Microtubular Density Research Articles

Abstracts of the 8th Meeting of the Italian Peripheral Nerve Study Group: 68

Colchicine is an anti‐inflammatory drug commonly used for the treatment of gouty arthritis, Behçet's disease and amyloidosis. Reported side effects, such as blood dyscrasias and gastroenteritis, are probably ascribed to its antimitotic properties. Colchicine‐induced myopathy with vacuolar changes and frequently associated sensory‐motor axonal neuropathy have also been described. A 52‐yr‐old woman, previously treated for gouty arthritis, persisted in self‐administering colchicine intermittently for three years, in order to reduce body weight by inducing diarrhea. The patient had weakness and hypotrophy in upper and lower girdles and proximal muscles, “stocking and glove” hypoesthesia and loss of deep tendon reflexes in lower limbs. EMG examination showed a neurogenic/myogenic mixed pattern and nerve conduction studies were consistent with sensory‐motor axonal polyneuropathy. Muscle biopsy showed a vacuolar myopathy, with autophagic vacuoles and associated neurogenic changes. A moderate loss of myelinated fibers and some degenerating axons were observed in sural nerve biopsy. There were neither demyelinating changes nor autophagic vacuoles within axons. Muscle and nerve findings were thus suggestive of colchicine neuromyopathy. The drug was withdrawn and clinical‐electrophysiological data improved. Colchicine muscular toxicity is presumably due to inhibition of tubulin monomer polymerization. The microtubular network destabilization might cause cytoplasmic deposits of autophagic debris in skeletal muscle. We thus evaluated number, density and spatial array of microtubules in myelinated and unmyelinated axons by transmission electron microscopy. Quantitative ultrastructural analysis was carried out on high magnification photographs in our patient biopsy and in control nerves (normal and with axonal loss). Decrease of microtubular density and increase of tangentially oriented microtubules were observed both in myelinated and unmyelinated axons. No neural deposits of tubulin‐like material, as described in some cases of colchicine myopathy, were found. As in experimental vincristine neuropathy, cytoskeletal damage induced by colchicine seems related to effects of primary microtubular disarray on axonal transport and caliber, rather than to the extent of axonal loss.

Cardiac microtubules are more resistant to chemical depolymerisation in streptozotocin-induced diabetes in the rat.

Evidence exists for a specific diabetic cardiomyopathy independent of concurrent vascular disease. Our aim was to test the hypothesis that a change in the microtubular cytoskeleton may contribute to cardiac dysfunction in type-1 diabetes. Resting sarcomere length and characteristics of unloaded shortening were measured in ventricular myocytes from rats 2 months after injection of streptozotocin (STZ). Microtubular density and organisation were assessed using immunofluorescence confocal microscopy and the effects of microtubule disruption by colchicine on shortening and microtubules were examined. Diabetic myocytes showed a significant reduction in resting sarcomere length and a 30% increase in time to peak shortening. The microtubule disruptor colchicine (10 micromol/l) had no effect on the amplitude or kinetics of shortening in myocytes from control or diabetic rats. Cardiac microtubular density and organisation were similar in control and diabetic animals, yet although colchicine significantly reduced microtubule density in control myocytes, microtubules in diabetic myocytes were resistant to its effects. These observations of an increase in microtubular stability in STZ-diabetes of 2 months duration imply a disruption to the normal balance between populations of dynamic and drug-stable microtubules. Such disruption has been observed in other pathological conditions and may contribute to diabetic cardiomyopathy.

Assembly and disassembly of axonal microtubules of the toad Xenopus laevis under the effect of temperature.

In toads Xenopus laevis living at 11 degrees (winter), the microtubular density of 4-microns myelinated axons of lumbosacral nerves was assessed with the electron microscope. In controls, the density was 11.2 microtubules/microns2. In nerves incubated at 0 degrees, microtubules decreased following a simple exponential curve with a half time of 4.7 min (k = 0.149 min-1); residual microtubules were 4.5%. After rewarming, the full complement of microtubules reappeared within 60 min. In steady state, the microtubular density exhibited a linear relationship with temperature (range: 0-22 degrees; slope 0.94 microtubules/microns 2 per degree; r, 0.96). After heating the nerve by 11 degrees above the physiological temperature, microtubules increased by 83%, whereby the pool of unpolymerized tubulin was at least 2.7 mg/ml of axoplasm. A seasonal variation of the microtubular density was observed which accorded with the environmental temperature. The macroscopic kinetics of microtubule disassembly in the axoplasm is similar to that reported for purified tubulin but that of assembly is slower. Microtubules of peripheral axons of Xenopus are cold-labile and vary during the annual cycle.

The effects of nerve growth factor and dibutyryl cyclic AMP on cytoskeletal densities in cultured sensory ganglia

The effects of nerve growth factor (NGF) and dibutyryl cyclic AMP (DBC) on the density of cytoskeletal structures in cultured dorsal root ganglia were examined using morphometric techniques. After 24 hr in culture, NGF-treated neurites were longer than either DBC-treated or control neurites. At 48 hr, neurites produced in response to NGF and DBC were of equivalent length, while controls were considerably shorter. Comparison of electron micrographs of neuritic profiles revealed some differences of area and cytoskeletal density between treatment groups. Morphometric analysis was used to determine these differences under several growth conditions, at various rates of elongation and at different neurite lengths. As shown by analysis of variance, both NGF-treated and control neurites tapered in diameter at 48 hr in vitro, while DBC-induced neurites increased in area. An increase in cytoskeletal density for all treatment groups indicated that density was not always correlated with changes in area. An increased density of microtubules as compared to neurofilaments was seen at 24 hr, with equal densities of both cytoskeletal elements present after 48 hr in vitro. Comparisons between individual groups of data indicated that NGF-treated neurites relied primarily on microtubular density at 24 hr in vitro, when NGF induced longer, faster growing neurites. At 48 hr, there was an increase in neurofilaments proximal to the explant in the presence of DBC, implying that DBC may cause increased synthesis and/or transport of these structures. A comparison of microtubule to neurofilament ratios indicated that at 24 hr, there was always a greater density of microtubules. However, after 48 hr, neurofilament density increased such that there were equivalent densities of both cytoskeletal elements, possibly due to the overall increase in length observed in each treatment group. These data imply that 1) neurites with different rates of elongation may exhibit differences in cytoskeletal density; 2) neurites of equivalent lengths may be of differing stabilities; 3) NGF and DBC produce neurites with different cytoskeletal densities, implying divergent mechanisms of neurite induction; 4) the presence or absence of NGF may be partially responsible for variations in cytoskeletal densities observed between peripheral and central processes of DRG during development.

Cytoskeletal components and calibers in developing fish Mauthner axon (Salmo gairdneri Rich)

In developing axons of many vertebrates, microtubular density is inversely correlated with fiber caliber. It is suggested that microtubules are causally related to axonal caliber. For this reason, cytoskeletal analysis during development of the fish Mauthner axon, which displays a giant caliber, is of particular interest. The Mauthner axon originates from the Mauthner cell in the medulla and runs in the fasciculus longitudinalis medialis in the spinal cord. At embryonic, larval and postlarval stages in trout (Salmo gairdneri Rich.), the following parameters were measured on conventional electron micrographs of Mauthner axon cross sections; axonal caliber, number of microtubules per axons, and microtubular and neurofilament densities. Results at each stage point to an inverse correlation between axonal caliber (x) and microtubular density (y) expressed by the equation y = axb (R = 0.932). Furthermore, three periods of Mauthner axon development are identified on the basis of the cytoskeletal content: (1) embryonic; the Mauthner axon has small caliber with a high microtubule density, (2) elongation period (larval stages); the axon enlarges and a transient peak of microtubules, corresponding to the caliber increment, is observed, and (3) postlarval; the axon enlarges still further (greater than 500 microns 2) but has the lowest microtubular content. During this period neurofilaments are the main axonal component.

Axons Sprout and Microtubules Increase After Local Inhibition of RNA Synthesis, and Microtubules Decrease after Inhibition of Protein Synthesis: A Morphometric Study of Rat Sural Nerves.

Drugs that inhibit RNA or protein synthesis are known to affect some functional properties of axons. In this context, we studied the ultrastructural effects of actinomycin-D, an inhibitor of RNA synthesis, and cycloheximide and emetine, inhibitors of protein synthesis, in rat sural nerves. A silicone sleeve (4 mm long) loaded with drug was placed around the nerves and left for about a week. The ultrastructural alterations of axons and Schwann cells progressed over this period. After cycloheximide and emetine, the cytoplasm of Schwann cells was enlarged and the rough endoplasmic reticulum was prominent. After actinomycin-D, the Schwann cells reached the stage of lysis. Nonmedullated were more affected than myelinated axons. After cycloheximide and emetine, the axoplasmic matrix decreased substantially but reversibly. Microtubules of nonmedullated fibres decreased by about 50%. Actinomycin-D determined sprouting of axons and a rise of axonal microtubules; in nonmedullated axons, the normal inverse correlation between microtubular density and calibre gave way to a high and constant density for all axonal sizes. A few millimetres proximal and distal to the sleeve, the nerve tissue and the axonal microtubular content were close to normal, i.e. the effects of drugs were local. Present results suggest that the local turnover of amino acids in the axon is necessary to maintain the integrity of microtubule and neurofilament proteins. We propose that the Schwann cell down-regulates the axonal cytomatrix.

Ultrastrukturele kwantifisering van mikrotubuli in volledige selprofiele

The ultrastructural morphometric quantification of cytoplasmic microtubular distribution densities requires electron micro­graph magnifications whereby only small cytoplasmic areas are included per negative. Consecutive electron micrographs can be composed in montages to attain complete profiles of high magnification cell sections. Conventional morphometric methods are not suitable for application on areas that may be as great or even greater than 1 m² in size. Modifications of conventional morphometric technology to facilitate investigations of this nature are described. Statistical comparison of results obtained with this method and a standard method reflect the soundness of the modifications.

The Microtubular Pattern Changes at the Spinal Cord-Root Junction and Reverts at the Root-Peripheral Nerve Junction in Sensory and Motor Fibres of the Rat.

In the rat, we studied the microtubular content of central nervous system (CNS) axons (pyramidal tract, dorsal funiculus, and intracord domain of motor axons), of radicular axons (ventral and dorsal roots), and of peripheral axons (sural and lateral gastrocnemius nerves). The microtubular density had an inverse relationship with the size of the axon. Within the CNS, values ranged from over 120 microtubules/microm2 for axons smaller than 0.1 microm2 of the pyramidal tract and dorsal funiculus to 24 for 3-microm motor axons (area, 7 microm2) in their spinal cord domain. Peripheral nerve and CNS axons of the same size had comparable microtubular densities. In contrast, the microtubular density of dorsal and ventral root axons was one half that of CNS or peripheral nerve axons of equal calibre. Considered along the axon, the microtubular density of motor and sensory fibres is high in the CNS domain, low in the root, and high again in the peripheral nerve domain. These observations are inconsistent with the notion that the cytoskeleton moves coherently away from the perikaryon. We conclude that the axonal microtubular content accords with the calibre of the fibre and with the anatomical region where it courses. We propose that axonal microtubules are regulated by local cues.

Calibre and Microtubule Content of the Non-Medullated and Myelinated Domains of Optic Nerve Axons of Rats.

Calibres and microtubule contents of the non-medullated and myelinated domains of optic nerve axons of adult rats were studied with the electron microscope. The cross-sectional areas of the non-medullated domain was 0.25 microm2, and that of the myelinated domain 0.40 microm2, that is, greater by 59%. The increase in size was uneven across the axonal population; it was marked in fine and medium sized axons, and modest in the largest axons. The number of microtubules increased with axonal size; the density, however, decreased from 85 mirotubules/microm2 in 0.1 microm2 axons to about 20 in 1.2 microm2 axons. In axons of equal cross sectional area, the microtubular density of the myelinated and non-medullated domains was the same. Microtubular density values of optic axons resemble those of dorsal roots more than those of peripheral nerve axons of equal calibre. The facts that optic axons increase in size and gain microtubules behind the eyeball while the microtubular packing decreases suggest a local regulation of the axonal cytoskeleton.

Microtubular packing varies along the course of motor and sensory axons: Possible regulation of microtubules by environmental cues

In the toad Xenopus laevis, the microtubular density of 3-μm myelinated fibres was assessed in peripheral nerves, dorsal and ventral roots, and dorsal and ventral funiculi of the spinal cord. In the roots, the axonal microtubular density was 6 microtubules/μm 2 and twice as much at the other sampling sites. This indicates that the pattern of the microtubular packing may vary along the course of the axon. We propose that axonal microtubules are regulated by local cues.

On the asymmetry of the primary branching of vagal sensory axons: possible role of the supporting tissue.

Central and peripheral nonmedullated processes of vagal nodosal neurons of the cat were studied in normal nerves and after regeneration along their anatomical course and along the hypoglossal nerve. Nonmedullated fibers above the ganglion and in the root had comparable sizes (approximately 0.37 micron2) and caliber distribution. Below the ganglion, the cross-sectional area increased to 1.0 micron2. In axons of equal caliber, supranodosal and radicular fibers had similar microtubular densities while infranodosal fibers had two- to threefold that of the former. Regenerated fibers were studied after a recovery period of 6-9 months. Regrown axons were smaller than their parent axons; in turn, these were smaller than normal axons. This holds for central and peripheral nodosal branches, for homologous and heterologous regeneration. Regrown peripheral branches, either along their anatomical pathway or along the hypoglossal nerve, showed no change in microtubular density. Central branches exhibited their characteristic microtubular content when they regenerated along their anatomical course, but when regrowth took place along the hypoglossal nerve, the original low microtubular content of these branches increased to match the high content of peripheral fibers; parent central axons also shifted their microtubular content toward the pattern of peripheral fibers. We propose that the supporting tissue participates in specifying the organization of axonal microtubules.

Dissociation of the inhibition of fast axonal transport by chlorimipramine from an effect on axonal microtubules

The effect of in vitro exposure of bullfrog spinal nerves to 0.2 mM chlorimipramine on the density of axonal microtubules was studied in an attempt to clarify the mechanism by which chlorimipramine inhibits fast axonal transport. A 17-h exposure to chlorimipramine reduced the density of microtubules in unmyelinated axons by only 18%; this microtubular loss does not reach the upper limit of the range of microtubule reduction associated with inhibition of fast axonal transport. A 23-h exposure to chlorimipramine, which had decreased microtubular density in unmyelinated axons by 40% in a previous study, did not decrease microtubular density in myelinated axons in the present study. These results rule out microtubular destruction as the mechanism responsible for inhibition of fast orthograde axonal transport by chlorimipramine, and greatly reduce the likelihood that microtubular destruction plays a significant role in the inhibition of fast retrograde transport by chlorimipramine.

Do axons grow during adulthood? A study of caliber and microtubules of sural nerve axons in young, mature, and aging rats.

Calibers and microtubules of sural nerve axons were studied in young (6-week-old), mature (14-week-old), and aging (2-year-old) rats. The mean cross-sectional area of nonmedullated fibers was about 0.50 micron 2 (range: 0.47-0.52) in the three age groups. Their caliber spectra were also similar. In contrast, myelinated axons grew from 6.6 to 16.7 micron 2 between the sixth and 14th week of age. The increase of cross-sectional area was greater, the greater the initial caliber of axon (range 44-154%). No further change of caliber was observed in the aging rat. The cross-sectional area of nerve allotted per myelinated fiber was 42, 66, and 97 micron 2 in young, mature, and aging rats, respectively. The fraction of nerve tissue occupied by the axoplasm, though, did not change substantially; it was 20, 28, and 21%, respectively. The microtubular density of 3-micron myelinated axons had a general average of 21 microtubules/micron 2. Differences between groups were not significant. In nonmedullated fibers, the microtubular density decreased as the size of the axon increased. No differences were observed between age groups. We conclude that nonmedullated fibers of the sural nerve stop growing before the sixth week whereas myelinated fibers keep growing until the 14th week of age. The correlation between microtubular content and axonal caliber is a lifelong feature of axons.

Axons grow in the aging rat but fast transport and acetylcholinesterase content remain unchanged

Caliber and microtubular density of myelinated fibers, acetylcholinesterase (AChE) content and its accumulation at a ligature were studied in the phrenic nerve of mature (3–4 months) and aging (2-year-old) rats. The number of axons remained constant. The cross-sectional area of the nerve was 67% greater in the older group; the axoplasm, though, constituted about 20% of the nerve tissue irrespective of age. The mean cross-sectional area of myelinated axons was twice as big in aging compared to mature rats. All axons grew in the same proportion irrespective of their original caliber. The microtubular density of 3-μm axons was about 22 microtubules/μm 2 in mature and aging rats. The AChE activity of aging rats was half as much as that of mature rats if it was expressed per wet weight of nerve tissue but did not change if it was expressed per nerve fiber. Twenty-four hours after ligation of the nerve, total AChE activity rose in mature and aging rats by ca. 168%; the molecular forms - asymmetric and globular — accumulated in the same proportion in both age groups. We conclude that myelinated axons grow in the adult stage of life but the structure of axoplasm, content of AChE per axon, and rate of fast transport remain lifelong features of nerve fibers.

Inhibition of fast axonal transport in bullfrog nerves by dibenzazepine and dibenzocycloheptadiene calmodulin inhibitors

The effects of the calmodulin inhibitors amitriptyline, desipramine, imipramine, and clomipramine on fast axonal transport, oxidative metabolism, and density of axonal microtubules were measured in bullfrog spinal nerves in vitro. The four drugs tested inhibited the fast orthograde transport of [3H]leucine-labelled proteins and the fast retrograde transport of acetylcholinesterase at a concentration of 0.2 mM. Amitriptyline, desipramine, and imipramine were equipotent inhibitors of transport, and clomipramine was a more potent inhibitor than imipramine. The adenosine triphosphate content of the nerves was reduced by at most 19% by the compounds under study; such a reduction cannot account for the inhibition of fast axonal transport. Desipramine and imipramine had no significant effect on the density of microtubules in unmyelinated axons, whereas amitriptyline only reduced it by 18%; the inhibition of axonal transport by these three drugs can therefore not be explained by microtubule disruption. Clomipramine reduced microtubular density by 40%, and this effect may have contributed to the inhibition of fast axonal transport. The inhibition of fast axonal transport by desipramine, imipramine, and amitriptyline may be related to the inhibition of calmodulin function by these drugs. The similar potency of these three drugs as inhibitors of fast axonal transport goes in parallel with their known similar potency as calmodulin antagonists.

Microtubules and calibers in developing axons.

The caliber and microtubular content of growing axons were assessed with the electron microscope in the sural nerve of the rat from birth until the age of 63 days (young adult). The caliber of both nonmedullated and myelinated fibers increased throughout the period of observation. At birth, nonmedullated fibers smaller than 0.2 micron2 represented 69% of the population; at day 63 less than 7% were that small. Irrespective of the age of the rat, the number of microtubule profiles of nonmedullated fibers increased with the cross-sectional area of the axon although their packing decreased. In myelinated fibers of a given caliber, the packing of microtubules increased with time, and by day 63 the density had reached its final value. In nonmedullated fibers of a given caliber, a similar trend was observed after day 31; i.e., fibers showed a small but consistent increase in density. However, before that, and in contrast to myelinated fibers, nonmedullated fibers of defined calibers exhibited a transient increase in the microtubular density. Notwithstanding, during the history of an individual fiber the packing of its microtubules may decrease continuously until stabilizing, because the developing fiber is increasing its caliber and hence decreasing its microtubular density. In the 5-day-old rats, the caliber spectra of myelinated and nonmedullated axons overlapped in the 0.49-0.83 micron2 range and their microtubular densities were similar. We conclude that the microtubular content of developing axons correlates with caliber, an architectural feature, and is largely independent of growth and of its myelinated condition. We propose that the cytoskeletal rather than the transport function commands the organization of axonal microtubules.

Microtubules and calibers in normal and regenerating axons of the sural nerve of the rat.

The calibers and microtubular content of axons were studied in normal and regenerating fibers of the sural nerve from 17 to 122 days after a lesion of the sciatic nerve of young adult rats. During this period (70-175 days of age), the cross-sectional area of control myelinated axons almost doubled but that of nonmedullated axons did not change. In regenerating nerves, after 122 days of recovery, the cross-sectional area of myelinated fibers was still 38% below that of the normal side. In contrast, the regenerating nonmedullated population was richer in fine (less than 0.2 micron2) and in coarse (greater than 0.9 micron2) fibers than on the control side; the cross-sectional area averages were 0.50 and 0.54-0.70 micron 2 for the normal and regenerating populations, respectively. The microtubular density of normal 3-micron myelinated fibers averaged 24.0 microtubules/micron2. In regenerating fibers of the same size the density varied between 19.2 and 23.2 microtubules/micron2. Microtubular density values of normal and regenerating fibers were not statistically different. In nonmedullated fibers, the microtubular content (expressed as microtubular density or number of microtubules per axon) correlated with the caliber of the fiber. In these correlations, only minor differences were observed between regenerating and uninjured fibers. Our results indicate that nonmedullated fibers terminate their radial growth well before myelinated fibers do, and that axonal microtubular content correlates with the local size of the fiber and is largely insensitive to regeneration.

Ventral root nonmedullated fibers: proportion, calibers, and microtubular content.

Proportion, caliber, and microtubular content of the L7 ventral root nonmedullated fibers were studied in the cat. Nonmedullated fibers constituted 28% of the axonal population at the far end of the root. The number of myelinated profiles at the far end of the root and in the vicinity of the ventral surface of the cord (1-2 mm distance) was the same whereas the number of nonmedullated fibers decreased toward the proximal site of the root by 42%. Caliber and microtubular content of nonmedullated fibers were assessed only at the far end of the ventral root. Nearly 90% of the axons were smaller than 0.3 micron2. The average cross-sectional area was 0.15 micron2, a value 35% below that of the L7 dorsal root fibers. The microtubular density was highest in the finest fibers (116 microtubules/micron2 in fibers smaller than 0.1 micron2) and decreased with the increase in cross-sectional area (25 microtubules/micron2 for 0.7-0.8 micron2 axons). The number of microtubules per axon in axons of both roots was similar in fibers smaller than 0.3 micron2; in larger axons, composing about 10% of the population, ventral root fibers had more microtubules than dorsal root fibers. The ventral and dorsal nonmedullated fibers differ slightly but significantly in caliber and microtubule content. However, they are similar in contrast to peripheral nonmedullated fibers, which are three to four times as big and contain two to three times as many microtubules as radicular fibers. Our results confirm the presence of a large admixture of nonmedullated profiles in the L7 ventral root of the cat, support the notion that a number of these fibers make a U turn in the ventral root, and suggest that these arise from the central process of the primary sensory axon.

The role of microtubules in axoplasmic transport in vivo

To verify whether microtubules are involved in the mechanism of axoplasmic transport in vivo, [ 3H]leucine was injected into ventral horns of rats, and 3 h later Ca 2+ or other drugs injected into sciatic nerves. The injection of 50–200 mM Ca 2+, raising intra-axoplasmic Ca 2+ levels, blocked transport above the intraneural injecting site and decreased microtubular density. Conversely, injection of 10 mM EGTA lowering the intra-axoplasmic Ca 2+ induced the same changes. By combining the injection of 50 mM colchicine with 25 mM Ca 2+ or 5 mM EGTA, the effects were additive in that transport was weakened further or even blocked and microtubules disappeared. Therefore, microtubules seemed to be a mediator between the injected drug and the blockade of transport and Ca 2+ to be a regulator of axoplasmic transport in vivo. Tubulin, a subunit of microtubules, contains SH groups and Cd 2+ is a chelate of them. By injection of 50–100 mM Cd 2+, transport was weakened or blocked. The sulfuhydryl inhibitor, N-ehtylmaleimide increased, but the sulfhydryl donor, dimercaptosuccinate, abolished the effect of Cd 2+ on transport. N-ethylmaleimide also amplified the Cd 2+ effect on decreasing SH group content of sciatic nerve homogenate. There were 8.7 SH groups per tubulin monomer isolated from rabbit brain. The SH groups of tubulin in vitro and microtubular density in vivo were decreased with the increase of Cd 2+ concentration. All these results indicated that microtubules play a role in the mechanism of axoplasmic transport.

Microtubules in unmyelinated and myelinated axons: a quantitative analysis

The average density of microtubules was compared in unmyelinated and myelinated axons from the following regions of the peripheral nervous system of lizard ( Lacerta muralis ): ventral spinal root, the segment of the dorsal root just peripheral to the spinal ganglion and the segment of the dorsal root just central to the spinal ganglion. In both unmyelinated and myelinated axons, the microtubular density was found to decrease with an increase in the cross-sectional area of the axon, while no significant difference in the microtubular density was observed between unmyelinated and myelinated axons of corresponding size. The results obtained in the present research suggest that in the axons of the peripheral nervous system of lizards the microtubular density is related to axonal size rather than to the absence or presence of a myelin sheath.