Retardation in the slow axonal transport of cytoskeletal elements during maturation and aging

Using the pulse-labeling method, the rate of the slow component (SC) of axonal transport was analyzed during maturation and aging. Ventral motor neurons and retinal ganglion cells of 3-, 6-, and 24-month-old Fischer 344 rats were radiolabeled with 35S-methionine. To measure the rates of SCa and SCb subcomponents, distributions of the total radiolabeled proteins and certain cytoskeletal proteins (actin, clathrin, tubulin, and the neurofilament proteins) were analyzed in the ventral root-sciatic nerve and optic nerve. Our results show that the rate of transport for both SCa and SCb proteins decreases with age in ventral motor axons and optic axons. For example, in ventral motor axons the rates of both SCa and SCb decreased 40% between 6 and 24 months. These results, with those of others, show that the rate of slow transport gradually decreases in the neurons of adult rats (7,11) The factors that may contribute to the slowing are discussed.     

Quantification of the mononuclear phagocyte response to Wallerian degeneration of the optic nerve

Summary We investigated the numbers, origin and phenotype of mononuclear phagocytes (macrophages/microglia) responding to Wallerian degeneration of the mouse optic nerve in order to compare it with the response to Wallerian degeneration in the PNS, already described. We found macrophage/microglial numbers elevated nearly four fold in the distal segments of crushed optic nerves and their projection areas in the contralateral superior colliculus 1 week after unilateral optic nerve crush. This relative increase in mononuclear phagocyte numbers compared well with the four-to five-fold increases reported in the distal segments of transected saphenous or sciatic nerves. Moreover, maximum numbers are reached at 3, 5 and 7 days in the saphenous, sciatic and optic nerves respectively, suggesting that the very slow clearance of axonal debris and myelin in CNS undergoing Wallerian degeneration is not simply due to a slow or small mononuclear phagocyte response. The apparent delay in the response in the CNS occurs because the mononuclear phagocytes respond to the Wallerian degeneration of axons, which is slightly slower in the CNS than the PNS, rather than to events associated with the crush itself, such as the abolition of normal electrical activity in the distal segment. This was demonstrated by the protracted time course of the mononuclear phagocyte response in the distal segment following optic nerve crush in mice carrying theWld ^smutation which dramatically slows the rate at which the axons undergo Wallerian degeneration. By³H-Thymidine labelling or by blocking microglial proliferation by X-irradiation of the head prior to optic nerve crush, we showed that the majority of macrophages/microglia initiating the response to Wallerian degeneration were of local, CNS origin but these cells rapidly (from 3 days post crush) upregulate endocytic and phagocytic functional markers although they do not resemble rounded myelin-phagocytosing macrophages observed in degenerating peripheral nerves. We speculate that the poor clearance of myelin in CNS fibre tracts undergoing Wallerian degeneration compared to the PNS, in the face of a mononuclear phagocyte response which is similar in relative magnitude and time course, is because Schwann cells in degenerating peripheral nerves promptly modify their myelin sheaths such that they can be recognized and phagocytosed by macrophages, whilst in the CNS oligodendrocytes do not.     

Inhibition of fast axonal transport and microtubule polymerization in vitro by colchicine and colchiceine

The effects of colchicine and colchiceine on fast axonal transport in frog sciatic nerves were studied in vitro. Colchiceine inhibited the transport to about the same extent as colchicine. Preincubation at low temperature potentiated the inhibitory effect of either drug. The polymerization of purified brain tubulin was inhibited by colchiceine at 5–10 times higher concentrations than colchicine. The similarity of the effects obtained with colchicine and colchiceine indicates that both drugs arrest axonal transport by interfering with microtubule function. Colchicine and colchiceine did not affect the levels of high energy phosphates (ATP and CrP) in frog nerves indicating that a reduced energy supply was not responsible for the arrested transport.     

Rapid axoplasmic transport of labelled proteins in the vagus and hypoglossal nerves of the rabbit

Summary Axoplasmic transport of labelled proteins was studied in the vagus and hypoglossal nerves of the adolescent rabbit after an intramedullary injection of ³H-leucine.As demonstrated by radioautography, the incorporated activity found in the intramedullary axons had a perikaryal origin and was transported distally at a rate of several mm per hour.The migration of labelled proteins along the axons outside the medulla was followed by measuring the specific radioactivity of the proteins in the hypoglossal and vagus nerves. A rapid transport of labelled proteins in proximo-distal direction was demonstrated at rates of 16–17 mm/h in the vagus nerve and 10–15 mm/h in the hypoglossal nerve. The effect of puromycin and nerve ligature on the rapidly moving component is described.     

Axoplasmic transport of acetylcholinesterase and choline acetyltransferase in the vagus and hypoglossal nerve of the rabbit

Summary Acetylcholinesterase (AChE) and choline acetyltransferase (ChAc) were studied in the vagus and hypoglossal nerve of the rabbit. Under normal conditions the activity of AChE was about twice as high in the vagus as in the hypoglossal nerve. The activity of ChAc, however, was about six times as high in the hypoglossal nerve as in the vagus.When the axons had been crushed by a ligature, the enzymes accumulated proximal to the ligature, presumably a reflection of a proximo-distal transport from the nerve cell soma. The transport velocity, as calculated from the rate of accumulation was highest in the vagus nerve both for AChE and ChAc. Approximately the same rates were observed for both enzymes, i.e., 15 mm/24 h in the vagus nerve and 5 mm/24 h in the hypoglossal nerve. These rates were of the same order as those previously observed for the slow axoplasmic transport of radioactively labelled proteins in unligated nerve.     

Inhibition of fast axonal transport and microtubule polymerization in vitro by colchicine and colchiceine.

The effects of colchicine and colchiceine on fast axonal transport in frog sciatic nerves were studied in vitro. Colchiceine inhibited the transport to about the same extent as colchicine. Preincubation at low temperature potentiated the inhibitory effect of either drug. The polymerization of purified brain tubulin was inhibited by colchiceine at 5-10 times higher concentrations than colchicine. The similarity of the effects obtained with colchicine and colchiceine indicates that both drugs arrest axonal transport by interfering with microtubule function. Colchicine and colchiceine did not affect the levels of high energy phosphates (ATP and CrP) in frog nerves indicating that a reduced energy supply was not responsible for the arrested transport.     

Effects of Colchicine and Vinblastine on Axonal Transport of Choline Acetyltransferase in Rat Sciatic Nerve1

The effects of colchicine (0.5–10^-2 M) and vinblastine (10^-2-10^-5 M) upon axonal transport of choline acetyltransferase (CAT) and on nerve impulse conduction have been investigated in the rat sciatic nerve. High concentrations of colchicine (0.5 M) and vinblastine (10^-2 M) blocked completely both axonal transport of CAT and impulse conduction. 10^-3 M vinblastine did not affect impulse conduction until 20–22 h after injection, but this concentration of vinblastine did block CAT transport; 5 × 10^-5 and 10^-4 M vinblastine blocked CAT transport but not impulse conduction. 10^-2 M and 10^-1 M colchicine were without effect on impulse conduction, but did produce substantial, although incomplete, block of CAT transport. The results are discussed in relation to the possible involvement of microtubules in transport of CAT.     

Peripheral axon crush elevates transport of p75NTR in the central projection of sensory neurones of rats

The effect of peripheral axon crush on the axonal transport of the neurotrophin receptors, p75(NTR) and trkA, was studied in dorsal roots of adult rats. Lumbar dorsal roots were crushed for 3-6 h to cause accumulation of p75(NTR) and trkA. Immunohistochemistry showed the presence of the NGF receptors in axons, indicating retrograde and anterograde axonal transport in the dorsal root. Western blots confirmed that the time course of accumulation of p75(NTR) was consistent with fast axonal transport. However, trkA accumulation was too low to indicate significant levels of axonal transport. Sciatic nerve crush induced a 2-fold increase (P<0.05) in the bidirectional axonal transport of p75(NTR) in the dorsal root while trkA transport remained below detectable levels.     

Re-expression of p75NTR by adult motor neurons after axotomy is triggered by retrograde transport of a positive signal from axons regrowing through damaged or denervated peripheral nerve tissue.

To investigate different types of potential signalling mechanisms that regulate neuronal reactions to axotomizing injury, we compared the re-expression of the low-affinity neurotrophin receptor, p75NTR, and the down-regulation of choline acetyltransferase expression, after various combinations of axotomy, crush injury and blockade of axonal transport in adult hypoglossal motor neurons in the rat. We found that pure axotomy in the absence of crush injury down-regulated choline acetyltransferase, but did not induce p75NTR re-expression. Blockade of axonal transport with colchicine had an identical effect. In contrast, both a crush injury on its own, or a crush injury proximal to a complete axotomy, induced p75NTR re-expression and down-regulated expression of choline acetyltransferase. Blockade of axonal transport with colchicine or tight ligation proximal to a crush prevented the crush injury-induced re-expression of p75NTR. Infusion of vehicle, nerve growth factor or ciliary neurotrophic factor induced low levels of p75NTR re-expression that were not significantly different from each other and were substantially lower than crush-induced levels. These findings confirm previous suggestions that the loss of choline acetyltransferase expression is due to the interruption of a constitutive retrograde signal, and show that the re-expression of p75NTR by adult motor neurons after axotomy is triggered by the retrograde transport of a positive signal derived from axons that are regrowing through damaged or denervated peripheral nerve tissue. The precise source and nature of this signal are not yet clear.     

The Effect of Local Application of Vinblastine or Colchicine on Acetylcholine Accumulation in Rat Sciatic Nerve1

The accumulation of acetylcholine (ACh) following nerve crush was inhibited by high concentrations of colchicine (10^-1, 10^-2 M) and vinblastine (10^-2-10^-4 M) in a dose-dependent manner. The two mitotic inhibitors were effective both when applied to axons (subepineural injections) and to perikarya (injections into the lumbar intumescence). The results indicate that mitotic inhibitors, when applied locally to cholinergic motor neurons, may block the proximo-distal transport of ACh. The possibility is discussed that this blockade is caused by an interference with a microtubule mechanism in the axons. Evidence for an increased local synthesis of ACh near the site of injection into nerves was also obtained with the highest concentrations of either substance used.     

Inhibition of axoplasmic transport in the developing visual system of the rat—I. Structural changes in the retina and optic nerve with graded doses of intraocular colchicine

In order to study the role of axonal transport in the mediation of transneuronal metabolic stimulation upon a population of differentiating neurons, colchicine, a potent inhibitor of rapid and slow phases of axonal transport, was injected into the eye of albino rats at 1,3,5, 10, 15 and 20 days postnatal in concentrations ranging from 10^?5 M to 2 × 10²M and in quantities of 0.3 to 0.5μl. Quantitative light and electron microscopy were subsequently employed to assess reactive alterations in the developing retina and optic nerve. Application of colchicine severely retarded the development of the sensory elements, with disappearance of synaptic ribbons of sensory cell axons, a significant reduction in the thickness of the inner plexiform layer, due to the presence of numerous shrunken synaptic elements and the appearance of rosettes of sensory cells displaced to the inner nuclear layer. These alterations were found to be dose-dependent. Counts of ganglion cell populations at various times after application of colchicine demonstrated optimal concentrations which could be injected at each postnatal age without causing ganglion cell degeneration. Ultrastructural examination of such cells revealed varying degrees of disorganization and dissolution of the endoplasmic reticulum with the formation of occasional small cytoplasmic vacuoles. Higher concentrations of colchicine caused extensive vacuole formation in all classes of retinal neurons, scattered hyperchromic cells and widespread degeneration and autolysis.The diameter of the optic nerve was reduced to 60–95% of normal following intraocular colchicine. depending on the concentration employed, but electron microscopy revealed normal patterns of distribution of axoplasmic microtubules and filaments in control and experimental animals and quantitative analysis revealed no significant loss of axons. While no reactive changes took place in individual elements, the periphery of the nerve was often indented by a highly-folded glia limitans.Maximal doses of intraocular colchicine for each age level were established by this study. These were: 1 day, 10^?3 M: 5 days, 5 × 10^?3M; 10 days, 5 × 10^?3M; 15 and 20 days, 10^?2 M. The information derived from this morphological analysis provides the foundation for subsequent measurements of axonal transport inhibition in the developing visual system to be reported in the second article of this series.     

Distribution and intraneuronal trafficking of a novel member of the chromogranin family, NESP55, in the rat peripheral nervous system

NESP55 (neuroendocrine secretory protein of M_r 55 000) is a novel member of the chromogranin family. In the present study, we have investigated the distribution, axonal transport and proteolytic processing of NESP55 in the peripheral nervous system. The amount of NESP55 immunoreactivity in adrenal gland was more than 240 times higher than that in the vas deferens. Double or triple immunostaining demonstrated that NESP55 immunoreactivity was highly co-localized with tyrosine hydroxylase immunoreactivity in bundles of thin axons and postganglionic sympathetic neurons; that NESP55 immunoreactivity also co-existed with vesicular acetylcholine transporter immunoreactivity in large-sized axons in sciatic nerves, and that NESP55 immunoreactivity overlapped with calcitonin gene-related peptide immunoreactivity in some large-sized axons, but NESP55 immunoreactivity was not detected in sensory neurons. Strong NESP55 immunoreactivity was found in cell bodies and axons, but it was not detectable in any terminal region by immunohistochemistry. In crush-operated sciatic nerves, NESP55 immunoreactivity could be found as early as 1 h after operation, and accumulated amounts increased substantially with time. However, NESP55 immunoreactivity was only observed in axons proximal to the crush, but none or very little distal to the crush, which was consistent with the data from radioimmunoassay. Finally, extracts of the normal and crushed sciatic nerve and vas deferens were subjected to high-performance liquid chromatography followed by radioimmunoassay. The results indicate that NESP55 is processed slowly to small peptides (GAIPIRRH) during axonal transport. NESP55 immunoreactivity was only detected in axons proximal to the crush.

The data in the present study indicate that NESP55 immunoreactivity is widely distributed in adrenergic, cholinergic, and peptidergic neurons, but not in sensory neurons, and that this peptide is anterogradely, but not retrogradely, transported with fast axonal transport and slowly processed to smaller peptides during axonal transport in the peripheral nervous system.     

Quantification of N-CAM and N-cadherin expression in axotomized and crushed rat sciatic nerve

Adhesion molecules are important in supporting axonal regeneration. Qualitative studies have described increased expression of neural cell adhesion molecule (NCAM) and N-cadherin in models of nerve injury allowing active regeneration. In this study we have used quantitative immunohistochemistry to compare expression of NCAM and N-cadherin after nerve injury either with active regeneration (crush) into the distal stump or without (axotomy and capping). Quantification was performed 15 days after axotomy in proximal and distal stumps. Quantification after crush either proximal, distal or within the crushed area was performed at 2, 7, 15 and 30 days after injury. Axotomy induced increases in expression in proximal stumps between two and three times those in uninjured nerves for both molecules. In distal stumps, N-cadherin levels increased seven-fold, yet NCAM levels did not exceed control values. After crush, NCAM immunoreactivity increased in the crushed area and distal stump in contrast to axotomy and NCAM-positive axons co-localized with PGP9.5. N-cadherin levels in the distal stump increased above control levels, but the magnitude of the increase seen after crush was different to those seen after axotomy. In conclusion, increased expression of adhesion molecules, particularly NCAM, in the distal stump of injured nerves is dependent upon the presence of regenerating axons.     

Influence of peripheral inflammation on growth-associated phosphoprotein (GAP-43) expression in dorsal root ganglia and on nerve recovery after crush injury

An experimental study was performed to investigate the influence of the inflammation in peripheral target tissue on growth-associated phosphoprotein (GAP-43) expression in dorsal root ganglia (DRG) and on recovery of crushed nerve. Fifty-four male Wistar rats were used for this study. The sciatic nerve was operatively crushed unilaterally with an aneurysm clip. Inflammation in peripheral target tissue was induced by injection of complete Freund's adjuvant (CFA) at 1 week before crush. In crushed or crushed with arthritis rats DRGs were examined in immunohistochemistry for GAP-43 and the sciatic nerves were observed in Epon embedded sections with toluidine blue stain. In addition, electrophysiological studies of the nerves were performed to evaluate the recovery of function. Immunohistochemical studies showed the ratio of GAP-43 immunopositive cells in crushed with arthritis rats was significantly lower than that in crushed rats at 1 week after crush (P<0.01). Electrophysiological studies at 4 weeks after crush showed functional nerve recovery in crushed with arthritis rats was significantly suppressed compared with that in crushed rats (P<0.01). Histological studies showed the mean diameter of the axons in crushed with arthritis rats was significantly smaller than that in crushed rats (P<0.01). All these findings indicate that inflammation in peripheral target tissue suppresses GAP-43 expression in DRG and eventually suppresses functional and morphological recovery of the crushed nerve.     

Further evidence for the involvement of microtubules in the proximo-distal intra-axonal transport of acetylcholine and related enzymes in rat sciatic nerve

The two mitotic inhibitors colchicine (COL) and podofyllotoxin (POD) and their respective isomers, IumiCOL and picropodofyllin (picPOD) were tested for their effect on the intraaxonal transport (AXT) of acetylcholine (ACh) and the cholinergic enzymes in rat sciatic nerve. The mitotic inhibitors and their isomers were dissolved in saline + 10%, ethanol (COL and lumiCOL) or dimethylformamid (DMFA) (POD and picPOD) and injected (3–5 γl) subepineurally into the sciatic nerve. As controls the vehicle (saline+ 10% ethanol or DMFA) alone were injected into some rats. 2 h later a crush operation was performed 15 mm distal to the site of injection. The accumulation of ACh or the two enzymes, ACh-esterase (AChE) and cholineacetyltransferase (CAT), in the nerve segment proximal to the crush (12 h before death) was used as a measure of the AXT. COL and POD were very effective in inhibiting AXT of all 3 substances, while their isomers, lumiCOL and picPOD, were essentially without effect on AXT in equimolar concentrations (0.1 M). The effects on AXT of the 4 test substances thus appear related to their affinity to bind to tubulin, which is several orders of magnitude higher for COL and POD than for their isomers. The results further support the view that intact microtubules are essential for AXT of both membrane-bound (AChE) and soluble (CAT) enzymes, as well as of ACh in rat motor nerves.     

Increased expression, axonal transport and release of pituitary adenylate cyclase-activating polypeptide in the cultured rat vagus nerve

The expression and axonal transport of pituitary adenylate cyclase-activating polypeptide (PACAP) was studied in the cultured vagus nerve of the rat by immunocytochemistry and in situ hybridization. The number of neurons immunoreactive for PACAP increased markedly within the nodose ganglion during a 24-48 h culture period, as did the number of cells containing messenger RNA for PACAP. PACAP was found to be axonally transported and accumulated at the site of a crush injury. The peptide was also released at this site. Addition of PACAP to regenerating nerves in culture did not affect axonal outgrowth, neither did antibodies against PACAP. Separate experiments showed that neither PACAP-27 nor PACAP-38 affected proliferation of non-neuronal cells measured as the incorporation of [3H]thymidine. In contrast, forskolin, another potent stimulator of adenylate cyclase besides PACAP, dramatically decreased [3H]thymidine incorporation. The results showed that, during regeneration of peripheral nerves, PACAP expression increases and the peptide is transported into the regenerating nerve, where it is released. The functional significance of this release is unknown, but it does not seem to be directly related to the initiation of proliferation of Schwann cells or initial axonal outgrowth.     

A calmodulin inhibitor with high specificity,compound 48/80, inhibits axonal transport in frog nerves without disruption of axonal microtubules

The calmodulin inhibitor compound 48/80 has previously been shown to arrest axonal transport in vitro in the regenerating frog sciatic nerve. The inhibition was limited to the outgrowth region of nerves, which had been allowed to regenerate in vivo for 6 days after a crush lesion, before they were incubated with or without drugs in vitro overnight. The effects of compound 48/80 on the regenerating nerve were further investigated. A concentration of compound 48/80 (50 μg ml^-1), which effectively inhibits axonal transport, did not cause observable changes of the microtubules of regenerating axons in the outgrowth region as judged by electron microscopy. Furthermore, it was shown that also a lower concentration (25 μg ml^-1) inhibited axonal transport. As a measure of possible metabolic effects, the level of ATP was assessed in the regenerating nerve after exposure to compound 48/80. Compound 48/80 at 25 μg ml^-1 did not change the level of ATP in the nerve. The assembly of bovine brain microtubule proteins in a cell-free system was unaffected by 25 μg ml^-1 of compound 48/80 and slightly inhibited by 50 μg ml^-1. At higher concentrations (> 100 μg ml^-1) assembly of microtubules appeared stimulated, and microtubule spirals as well as closely aligned microtubules could be seen. These effects appeared to be unrelated to the transport effects. The present results indicate that compound 48/80 arrests axonal transport via mechanisms other than destruction of axonal microtubules or interference with the energy metabolism. It is possible that these mechanisms involve inhibition of calmodulin-regulated events essential to the transport.     

Uptake and retrograde axonal transport of horseradish peroxidase in regenerating facial motor neurons of the mouse

Summary Uptake and retrograde axonal transport of intravenously injected horseradish peroxidase (HRP) was studied during regeneration after a crush injury of the facial nerve of the mouse. The circulation time of HRP was 12 to 24 h. HRP injected immediately after the crush diffused into injured axons in the crushed region and accumulated subsequently in perikarya of facial neurons in the brain stem. After a time interval of 1 h or 5 days between the crush and the injection only a faint HRP accumulation occurred in a few facial neurons. After an interval of 7 days a moderate number of neurons had incorporated the tracer, while after more than 9 days the HRP activity in the regenerating neurons was more pronounced than in the contralateral neurons. Ultrastructurally, muscles of the vibrissae showed denervated subneural apparatuses 6 days after the crush. 8 days after the crush regenerating axon terminals containing small clusters of synaptic vesicles, dense cored vesicles and some HRP-labelled vesicles, were found over some gutters and after 10 to 13 days all examined gutters contained axon terminals with large numbers of synaptic vesicles and some HRP-containing vesicles. More than one axon terminal profile was seen in the same synaptic gutter. 32 and 64 days after the crush the neuromuscular junctions had regained a more mature appearance. The calibre spectra of the crushed facial nerves still showed a shift towards smaller diameters 134 days after the crush, at a time when a slight increase in HRP activity in the facial neurons persisted.     

The turnover of acetylcholine in ligated sciatic nerves of the rat

The accumulation of acetylcholine (ACh) and choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities proximal to a crush on rat sciatic nerves was investigated after superfusion of the nerves in situ with or without inhibitors of ACh synthesis and/or AChE. 9 h after crushing of nerves, the ACh-content of the 5 mm segment of nerve immediately proximal to the crush was increased from 37±5 to 80±4 pmol (mean ± SE), while ChAT-activity was increased to 112±10% and AChE-activity to 198±19% over that in non-ligated nerves. Superfusion of the nerves for 8 h with Krebs' bicarbonate medium had no effect on enzyme accumulations, but reduced the ACh content to 59±4 pmol. The presence of hemicholinium 3 (HC-3) (2×10^-5 M) in the superfusion medium reduced the ACh content markedly (to 17±2 pmol), but had no effect on enzyme accumulations at the crush. Adding eserine (10^-5 M) or soman (10^-6 M) to the superfusion medium increased ACh content to 133±8 pmol and 101±8 pmol, respectively, and markedly reduced AChE-activity; ChAT activity was not effected. Superfusion with a combination of HC-3 and eserine caused a marked reduction in ACh content compared with eserine alone; the effect was less with soman. The results are consistent with the view that the ACh which accumulates proximal to crush exists in a protective organelle, but that there is a continuous turnover of ACh due to leakage of ACh from the organelle, both during axonal transport and after accumulation.     

Effect of denervation and colchicine treatment of the nerve on electrical and chemical excitability of frog tonic muscle fibre

In order to investigate the role of axoplasmic transport in controlling the acetylcholine sensitivity and action potential generation in frog tonic muscle fibres, a comparison of changes produced in these fibres by denervation or by application of colchicine to the nerve was performed. Extrajunctional cholinergic sensitivity appeared both after colchicine treatment and after denervation. However, action potential generation appeared after denervation but not after colchicine treatment. Insensitivity of the factor controlling action potential generation to colchicine, which is known to interrupt fast axonal flow, and the low rate of its transport (determined by the time difference in action potential occurrence at various lengths of the nerve stump after denervation and coming to about 1.6 mm/day) suggest that the tropic factor responsible for the suppression of action potentials in tonic fibres is moved by slow axonal transport.It is concluded that the action potential mechanism in tonic fibres is controlled by the axonal flow of substances which differ from these controlling the acetylcholine receptors.