Removal of retrogradely transported material from rat lumbosacral alpha-motor axons by paranodal axon-schwann cell networks

The aim of this study was to investigate the potential ability of Schwann cells to sequester axonally transported material via so called axon-Schwann cell networks (ASNs). These are entities consisting of sheets of Schwann cell adaxonal plasma membrane that invade the axon and segregate portions of axoplasm in paranodes of large myelinated mammalian nerve fibres. Rat hindlimb alpha-motor axons were examined in the L4–S1 ventral roots using light/fluorescence, confocal laser, and electron microscopy for detection of retrogradely transported red-fluorescent latex nanospheres taken up at a sciatic nerve crush, and intramuscularly injected horseradish peroxidase endocytosed by intact synaptic terminals. Survival times after tracer administration ranged from 27 hours to 4 weeks. During their retrograde transport toward the motor neuron perikarya, organelles carrying nanospheres/peroxidase accumulated at nodes of Ranvier, where they often appeared in close association with the paranodal myelin sheath. Serial section electron microscopy showed that many of the tracer-containing bodies were situated within ASN complexes, thereby being segregated from the main axon. Four weeks after nanosphere administration, several node-paranode regions still showed ASN-associated aggregations of spheres, some of which were situated in the adaxonal Schwann cell cytoplasm. The data establish the ability of Schwann cells to segregate material from motor axons with intact myelin sheaths, using the ASN as mediator. Taken together with our earlier observations that ASNs in alpha-motor axons are also rich in lysosomes, this process would allow a local elimination and secluded degradation of retrogradely transported foreign substances and degenerate organelles before reaching the motor neuron perikarya. In addition, ASNs may serve as sites for disposal of indigestable material. GLIA 20:115–126, 1997. © 1997 Wiley-Liss Inc.     

Ultrastructural analysis of the paranodal junction of myelinated fibers in 31-month-old-rats

Recent studies have revealed a significant decrease in white matter volume, including loss of myelin, with age but minimal decrease in gray matter volume (Guttmann et al., [1998] Neurology 50:972-978). Myelin is necessary for the rapid conduction of impulses along axons. Myelinated nerve includes various domains, the node of Ranvier, the paranodal region, the juxtaparanodal region and the internode. The paranodal junction may serve to anchor the myelin sheath to the axon. We analyzed the ultrastructure of the paranodal region in myelinated fibers from the aged rat brain. Severe alterations of myelinated fibers were observed in 31-month-old rats, resulting in the appearance of macrophages, splitting of the myelin sheath, myelin balloon formation and separation from the axon. Many paranodal retractions of myelinated axons occurred in the aged rats. It should be noted that the paranodal junction is functionally important, serving to anchor the myelin to the axon and that there is a diffusion barrier in the paranodal region. We analyzed myelin-related proteins from young and aged rat brains. The 21.5-kDa isoform of myelin basic protein (MBP) almost disappeared in the 31-month-old rats, whereas other myelin proteins were not significantly changed between young and aged rats. These results suggest that this isoform, a highly cationic charged major dense component protein that binds lipid bilayer in the membrane, may participate in the formation of a paranodal diffusion barrier at the myelin/noncompact membrane border.     

Nodes of ranvier above a neuroma in the rat sciatic nerve: Voltage clamp analysis and electron microscopy

Neuroma formation was induced in adult rat sciatic nerves and the animals were allowed to survive for 1-10 months. In 10 animals single large myelinated fibres from the nerve segment above the neuroma were subjected to voltage clamp analysis. Six animals were fixed by glutaraldehyde perfusion and nodes of Ranvier or large myelinated fibres above the neuroma were examined in the electron microscope (EM). Most fibres exhibited normal action potentials, but a few had a reduced excitability and small action potentials. Some fibres had increased membrane time constant and leak conductance and a markedly increased membrane capacitance. Most of the examined nodes of Ranvier exhibited abnormally large delayed K currents, which could be blocked with 4-aminopyridine (4-AP). The Na current was normal. In the EM most large cross-cut myelinated axons were markedly atrophic, particularly after long survival times. Evaginations from the paranodal region of these axons penetrated between the terminating paranodal myelin lamellae. The nodal axolemmal undercoating could be very prominent and in some cases the nodal axon was irregular. These findings show that large myelinated peripheral nerve fibres, which are chronically disconnected from their peripheral targets, exhibit specific structural and functional abnormalities of the nodes of Ranvier.     

Lysosomal activity at nodes of Ranvier in dorsal column and dorsal root axons of the cat after injection of horseradish peroxidase in the dorsal column nuclei.

The occurrence of acid phosphatase (AcPase)-positive bodies, i.e. lysosomes, in dorsal column and dorsal root axons of the spinal cord segments C8 and L7 in adult cats was analyzed by light and electron cytochemical methods after injection of horseradish peroxidase (HRP) in the dorsal column nuclei. Axonal lysosomes were, with few exceptions, concentrated at the nodes of Ranvier. We found no changes in nodal occurrence and distribution of lysosomes in axons of the HRP-injected sides, as compared to axons of the uninjected sides or of animals not exposed to HRP. Axonal lysosomes were very rare in the dorsal columns, where the frequency of nodes containing light microscopically detectable AcPase-positive bodies was 0-5% at the HRP-injected sides, 0-6% at the contralateral sides, and 0-3% in control animals. The corresponding values in the cervical and lumbar dorsal roots were 6-23%, 9-20%, 10-12% and 19-37%, 21-40%, 26-43%, respectively. In view of our recent observations in alpha-motor neurons, the results point at a noteworthy difference in local degradative ability between dorsal column axons and alpha-motor axons, the latter being able to accumulate intramuscularly injected and retrogradely transported HRP at their PNS nodes of Ranvier for 48-60 h, during which period the axoplasmic AcPase activity/concentration increases at some nodes. Such a degradative activity, which could protect the motor neurons by restricting axoplasmic transport of exogenous materials imbibed by their axon terminals outside the CNS, may not be of the same significance for neurons, e.g. dorsal root ganglion neurons, the axon terminals of which are located within the CNS.     

Lysosomal activity in developing cat alpha-motor axons under normal conditions and during retrograde axonal transport of horseradish peroxidase.

The occurrence of acid phosphatase (AcPase)-positive bodies, i.e., lysosomes, in lumbosacral alpha-motor axons of kittens, 0-16 weeks of age, was analyzed by light and electron cytochemical methods under normal conditions and after intramuscular injection of horseradish peroxidase (HRP). Axonal lysosomes were rare early postnatally. In 3-week-old animals, a few AcPase-positive bodies appeared in the axoplasm at some nodes of Ranvier in the peripheral nervous system (PNS) and internodally in the intrafunicular motor axon parts within the central nervous system (CNS). From 6 weeks postnatally, a nodal concentration of AcPase-positive bodies was also noted in the CNS. The number of AcPase-positive bodies continued to increase gradually in the course of neuronal maturation. In 16-week-old animals, axonal AcPase activity was still at considerably lower levels than at adult stages. At all ages, acid hydrolase-containing organelles were most commonly found at ventral root nodes. After injection of HRP in the medial gastrocnemius muscle, accumulations of AcPase-positive bodies were seen in the axoplasm at some PNS nodes of the HRP-injected sides of kittens aged 8, 12, and 16 weeks. Incubation for demonstration of both HRP and AcPase activity showed that some organelles at HRP-transporting nodes contained both types of reaction product. The nodal AcPase activity in the intrafunicular, CNS parts of alpha-motor axons of the HRP-exposed sides did not differ from that of the contralateral, uninjected sides. In view of our previous observations in alpha-motor neurons of adult cats in which a lysosome-mediated degradation of axonally transported materials may take place at PNS nodes of Ranvier, the present study illuminates possible differences in the ability to interfere with axonal transport between developing and mature neurons. The infrequent presence of lysosomes in developing alpha-motor axons and the implied disability of their nodal regions to interfere with axonally transported constituents in a way similar to that seen in adult animals may be of significance in that trophic and chemical signals can pass unhindered between the periphery and perikaryon. However, this could also have negative consequences for the vulnerable immature neuron in that various materials retrieved at the axon terminals outside the CNS are permitted a more-or-less free access to the perikaryon.     

Differential expression of gangliosides on the surfaces of myelinated nerve fibers

The binding of cholera and tetanus toxins to receptors on the surfaces of teased nerve fibers was used to localize GM1 and G1b-series gangliosides, respectively, by immunocytochemical methods. Native fibers and fibers treated with various hydrolytic enzymes to degrade specific surface components were studied. With native fibers, both toxins bound abundantly to nodes of Ranvier and poorly to the most external, internodal Schwann cell surfaces. Treatment of the fibers with proteases, hyaluronidase, and chondroitin ABC lyase neither eliminated receptors at the nodes nor unmasked receptors over the internodes. The axolemma underlying the paranodal or internodal myelin, exposed by extensive treatment with protease, bound both toxins in large amounts. Neuraminidase action induced cholera toxin receptors on the Schwann cell surface; these receptors were insensitive to protease. The results indicate that GM1 and G1b-series gangliosides are predominantly localized to axonal and glial structures of the node of Ranvier and to paranodal/internodal Axolemma, and that polysialogangliosides not of the G1b-series are present on the internodal Schwann cell surface.     

Regeneration of the node of Ranvier: a light and electron microscope study

Summary Regeneration of the node of Ranvier was investigated in the rat peroneal nerve 10–60 days after nerve crush, by light and electron microscopy. At 10 and 20 days after crush nodes of Ranvier were clearly identifiable by electron microscopy but had a relatively simple structure. At 40 days after crush however nodes were highly differentiated showing specialised features such as paranodal bulbs, nodal constriction of the axon, paranodal Schwann cell mitochondria, nodal Schwann cell microvilli, and nodal gap substance. By light microscopy some nodes were identifiable as early as 20 days after crush. At both 30 and 60 days after crush regenerated internodes were uniformly short (means of 275 m and 339 m respectively).     

ATYPICAL AXON-SCHWANN CELL RELATIONSHIPS IN THE COMMON PERONEAL NERVE OF THE DYSTROPHIC MOUSE: AN ULTRASTRUCTURAL STUDY

Abstracts Jaros E. & Bradley W.G. (1979) Neuropathology and Applied Neurobiology 5, 133–147
Atypical axon-Schwann cell relationships in the common peroneal nerve of the dystrophic mouse: an ultrastructural study
Several atypical features of myelination of the peripheral nervous system are reported in common peroneal nerve of dystrophic mice (129 Re J dy/dy): ( i ) central nervous system-like contact between myelin sheaths of adjacent nerve fibres; ( ii ) nodes and internodes of myelinated fibres enwrapped with cytoplasmic processes of Schwann cells from adjacent nerve fibres; ( iii ) Schwann cells of adjacent nerve fibres co-operating in formation of a single myelin sheath; and ( iv ) a single Schwann cell myelinating two separate axons. In view of the presence of similar features of myelination in the central nervous system, where the myelin producing cells lack basement membrane, we suggest that in the dystrophic peripheral nerves the development of these features can be attributed to the partial deficiency of the Schwann cell basement membrane. Two types of widened nodes of Ranvier are also identified: ( i ) nodes with paranodal damage; and ( ii ) nodes without paranodal damage. In addition, abnormal features of myelination are described which are likely to represent altered Schwann celliaxon relationships during demyelination and remyelination and/or decreased myelinating ability of Schwann cells. We interpret these findings as indicating a metabolic disorder of Schwann cells. They provide an experimental model for the investigation of factors involved in the origin and maintenance of the structural organization of peripheral nerve.     

Ion channels in axon and Schwann cell membranes at paranodes of mammalian myelinated fibers studied with patch clamp.

While recent studies have established the presence of voltage-gated ion channels on Schwann cells in culture and on freshly isolated fibers from mature mammals, an important issue not yet explored is whether Schwann cell channels are regionally specialized. In the nodal region, the intimate association between the Schwann cell and its axon suggests that this is a likely site for functional specialization. Here, we examine whether there is a localized expression of channels in the Schwann cell paranodal regions, in a manner similar to that already shown for the nodal axon. Cell-attached and outside-out patch-clamp recordings were made from paranodal regions of rat myelinated sciatic nerve fibers where the myelin on both sides of the node was retracted by enzymatic treatment. Even though no myelin was visible on the surface of the retracted paranode, significant portions of this surface were found to stain positively with a marker (anti-galactocerebroside) for Schwann cell membranes, suggesting that part of the axon still was covered by glial membranes. Using Lucifer yellow in the recording pipettes, we observed that the dye diffused into either axons or Schwann cells when the membrane under the tip was ruptured. Using this as a criterion to identify membranes obtained from retracted paranodes, we found delayed and inwardly rectifying potassium channels on both axon- and Schwann-derived patches. However, sodium channels were detected only in axon patches. This is the first report that voltage-gated glial channels are present in immediate vicinity to axons of the PNS. This finding, coupled with earlier reports that functional channels are absent in soma of mature myelinating Schwann cells, suggests that ion channels in these cells are regionally specialized for functional interaction with axons.     

Gangliosides contribute to stability of paranodal junctions and ion channel clusters in myelinated nerve fibers

Paranodal axo-glial junctions are important for ion channel clustering and rapid action potential propagation in myelinated nerve fibers. Paranode formation depends on the cell adhesion molecules neurofascin (NF) 155 in glia, and a Caspr and contactin heterodimer in axons. We found that antibody to ganglioside GM1 labels paranodal regions. Autoantibodies to the gangliosides GM1 and GD1a are thought to disrupt nodes of Ranvier in peripheral motor nerves and cause Guillain-Barré syndrome, an autoimmune neuropathy characterized by acute limb weakness. To elucidate ganglioside function at and near nodes of Ranvier, we examined nodes in mice lacking gangliosides including GM1 and GD1a. In both peripheral and central nervous systems, some paranodal loops failed to attach to the axolemma, and immunostaining of Caspr and NF155 was attenuated. K(+) channels at juxtaparanodes were mislocalized to paranodes, and nodal Na(+) channel clusters were broadened. Abnormal immunostaining at paranodes became more prominent with age. Moreover, the defects were more prevalent in ventral than dorsal roots, and less frequent in mutant mice lacking the b-series gangliosides but with excess GM1 and GD1a. Electrophysiological studies revealed nerve conduction slowing and reduced nodal Na(+) current in mutant peripheral motor nerves. The amounts of Caspr and NF155 in low density, detergent insoluble membrane fractions were reduced in mutant brains. These results indicate that gangliosides are lipid raft components that contribute to stability and maintenance of neuron-glia interactions at paranodes.     

Peroxidase activity at CNS nodes of Ranvier and in initial axon segments of lumbosacral alpha-motoneurons after intramuscular administration of horseradish peroxidase

The occurrence of peroxidase activity in central (CNS) and peripheral nervous system (PNS) parts of alpha-motor axons was studied by light and electron microscopy in adult cats after injection of horseradish peroxidase (HRP) into the medial gastrocnemius muscle. The intrafunicular parts of the axons were virtually free of HRP-positive bodies except at a few nodes of Ranvier. Most of these nodes were weakly HRP-positive and contained, irrespective of a survival time between 25 and 48 h, only a few HRP-positive bodies randomly scattered in the nodal axoplasm. In contrast to this and as described elsewhere (J. Neurocytol., 15 [1986] 253-260), the nodal regions of alpha-motor axons at the level of the ventral root showed strong and characteristic accumulations of HRP-activity. The initial axon segments and adjoining axonal parts contained many HRP-positive bodies. We conclude that the CNS and the PNS parts of an alpha-motor axon differ with regard to the way nodal regions interact with retrogradely transported HRP. Possible mechanisms behind this difference are discussed.     

Schwann Cells Provide Iron to Axonal Mitochondria and Its Role in Nerve Regeneration

Iron is an essential cofactor for several metabolic processes, including the generation of ATP in mitochondria, which is required for axonal function and regeneration. However, it is not known how mitochondria in long axons, such as those in sciatic nerves, acquire iron in vivo. Because of their close proximity to axons, Schwann cells are a likely source of iron for axonal mitochondria in the PNS. Here we demonstrate the critical role of iron in promoting neurite growth in vitro using iron chelation. We also show that Schwann cells express the molecular machinery to release iron, namely, the iron exporter, ferroportin (Fpn) and the ferroxidase ceruloplasmin (Cp). In Cp KO mice, Schwann cells accumulate iron because Fpn requires to partner with Cp to export iron. Axons and Schwann cells also express the iron importer transferrin receptor 1 (TfR1), indicating their ability for iron uptake. In teased nerve fibers, Fpn and TfR1 are predominantly localized at the nodes of Ranvier and Schmidt-Lanterman incisures, axonal sites that are in close contact with Schwann cell cytoplasm. We also show that lack of iron export from Schwann cells in Cp KO mice reduces mitochondrial iron in axons as detected by reduction in mitochondrial ferritin, affects localization of axonal mitochondria at the nodes of Ranvier and Schmidt-Lanterman incisures, and impairs axonal regeneration following sciatic nerve injury. These finding suggest that Schwann cells contribute to the delivery of iron to axonal mitochondria, required for proper nerve repair.SIGNIFICANCE STATEMENT This work addresses how and where mitochondria in long axons in peripheral nerves acquire iron. We show that Schwann cells are a likely source as they express the molecular machinery to import iron (transferrin receptor 1), and to export iron (ferroportin and ceruloplasmin [Cp]) to the axonal compartment at the nodes of Ranvier and Schmidt-Lanterman incisures. Cp KO mice, which cannot export iron from Schwann cells, show reduced iron content in axonal mitochondria, along with increased localization of axonal mitochondria at Schmidt-Lanterman incisures and nodes of Ranvier, and impaired sciatic nerve regeneration. Iron chelation in vitro also drastically reduces neurite growth. These data suggest that Schwann cells are likely to contribute iron to axonal mitochondria needed for axon growth and regeneration.     

Ultrastructural evidence of a peripheral nervous system pattern of myelination in the avascular retina of the guinea pig

Summary Myelination of axons in the nerve fiber layer (NFL) of the retina occurs as a sporadic abnormality in several mammalian species including man, monkey, cat and rat. All of these species have vascularized retinae and, in the latter three, ultrastructural studies have demonstrated the pattern of medullation to be similar to Schwann cell myelination in the peripheral nervous system. This contrasts with an oligodendrocytic pattern of myelination normally present in the avascular retina of the rabbit. One possible explanation for this difference is that the pattern of myelination is related to the presence or absence of retinal blood vessels. The present investigation provides the first evidence of NFL myelination in another avascular retina, that of the guinea pig. Myelination in the guinea pig retina was observed in a single bundle of axons and involved only large diameter fibers. With several axons, myelin sheaths terminated at hemi-nodes of Ranvier and in all such cases this occurred in association with marked paranodal infolding. Morphological characteristics of the myelination include (1) a one to one relationship between axon and myelinating cell, (2) cytoplasm between myelin sheath and plasma membrane, (3) basal lamina surrounding the myelinating cell, (4) collagen fibers in the adjacent extracellular space and (5) double intraperiod lines. These morphological features are characteristic of peripheral nerve myelination by Schwann cells. Thus, in all species so far described in which retinal medullation is abnormally present, the pattern of myelination has been Schwann cell in nature rather than oligodendrocytic. The reasons for this Schwann cell predominance remain undefined.Supported by the Medical Research Council of Canada     

Features associated with paranodal demyelination at a specialized site in the non-pathological nervous system

Experimental demyelination in the CNS and PNS have been shown in some cases to exhibit a paranodal distribution. The electric organ of the gymnotid Sternarchus is composed of specialized axons which generate external electric fields. The structure of the nodes of Ranvier changes characteristically along the course of these specialized non-pathological axons. The nodes of Ranvier in two locations along the fibers are markedly enlarged. At the enlarged nodes, but not at normal nodes from the same fibers, the paranodal myelin exhibits morphological features associated with paranodal demyelination. These features include termination of the innermost myelin lamellae at distances of up to 200 μm from the nodal gap. The results indicate that these morphological findings are not necessarily associated with pathological demyelination, and suggest that remodeling of the myelin sheath, including programmed demyelination, may play a role in the development of certain specialized neural systems.     

Immunolocalization of ciliary neuronotrophic factor in adult rat sciatic nerve.

Two rabbit polyclonal antibodies were raised against synthetic peptides corresponding to residue numbers 45-59 and 181-200 of rat ciliary neuronotrophic factor (CNTF). The resulting antibodies were purified by affinity chromatography and both purified antibodies reacted by enzyme-linked immunoassay (ELISA) and immunoblotting with rat sciatic nerve CNTF. The anti-CNTF peptide antibodies were used to immunostain sections of adult rat sciatic nerve, previously known as the richest tissue source of CNTF. By light microscopy both antibodies appeared to stain exclusively Schwann cells and axons and both did so with the same pattern of specific staining. Immunostaining was eliminated by absorption of the anti-peptide antibodies with either their corresponding peptide or with purified rat nerve CNTF or by using purified nonspecific IgG. Schwann cells were stained and in semi-thin sections this staining appeared to be in the Schwann cell cytoplasm. Axons could be stained in addition to Schwann cells providing higher concentrations of antibodies were used. Epineurial, endoneurial and endothelial cells appeared unstained. Since all Schwann cells and axons appear to contain CNTF and since CNTF is known to act in vitro to support sensory and sympathetic ganglionic and motor neurons, we suggest that Schwann cells may normally provide CNTF to those neurons contributing axons to the peripheral nerve.     

Schwann cell proliferation and migration during paranodal demyelination

This study examined Schwann cell behavior during paranodal demyelination induced by beta,beta'-iminodipropionitrile (IDPN). The stimuli for Schwann cell proliferation, extensively studied in vitro, are less well understood in vivo. Most in vivo systems previously used to examine Schwann cell proliferation in disease are dominated by loss of internodal myelin sheaths. As used in this study, IDPN administration produces neurofilamentous axonal swellings and paranodal demyelination, without segmental demyelination or fiber degeneration. We asked whether Schwann cells would proliferate following the restricted paranodal demyelination that accompanies the axonal swellings, and if so what the sources and distributions of new Schwann cells might be. IDPN was given as a single large dose (2 ml/kg) to 21-d-old rats. Neurofilamentous axonal swellings formed in the proximal regions of motor axons, reaching their greatest enlargement in the root exit zone 8 d after IDPN administration. These swellings subsequently migrated distally down the nerves at rates approaching 1 mm/d. The axonal enlargement was consistently associated with displacement of the myelin sheath attachment sites into internodal regions, and consequent paranodal demyelination. This stage was associated with perikaryal changes, including nucleolar enlargement, "girdling" of the perikaryon, and formation of attenuated stalks separating the perinuclear region from the external cytoplasmic collar. Schwann cells proliferated abundantly during this stage. Daughter Schwann cells migrated within the endoneurial space (outside the nerve fiber basal laminae) to overlie the demyelinated paranodes of swollen nerve fibers. In these regions, local proliferation of Schwann cells continued, resulting in large paranodal clusters of Schwann cells. As the axonal calibers subsequently returned to normal, the outermost myelin lamellae of the original internodes returned to their paranodal attachment sites and the supernumerary Schwann cells disappeared. Formation of short internodes, segmental demyelination, and nerve fiber loss were rare phenomena. These results indicate that paranodal demyelination is a sufficient stimulus to excite abundant Schwann cell proliferation; neither internodal demyelination nor myelin breakdown is a necessary stimulus for mitosis. The 3H-thymidine incorporation studies indicated that the sources of new Schwann cells included markedly increased division of the Schwann cells of unmyelinated fibers and, as they formed, supernumerary Schwann cells. In addition, there were rare examples of 3H-thymidine incorporation by Schwann cells associated with myelinated nerve fibers.(ABSTRACT TRUNCATED AT 400 WORDS)     

Molecular domains of myelinated axons in the peripheral nervous system

Myelinated axons are organized into a series of specialized domains with distinct molecular compositions and functions. These domains, which include the node of Ranvier, the flanking paranodal junctions, the juxtaparanodes, and the internode, form as the result of interactions with myelinating Schwann cells. This domain organization is essential for action potential propagation by saltatory conduction and for the overall function and integrity of the axon.     

Distribution of sodium channels in chronically demyelinated spinal cord axons: immuno-ultrastructural localization and electrophysiological observations

The immuno-ultrastructural localization of voltage-sensitive sodium channels was demonstrated within a central demyelinating lesion induced in the rat spinal cord by ethidium bromide/irradiation using polyclonal antibody 7493. Antibody 7493 has previously been shown to immunostain intensely axon membrane at nodes of Ranvier, and also perinodal astrocyte processes. At 25–35 days post injection/irradiation, the central portion of the demyelinating lesion is populated with chronically demyelinated axons and there is an absence of glial processes. Sodum channel immunoreactivity was not observed on the chronically demyelinated axolemma within this central portion of the lesion. Within the peripheral portion of the lesion demyelinated axons were occasionally abutted by astrocyte and Schwann cell processes. At these focal sites of apposition, the axon membrane displayed intense sodium channel immunoreactivity, while the abutting astrocyte and Schwann cell processes did not exhibit immunostaining. Also in the periphery of the lesion, some axons become ensheathed and myelinated by oligodendrocytes and Schwann cells. The axon membrane of circumferentially ensheathed axons displayed antibody 7493 immunostaining, and this immunoreactivity persisted on the axolemma until the ensheathing cytoplasmic processes compacted into myelin. Internodal axon membrane beneath the myelin sheath did not display sodium channel immunoreactivity, though (putative) developing nodal axon membrane adjacent to terminal paranodal loops exhibited robust sodium channel staining. Electrophysiological recordings within the ethidium bromide/irradiation lesion demonstrated that at least some axons conducted action potentials within the lesion, while others exhibited conduction block. These results indicate that there is a reorganization of sodium channels within the axon membrane of chronically demyelinated central axons.     

Ultrastructural observations on nodes of ranvier from isolated single frog peripheral nerve fibers

A preparative procedure is described by which well-preserved nodes of Ranvier from isolated frog peripheral nerve fibers may be obtained. The following steps are crucial: careful and gentle dissection and isolation of a single nerve fibre, mounting in a chamber limiting lateral movements of the fibre during the fluid exchange, simultaneous glutaraldehyde-OsO₄ fixation and embedding in the same chamber used for fixation. Serial sectioning of individual nodes from both motor and sensory fibres made it possible to reconstitute three-dimensional models of several nodes and to study their morphology extensively. In addition to well-known ultrastructural features of the nodal and paranodal architecture, evaginations of a nodal membrane containing mitochondria and outpouchings of the paranodal axoplasm containing vesicles are described.