Neurofilament Density Research Articles

Neurofilaments assume a less random architecture at nodes and in other regions of axonal compression

Neurofilament distributions were mathematically characterized in four chicken somatic motor axons at each of four histologically distinct regions: compact myelinated regions, compact myelinated regions associated with Schwann cell nuclei, Schmidt-Lanterman clefts, and nodes of Ranvier. Compact myelinated regions had the largest cross-sectional areas, the lowest neurofilament densities, and the most random neurofilament organizations — nodes of Ranvier had the smallest cross-sectional areas, the highest neurofilament densities, and the most ordered architectures. In these myelinated axons, the closest natural neurofilament spacing was 25 nm. Mathematical analyses of serial sections suggested that neurofilament interactions are sufficiently weak and transient to permit a full range of variation from random to ordered cytoskeletal architectures as the neurofilaments move longitudinally through the few micron span of the paranodal-nodal region of a single axon.

Neurofilaments move apart freely when released from the circumferential constraint of the axonal plasma membrane.

Squid giant axons were used to obtain axonal cytoskeletons that had been separated from the confines of their plasma membranes. To remove the plasma membrane, axoplasm was extruded from the giant axon directly into an artificial axoplasm solution (AAS). This procedure produces a smooth axoplasmic cylinder in which neurofilaments (NFs) are the most prevalent cytological elements. The NFs scatter light strongly and thus dark-field light microscopy can be used to quantify the volume occupied by these polymers. Measurements of the widths of the dark-field images of the axoplasmic cylinders showed that the cross-sectional area of the NF population increased by 60-110% (n = 8) between 1-100 min after plasma membrane removal, and then continued to increase more slowly for many hours. After 1,000 min, the cross-sectional area was 75-160% (n = 8) larger than at 1 min. These light microscopic measurements of axoplasm suggest that the NF population disperses to occupy a continuously increasing volume after removal of the plasma membrane and immersion in AAS. This inference was confirmed by quantitative ultrastructural studies of NFs in axoplasmic cross-sections, which demonstrated that the spacing between the NFs increased between 1-1,000 min after plasma membrane removal. Comparison of the NF density distribution after 1,000 min with a theoretical distribution calculated using the Poisson theorem indicated that the NFs dispersed randomly. These studies on NFs in isolated axoplasm suggest that ordinary thermal forces of Brownian motion are sufficient to move axonal NFs apart independently and thereby to disperse them. We propose that, in the intact axon, the dispersive movements of the NFs spread the NF cytoskeleton radially and expansively to fill out the cylindrical space contained by the axonal plasma membrane and its surrounding connective tissue elements.

Local modulation of neurofilament phosphorylation, axonal caliber, and slow axonal transport by myelinating Schwann cells

Studies in Trembler and control mice demonstrated that myelinating Schwann cells exert a profound influence on axons. Extensive contacts between myelin and axons have been considered structural. However, demyelination decreases neurofilament phosphorylation, slow axonal transport, and axonal diameter, as well as significantly increasing neurofilament density. In control sciatic nerves with grafted Trembler nerve segments, these changes were spatially restricted: they were confined to axon segments without normal myelination. Adjacent regions of the same axons had normal diameters, neurofilament phosphorylation, cytoskeletal organization, and axonal transport rates. Close intercellular contacts between myelinating Schwann cells and axons modulate a kinase-phosphatase system acting on neurofilaments and possibly other substrates. Myelination by Schwann cells sculpts the axon-altering functional architecture, electrical properties, and neuronal morphologies.

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.

A useful programme in BASIC for axonal morphometry with introduction of new cytoskeletal parameters

Interest in the structure of axons and quantification of their components has been growing over the last years. However, the existing literature contains few reports of available computer programmes to facilitate such studies. This paper presents a fully comprehensive BASIC programme for the morphometric analysis of electron micrographs of cross-sectional nerve fibres. From drawings of fibre and axonal contours and dots of the microtubules and neurofilaments, the programme calculates the following parameters: area, diameter and form factor of the fibres and axons, number and density of microtubules and neurofilaments, proportion between microtubules and neurofilaments (R-proportion), myelin thickness and the diameter of the axon relative to its sheath (g-ratio). The programme also introduces three new parameters to analyse the degree of uniformity of microtubule and neurofilament distribution: distances between microtubules and between neurofilaments, equilateral index and cytoskeletal intermingling index. The programme is written in Microsoft BASIC Interpreter for Apple Macintosh (Microsoft Corporation) but can be used on other computers. Although the programme has been tested on adult rat optic nerve fibres, it can be used for different projects concerning axonal morphometry.

Immunohistological study of neuronal markers in inflamed gingiva obtained from children with Down's syndrome

The histological appearance of the gingiva in children with Down's syndrome (DS) was studied with special reference to inflammatory involvement and innervation. A dense infiltration of inflammatory cells was seen in the propria of most of the DS patients, including a few polymorphonuclear leucocytes. A hyperplasia of the epithelium was also found. The innervation of the gingiva was studied using immunohistochemistry. Nerve fibers as well as nerve bundles immunoreactive to neurofilament (NF) were seen in the propria, while occasionally intraepithelial NF fibers were observed. Calcitonin gene-related peptide (CGRP)-immunoreactive fibers and fiber bundles were also visualized, but they were less abundant than NF fibers. The density of NF and CGRP fibers and fiber bundles was estimated by semiquantitative evaluation. A higher density of NF and CGRP immunoreactive structures was observed in the propria of DS patients compared to the control subjects, while no obvious alteration was seen in their distribution in the propria. In addition, sparsely distributed fibers immunoreactive to peptide histidine isoleucine amide (PHI) and vasoactive intestinal polypeptide (VIP) fibers as well as neuropeptide Y (NPY) and tyrosine hydroxylase (TH) were seen, mainly surrounding blood vessels. A few substance P (SP) fibers were also found, mostly close to the epithelium. No obvious differences of these sparsely distributed fibers were seen in the DS patients compared to controls. Thus, a profound inflammatory involvement of the gingiva of DS patients is seen concomitant with a hyperinnervation of the presumed sensory component of the gingival innervation. In contrast, no alterations were seen in the density of neuronal markers related to autonomic nerve fibers. The sensory hyperinnervation observed is probably not specifically related to DS, but may be due to a sprouting of afferent nerves induced by the inflammatory reaction. However, factors released from the sensory afferents could contribute to the gingival inflammation seen in DS.

Phosphorylation‐Dependent epitopes on neurofilament proteins and neurofilament densities differ in axons in the corticospinal and primary sensory dorsal column tracts in the rat spinal cord

The highest molecular weight neurofilament protein (NF-H) is multiply phosphorylated at epitopes which can be distinguished by specific monoclonal antibodies on Western blots. Eight characterized antibodies were used in immunocytochemistry to examine the tissue distributions of phosphorylated variants of NF-H in axons of the adult rat spinal cord. The most striking difference in staining was found between axons in the cuneate tract and those in the neighboring dorsal corticospinal tract. Axons in the cuneate tract reacted intensely with antibodies to phosphorylated epitopes of NF-H and poorly with antibodies to dephosphorylated epitopes of NF-H, whereas the reverse was the case for the axons of the dorsal corticospinal tract. These differences showed that systematic variations in the phosphorylation of NF-H in long-tract axons in the central nervous system occur as a function of cell type. When the cytoskeletons of these axons were compared by electron microscopy, the neurofilaments of the cuneate fibers were seen to be more abundant and formed a latticework, more compactly organized than the neurofilaments of the dorsal corticospinal axons. By comparison, the dorsal corticospinal axons were relatively richer in microtubules than the cuneate axons. Although the cuneate fiber tract contained many more large (greater than 2.0 microns 2 in cross section) axons than did the dorsal corticospinal tract, these differences in cytoskeletal organization were apparent even when myelinated axons of similar sizes (0.4 micron 2 to 2.0 microns 2) were compared. In addition, the number of neurofilaments in cuneate axons in the 0.4 to 2.0 microns 2 size range was significantly better correlated with axon size than was the case for this size range of dorsal corticospinal axons. Thus, the differences seen in the organization of the neurofilament latticework and the phosphorylation of NF-H between axons found in these two tracts both appeared to be correlated with cell type, and were independent of length or caliber of the axons.

Internal axonal cytoarchitecture is shaped locally by external compressive forces

The cross-sectional architecture of the axon and the area of its surrounding Schwann cell were quantified at selected histological regions along the length of avian myelinated axons. The number of neurofilaments (NFs), the density of NFs, axoplasmic area, and Schwann cell cross-sectional area were measured. These parameters were examined at Schmidt-Lanterman (S-L) clefts, at paranodal-nodal regions, and at regions of compact myelin Schwann cell nuclei. The results were then compared with the same parameters in adjacent compact myelinated regions of the same axons. At S-L clefts, paranodal-nodal regions, and Schwann cell nuclei, the axonal areas were smaller and the NF densities were higher than at compact myelinated regions. From other studies, it has been suggested that NF organization is responsive to local compressive forces — NF packing density tends to increase with increasing compression of the axon. We found that the NF packing densities were relatively small and the axon diameters were relatively large in the compact myelinated regions; this result suggests that in these axonal regions external constraints on axonal architecture are minimal. The higher NF packing densities and smaller axon diameters in the other histological regions suggest that external compressive effects on the axon increase in the following order: simple compact myelin < Schwann cell nucleus < S-L cleft < paranodal-nodal region. Ultrastructural comparisons of these 4 histological regions show that the Schwann cell cross-sectional areas differ reproducibly, and this is consistent with the idea that variations in the organization of extra-axonal elements that envelop the axon produce different amounts of physical constraint on the axon and that this can affect the amount of external pressure on the internal architecture of the axon.

Expression of NF-L in both neuronal and nonneuronal cells of transgenic mice: increased neurofilament density in axons without affecting caliber.

We have generated transgenic mice containing additional copies of the murine NF-L gene in order to examine the consequences of neurofilament-L overexpression on axonal morphology. Founder mice were constructed to carry a transgene in which the presumptive 5' promoter sequences of NF-L were replaced with the strong murine sarcoma virus long terminal repeat promoter. The transgenes were expressed prominently in several tissues, including skeletal muscle and kidney where NF-L accumulated to approximately 2% of cell protein. This was not accompanied by an overt phenotype, except that expression in lens led to cataract formation. In the brains of these animals, transgene RNA levels exceeded the endogenous NF-L RNAs by up to 20-fold, although no additional protein accumulated, indicating posttranscriptional regulation of NF-L expression. However, in peripheral neurons transgene RNA was approximately fourfold higher than endogenous NF-L mRNAs, and a corresponding increase in NF-L subunits was found in axons arising from these neurons. Myelinated nerve fibers of transgenic animals contained increased numbers of NFs, assembled predominantly of NF-L. This was reflected in an increase in the density of axonal NFs; axonal caliber was not affected.

Microtubule number increases locally within regions of axonal compression

Along the length of myelinated axons, stereotyped variations in the interaction with the enveloping Schwann cell produce different amounts of compressive force on the axon and its internal elements. Comparisons of histologically distinct axonal regions indicate that compression of the axon by the Schwann cell increases in the following order: internodal regions of compact myelin &lt; Schmidt-Lanterman (S-L) clefts &lt; nodes of Ranvier. Quantitative morphometry has shown that regions of greater Schwann cell compression are associated with regions of smaller axon diameter and greater neurofilament (NF) packing density. By locally decreasing axon diameter and increasing NF density, Schwann cell compression (especially at the node of Ranvier) may slow the transport of those axonal structures - such as microtubules (MTs) - that move by a polymer sliding mechanism. Under this hypothesis, a local slowing of MT transport should produce a local increase in MT number, and here we have studied whether such a local increase in MT number can be quantitatively documented.A twenty-eight day old chicken was perfused by cardiac puncture with 4 1 of fixative (4% paraformaldehyde, 2% glutaraldehyde, 0.005% CaCl2, in 0.1M cacodylate buffer, pH 7.4). The somatic motor branch of the oculomotor nerve was removed (noting the proximo-distal orientation) and fixed overnight at 4°C, followed by a buffer rinse in 0.1M cacodylate buffer at room temperature and postfixation in OSO4. The nerve was then dehydrated in a graded ethanol series and embedded in PolyBed/812. Starting at the proximal end of the nerve segment, l00nm sections were cut at consecutive 2-3 um intervals over a 225 um length of nerve, stained with an LKB Ultrostainer, and photographed on a JEOL 100CX microscope. We selected two axons with normal morphology, in which the cytoskeletal polymer profiles were consistently cross-sectioned and in which nodes were located approximately 120 um distal to the first section taken. All sections from these 2 axons were photographed, and MTs were counted on 58,000X micrographs.

Rapid changes in ultrastructure during deafferentation-induced dendritic atrophy.

This study describes qualitative and quantitative changes in dendritic ultrastructure during the rapid atrophy of nucleus laminaris (NL) dendrites following deafferentation. The dendrites of n. laminaris neurons in the chick auditory system are segregated into dorsal and ventral dendritic tufts, which receive spatially separated innervation from the ipsilateral and contralateral nucleus magnocellularis, respectively. We have previously shown that removing the input to the ventral side of NL results in the rapid atrophy of the ventral dendrites, whereas the nondeafferented dorsal dendrites of the same cells do not change in length. The ultrastructure of NL was examined in normal animals and after deafferentation. Changes in dendritic ultrastructure were not qualitatively apparent 4 hours after deafferentation. Between 12 and 48 hours the cytoplasm of the ventral dendrites became progressively more lucent, and a gap formed in the transition between the soma and ventral dendritic cytoplasm. Many of the dendrite tips, however, appeared normal even 2 days after deafferentation. Degeneration of dendrite plasma membrane was not visible until 2 days after deafferentation. On the other hand, quantitative measurements revealed a 30% decrease in microtubule density in the initial portion of the ventral dendrite by 4 hours, and a 50-60% decrease from 12 to 48 hours after deafferentation. Neurofilament density in the initial ventral dendrites decreased 50% by 12 hours, and 70% by 2 days after deafferentation. Many of the terminals of the severed afferents remained attached to the atrophying dendrite until 2 days after surgery, when they were in advanced stages of degeneration. Glia apparently were not involved in dendrite loss. The implications of these results on the role of cytoskeleton in the production and maintenance of dendritic shape are discussed.

The effects of dephosphorylation on the structure of the projections of neurofilament

Carboxy-terminal tail domains of larger molecular mass subunits (NF-M and NF-H) of neurofilaments (NFs), which are the highly phosphorylated moieties, were observed as thin flexible filaments projecting from NF core filaments by rotary shadowing (Hisanaga and Hirokawa, 1988). Dephosphorylation of NFs has been suspected to affect the structures and the functions of the carboxy-terminal tail projections. We report here the effects of the dephosphorylation on the structure of NFs studied by electron microscopy. (1) The structures of carboxy-terminal tail projections after dephosphorylation were compared with those of the control NFs by low-angle rotary shadowing. This was examined with 2 samples; the isolated neurofilaments and the short filaments assembled from NF-H. Both the dephosphorylated NFs and the short filaments showed many projections laterally extending from core filaments similar to those observed in the control samples. (2) With respect to the structure of NF in physiological solution, the density of NFs in the precipitates was examined by thin-section electron microscopy. No difference in the density was noted between control and dephosphorylated NFs. (3) The ability to form cross-bridges in vitro was examined by quick-freeze, deep-etch electron microscopy. The structure and frequency of cross-bridges appeared to be similar in both control and dephosphorylated NFs. (4) Phosphate determination revealed that about 90% of the phosphate groups of NF-H subunit were removed by treatment with E. coli alkaline phosphatase. These results indicated that the dephosphorylation of NF did not affect the structure and the ability to form cross-bridges of the carboxy-terminal tail projections in vitro.

Postural sway during retinal image stabilisation.

Posturographic measurements using a piezoelectric platform were made in normal subjects while wearing a combination of spectacle and contact lens providing partial stabilisation of the retinal image (RIS). The amount of postural sway seen while wearing the device at rest is intermediate between the "normal vision" and "eyes closed" conditions, and increases with increasing amounts of RIS. However, when large active head-and-eye movements are performed, postural sway is dramatically increased when using RIS, and is then worse than while performing the same task in the "eyes closed" condition. It is concluded that patients who use the partial-RIS device for the treatment of severe oscillopsia may benefit only when performing tasks in which the head is relatively still, such as reading, writing or watching TV. It is also proposed that the partial-RIS device can serve as a model in normal free-standing subjects for the postural effects of oculomotor disorders.

Different neurofilament densities show identical random distributions in side-by-side axons

Neurofilaments (NF) are architectural polymers that contribute to the radial dimensions of an axon. In general, the diameter of an axon Increases as a function of the number of neurofilaments. We have studied the space-filling properties of neurofilaments in two different types of axons that have a large neurofilament content -- somatic motor axons and parasympathetic axons of the oculomotor system. Both types of axons originate from neurons in the oculomotor nucleus and they course together (side-by-side) through the oculomotor nerve; nonetheless, the packing density of neurofilaments in the parasympthetic axons is 30% greater than the neurofilament density in the somatic motor axons. Visual inspection of electron micrographs of parasympathetic and somatic motor axons suggests that the neurofilaments are uniformly distributed in both types of axons. To test this possibility, we quantified the distributions of the neurofilaments and then compared those distributions with a randomly generated (Poisson) distribution.

Effect of changes in neurofilament content on caliber of small axons: the beta,beta'-iminodipropionitrile model

The structural role of neurofilaments in the normal axon and the consequences of altered axonal transport of neurofilaments have been extensively studied in large axons. These studies suggest that neurofilament numbers and interneurofilament spacing are major determinants of axonal cross-sectional area. In contrast, in small axons and dendrites, microtubules and membranous organelles appear to be the most closely correlated with size and shape of the cell process. In this study we have examined the effect of impairment in neurofilament transport on small axons, typical of most CNS pathways. Neurofilament transport was impaired by administration of beta,beta'-iminodipropionitrile (IDPN), resulting in proximal accumulation and distal depletion of neurofilaments. The evolution of these changes was studied in the optic nerves of guinea pigs treated with IDPN, 1-35 weeks following intoxication. The effect of this redistribution of neurofilaments on cross-sectional area of small axons was evaluated using quantitative ultrastructural methods. Our results show that with the alteration in neurofilament transport seen with IDPN intoxication, there is a wide spectrum of neurofilament densities, ranging from a 5-fold increase above normal in the proximal axon, to a 5-fold decrease below normal in the distal axon. Although the optic nerve fibers enlarge with the increase in neurofilament content, they do not atrophy significantly with the continued loss of neurofilaments. We conclude that factors other than neurofilament content are capable of maintaining size and shape of these small axons. Candidate organelles include microtubules and membranous organelles and possibly other axonal elements.

The density of neurofilaments differs in the somatic motor and parasympathetic oculomotor axons of chickens

The rate of neurofilament transport in somatic oculomotor axons is 2-3 times faster than 1t 1s in the preganglionic parasympathetic oculomotor axons of young chickens. The rate of neurofilament transport may be influenced by the lateral interactions between neurofilaments. To determine whether the lateral Interactions between neurofilaments differs between the somatic motor and the parasympathetic axons, we determined the neurofilament density 1n these axons.

Axonal caliber and neurofilaments are proportionately decreased in galactose neuropathy.

Feeding galactose to rats induces nerve conduction abnormalities, increased levels of nerve galactitol, endoneurial edema, elevated pressure and hypoxia of endoneurial fluid, and pathological abnormalities of nerve fibers. To investigate the cellular mechanisms of the fiber lesions and their possible relationship to alterations in the nerve microenvironment, rat peroneal nerves were morphometrically evaluated eight months after the commencement of galactose feeding. Whereas the density of neurofilaments (NF/micron2) in the transverse axonal area of myelinated fibers was not significantly different between the nerves of galactose-fed and control rats, axonal areas and the number of NF/axon, when related to myelin spiral length, were significantly less in nerves of galactose-fed rats. Myelin alterations, characteristic of axonal atrophy, were also significantly increased. The present data provide evidence of a proportionate decrease in axonal caliber and the number of NF/axon in myelinated fibers in experimental galactose neuropathy, suggesting that galactose induces fibers in experimental galactose neuropathy, suggesting that galactose induces either decreased NF synthesis, assembly or transport. The possible role of microenvironmental alterations, including endoneurial hypoxia and hyperosmolarity, in the production of this axonal atrophy is discussed.

Morphometric effects of vincristine on nerve regeneration in the rat

Administration of vincristine (200, 100 or 50 μg/kg/week) for 6 months during regeneration of the sciatic nerve after crush injury caused a dose-dependent reduction in nerve fibre size and failure of removal of myelin debris. Successfully regenerating neurites showed an unusual amount of shape distortion. The ratio of myelin sheath thickness to axon circumference was reduced, but the ratio of myelin sheath thickness to axon area was normal. Microtubule concentration was diminished in axons, but neurofilament density was unaffected. Unmyelinated axons were reduced in number but their axon diameter distribution was not affected. Fibres on the non-crushed side appeared normal. The toxicity of vincristine to regenerating nerves is probably related to increased blood-nerve permeability occurring both at the site of crush and along the degenerating nerve.

Slowing of neurofilament transport and the radial growth of developing nerve fibers

Several lines of evidence indicate that neurofilaments are major intrinsic determinants of axonal caliber in myelinated nerve fibers, and that the delivery of neurofilaments by slow axonal transport is an important mechanism by which neurons regulate axonal caliber. To further clarify the relationship between neurofilament transport and axonal caliber, we examined transport in developing motor fibers of rat sciatic nerve. In 3-, 10-, 12-, and 20-week-old rats, lumbar motor neurons were labeled by the intraspinal injection of radioactive amino acids, and the distributions of labeled cytoskeletal proteins within the sciatic nerve were analyzed at various times afterwards using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel fluorography, and liquid scintillation spectroscopy. There was a progressive decline in the velocity of neurofilament transport with increasing distance along axons undergoing radial growth. By examining transport in different regions of the nerve in animals of the same age, we separated age-dependent reductions in velocity from those related to position along the nerve. The cross-sectional areas of these motor axons (in the L5 ventral root) increased linearly between 3 and 18 weeks of age. Quantitative electron microscopic analysis at 3 and 10 weeks of age revealed that neurofilament density was comparable in fibers of all calibers, indicating that the radial growth of these myelinated nerve fibers correlates with a proportional increase in neurofilament content. We propose that progressive reduction in the velocity of neurofilament transport along the nerve provides for radial growth during development.