The amount of slow axonal transport is proportional to the radial dimensions of the axon

Summary Axons are fundamentally cylindrical and their geometry is defined by two basic parameters, i.e. diameter and length. The average cross-sectional diameter of an axon is determined primarily by the number and density of cytoskeletal structures (i.e. microtubules and neurofilaments) in the axon. The proteins that constitute these structures are synthesized in the nerve cell body and are conveyed through the axon by slow axonal transport. In particular, slow component a (SCa) supplies all of the axonal neurofilament proteins and most of the microtubule proteins to the axon. To study the relationship between slow axonal transport and axonal diameter, the slowly transported proteins were radiolabelled in rat dorsal root ganglion (DRG) cells. The amount of radiolabelled SCa proteins transported in individual unmyelinated and myelinated DRG axons was measured by the electron microscopic autoradiographic method. We found that the amount of SCa transported in the axons is proportional to axonal cross-sectional area. These results indicate that slow axonal transport of microtubules and neurofilaments is a primary determinant of axonal diameter.     

Axonal microtubules: comparative anatomy in vertebrates, including man

The microtubular density was assessed with the electron microscope in 3 microns myelinated fibers, myelin excluded, of 11 species from the following classes: Osteichthyes, Amphibia, Reptilia, Aves, and Mammalia. The average for all species was 20.6 microtubules/microns 2. Dispersion of values was restricted as shown by a coefficient of variation of 15.8. The microtubular content of nonmedullated axons was assessed in trout, lizard, finch, and man. In the four species, the number of microtubules increased with the cross sectional area of the axon. In trout, lizard and finch, the microtubular density decreased from over 100 microtubules/microns 2 in fibers smaller than 0.1 micron 2 to about 30 in 1 micron 2 fibers; in axons of equal size, the packing of microtubules of nonmedullated was similar between them, and with reported values for peripheral axons of cat and rat. In man, the microtubular density of nonmedullated fibers exhibited only a mild decrease with the axonal size. In the finch, myelinated and nonmedullated axons overlapped in the range 0.23-0.60 micron 2 and both groups exhibited similar microtubular densities. We conclude that the packing of microtubules of the vertebrate peripheral axon is a feature largely conserved during evolution.     

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-microns 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/microns 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.     

A-alpha-nerve-fiber: number of neurotubules in the stem fibre and in the terminal branches

Summary Cross sections of A--motor stem fibres of the rat accessory nerve and of one of its branches, the N. musculi sternomastoidei were compared with cross sections of terminal branches of the same nerve, with respect to the axonal cross sectional areas and the number of neurotubules. The absolute number of neurotubules in a stem fibre was found to be on average five times that of one of its terminal branches, corresponding to the ratio of their axonal cross-sectional areas. Thus no significant differences could be found in the tubular density of large stem fibres and of small final branches. Taking into account that in the course of the terminal ramification the total axonal cross-sectional area increases (Zenker and Hohberg, 1973), the combined total of the numbers of neurotubules in all terminal branches of a single A--fibre of the nerve innervating the sternomastoid muscle surpasses the amount of tubules in the stem fibre on average about 11 times. These findings are incompatible with the idea of a constant number of neurotubules within a given axon and its branches. In accordance with recent biochemical studies of microtubular protein, our results indicate that neurotubules may be formed in axon branches far from the perikaryon.     

Computer model for action potential propagation through branch point in myelinated nerves

A mathematical model is developed for simulation of action potential propagation through a single branch point of a myelinated nerve fiber with a parent branch bifurcating into two identical daughter branches. This model is based on a previously published multi-layer compartmental model for single unbranched myelinated nerve fibers. Essential modifications were made to couple both daughter branches to the parent branch. There are two major features in this model. First, the model could incorporate detailed geometrical parameters for the myelin sheath and the axon, accomplished by dividing both structures into many segments. Second, each segment has two layers, the myelin sheath and the axonal membrane, allowing voltages of intra-axonal space and periaxonal space to be calculated separately. In this model, K ion concentration in the periaxonal space is dynamically linked to the activity of axonal fast K channels underneath the myelin in the paranodal region. Our model demonstrates that the branch point acts like a low-pass filter, blocking high-frequency transmission from the parent to the daughter branches. Theoretical analysis showed that the cutoff frequency for transmission through the branch point is determined by temperature, local K ion accumulation, width of the periaxonal space, and internodal lengths at the vicinity of the branch point. Our result is consistent with empirical findings of irregular spacing of nodes of Ranvier at axon abors, suggesting that branch points of myelinated axons play important roles in signal integration in an axonal tree.     

The number, size, and type of axons in rat subcortical white matter on left and right sides: a stereological, ultrastructural study

Abundant evidence indicates important functional differences between the two cerebral hemispheres of humans, although the cellular basis of these differences is unknown. A recent hypothesis proposes that these functional differences depend on differences between sides in the "repertoire" of axonal conduction delays for cortico-cortical axons. In morphological terms this corresponds to differences in caliber, or proportion, of myelinated versus unmyelinated axons. Several behavioural studies have indicated that cerebral asymmetry occurs in rodents, in which rigorous morphological analysis is possible. The hypothesis was therefore tested for the first time in adult male Wistar rats, using transmission electron microscopy and stereological methods. Subcortical white matter was compared between left and right sides in three regions (frontal, parietal, and occipital). The average caliber and numerical density of unmyelinated and myelinated axons was compared between sides and between regions. All data were corrected for shrinkage. No significant differences between sides were found in the average caliber of either type of axon in any region. The numerical density of either type of axon also yielded no significant differences between sides in any region. Significant differences were evident between regions in both caliber and numerical density of the two axonal types, and these quantitative data are reported. The proportion of unmyelinated axons in the lateral white matter was also higher than in previous studies of hemispheric white matter that studied the corpus callosum. The present study provides no evidence supporting the hypothesis that functional hemispheric specialization is due to differences in axonal number, caliber or type.     

Irregular geometries in normal unmyelinated axons: A 3D serial EM analysis

Summary Axons have generally been represented as straight cylinders. It is not at all uncommon for anatomists to take single cross-sections of an axonal bundle, and from the axonal diameter compute expected conduction velocities. This assumes that each cross-section represents a slice through a perfect cylinder. We have examined the three-dimensional geometry of 98 central and peripheral unmyelinated axons, using computer-assisted serial electron microscopy. These reconstructions reveal that virtually all unmyelinated axons have highly irregular axial shapes consisting of periodic varicosities. The varicosities were, without exception, filled with membranous organelles frequently including mitochondria, and have obligatory volumes similar to that described in other neurites. The mitochondria make contact with microtubules, while the other membraneous organelles were frequently found free floating in the cytoplasm. We conclude that unmyelinated axons are fundamentally varicose structures created by the presence of organelles, and that an axon's calibre is dynamic in both space and time.These irregular axonal geometries raise serious doubts about standard two dimensional morphometric analysis and suggest that electrical properties may be more heterogeneous than expected from single section data. These results also suggest that the total number of microtubules contained in an axon, rather than its single section diameter, may prove to be a more accurate predictor of properties such as conduction velocity. Finally, these results offer an explanation for a number of pathological changes that have been described in unmyelinated axons.     

Evidence for loss of myelinated input to the spinal cord in senescent rats.

Impaired sensory perception is a well-established stigma of aging and whereas loss of dorsal root ganglion (DRG) neurons is marginal there is a specific pattern of reduced peripheral sensory innervation. To resolve if similar regressive processes occur in the central innervation, peripheral nerves were injected with markers for unmyelinated (isolectin B4) or myelinated (cholera toxin B subunit; CTB) DRG neurons. The results were a dramatic decrease of primary sensory endings in the spinal cord of aged rats following transganglionic labeling with CTB, and also to a lesser degree with B4. Profile counting and frequency estimates showed that the reduction of CTB labeled profiles not was caused by impaired axonal uptake, slowed axonal transport of CTB, or by a loss of myelinated fibers in the peripheral nerve. At the ultrastructural level, peripheral nerves showed the classical hallmarks of aging, with more pronounced alterations in myelinated than unmyelinated axons. Taken together, sensory deprivation in senescence appears to be a distal process in DRG neurons involving both peripheral and central target disconnection. Finally, preliminary data indicates that the substantial reduction in mechanoreceptive input to the central nervous system co-varies with the degree of sensorimotor impairment of the aged individuals.     

Effect of peripheral axotomy on the fine structure and histochemistry of the Rolando substance: Degenerative atrophy of central processes of pseudounipolar cells

Summary Disappearence of fluorid-resistant acid phosphatase activity from the ipsilateral Rolando substance after transection of the peripheral nerve, is shown to be due to the cessation of enzyme supply from dorsal root ganglion cells to their central terminals. This is accompanied by (or ensues in consequence of) a fine structural derangement of these terminals (degenerative atrophy). Fine structural alterations of axon terminals undergoing degenerative atrophy, though similar to some extent to those seen during early phases of a Wallerian degeneration, are markedly different. Also myelinated nerve fibers, both in the dorsal horn and in dorsal columns, are affected by degenerative atrophy. This important, new trophical feature of sensory ganglion cells suggests a delicate metabolic balance between peripheral and central axonal branches of bipolar (pseudounipolar) cells. Degenerative atrophy raises serious implications in evaluating hodological experiments based upon Wallerian degeneration and offers new perspectives for theoretical and clinical neurology.     

Electron microscopic nerve fiber caliber analysis in the nerve branch to the venter rostralis of the M. digastricus in mice

Nerve fiber counts and caliber analysis were performed with an electron microscope on 5 nerve branches to the venter rostralis of m. digastricus (VRD) from 3 mice. Branches contained an average of 42.4 (79.4%) myelinated fibers and 11.0 (20.6%) unmyelinated fibers. 68.4% of the sheaths of the myelinated fibers in the nerve branches were 6.5-8.5 microns (peak 7.0-7.5 microns) in major diameter and 76.4% were 5.0-7.5 microns (peak 6.0-6.5 microns) in minor diameter, 68.8% of the axons of the myelinated fibers were 4.5-6.5 microns (peak 5.5-6.5 microns) in major diameter and 76% were 3.5-5.5 microns (peak 4.0-5.0 microns) in minor diameter. The frequency distribution of the diameters showed that the nerve branch to VRD consisted of extra large myelinated fibers as in the r. digastricus to the venter caudalis of m. digastricus (VCD). Average ratios of the areas of the myelin sheaths to those of axons of the myelinated fiber transverse sections in the various axon area classes of the nerve branches were 0.7-2.9, and those ratios tended to decrease in the larger axon classes.     

Three-dimensional analyses of touch domes in the hairy skin of the cat paw reveal morphological substrates for complex sensory processing

The three-dimensional morphology of the innervation of touch domes in the hairy skin folds of cat forepaws was investigated by the confocal laser scanning microscopic analyses of sections stained immunocytochemically with primary antibodies for protein gene product 9.5, neurofilament 200 and cytokeratin 20 in combination with transmission electron microscopic observations. One square centimeter of interdigital skin can contain as many as 68 touch domes. Each touch dome can have up to 150 Merkel cells and all are innervated by a single large-caliber afferent myelinated nerve fiber at the level of the palisade endings around the guard hair. It gives rise to multiple long, myelinated branches. Each final myelinated branch gives rise to several short and fine unmyelinated branches, supplying approximately 15 Merkel cell–axon complexes. Each Merkel cell is typically contacted by multiple small discoid endings instead of by a large single one. Discoid endings on separate Merkel cells were usually the distal ends of the unmyelinated branches, although, some were en-passant swellings of the branches. Only a few Merkel cell–axon complexes at the marginal zone of each territory could also be supplied by adjacent final myelinated branches. Each Merkel cell is surrounded by protrusions of keratinocytes that are penetrated by several collagen bundles of the dermis. This intricate pattern of innervation may explain the unique irregular discharges of action potentials typical for this type of mechanoreceptor.     

The relation of axonal transport of mitochondria with microtubules and other axoplasmic organelles.

Axonal transport of mitochondria was studied in frog sciatic nerves incubated in agents selected for their known or alleged effect on microtubules or axonal flow. Quantitative data on mitochondria, microtubules, neurofilaments, endoplasmic reticulum, and cross-sectional area of the axon indicate that axonal transport of mitochondria is dependent on microtubules. When more than half of the microtubules are destroyed, the axonal transport of mitochondria is diminished in proportion to the destruction of microtubules. Axonal transport of mitochondria is not related to neurofilaments and endoplasmic reticulum. Changes in the cross-sectional area of axons, even upon reduction to half the normal size, do not noticeably affect mitochondrial transport. Cyanide which blocks oxidative metabolism also blocks axonal transport of mitochondria, but analysis of fine structure indicates that cyanide is destructive to microtubules as well.     

Regulation of axonal microtubules: Effect of sympathetic hyperactivity elicited by reserpine

The density of microtubules in sympathetic postganglionic fibres of the cat was studied with the electron-microscope before and after administration of reserpine. The microtubule density was 56 microtubles per square micron under basal conditions. Six hours after reserpine administration, the density rose by 46%. This change was still present 55 h later. At least 31% of the total microtubular protein in the axoplasm of sympathetic fibres of the unrestrained cat was estimated to be in the soluble form. The increase in microtubule density was prevented by a section of the preganglionic fibres. Microtubules of the unmyelinated fibres of the cutaneous sural nerve were unaffected by reserpine treatment.Since reserpine is known to produce hyperactivity of sympathetic nerves, it is concluded that this hyperactivity is instrumental in the increase of the number of axonal microtubules. It is proposed that the electrical activity of nerves regulates axonal microtubules in the living animal.     

Study on the ultrastructure and acetylcholinesterase activity in von Recklinghausen's neurofibromatosis.

The cutaneous nodules obtained from seven patients with von Recklinghausen's neurofibromatosis were investigated by electron microscopy, and ultrastructural localization of acetylcholinesterase activity was demonstrated in the nerve fibers of this tumor for the first time using Karnovsky's thiocholine method. The enzymatic activity was mainly found in unmyelinated fibers, exactly associated with their axonal membranes, the interspace between the apposing axonal and Schwann cell membrane, and some different mesaxons, which indicated their cholinergic nature. Almost all myelinated fibers and some unmyelinated fibers did not possess the activity. The relationship between axon and Schwann cell was quite similar to that of normal peripheral nervous system, but two striking alterations of the nerves existed: One is the dissociation of unmyelinated fibers, and the other is the degenerative changes of the axon and the myelin sheath. As the evidence of schwannian proliferation, onion bulb formations and collagen pockets were observed. Some signs of fibroblastic proliferation were also found. From the present study and the review of the literature, the probable histogenesis of this disease was discussed.     

Axonal transport in the central axon of sensory neurons during regeneration of their peripheral axon

Following sciatic nerve injury (which provokes prompt regeneration of peripheral sensory axons) there was no change in the ratio between amounts of labeled protein conveyed by fast axonal transport into the central and peripheral axons. A change in composition of the transported protein, characteristic of regenerating peripheral axons, also occurred in the central axons. When the peripheral axon is injured, changes in axonal transport thus affect both processes, and there is no routing of fast-transported protein into or away from the regenerating process.     

STUDY ON THE ULTRASTRUCTURE AND ACETYLCHOLINESTERASE ACTIVITY IN VON RECKLINGHAUSEN'S NEUROFIBROMATOSIS

The cutaneous nodules obtained from seven patients with von Reckling-hausen's neurofibromatosis were investigated by electron microscopy, and ultrastructural localization of acetylcholinesterase activity was demonstrated in the nerve fibers of this tumor for the first time using Karnovsky's thiocholine method. The enzymatic activity was mainly found in unmyelinated fibers, exactly associated with their axonal membranes, the interspace between the apposing axonal and Schwann cell membrane, and some different mesaxons, which indicated their cholinergic nature. Almost all myelinated fibers and some unmyelinated fibers did not possess the activity. The relationship between axon and Schwann cell was quite similar to that of normal peripheral nervous system, but two striking alterations of the nerves existed: One is the dissociation of unmyelinated fibers, and the other is the degenerative changes of the axon and the myelin sheath. As the evidence of schwannian proliferation, onion bulb formations and collagen pockets were observed. Some signs of fibroblastic proliferation were also found.
From the present study and the review of the literature, the probable histogenesis of this disease was discussed.     

Fiber composition of the rat sciatic nerve

The rat sciatic nerve originates from the spinal segments L4-L6. It is unifascicular at the trochanter; 5-7 mm distally, the nerve splits into two and then into four fascicles. The tibial portion gives rise to the tibial and the sural nerves, and the peroneal portion gives rise to the peroneal nerve and a cutaneous branch that perforates the lateral hamstring muscles to innervate the proximolateral face of the calf. The number and type of the axons in these branches were determined in light and electron micrographs of normal nerves, and after de-efferentation or sympathectomy. Deafferentation was technically not feasible because spinal ganglia and ventral roots were supplied by the same vascular plexus. The tibial nerve contained 1,000 motor and 3,500 myelinated afferent axons, 3,700 sympathetic axons, and 5,400 unmyelinated afferent axons. The peroneal nerve contained 600 motor and 1,300 myelinated afferent axons, 1,100 sympathetic axons and 3,000 unmyelinated afferent axons. The sural nerve contained 1,100 myelinated and 2,800 unmyelinated afferent axons; in addition, there were 1,500 unmyelinated sympathetic axons. The cutaneous branch consisted of 400 myelinated and 1,800 unmyelinated afferent axons. Thus, the entire sciatic nerve at midthigh is composed of about 27,000 axons; 6% are myelinated motor axons, 23% and 48% are myelinated and unmyelinated sensory axons, respectively, and 23% are unmyelinated sympathetic axons. The techniques used did not demonstrate sympathetic axons in the cutaneous branch and did not reveal the few motor axons contained in the sural nerve.     

Beyond the initial axon segment of the spinal motor axon: fasciculated microtubules and polyribosomal clusters

Dense undercoating, microtubular fascicles and scattered polyribosomal clusters have until now been considered to be the three structural features of the initial segment, and were thought not to extend beyond the initial segment into the myelinated parts of the axon. The aim of the present study was to make clear whether there is a sudden change in morphology between the unmyelinated and myelinated part. We followed spinal motor axons from the initial segment to the first internode by conventional electron microscopy and serial sectioning, and found that the microtubular fascicles and polyribosomal clusters do exist in the internodal axoplasm. The fasciculated microtubules were observed mainly in the first paranode. The polyribosomal clusters were found along the course of the first internode at a random distance, however, they occurred mainly in the proximal part of the first internode. The proportion of sections in which ribosomes were found, i.e. the incidence of ribosomes, in the first 30-microm-long portion was 71 +/- 24% (mean +/- SD, n = 4), and significantly different from that in the second 30-microm-long portion (3.2 +/- 1.3%) (mean +/- SD, n = 4) (P < 0.005). The more distal part of the first internode was not investigated.     

On the organization of the thoracic spinal ganglion and nerve in the rat

Summary The location of spinal ganglion cells projecting into various thoracic spinal nerve branches in the rat has been investigated using retrograde transport of horseradish peroxidase (HRP). In addition, total numbers of myelinated and unmyelinated axons in three consecutive pairs of thoracic spinal nerves have been analyzed. The animals were perfused one to 2 days after application of HRP to thoracic spinal nerve branches and several ipsilateral spinal ganglia sectioned and processed for HRP histochemistry. Labeled cells were found in ganglia corresponding to the level of operation, as well as several adjacent rostrally and caudally located ganglia. No somatotopic organization was found within the ganglia, but occasionally a clustering of labeled cells belonging to the same peripheral nerve was observed. Pieces from three consecutive pairs of ventral and dorsal spinal nerve rami were removed from two normal rats and embedded in Vestopal. Semithin sections including the entire cross-sectional area of the rami were used to count the total number of myelinated axons. The number of unmyelinated axons was estimated by sample counting in the electron microscope. The ventral ramus of the spinal nerve contained about twice as many axons and a lower proportion of unmyelinated fibers in comparison with the dorsal ramus. Large differences in the number of axons between the two sides in pairs of spinal nerve rami and entire spinal nerves were found. These differences diminished when comparing the total number of axons from three consecutive rami or nerves on each side. The results of the present study are compatible with the notion that although there is a strict somatotopic organization of the periphery in the spinal cord dorsal horn, the pathway between these two sites is relatively diffusely organized. It is possible that there is instead a modality-related organization in the peripheral nerve pathway.     

Vagal nerve maturation in the fetal lamb: An ultrastructural and morphometric study

The maturation of the left vagal nerve was studied in the fetal lamb by transmission electron microscopy and by computer-assisted morphometry of sections of the entire nerve at seven gestational ages between 79 and 145 days (term is 147 days) and in the adult ewe. The number of unmyelinated axons per Schwann cell progressively decreased from 25 to 55 at 79 days to 1 to 5 at near-term. Unmyelinated axons of various sizes were enclosed within a single Schwann cell at all ages, but the mean axonal diameter increased in inverse relation to the number of unmyelinated axons. A few Schwann cells enclosed two myelinated axons, but in most instances myelination did not begin until a 1:1 ratio was achieved; some single axons with a Schwann cell remained unmyelinated in the adult. Myelinated fibers were rare at 79 days but myelination progressed rapidly thereafter until the adult ratio of myelinated: unmyelinated fibers was reached at about 100 days; myelinated axons were not uniformly distributed. The myelin sheaths and axons of small fibers progressively increased in diameter in late gestation, but new large fibers were not added. Early myelinating fibers and immature unmyelinated axons contained more microtubules than neurofilaments; neurofilaments predominated in mature axons with or without myelin. Cross-linkages between neurofilaments were already evident by 79 days. Maturation of the vagal nerve thus occurs first by an increase in number of myelinated fibers and then by an increase in the size of each fiber in this fixed population. The bimodal distribution in the size histogram of myelinated fibers is not achieved until 134 days gestation and correlates well with physiological maturation of respiratory patterns. © 1993 Wiley-Liss, Inc.