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PubMed1986

Prenatal development of retinal ganglion cell axons: segregation into eye-specific layers within the cat's lateral geniculate nucleus.

The morphological changes in individual retinal ganglion cell axons associated with the formation of the eye-specific layers in the dorsal lateral geniculate nucleus (LGN) were studied during the prenatal development of the cat's visual system. Previous work has shown that the pattern of segregated eye inputs found in the adult arises from an immature state in which inputs from the two eyes are intermixed within the nucleus (Shatz, 1983). Here, this developmental process is examined at its fundamental unit of connectivity--the individual retinal ganglion cell axon. To do so, an in vitro method was used to label fetal cat optic tract axons with HRP at various times during development between embryonic day 38 (E38) and postnatal day 2 (P2) (gestation = 65 d). The results presented here are based on reconstructions of 172 axons. During the initial period of intermixing (E38-43), axons are relatively simple in morphology. Many axons studied at the earliest ages (E38) end in growth cones and have very few branches along the main axon trunk as they traverse the nucleus. By E43, the number of side branches given off along the main axon trunk has increased and most axons also have a simple terminal arbor. Over the next 2 weeks (E43-55), the majority of axons are studded with side branches and the terminal arbor is well defined. Then, between E55 and birth, axons lose their side branches and the eye-specific layers appear. By birth, nearly all axons have a smooth trunk and an elaborate terminal arbor restricted to the LGN layer appropriate to the eye of axon origin. When the number of side branches per axon was quantified, the time course of appearance and subsequent loss of side branches was found to parallel the time course of the initial intermixing of inputs and subsequent reduction in territory shared by the two eyes as determined from previous intraocular injection experiments. Our results also showed that the side branches along each axon were located primarily within LGN territory destined to be occupied by the other eye. Thus, the side branches are likely to represent a morphological substrate for the intermixing of inputs from the two eyes. These observations suggest that the segregation of eye input to the LGN involves two fundamental and simultaneous events. One event is the remodeling of the branching pattern along the length of the main axon trunk so that the side branches present early on are eliminated and the main axon trunk becomes smooth.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1998

Axon order in the visual pathway of the quokka wallaby.

Axon order throughout the visual pathway of the quokka wallaby (Setonix brachyurus) was determined after localised retinal applications of the tracers DiI and/or DiASP. Postnatal days (P) 22-90 were studied to encompass the development and refinement of retinal projections. Order was essentially similar at all stages. Axons entered the optic nerve head true to their sector of retinal origin. In the optic nerve, nasal and temporal axons continued to reflect their retinal origin, dominating, respectively, the medial and lateral halves. By contrast, dorsal and ventral axons exchanged locations between the retrobulbar level and one-third the distance along the nerve; thus, the inversion of the dorsoventral retinal axis, imposed by the lens, was corrected. Decussating axons maintained their relative locations through the chiasm. At the base of the optic tract, nasal and temporal axons underwent an axial rotation to lie on the medial and lateral sides, respectively; thus nasal overlapped with ventral axons and temporal with dorsal axons. Axons maintained their alignments throughout the tract, and as a result, nasal and ventral axons invaded the superior colliculus medially, whereas temporal and dorsal axons invaded laterally. Each retinal quadrant terminated preferentially in its retinotopically appropriate sector of the colliculus. The arrangement of axons in the quokka visual pathway displays several novel features. Axon order is distinct throughout, involving a well-demarcated exchange of dorsal and ventral axons in the nerve and an axial rotation of nasal and temporal axons at the base of the tract; these relocations suggest decision regions for growing axons. The organisation presumably underlies the less extensive searching within the developing superior colliculus to generate retinotopic maps in the quokka and also in tammar wallaby [Marotte, J. Comp Neurol. 293:524-539, 1990] than in the rat [Simon and O'Leary, J. Neurosci. 12:1212-1232, 1992].

Animals↗
PubMed2003

Ascending sensory, but not other long-tract axons, regenerate into the connective tissue matrix that forms at the site of a spinal cord injury in mice.

Mice exhibit a unique wound healing response following spinal cord injury in which the lesion site fills in with a connective tissue matrix. Previous studies have revealed that axons grow into this matrix, but the source of the axons remained unknown. The present study assesses whether any of these axons were the result of long tract regeneration. C57Bl/6 mice received crush injuries and were allowed to survive for 6 weeks to 7 months. Biotinylated dextran amine (BDA) was injected into the somato-motor cortex to trace descending corticospinal tract (CST) axons, into the midbrain to label descending brainstem pathways including the rubrospinal and reticulospinal tracts, or into the L5 dorsal root ganglion to trace ascending projections of first-order sensory neurons. Spinal cords from other mice were prepared for immunocytochemistry using antibodies against neurofilament protein (NF), 5-HT to reveal descending serotonergic axons, calcitonin gene-related protein (CGRP) to reveal ascending sensory axons, and chondroitin sulfate proteoglycan (CSPG) to assess the distribution of molecules that are inhibitory to axon growth. NF immunostaining revealed axons in the connective tissue matrix at the lesion site, confirming previous studies that used protargol staining. CST axons did not enter the connective tissue matrix, but did sprout extensively in segments adjacent to the injury site. Rubrospinal and reticulospinal tract axons also did not grow into the lesion site. 5-HT-positive axons extended to the edge of the lesion, and a few axons followed astrocyte processes into the margins of the lesion site. In contrast to the other pathways, BDA-labeled ascending sensory axons did extend into and arborized extensively within the connective tissue matrix, although the subgroup of ascending axons that are positive for CGRP did not. These results indicate that the connective tissue matrix is permissive for regeneration of some classes of ascending sensory axons but not for other axonal systems.

Animals↗
PubMed1978

Growth pattern of axons in the optic nerve of chick during myelogenesis.

The purpose of this experiment was to study the diameter of axons at the time of the initiation of myelin and the pattern of growth of axons in the optic nerve of the chick. Embryos between 15 and 20 days and chicks 3, 5, 22 and 60 days of age were studied on the electron microscopic level. Based on axon diameter a unimodal distribution of unmyelinated axons is present through day 20 of incubation with a mean of approximately 0.35 micrometer. This population is represented through 22 days of age but from day 3 on, a second distinct population of unmyelinated axons is present which has a mean diameter that is approximately twice that of the smaller unmyelinated axons. All axons do not increase simultaneously in diameter but once growth starts, the unmyelinated axons apparently double in diameter at a relatively rapid rate prior to myelination. On incubation day 17 less than 1% of the axons in the optic nerve is myelimated. The number of axons in this group and their diameter (mean approximately 1.2 micrometer) remain relatively constant through day 3 but from days 5 through 22, two distinct populations of myelinated axons are present. By day 60, three distinct distributions of myelinated axons are present with mean diameters of 0.51 micrometer, 1.76 micrometer, and 3.90 micrometer. These populations represent approximately 20%, 67%, and 13% respectively of the total fiber population. As age increases the diameter of some myelinated axons is as small as or smaller than the unmyelinated axons at an earlier period in development. This suggests that factors other than axon diameter might be involved in the start of myelination. It appears that the increase in axon diameter does not occur in a continuous manner but in a saltatory manner from one size to another.

Animals↗
PubMed1983

The morphology of corticofugal axons to the dorsal lateral geniculate nucleus in the cat.

The structural features of corticogeniculate axons were studied in adult cats after labeling them with horseradish peroxidase (HRP). Injections of HRP into the optic radiations near the dorsal lateral geniculate nucleus result in Golgi-like filling of both geniculate relay neurons and corticogeniculate axons. In the present material at least two main types of axons could be defined. The most common type is called the type I axon because it so closely resembles the type I axons described by Guillery ('66, '67) in Golgi preparations. These fine axons have smooth surfaces and consistent fiber diameter. Most terminal swellings are at the ends of short collateral branches and these swellings form asymmetric synaptic contacts onto small and medium-sized dendrites. Type I axons typically innervate more than one lamina as well as interlaminar zones and they clearly arise from the cerebral cortex. The second type of axon is called the beaded axon because of its numerous swellings, en passant. These swellings frequently are larger than those on type I axons and they differ from previously described corticogeniculate axon terminals in their ultrastructural features. That is, their synaptic contacts appear symmetrical and they form axosomatic contacts. Because of these differences, the possibility that beaded axons are of subcortical origin, particularly from the perigeniculate nucleus, is discussed. When type I axons and geniculate relay neurons are filled in the same region of the nucleus it is possible to identify probable sites of synaptic contact by using the light microscope. Such analyses indicate that corticogeniculate axons synapse directly onto relay cells, primarily on peripheral dendritic branches. Further, it appears that single axons contact many geniculate neurons and that single neurons are contacted by many axons.

Animals↗
PubMed1987

Distribution of axons according to diameter in the monkey's optic tract.

The distribution of axonal diameters in the optic tract of Old World monkeys was examined by light and electron microscopy. Axon diameters were measured in samples of 100 axons taken from several locations in a cross section of the tract about 5 mm behind the optic chiasm. Fine-caliber axons (less than 1.75 micron in diameter) were found in all parts of the tract. Dorsally no coarse axons were present. Further ventrally, coarse axons gradually appeared and increased steadily in proportion. The largest optic axons (greater than 2.5 micron) were found in the most ventral parts of the tract, near the pial surface. This pattern of segregation of axons of differing diameters in the optic tract is a rearrangement of the distribution of axon diameters seen in the nerve rather than a continuation of the same pattern. Examination of axon diameters in the optic nerve has shown that there is a preponderance of fine axons centrally, while coarser axons are found in the periphery, near the pial surface; however, histograms from central parts of the nerve contain a greater proportion of coarse axons than the dorsal parts of the optic tract, while histograms from the periphery of the optic nerve contain a conspicuously greater proportion of fine axons than do histograms from the most ventral parts of the tract. This relatively greater segregation of axons according to diameter in the optic tract demonstrates that the distribution of axons in the tract cannot be formed by the simple combination of two hemiretinal maps contained in each optic nerve, as suggested in classic descriptions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

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.

Animals↗
PubMed1992

Central projections of axons from taste hairs on the labellum and tarsi of the blowfly, Phormia regina Meigen.

Taste hairs are located on the labellum and tarsi of blowflies. These multimodal hairs consist of four functionally distinct chemoreceptors and a mechanoreceptor. By staining selected multimodal hairs, we sought to identify the central projection patterns of multiple and single axons from those hairs. On each side of the labellum there are 11 "largest" hairs (LH). The neurons associated with the anteriormost (LH-1), posteriormost (LH-11), and one lateral (LH-6) hair on the labellum were stained selectively with cobaltous sulfide. The overall projection pattern in the central nervous system (CNS) for axons from LH-1 and LH-11 is similar and differs markedly from axons from LH-6. At least three individual axon-projection patterns were determined for each labellar hair filled, indicating a partial functional organization for axons from multimodal hairs. One identified axon, the dorsalmost axon, has terminal arborizations that do not differ with the location of its associated hair. Another axon, thicker than the others, projects to a region that is distinct from the four thin axons. Within this region the arborizations of the thick axons occupy different areas depending on the location of their associated hair. Neurons from the largest hairs on the distalmost tarsomere (D5) of each leg were also stained and consisted of one thick and four thin axons. All axons except one thin axon from tarsal D5 hairs terminate in their respective leg neuromeres. The remaining thin axon projects to the suboesophageal ganglion ipsilateral to the hair filled and terminates in the same region as a branch of the labellar dorsalmost axon. These data suggest that axonal arbors from multimodal hairs have a limited functional and somatotopic organization in the blowfly CNS.

Animals↗
PubMed1995

Differential regenerative response of Purkinje cell and inferior olivary axons confronted with embryonic grafts: environmental cues versus intrinsic neuronal determinants.

Regeneration of severed central axons is supposed to depend on two factors: a permissive local environment and the particular intrinsic properties of axotomized neurones. To assess the role of each of these factors in axonal regeneration, the capability of two particular axon populations of the adult mouse cerebellum to grow into target-specific (cerebellum) and target-unspecific (neocortex) embryonic grafts was determined. Purkinje cell and inferior olivary axons were transected by passing a microscalpel through the axial white matter of the cerebellar folia, particularly those of the anterior lobe. Immediately after the injury, solid transplants were placed in the lesion cavity. Purkinje cell axons were labelled by using anticalbindin immunocytochemistry, and olivocerebellar fibres were visualized by biotinylated dextran amine anterograde axonal tracing. Following axotomy, Purkinje cell axons appeared as thickened processes ending with large terminal clubs. Their morphology and number did not change up to the longest survival time considered (2 months), thereby confirming previous demonstrations that Purkinje cells survive axon injury (I. Dusart and C. Sotelo, 1994, J. Comp. Neurol. 347:211-232). Inferior olivary axons were thinner and bore smaller terminal bulbs. When embryonic cerebellar grafts, containing cortical and deep nuclear precursors, were placed close to the injured axons, olivocerebellar fibres vigorously regenerated into the transplants and ended in new climbing fibres along the dendrites of grafted Purkinje cells. By contrast, host Purkinje cell axons never showed any outgrowth towards the graft. Similarly, these axons failed to regenerate into grafts containing solely the rostromedial portion of the cerebellar anlage, mostly consisting of deep nuclear neurones, their main targets. Comparable results were obtained by transplanting embryonic neocortical tissue: inferior olivary axons also regenerated into the grafts, although with distinct terminal arbours without the climbing fibre phenotype, whereas Purkinje cell axons always failed to grow. These results provide the first direct demonstration that severed inferior olivary axons are able to regenerate. In addition, they show that the growth-permissive/-promoting conditions created by embryonic nervous tissue are not sufficient to induce the regeneration of every axonal type and allow us to hypothesise that successful regeneration depends on the interplay between environmental cues and intrinsic properties of the axotomized neurones.

Animals↗
PubMed1986

Axonal contribution to subthreshold currents in Aplysia bursting pacemaker neurons.

The contribution of axonal activity to the ionic currents which generate bursting pacemaker activity was studied by using the two-electrode voltage-clamp technique in Aplysia bursting neuron somata in conjunction with intraaxonal voltage recordings. Depolarizing voltage-clamp pulses applied to bursting cell somata triggered axonal action potentials. The voltage-clamp current recording exhibited transient inward current "notches" corresponding to each of the axonal spikes. The addition of 50 microM tetrodotoxin (TTX) to the bathing medium blocked the fast axonal spikes and current notches, revealing a slower axonal spike which was blocked by the replacement of external Ca2+ with Co2+. The inward current evoked by applying a depolarizing voltage-clamp pulse in the soma is distorted by the occurrence of the axonal Ca2+ spike. Elimination of the axonal spike, by injecting hyperpolarizing current into the axon, changes both the time course and the magnitude of the inward current. The axonal Ca2+ spikes are followed by a series of Ca2+-dependent afterpotentials: a rapid postspike hyperpolarization, a depolarizing afterpotential (DAP) and, finally, a long-lasting postburst hyperpolarization. The long-lasting hyperpolarization is not blocked by 50 mM external tetraethyl ammonium, an effective blocker of Ca2+-activated K+ current [IK(Ca)], and does not appear to reverse at EK. Hence, the axonal long-lasting hyperpolarization may not be due to IK(Ca). Somatic voltage-clamp pulses in bursting neurons are followed by a slow inward tail current, which is sometimes coincident with a DAP in the axon. In some cells, the amplitude of the slow inward tail current is greatly reduced if axonal spikes and DAPs are prevented by hyperpolarization of the axon, while, in other cells, elimination of axonal activity has little effect. Therefore, the slow inward tail current is not necessarily an artifact of poor voltage-clamp control over the axonal membrane potential but probably results from the activation of an ionic conductance mechanism located partly in the axon and partly in the soma.

Action Potentials↗
PubMed1998

Distribution, size and number of axons in the optic pathway of ground squirrels.

The present study has examined the distribution of axons of differing sizes in the optic pathway of the ground squirrel. Axon diameters were measured from electron micrographs at various locations across sections of the optic nerve and tract, and total distributions and numbers were estimated. In both the nerve and tract, roughly 1.2 million optic axons were present. The population of optic axons had a unimodal size distribution, peaking at 0.9 microm in diameter and having an extended tail toward larger diameters. Local axon diameter distributions in the optic tract indicated distinct (though partially overlapping) axon diameter classes, including one of fine sizes peaking at 0.8-0.9 microm, a second of medium sizes peaking around 1.7-1.8 microm, and a third composed of the larger fibers with diameters up to 4.8 microm. The fine-caliber axons were found at all locations in the tract, and were the only axons present immediately adjacent to the pia, while the medium- and coarse-caliber axons were found at deeper locations. Curiously, the larger axons were found primarily in the medial parts of the tract, where axons from the dorsal retina normally course. A similarly restricted distribution of the larger axons was observed in the dorsotemporal parts of the optic nerve, suggesting that this difference in the tract may relate to an asymmetric distribution of ganglion cells on the retina giving rise to these axons. Measurements of axonal size taken within the optic fiber layer in dorsal and ventral parts of the retina confirmed this asymmetry, consistent with previous demonstrations of soma size differences in the dorsal versus ventral retina. The partial segregation of axons by size in the optic tract of the ground squirrel then reflects both the asymmetric distribution of retinal ganglion cell classes and the chronotopic reordering of optic axons that occurs within the chiasmatic region.

Animals↗
PubMed1987

Changes in the numbers of retinal ganglion cells and optic nerve axons in the developing albino rabbit.

In albino rabbits aged from the 16th postconceptional day (16PCD) to adulthood, the number of axons in the optic nerves were estimated from sample areas totalling 1-12% of the cross-sectional area of the nerve. On the 16PCD there are about 20,000 axons in the optic stalk. The number of axons in the retrobulbar part of the optic nerve reaches a peak value of 766,000 on the 23PCD, and then decreases to about 350,000 by the 32PCD (the day of birth). The number of axons does not change between the 32PCD and 50PCD, but thereafter it slowly decreases, reaching the adult number (294,000) by the 84PCD. A similar trend is apparent in pigmented animals. Thus, on the 25PCD there are 736,000 axons in the retrobulbar part of the optic nerve and the number decreases to 428,000 by the 31PCD. In the adult pigmented rabbit there are 280,000 axons in the optic nerve. In animals younger than the 32PCD, growth cones are present, and the number of axons in the prechiasmal part of the optic nerve was 8-22% lower than in the retrobulbar part of the same nerve. These observations suggest that there is a continued outgrowth of axons from the eye towards the target nuclei. By the 32PCD, the numbers of axons in the retrobulbar and prechiasmal parts of the nerve were very similar, suggesting that by this age all axons had reached the chiasm. The numbers of retinal ganglion cells (RGCs) labelled by massive injections of horseradish peroxidase into the retino-recipient nuclei were estimated in albino rabbits aged from the 24PCD to adulthood. RGCs were counted in evenly spaced sample areas totalling 4-11% of the retinal area. On the 24PCD, the number of labelled RGCs (500,000) was lower than the number of axons in the optic nerve (probably because not all RGC axons had reached their target nuclei by this age). However, by the 27PCD the number of labelled RGCs (550,000) was very similar to the number of prechiasmal axons (568,000). At all ages thereafter, the numbers of both RGCs and axons were very similar, with adult RGC numbers (about 291,000) being reached by the 85PCD. We conclude that axon loss in the rabbit optic nerve after the 27PCD is almost certainly due to the elimination (presumably death) of the parent RGCs, and we suggest that RGC death is also the most likely cause of axon loss prior to the 27PCD.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗
PubMed1996

Expression of a developmentally regulated, phosphorylated isoform of microtubule-associated protein 1B in sprouting and regenerating axons in vitro.

We have developed a novel culture system for studying axonal regeneration. Short lengths of spinal nerves with their attached dorsal root ganglia were removed from adult mice, explanted into Matrigel and maintained in serum-free medium for up to eight days. Profuse outgrowth of unfasciculated, naked axons occurred within 6 h from the cut ends of the peripheral nerve, dorsal roots and eventually from the ganglion itself, and continued to grow throughout the observation period. Some axons were entirely smooth, whilst others showed prominent varicosities. The former stained with antibody RT97, a marker for large-calibre, myelinated axons, whilst the latter stained with antibodies to calcitonin gene-related peptide, predominantly a marker for unmyelinated and small-diameter myelinated sensory axons. All axons stained with a monoclonal antibody (150) that recognizes a developmentally regulated phosphorylated isoform of the microtubule-associated protein 1B [Gordon-Weeks P. R. et al. (1993) Eur. J. Neurosci. 5, 1302-1311]. Monoclonal antibody 150 staining was observed along the entire length of all axons growing out of the explant; the proximal regions of these axons within the explant itself did not stain. The staining extended to the growth cones, which had elaborate morphologies. Other antibodies (e.g. to growth-associated protein 43) labelled axons within the nerve, as well as those growing in Matrigel. In preparations where the peripheral nerve had been crushed half-way along its length at the time of explantation, monoclonal antibody 150 staining was absent from axons in the nerve proximal to the crush, but present in axons which had regenerated within the nerve distal to the crush. The results indicate that re-expression during axonal regeneration of the phosphorylated isoform of microtubule-associated protein 1B recognized by monoclonal antibody 150 is restricted to the newly formed lengths of regenerated axons. The correlation between its expression and axonal growth during development and regeneration suggests that it may play a role in axonal extension. Our observations also demonstrate the usefulness of these explant cultures for studying axonal regeneration.

Animals↗
PubMed1997

A mechanistic analysis of nondisruptive axonal injury: a review.

Axons are particularly at risk in human diffuse head injury. Use of immunocytochemical labeling techniques has recently demonstrated that axonal injury (AI) and the ensuing reactive axonal change is, probably, more widespread and occurs over a longer posttraumatic time in the injured brain than had previously been appreciated. But the characterization of morphologic or reactive changes occurring after nondisruptive AI has largely been defined from animal models. The comparability of AI in animal models to human diffuse AI (DAI) is discussed and the conclusion drawn that, although animal models allow the analysis of morphologic changes, the spatial distribution within the brain and the time course of reactive axonal change differs to some extent both between species and with the mode of brain injury. Thus, the majority of animal models do not reproduce exactly the extent and time course of AI that occurs in human DAI. Nonetheless, these studies provide good insight into reactive axonal change. In addition, there is developing in the literature considerable variance in the terminology applied to injured axons or nerve fibers. We explain our current understanding of a number of terms now present in the literature and suggest the adoption of a common terminology. Recent work has provided a consensus that reactive axonal change is linked to pertubation of the axolemma resulting in disruption of ionic homeostatic mechanisms within injured nerve fibers. But quantitative data for changes for different ion species is lacking and is required before a better definition of this homeostatic disruption may be provided. Recent studies of responses by the axonal cytoskeleton after nondisruptive AI have demonstrated loss of axonal microtubules over a period up to 24 h after injury. The biochemical mechanisms resulting in loss of microtubules are, hypothetically, mediated both by posttraumatic influx of calcium and activation of calmodulin. This loss results in focal accumulation of membranous organelles in parts of the length of damaged axons where the axonal diameter is greater than normal to form axonal swellings. We distinguish, on morphologic grounds, between axonal swellings and axonal bulbs. There is also a growing consensus regarding responses by neurofilaments after nondisruptive AI. Initially, and rapidly after injury, there is reduced spacing or compaction of neurofilaments. This compaction is stable over at least 6 h and results from the loss or collapse of neurofilament sidearms but retention of the filamentous form of the neurofilaments. We posit that sidearm loss may be mediated either through proteolysis of sidearms via activation of microM calpain or sidearm dephosphorylation via posttraumatic, altered interaction between protein phosphatases and kinase(s), or a combination of these two, after calcium influx, which occurs, at least in part, as a result of changes in the structure and functional state of the axolemma. Evidence for proteolysis of neurofilaments has been obtained recently in the optic nerve stretch injury model and is correlated with disruption of the axolemma. But the earliest posttraumatic interval at which this was obtained was 4 h. Clearly, therefore, no evidence has been obtained to support the hypothesis that there is rapid, posttraumatic proteolysis of the whole axonal cytoskeleton mediated by calpains. Rather, we hypothesize that such proteolysis occurs only when intra-axonal calcium levels allow activation of mM calpain and suggest that such proteolysis, resulting in the loss of the filamentous structure of neurofilaments occurs either when the amount of deformation of the axolemma is so great at the time of injury to result in primary axotomy or, more commonly, is a terminal degenerative change that results in secondary axotomy or disconnection some hours after injury.

Animals↗
PubMed1994

Differences in behaviour of sensory and motor axons following release of ischaemia.

The changes in excitability and supernormality of sensory and motor axons of the median (or ulnar) nerve were tracked during and following ischaemia at the wrist for periods of 5-20 min in normal human volunteers. Supernormality was defined as the fractional increase in excitability produced by a maximal conditioning stimulus, 10 ms before the test stimulus. With relatively brief periods of ischaemia (< 10 min), sensory and motor axons behaved similarly, with an increase in excitability (producing a decrease in threshold) and a decrease in supernormality during ischaemia and a long-lasting decrease in excitability (and increase in supernormality) following release of ischaemia. Most subjects reported paraesthesiae during brief periods of ischaemia but not after its release. No one experienced fasciculation. The threshold changes were generally similar during longer periods of ischaemia, but in the post-ischaemic phase the behaviour of sensory and motor axons diverged. After a rapid post-ischaemic increase, the threshold of sensory axons decreased, approaching the pre-ischaemic level, before rising again and then slowly returning to the control level. Sensory axons of different threshold behaved in a qualitatively similar manner, with no evidence of a bimodal distribution of thresholds in the post-ischaemic phase (as occurs with motor axons when the ischaemia is sufficient to produce fasciculation; see Bostock et al. J. Physiol (Lond) 1991; 441: 537-57). The 'notch' on the threshold plot for sensory axons lasted 20-40 min and was accompanied by a relatively small but appropriate change in supernormality. No such 'notch' was seen with motor axons. The changes in latency were generally similar for sensory and motor axons, largely paralleling the supernormality plots, except at the time of the 'notch'. To test the hypothesis that the differences in behaviour of sensory and motor axons resulted from differences in inward rectification activated by hyperpolarization, the changes in threshold produced by long-lasting (300 ms) depolarizing and hyperpolarizing current pulses were compared for sensory and motor axons. In seven of eight subjects, there was evidence of more inward rectification in sensory axons. In the eighth subject, motor axons behaved similarly to sensory axons. It is concluded that a difference in inward rectification contributes to but is insufficient by itself to account for the differences in behaviour of sensory and motor axons and that the greater propensity of sensor y axons to discharge ectopically cannot be attributed to a single factor.

Action Potentials↗
PubMed1989

Experimental increase of neurofilament transport rate: decreases in neurofilament number and in axon diameter.

In 2,5-hexanedione (2,5-HD)-induced axonal neuropathy, the rate of neurofilament (NF) transport increases in optic axons. To test the prediction that increases in the rate of polymer transport in any one locality of the axon lead directly to a decrease in the number of NF in that locality, NF and microtubules (MT) were quantitatively analyzed in axonal cross sections. In 2,5-HD axons the number of NF was 38% of that in control axons while the number of MT was not significantly changed; it appears that the drug treatment decreases NF number in the proximal axon regions, most directly through an increase in rate of NF transport. In those regions, the cross-sectional areas of the 2,5-HD-treated axons were 45% smaller than those of control axons; although the axons had shrunk in diameter, they retained their normal cylindrical shapes as measured by the index of circularity. Reduced internal expansive forces in the axon, working in conjunction with the normal external compressive forces, appear to reduce the radius of the axon. Quantitative analyses demonstrated that the average and the maximum lateral spacings between NF-NF, NF-MT, and MT-MT were all 30% larger in 2,5-HD-treated axons than in control axons. This suggests that polymers are relatively free to move laterally away from one another and to fill the available space within the axon. These observations are not consistent with models wherein 2,5-HD acts to crosslink the NF into an immobile network that can no longer advance within the axon. Instead, it appears more likely that 2,5-HD acts selectively on the interaction between some NF and the slow transport mechanism to increase the rate of NF transport.

Animals↗
PubMed1996

An analysis of an axonal gradient of phosphorylated MAP 1B in cultured rat sensory neurons.

The present study investigated the cellular distribution of a developmentally regulated phosphorylated form of MAP 1B recognized by monoclonal antibody (mAb) 150 in cultures of dorsal root ganglia. The cell soma and the whole axon, when it first appears, are labelled, but longer axons label with a proximodistal gradient, such that the cell soma and proximal axon become unlabelled, whilst the distal axon and growth cone label strongly. Double-labelling experiments with mAb 150 and a polyclonal antibody (N1-15) that recognizes all forms of MAP 1B demonstrated that MAP 1B is distributed along the entire length of axons with gradients, so the gradient of phosphorylated MAP 1B is not due to a loss or absence of MAP 1B from the proximal axon. The proportion of axons from 20 h cultures that were labelled with a mAb 150 gradient was at least 80% and this proportion was independent of the nerve growth factor concentration of the culture medium. Analysis of axons ranging in length from 100 to 700 microm and labelled with a gradient showed that the unlabelled proximal portions of axons increased in length more slowly than the labelled distal axon. Axons labelled along their entire length accounted for no more than 19% of th axonal population and analysis of these showed them to be frequently < 400 microm long. After simultaneously fixing and detergent-extracting cultures this proportion rose significantly to 93%, suggesting that in the proximal axon the mAb 150 epitope is masked by some factor(s) that is removed by detergent extraction. The possibility that mAb 150 could not access the epitope in the proximal axon was discounted because another IgM, mAb 125, which recognizes a different phosphorylation epitope on MAP 1B, labelled the proximal axon of conventionally fixed cultures. In growth cones of fixed and extracted neurons examined by immunofluorescence, the mAb 150 labelling strongly colocalized to bundled microtubules in the distal axon shaft and the C-domain. In the P-domain, mAb 150 staining was weaker and more widely distributed than the microtubules. Immunogold electron microscopy confirmed that antibody N1-15 and mAb 150 strongly labelled the bundled microtubules in the C-domain and also showed that individual microtubules in the P-domain, some of which lie alongside actin filament bundles of filopodia, were labelled lightly and discontinuously with both antibodies. This suggests that the phosphorylated isoform of MAP 1B recognized by mAb 150 may be microtubules and actin filaments in the P-domain.

Animals↗
PubMed1987

Sodium currents in axon-associated Schwann cells from adult rabbits.

1. Patch-clamp and electron-microscopic studies were carried out on individual axon-Schwann-cell complexes 2-6 h after they were isolated from the sciatic nerves of rabbits 5, 10 and 20 weeks old. 2. Under Hoffman modulation contrast optics Schwann cells associated with both myelinated and non-myelinated axons could be seen. Frequently, fine cable-like structures about 1 micron in diameter, which are presumably axons, could be seen in isolation from a Schwann cell. 3. Cross-sectional electron-microscopic studies directly demonstrated the presence of axons engulfed by Schwann cells. For Schwann cells associated with non-myelinated axons, multiple fine axons (approximately 1 micron) could be seen enclosed by one or few turns of spiralling tongues of Schwann cells. Schwann cells associated with a single large myelinated axon showed characteristic compact myelin wrappings. No membrane fusion between Schwann cells and the axons could be detected. 4. Giga-seals could readily be formed when a patch pipette was pressed against the body region of a Schwann cell associated with either non-myelinated or myelinated axons. In contrast, giga-seals were only infrequently obtained on fine cable-like structures (1 micron) visually identified to be separated from the Schwann cell body. 5. Whole-cell recordings made from the body region of a Schwann cell revealed a TTX-sensitive fast inward current. Intriguingly, the expression of this current appeared to be dependent on the type of associated axon; this current was detectable in virtually all recordings made at the body region of Schwann cells associated with small non-myelinated axons, but not from those associated with large myelinated axons. 6. The inward current was like a neuronal sodium current; it had voltage-gated kinetics similar to the Hodgkin-Huxley sodium current, and exhibited a reversal potential close to the expected Nernstian potential for sodium ions. 7. From the observed size of the whole-cell membrane capacity and the electron-microscopic observations that the surface area of the Schwann cell at the body region was much larger than that of a 1 micron non-myelinated axon, it was argued that the whole-cell recordings were from Schwann cells rather than from single axons. Furthermore, the peak sodium current density was similar to that of Schwann cells cultured from new-born rabbits in which axons were presumed to be absent. 8. The results suggested that Schwann cells normally associated with non-myelinated axons in the rabbit sciatic nerves maintain an active synthesis of neuronal-like sodium channels throughout normal development.

4-Aminopyridine↗