.5 microns/s. Incidental findings which are also of significance to the study of axonal transport were: large particulate material may reverse its direction of movement at an axonal obstruction, and organelles which accumulate on either side of a lesion do so in rows which are associated with microtubules.

Animals↗
PubMed1976

Reversible inhibition of rapid axonal transport in vivo by lidocaine hydrochloride.

Rats were given standardized injections of 3H-L-proline into the trigeminal ganglion and 14C-lidocaine hydrochloride at the infraorbital foramen. The 3H-L-proline was always injected 2.5 h before removal of the nerve. Lidocaine, 1, 2, and 4%, produced a concentration-related inhibition of entry of 3H-labeled rapid axonal transport into the distal portions of the nerve. Addition of epinephrine, 1:200,000, doubled the intensity of the effect. The time delay of recovery was also concentration-related, and with 4% lidocaine recovery still seemed incomplete after 4.5. h. It is concluded that inhibition of rapid axonal transport is probably a usual byproduct of nerve block with local anesthetics such as lidocaine. The inhibition seems attributable in part to a disturbance of the energy metabolism of the nerve.

Animals↗
PubMed1978

Axonal transport of acetylcholine, choline acetyltransferase and cholinesterase in regenerating peripheral nerve.

1. The axonal transport of acetylcholine (ACh), choline acetyltransferase (ChAc) and cholinesterase (ChE) was estimated in the peroneal nerves of rabbits by measuring the accumulation of each against a nerve crush over a period of 20 hr. 2. Estimates were made of the amounts of these substances that were transported in nerves that had been regenerating for up to 111 days after being crushed or up to 13 days after being cut. 3. The initial response was the same whether the injury was a crush or a cut; the amount of ACh transported was increased, while ChAc and ChE transport was reduced. 4. The amounts of ACh, ChAc and ChE transported tended to return to normal levels when the nerves were allowed to reinnervate the denervated muscles. ChAc transport also showed an early recovery in the cut nerves. 5. The ACh content of the central nerve stump did not alter throughout regeneration but ChAc and ChE contents were reduced at the times when the transport of the enzymes was reduced. 6. These results are discussed in relation to the time course of nerve regeneration.

Acetylcholine↗
PubMed1980

Axonal transport of neuropeptides in the cervical vagus nerve of the rat.

Accumulations of the neuropeptides substance P (SP), somatostatin (ST), and vasoactive intestinal polypeptide (VIP) proximal to a crush in the cervical vagus nerve of the rat have been measured using sensitive radioimmunoassays. Each of the peptides was rapidly transport towards the peripheral terminals of vagal afferent fibres, with average rates of flow ranging from 0.8 to 2.7 mm h-1. In the rabbit vagus nerve, SP was transported with an average rate of 4 mm h-1, which is more than double the rate for this peptide in the rat. Double crush experiments in rabbit vagus nerves indicated that the rapidly transported proportion of the total content of SP in the nerve free was about 34%. From this, the rate of transport of SP in the rapidly transported pool in the rabbit vagus nerve can be calculated to be 12 mm h-1 (280 mm day-1). Since such double crush experiments were not possible in the rat, it is not clear whether the different average rates of transport of SP in the rat and the rabbit reflect real differences in the rate of rapid transport in the two species. In common with rapid axonal transport of other neurotransmitters, the transport of SP and ST in the rat vagus nerve was blocked by colchicine, a drug that disrupts microtubules.

Animals↗
PubMed1993

Altered spectrum of retrogradely transported axonal proteins in p-bromophenylacetylurea neuropathy.

The composition of retrogradely transported axonal proteins was examined by acrylamide gel electrophoresis and gel autoradiography in the experimental neuropathy induced in rats by p-bromophenylacetylurea (BPAU). Protein composition was normal during the early phase of retrograde transport but showed significant abnormalities during a later phase. The early phase consisted of proteins collected distal to a mid-thigh ligature of sciatic nerve between 15 and 24 hours after injection of [35S] methionine into lumbar ventral horn of the spinal cord. In terms of their relative labeling and electrophoretic mobility, these proteins were almost identical in experimental and control rats. Most of the labeled protein bands were also identical in the later phase, collected between 24 and 48 hours, but there were some consistent omissions and additions. Present in controls but missing in BPAU treated rats were three bands at 42, 41, and 25 KDa. In contrast, 4 bands (63, 56, 50, 26 KDa) were more prominent in the experimental rats than in controls. We suspect abnormal post-translational modification or proteolysis of rapidly transported proteins in the terminal or preterminal portion of the neurons exposed to BPAU. This abnormality, in addition to a previously reported premature processing of transported organelles, may underlie the development of peripheral neuropathy.

Animals↗
PubMed1986

Reduction of axonal transport in the rat optic system after direct application of methylmercury.

Fast axonal transport of proteins in the optic nerve and tract was quantified by scintillation counts of protein-bound radioactivity along the visual pathway after an intraocular injection of [3H]proline. In control rats the label traveled at a rate of about 60 mm/day, reaching the optic chiasm at 4 h and the lateral geniculate body at 8 h postinjection. When methylmercury was injected simultaneously with [3H]proline, the label traveled at a rate of about 30 mm/day. At 8 h postinjection, the labeled protein had reached the optic chiasm, but the more distal pathway was unlabeled. The same pattern was observed histologically by emulsion autoradiography of the pathway. Some label was detected in the lateral geniculate of methylmercury-treated animals at 8 h, but this may have resulted from local incorporation, as judged by a similar level of labeling in the contralateral visual pathway. Alternatively, it may be the case that a small fraction of the axons in the treated pathway continued to transport proteins in a normal fashion. The very heavy label observed throughout the pathway in controls was present only in the proximal half of the pathway in methylmercury-treated rats. Methylmercury significantly reduced incorporation of [3H]proline in the rat retina, but this reduction was not as great as the effect in the optic nerve. In contrast, cycloheximide, a potent protein synthesis inhibitor, reduced labeled protein in the optic nerve only to the same extent as it reduced incorporation. These results suggest that methylmercury's effect on transport is not dependent solely on its effects on protein synthesis, but represents a separate mechanism of neurotoxicity.

Animals↗
PubMed1979

The intra-axonal transport of acetylcholine and cholinergic enzymes in rat sciatic nerve during regeneration after various types of axonal trauma.

The proximo-distal intra-axonal transport of acetylcholine (ACh) and cholinergic enzymes (choline acetyltransferase, CAT, and ACh-esterase, AChE) in rat regenerating sciatic nerve was studied by accumulation technique. Four types of axonal trauma were performed: freezing with solid CO2, crushing, ligating the nerve with remaining tight silk ligature, and cutting the nerve. Normal and sham-operated rats were used as controls. One to twenty-nine days later, the nerves were crushed about 15 mm proximal to the trauma. The nerve segment proximal to this crush was dissected out 12 hr later and assayed for ACh-content and enzyme activities. The increase in this segment 12 hr after crushing was taken as an indication of proximo-distal transport in the regenerating nerves. ACh transport did not seem to vary during regeneration as compared to controls. In contrast, the transport of both CAT and AChE was initially markedly depressed. Towards the end of the observation period (29 days), a recovery of CAT-transport occurred in all groups. Recovery of AChE-transport was marked in the freeze and crush groups. In the cut group no recovery was seen and in the ligated group only a small recovery occurred. Thus, in the nerves where regeneration was facilitated by the presence of intact connective tissue sheaths (freezing and crushing) recovery of transport occurred earlier than in cut or ligated nerves.

Acetylcholine↗
PubMed1984

A study of the reciprocal connections between the septum and the entorhinal area using anterograde and retrograde axonal transport methods in the rat brain.

The reciprocal connections between the septum and the entorhinal area (EA) was studied in the rat brain using antero- and retrograde axonal transport methods. After injections of large volumes (2 X 100 nl) of horseradish peroxidase (HRP) conjugated to wheat-germ agglutinin (WGA) into the medial septum (MS) and the diagonal band of Broca (dbB), anterogradely transported HRP-WGA was found primarily in layers II and IV of the medial and lateral EA. Injections of HRP-WGA (50-100 nl) or fluorescent dyes (50-100 nl) into different parts of the retrohippocampal region resulted in labeling, by retrograde axonal transport, of cells in the MS and dbB, both ipsi- and contralateral to the injected hemisphere. The labeled cells were either small (long axis of soma: 10-15 micron), round, and oval, or medium (15-25 micron) to large (25-35 micron) of fusiform or multipolar shape. By using the method of retrograde fluorescent double labeling, the septal afferents to the EA were found to give off collaterals to other parts of the hippocampal region. A much smaller number of septal cells appeared to send bilateral projections to the EA of both hemispheres. Studies employing retrograde transport of HRP in combination with acetylcholinesterase (AChE) histochemistry on the same tissue section showed that, while a large number of cells projecting to the EA contain AChE, many projecting cells are devoid of AChE reaction products. These findings suggest that the septo-entorhinal projection consists of a cholinergic as well as a noncholinergic component. The entorhinal efferents to the septum were studied after injections of HRP-WGA into different parts of the retrohippocampal region. Labeled fibers could be traced through the fimbria to their terminal fields in the intermediate parts of the lateral septal nucleus and to the most lateral aspect of the vertical limb of the dbB. The cells giving rise to this projection were situated in layer IV of the medial and layers II through V of the lateral EA. Taken together, the present findings demonstrate a close anatomical relationship between the septum and the entorhinal area, in addition to the better known connections between the septum and the Ammon's horn.

Acetylcholinesterase↗
PubMed1986

Lectin affinity and PAGE analysis of soluble axonally transported glycoconjugates in the rat visual system.

Recent reports of the transsynaptic transfer of wheat germ agglutinin (WGA) in several systems are consistent with the hypothesis that WGA binds to endogenous glycoproteins(s) which undergo transsynaptic transfer. Such molecules might be involved in trophic interactions between neurons. Since these hypothetical glycoproteins must be soluble proteins (it is unlikely that integral membrane proteins are exchanged between neurons) this preliminary study of the soluble, axonally transported glycoconjugates of the visual system was undertaken. Soluble, tritium-labeled macromolecules which accumulated in the rat lateral geniculate nucleus (LGN) and visual cortex after intraocular injections of tritiated fucose were studied by lectin affinity chromatography and polyacrylamide gel electrophoresis (PAGE). After correction for hematogenously or cerebrospinal fluid transported label, 9.6% of the radioactivity in the LGN and 17.9% of the radioactivity in the visual cortex was found in the soluble fraction of a phosphate-buffered saline homogenate. In the LGN, 31.3% of the axonally transported soluble label was bound by concanavalin-A (Con-A) agarose. The corresponding figure in the visual cortex was 25.7%. Most (greater than 90%) of the label which did not bind Con-A was soluble in 10% trichloroacetic acid (TCA). Eighty-eight percent of the label which bound Con-A was precipitated by 10% TCA. On 7.5% polyacrylamide SDS reducing gels, the Con-A bound material from either the LGN or the visual cortex migrated as a single peak near the origin of the gel (apparent molecular weight greater than 300,000 Da).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Theoretical calculation methods for kinesin in fast axonal transport.

The method of making Monte Carlo calculations of the velocity of fast axonal transport is described and applied in a relatively simple case. These illustrative calculations are supplemented by a differential equation solution of the same problem, valid as an asymptotic limit. The latter treatment is closely related to the theory of muscle contraction.

Animals↗
PubMed1989

Slow axonal transport of structural polypeptides in rat, early changes in streptozocin diabetes, and effect of insulin treatment.

The synthesis and transport of slowly transported polypeptides in sciatic nerves of rats was investigated by [35S]methionine pulse labeling and gel electrophoresis in control, diabetic, and insulin-treated diabetic rats. To detect very early changes diabetes was induced by streptozocin only 5 days prior to the labeling of the dorsal root ganglion cells. Fourteen days were allowed for axonal transport. In this experimental system, the neurofilament triplet is transported at an apparent velocity of 1.1 +/- 0.1 mm/day (mean +/- SD). The actin-related complex, including actin and two polypeptides of 87 kilodaltons and 37 kilodaltons, was transported at a velocity of 2.6 +/- 0.2 mm/day. For alpha- and beta-tubulin we found an apparent transport velocity of 2.2 +/- 0.1 mm/day, placing it between actin and the neurofilament triplet. The diabetic rats had a selective 32% decrease in the amount of the heaviest neurofilament subunit: 0.47 +/- 0.19% of trichloroacetic acid-insoluble radioactivity versus 0.69 +/- 0.17% in controls; 2p less than 0.05. This decrease was associated with a proximal accumulation of the two lighter neurofilament subunits. Insulin treatment of a diabetic group failed to normalize the changes of axonal transport and additional changes suggesting a hypoglycemic injury was observed.

Actins↗
PubMed1998

Disruption of axonal transport from olfactory epithelium by 3-methylindole.

The effects of 150, 350, and 400 mg/kg intraperitoneal 3-methylindole (3-MI) on anterograde transport of horseradish peroxidase from the olfactory epithelium to the olfactory bulb were investigated. In 400 mg/kg 3-MI-treated rats sacrificed after 7 days only about 2% of all glomeruli had normal levels of the reaction product, and most glomeruli had no detectable reaction product. Lower doses of 3-MI produced correspondingly less disruption of axonal transport, with savings located primarily in the ventral to midlateral and the ventromedial region of the bulb. There was a gradual recovery of bulbar connections in 12-, 22-, and 92-day survival rats. In all cases, the increase in axonal transport was greatest in glomeruli on the lateral, ventral, and ventromedial areas of the bulb, and least evident or absent on the dorsal and dorsomedial areas.

Animals↗
PubMed1994

Changes in slow axonal transport of tubulin induced by local application of colchicine to rabbit vagus nerve.

The biochemical and morphological responses of the rabbit vagus nerve to local application of colchicine and to nerve crush were investigated. Fourteen days after the cervical vagus nerve had been crushed or subjected to local application of colchicine for 2 h, nodose ganglia of anaesthetized rabbits were either injected with [35S]methionine or [3H]leucine for studies of slow and fast axonal transport, respectively, or prepared for light microscopical examination. The radio-labelled proteins of the faster of the two slow transport groups (SCb; 25-30 mm day-1) were separated by one- or two-dimensional polyacrylamide gel electrophoresis and both radio-labelled tubulin and actin were quantified by densitometry from resulting fluorographs of gels. A relative increase in radio-labelled tubulin was found in SCb in the crushed and colchicine-treated nerves; this increase persisted for up to 50 days after nerve crush. Morphological changes in nerve cell bodies induced by colchicine were similar, but smaller in magnitude than those in crushed nerves. It is concluded that a temporary arrest of axonal transport produced by colchicine can lead to a redistribution of tubulin transport comparable with that found in regenerating nerve.

Animals↗
PubMedDate not supplied

Cytofluorometric quantification of somatopetal axonal transport: effects of a conditioning lesion and 2,5-hexanedione.

Cytofluorometric quantification of axonally transported fluorescein isothiocyanate (FITC)-labelled wheat germ agglutinin (WGA) from the injection site in the snout area was performed in the facial nucleus at various times during regeneration of one of the facial nerves. Measurements were made on single neurons on both operated and non-operated sides in three different groups of mice 8, 12 and 16 days after a nerve crush, Group 1: (control group) animals with a nerve crush, Group 2: animals with a conditioning lesion (nerve injury) made 3 days before the nerve crush, and Group 3: animals exposed daily to 2,5-hexanedione from 2 weeks before nerve crush until killing. A conditioning lesion caused a more rapid return of transport in regenerating nerves but there was no evidence for an increase in the total amount of transported FITC-WGA. For mice exposed to 2,5-hexanedione a transient increase of tracer transport in regenerating nerves could be demonstrated on day 12 after nerve crush. On day 16, however, a reduction of transport was seen in both operated and non-operated nerves. This study shows that it is possible experimentally to manipulate the influx of macromolecules to the nerve cell body from the periphery during nerve regeneration, and the present method offers the opportunity to study quantitatively the effects of various treatments on reinnervation of a muscle.

Animals↗
PubMed1994

Sciatic axotomy compromises axonal transport of transganglionic tracer BSI-B4 from the soma to the central terminals of C fibre afferents.

The C afferent specific lectin BSI-B4 was used to examine the effects of sciatic axotomy on axonal transport by the C afferent subpopulation. From about 4 days after sciatic nerve lesion, BSI-B4 injected into the peripheral nerve is transported only as far as the neuronal cell bodies in the dorsal root ganglion. The previous demonstrations of A beta afferent terminal sprouting into lamina II, and the atrophy of lamina II terminals, in response to sciatic lesions may be related to the inability of C afferents to maintain transganglionic transport.

Animals↗
PubMed1984

Postnatal changes in [3H]fucosyl glycoconjugates axonally transported into hamster optic nerve endings.

The subsynaptosomal distribution of [3H]fucosyl glycoproteins axonally transported into the optic nerve endings of neonatal and adult hamsters changed dramatically at eye-opening. In 12 day-old previsual hamsters, the highest concentration of incorporated fucose was in the axoplasmic reticulum/synaptic vesicle fraction (51%), with only 6% in the dense synaptic membrane fraction. By the end of the eye-opening period four days later proportional labeling of the dense synaptic membrane fraction had increased four-fold to 23% of total sub-synaptosomal radioactivity. Labeling of the synaptic membrane doubled again in adults (41%). Total synaptosomal radioactivity was greatest in 16 day-olds. These results imply that utilization o [3H]fucose by the retinal ganglion cells, as well as composition of the synaptic membrane, change in association with the onset of functional visual activity.

Aging↗