The effects of axotomy on electrophysiological properties of B cells of bullfrog sympathetic ganglia conditioned by a previous lesion

In bullfrog sympathetic B cells, axotomy decreases the amplitude and decay time of membrane afterhyperpolarization (AHP) and increases action potential (AP) duration. A second (test) axotomy, 7 days after an initial (conditioning) axotomy, did not amplify these changes. No recovery of AHP amplitude or AP duration occurred by 56 days post-axotomy, but AHP decay time recovered 21 days earlier than after test axotomy alone. Conditioning, previously shown to accelerate regeneration, speeds the return to normal of those membrane properties previously shown to recover after axotomy.     

The effect of age on motor neurone death following axotomy in the mouse.

The ability of mouse motor neurones to survive axotomy during the first month of life was studied. The motor neurones that lie in the dorsolateral columns of spinal segments C7 and C8 and supply the flexor muscles of the forepaw were axotomized by cutting and removing part of the median and ulnar nerves above the elbow. The number and position of cell bodies with axons in these nerves were confirmed by retrograde labelling of the cut axons with horseradish peroxidase. The ability of these neurones to survive axotomy varies with the age of the animal at the time of axotomy. When the axons are sectioned within the first four postnatal days, 80-90% of the cell bodies will die, more than half of this death occurring in less than one week after axotomy. If the animals are one week old at the time the nerves are cut, a significantly smaller number (50%) die (P = 0.013), and the time-course of death is different, with eight to ten days elapsing before half the death has occurred. 40% of the neurones will die if sectioned at two weeks of age, and it is not until four weeks of age that more than 90% of the cells can survive axotomy. We conclude, therefore, that the kinetics of motor neurone death, as well as the final extent of neuronal loss, are affected by the age at which the animal is axotomized.     

Differential time course of the response to axotomy induced by cut or crush in the leech AP cell

The time course of the reaction to axotomy in the leech AP cell was determined by measuring the duration of the spontaneous spikes at different times after the operation. The axotomy performed by section of the segmental roots containing the AP axon induced an increase of the spike duration, which persisted over 30 days. A different time course was found when the axotomy was performed by nerve crush: the changes in duration of the spontaneous spikes, which occurred during the early 2 weeks, were significantly reduced afterwards. Dye staining of some cells axotomized by crushing revealed that the reversion of the changes, which had been set up by axotomy, was in some cases concomitant with the reconnection between proximal and distal axon stumps. The section of a single axonal branch was never sufficient to affect the membrane properties of the AP cells. It is concluded that the changes observed in axotomized AP cells are not produced by simple axonal injury and that the maintainance of normal properties in the somatic membrane requires the presence of at least part of the distal axon arborization.     

Differential regulation of motor neuron survival and choline acetyltransferase expression following axotomy

Although it is well known that motor neuron survival following axotomy is enhanced with maturation, the ability of surviving neurons to express the cholinergic enzyme choline acetyltransferase (ChAT) following axotomy has not been closely examined. Moreover, the utility of the facial nucleus in studies of motoneuron response to injury and to trophic factors, coupled with the increasing importance of the mouse in gene targeting, compelled us to investigate the age dependence of neuronal survival and ChAT expression in the mouse facial nucleus following axotomy. We cut the facial nerve at postnatal day (P)4, 7, 14, 21, and 28 or in the adult and used Nissl staining and ChAT immunocytochemistry to quantitate survival and ChAT expression, respectively, following 1, 2, or 3 weeks' survival at each age. We confirm in this model that the rate and extent of motor neuron death following axotomy is reduced with increasing maturity. The surviving neurons maintain a high ChAT content through P21; however, axotomy from P28 through adulthood results in a striking reduction in ChAT immunoreactivity. That is, although axotomy at P21 results in 61% motor neuron survival, with virtually all of the surviving neurons being ChAT positive, axotomy in the adult results in 72% survival but only 9% of the neurons are ChAT positive. Thus, surviving motor neurons in the adult animals are only weakly cholinergic. These results indicate that a change in the regulation of ChAT expression occurs following P21 so that cell survival and enzyme levels are uncoupled. We suggest that the putative factor or factors that enhances motor neuron survival in maturity is not capable of maintaining ChAT expression. © 1995 John Wiley & Sons, Inc.     

The interaction between lipid derivatives of colchicine and tubulin: consequences of the interaction of the alkaloid with lipid membranes

Colchicine is a potent antimitotic poison which is well known to prevent microtubule assembly by binding tubulin very tightly. Colchicine also possesses anti-inflammatory properties which are not well understood yet. Here we show that colchicine tightly interacts with lipid layers. The physical and biological properties of three different lipid derivatives of colchicine are investigated parallel to those of membrane lipids in the presence of colchicine. Upon insertion in the fatty alkyl chains, colchicine rigidifies the lipid monolayers in a fluid phase and fluidifies rigid monolayers. Similarly X-ray diffraction data show that lecithin-water phases are destabilized by colchicine. In addition, an unexpectedly drastic enhancement of the photoisomerization rate of colchicine into lumicolchicine in the lipid environment is observed and further supports insertion of the alkaloid in membranes. Finally the interaction of colchicine with lipids makes the drug inaccessible to tubulin. The possible in vivo significance of these results is discussed.     

Effect of some drugs on colchicine uptake by colchicine-sensitive and resistant cells

The effect of several agents on 3H-colchicine, uptake by L cells and resistant to colcemide and colchicine L-53 cells was studied. Vinblastin to which L-53 cells are cross-resistant increases labeled colchicine uptake by L and L-53 cells 3- and 8-fold, respectively. The substances which decrease ATP level in the cells (olygomycin, etc.) enhance colchicine uptake by L and L-53 cells 2--4-fold. In the presence of these substances colchicine uptake by resistant cells is more intensive than by sensitive L cells. The structural analogue of colchicine, lumicolchicine, inactive in binding the microtubular protein tubulin enhances colchicine uptake by L and L-53 cells to about equal degree.     

Fast axonal transport in central nervous system and peripheral nervous system axons following axotomy

After axotomy, changes in the composition of fast axonally transported proteins ( FTP ) within the peripheral nervous system (PNS) axons have been reported. The most significant and reproducible changes involved polypeptides found within the molecular weight range of 31.0 to 14.5 kilodaltons ( Bisby , 1980). We wished to determine whether similar changes following axotomy occur in axons of the central nervous system (CNS). Intracranial axotomy of the left optic tract was performed stereotaxically in rats. Six days post axotomy 50 muCi 35[S]-methionine was injected into the vitreous body of both eyes. FTP were isolated within the optic nerves 2 h after isotope injection. The nerve segments were processed for SDS-PAGE, fluorography, and compared to similarly prepared fluorographs of normal and eight day post-axotomy sciatic nerve segments. The labelling of 5 major polypeptide bands (S1, MW congruent to 28,000; S2a , MW congruent to 25,000; S2b , MW congruent to 23,000; T1, MW congruent to 20,200; and T2, MW congruent to 17,000) was studied by laser densitometry. Band S2b showed a highly significant (p less than 0.001) increase in concentration, while bands S1 and T1 demonstrated highly significant decreases in concentration following axotomy of the sciatic nerve. In contrast, after axotomy of the retinal ganglion cell axons the only significant change was a decrease (p less than 0.05) in T1. We suggest that failure of CNS axons to respond similarly to PNS axons following axotomy may be related to the failure of CNS axons to regenerate.     

Using comparative anatomy in the axotomy model to identify distinct roles for microglia and astrocytes in synaptic stripping

The synaptic terminals' withdrawal from the somata and proximal dendrites of injured motoneuron by the processes of glial cells following facial nerve axotomy has been the subject of research for many years. This phenomenon is referred to as synaptic stripping, which is assumed to help survival and regeneration of neurons via reduction of synaptic inputs. Because there is no disruption of the blood-brain barrier or infiltration of macrophages, the axotomy paradigm has the advantage of being able to selectively investigate the roles of resident glial cells in the brain. Although there have been numerous studies of synaptic stripping, the detailed mechanisms are still under debate. Here we suggest that the species and strain differences that are often present in previous work might be related to the current controversies of axotomy studies. For instance, the survival ratios of axotomized neurons were generally found to be higher in rats than in mice. However, some studies have used the axotomy paradigm to follow the glial reactions and did not assess variations in neuronal viability. In the first part of this article, we summarize and discuss the current knowledge on species and strain differences in neuronal survival, glial augmentation and synaptic stripping. In the second part, we focus on our recent findings, which show the differential involvement of microglia and astrocytes in synaptic stripping and neuronal survival. This article suggests that the comparative study of the axotomy paradigm across various species and strains may provide many important and unexpected discoveries on the multifaceted roles of microglia and astrocytes in injury and repair.     

Retrograde axon reaction following section of asynaptic nerve fibers

Summary The asynaptic spinal neurons of the gymnotid teleost Sternarchus albifrons show several distinct characteristics of the retrograde reaction of the perikaryon (which corresponds to chromatolysis in mammals) following axotomy. Nuclei of affected cells are characteristically eccentric. Large bundles of neurofilaments, never seen in normal perikarya of these cells, become prominent following axotomy. There is a marked increase in the number and size of dense bodies in the affected perikarya. Large arrays of parallel rough endoplasmic reticulum, never seen in normal cells, are frequent in the axotomized neurons. These results demonstrate that disconnection from synaptic terminals is not a necessary condition for the retrograde reaction of the perikaryon following axotomy.     

Anion-induced increases in the affinity of colcemid binding to tubulin

Colcemid binds tubulin rapidly and reversibly in contrast to colchicine which binds tubulin relatively slowly and essentially irreversibly. At 37 degrees C the association rate constant for colcemid binding is 1.88 X 10(6) M-1 h-1, about 10 times higher than that for colchicine; this is reflected in the activation energies for binding which are 51.4 kJ/mol for colcemid and 84.8 kJ/mol for colchicine. Scatchard analysis indicates two binding sites on tubulin having different affinities for colcemid. The high-affinity site (Ka = 0.7 X 10(5) M-1 at 37 degrees C) is sensitive to temperature and binds both colchicine and colcemid and hence they are mutually competitive inhibitors. The low-affinity site (Kb = 1.2 X 10(4) M-1) is rather insensitive to temperature and binds only colcemid. Like colchicine, 0.6 mol of colcemid are bound/mol of tubulin dimer (at the high-affinity site) and the reaction is entropy driven (163 J K-1 mol-1). Similar to colchicine, colcemid binding to tubulin is stimulated by certain anions (viz. sulfate and tartrate) but by a different mechanism. Colcemid binding affinity at the lower-affinity site of tubulin is increased in the presence of ammonium sulfate. Interestingly, the lower-affinity site on tubulin for colcemid, even when converted to higher affinity in presence of ammonium sulfate, is not recognized by colchicine. We conclude that tubulin possesses two binding sites, one of which specifically recognized the groups present on the B-ring of colchicine molecule and is effected by the ammonium sulfate, whereas the higher-affinity site, which could accommodate both colchicine and colcemid, possibly recognized the A and C ring of colchicine.     

Characterization of a colchicine receptor protein in rat epididymal adipose tissue.

Colchincine was found to be taken up by adipose tissue and therein to bind to a soluble macromolecule not sedimented by centrifugation for 2 h at 100 000 x g. A similar binding occurred when soluble extracts of adipose tissue were incubated with colchicine. The binding reaction reaction is temperature dependent and shows a pH optimum between 6.8 and 7.0. Double reciprocal plots of colchicine concentration versus amounts of colchicine bound to protein in the steady state disclosed an apparent Km of 0.250 to 1.5 muM. The colchicine binding activity of soluble tissue extracts decreased when the extracts were incubated at 37 degree C. Addition of guanosine triphosphate and Mg-2+ retarded the loss of colchicine binding activity. The molecular weight of the colchicine complex was estimated to be 115 000 and its sedimentation coefficient 5.8 S. All of these characteristics are remarkably similar to those of the protein tubulin which has been isolated from other tissues. Since it is now well known that tubulin is a protein subunit of cytoplasmic microtubules, it is suggested that the previously reported metabolic effects of colchicine on adipose tissue result from the dissolution of microtubules by colchicine.     

Retinal ganglion cells lose trophic responsiveness after axotomy.

Whereas PNS neurons in culture are intrinsically responsive to peptide trophic factors, retinal ganglion cells (RGCs) are not unless they are depolarized, or their intracellular levels of cyclic AMP (cAMP) are elevated. We show here that depolarization increases cAMP in cultured RGCs sufficiently to enhance their responsiveness and that the trophic responsiveness of developing RGCs in intact retinas depends on physiological levels of activity and cAMP elevation. Responsiveness is lost after axotomy but is restored by cAMP elevation. The death of axotomized RGCs can be prevented if they are simultaneously stimulated by several trophic factors together with cAMP elevation. Thus, the death of RGCs after axotomy is not caused solely by the loss of retrograde trophic stimuli but also by a profound loss of trophic responsiveness.     

Androgen receptor mRNA regulation in adult male and female hamster facial motoneurons: effects of axotomy and exogenous androgens

Testosterone propionate (TP) administration at the time of facial nerve injury in the adult hamster augments the regenerative properties of the injured facial motoneurons (FMN), with the androgen receptor (AR) playing a key role in mediating the actions of TP on facial nerve regeneration. The purpose of the present study was to determine the effects of axotomy on AR mRNA expression in FMN. This was accomplished using in situ hybridization in conjunction with a (35)S-labeled AR riboprobe. Gonadally intact adult male and gonadectomized (gdx) adult female hamsters were subjected to a right facial nerve axotomy, with the left side serving as internal, unoperated control. Half the animals were subcutaneously implanted with a 10-mm TP Silastic capsule, and the other half were sham-implanted. An additional group of nonaxotomized, gonadally intact males was also included. Postaxotomy survival times were 1, 4, and 7 days. At 1 postoperative day 1, there were no effects of axotomy on AR mRNA levels. By postoperative days 4 and 7, axotomy caused a significant decrease in AR mRNA levels in FMN of gonadally intact males, relative to either the contralateral control FMN of the same animals or FMN from the group of gonadally intact males that were not subjected to facial nerve axotomy. There were no significant differences between AR mRNA levels in contralateral control FMN and FMN from the gonadally intact group of nonaxotomized males. TP administration at the time of axotomy had no effect on AR mRNA levels in either the axotomized or contrala(teral control FMN of gonadally intact males, relative to the nonaxotomized, gonadally intact male group. Corroborating our previous work, AR mRNA levels were reduced in the contralateral control FMN of gdx females, relative to the nonaxotomized, gonadally intact male group, with axotomy having no additional effects. The data are discussed in a mechanistic framework suggesting how TP acts to augment facial nerve regeneration.     

Ultrastructural changes in the shape of neurons and related structures in frog sympathetic ganglia after axotomy.

Changes in the shape of neuronal perikarya and other ganglionic structures were observed by electron microscopy in the frog sympathetic ganglia at different times after axotomy. Degenerating and hypertrophic profiles appeared to reflect a remodelling process affecting preganglionic fibres. The shape of neuronal perikarya was modified by the formation of infoldings occupied by preganglionic fibres and/or by that of short winding dendrites often bearing a synapse. The origin of these changes is discussed. In frog sympathetic ganglia, the period of recovery after axotomy was marked by specific reactions which affected neuronal shape and preganglionic fibres, and are not known to occur in the ganglia of mammals.     

The binding of isocolchicine to tubulin. Mechanisms of ligand association with tubulin

Isocolchicine is a structurally related isomer of colchicine altered in the methoxytropone C ring. In spite of virtual structural homology of colchicine and isocolchicine, isocolchicine is commonly believed to be inactive in binding to tubulin and inhibiting microtubule assembly. We have found that isocolchicine does indeed bind to the colchicine site on tubulin, as demonstrated by its ability to competitively inhibit [3H]colchicine binding to tubulin with a KI approximately 400 microM. Isocolchicine inhibits tubulin assembly into microtubules with an I50 of about 1 mM, but the affinity of isocolchicine for the colchicine receptor site, 5.5 +/- 0.9 x 10(3) M-1 at 23 degrees C, is much less (approximately 500-fold) than that of colchicine. Unlike colchicine, isocolchicine binds rapidly, and the absorption and fluorescence properties of the complex are only modestly altered compared to free ligand. It is proposed that the binding of isocolchicine to tubulin may be rationalized either in terms of conformational states of colchicinoids when liganded to tubulin or by the structural requirements for C-10 substituents for high affinity binding to the colchicine receptor.     

COLLAGEN SYNTHESIS AND CELL GROWTH IN CHICK EMBRYO FIBROBLASTS: INFLUENCE OF COLCHICINE,CYTOCHALASIN B AND CONCANAVALIN A

Culturing of chick embryo fibroblasts in the presence of colchicine or cytochalasin B with and without concanavalin A (Con A) demonstrated that colchicine induces greater neosynthesis of endocellular type I collagen, whereas cytochalasin B boosts secretion. The effects are modified by the addition of Con A, which increases α₂more than a1 chain production.³H-thymidine incorporation is unaffected by cytochalasin B, but stimulated by colchicine. Con A neutralizes the stimulatory action of colchicine. It would therefore seem that Con A exerts transmembrane control of effects induced by colchicine and cytochalasin B by binding to cell surface receptors and so triggering rearrangement of the cytoskeleton.     

Potentiation of lymphocyte activation by colchicine.

Proliferation of human lymphocytes induced by IO4- is potentiated by 30 min exposure to colchicine (10(-6)M), whereas the response to Con A is inhibited. Treatment with colchicine before or after IO4- modification has similar enhancing effects. Lumicolchicine does not alter proliferative responses. In addition to the proliferation of IO4--oxidized cells, irradiated IO4- modified lymphocytes induce proliferation when mixed with untreated lymphocytes. Enhancement occurs in both these conditions only when IO4--modified cells are treated with colchicine. Preliminary data indicate that proliferation in mixed lymphocyte cultures is also potentiated when either stimulating or responding cells are pretreated with colchicine. These findings suggest a selective stimulatory effect of colchicine on lymphocyte responses induced by cell-cell contact. Agents that modify microtubular assemblies might regulate the induction of immune responses that involve cellular interactions.     

Neurite Extension by Mouse Neuroblastoma: Evidence for Two Modes of Induction

The induction of neurite extension by mouse neuroblastoma cells in vitro can be reversed by adding colchicine or cytochalasin B (CB) to the culture medium. The relative sensitivity of the neurites to retraction is a function of the method used to induce extension. Cells incubated in the absence of serum or in the presence of serum and dibutyryl cyclic-AMP are sensitive to both colchicine and CB; cells incubated in serum-free medium containing either cycloheximide or dimethylsulphoxide are sensitive only to colchicine.     

Genetic study of the resistance of mouse somatic cells to colchicine (high resistance level)

The studies of the high level of colchicine resistance of mouse L cells have shown that two mutagens (EMS and NMM) do not induce cell variants resistant to 8 microgram/ml of colchicine in the population of mouse heteroploid L-53 cells (subline of L cells, the level of colchicine resistance 140) and that colchicine resistance of L-53 cells gradually diminishes when cells are propagated in non-selective conditions: after 1 month it diminishes 2-fold, after 3 month--9-fold. The extent of the decrease of the drug resistance was the same in 6 independent cultures obtained from the inoculum of 200 cells and in control cultures propagated by large quantities of cells. These data coincide with the results of the previous studies of lower level of colchicine resistance. In both studies the frequency of the occurrence of colchicine resistant variants in selective medium was about 2.10(-4). These data are consistent with the hypothesis that colchicine resistance of mouse L cells is not due to a gene mutation.