E-PTA stains oligodendroglial surface membranes and microtubules in optic nerves during myelination.

Aldehyde fixed Xenopus tadpole and frog optic nerves were stained en bloc with ethanolic phosphotungstic acid (E-PTA). During rapid myelination, intense staining was observed on cytoplasmic faces of paranodal terminal loops and loosely wrapped oligodendroglial membranes found along inner and outer surfaces of compact myelin sheaths. Oligodendroglial microtubules also were heavily stained. Where stained cytoplasmic faces fused to form a lamella of compact myelin, the intense staining was reduced to a thinner, fainter line. In optic nerves of adult frogs, the staining was less dense but the pattern was similar. The staining distribution and available histochemical evidence indicate that E-PTA stains positively charged proteins non specifically. Since myelin basic protein is found in oligodendroglia during myelination, we suggest that it is being stained by E-PTA while being transported along microtubules to sites where it is inserted into developing myelin lamellae.     

Morphological progression of myelin abnormalities in IgM-monoclonal gammopathy of undetermined significance anti-myelin-associated glycoprotein neuropathy

To characterize the morphological progression of neuropathy associated with immunoglobulin M-monoclonal gammopathy of undetermined significance with anti-myelin-associated glycoprotein antibody, we assessed histopathologic features of sural nerve specimens from 15 patients, emphasizing widely spaced myelin (WSM), demyelination, and tomaculous changes. The frequency of WSM correlated with that of demyelination and tomaculous appearance in teased-fiber preparations. In longitudinal sections at nodes of Ranvier and paranodal regions, the spaces between terminal myelin loops, particularly those adjacent to the node of Ranvier, were widened, indicating an early change before demyelination, and there was concomitant swelling of terminal myelin loops. Some conspicuously swollen terminal myelin loops were detached from the paranodal axolemma, thereby widening the nodes of Ranvier. Tomacula coexisted frequently with redundant myelin loops and WSM, particularly in the outermost layer of myelin sheaths, suggesting that loosening of the outer layers contributes to their formation. By immunofluorescence microscopy, immunoglobulin M and myelin-associated glycoprotein were colocalized in paranodal regions and Schmidt-Lanterman incisures. Confocal analysis revealed colocalization of immunoglobulin M and complement product C3d corresponding to the area of WSM. Thus, morphological changes in terminal myelin loops, formation of WSM at paranodes, and subsequent dissociation from paranodal axolemma (which may be associated with activation of the complement pathway) likely contribute to demyelination in this condition. Loosening of compact myelin seems to contribute to tomacula formation.     

Transfer of horseradish peroxidase from oligodendrocyte to axon in the myelinating neonatal rat optic nerve: Artefact or transcellular exchange?

In this paper we make the surprising observation that intracellular injection of horseradish peroxidase (HRP) into a single myelinating oligodendrocyte also resulted in localised HRP labelling at the nodes of Ranvier of some axons of the unit. It appeared that HRP had been transferred to the nodal axoplasm from the paranodal loops of the HRP-filled oligodendrocyte. Three HRP-filled oligodendrocytes from isolated optic nerves of 14-day-old rats were analysed by serial section electron microscopy, and HRP was observed in the axonal cytoplasm at three of the nodes of Ranvier delineated by one of the cells. At labelled nodes, HRP was of a uniform intensity throughout the nodal axoplasm. Axonal labelling gradually diminished along the paranodal regions and was not evident in the contiguous internodal axoplasm beyond 20 μm from the node. The myelin sheaths, paranodal loops, and axons appeared normal at labelled nodes, and the paranodal loops and astrocyte perinodal processes adjacent to those of the HRP-filled oligodendrocyte unit did not contain HRP. There was no evidence of extracellular HRP or tissue damage in the surrounding neuropil, and axons neighbouring those enwrapped by the HRP-filled oligodendrocyte did not contain HRP. The possibility that axonal labelling was an artefact of either iontophoretic injection or tissue preparation is discussed. This provocative finding is not definite proof of exchange, but the balance of evidence supports the possibility that there was transcellular exchange of HRP at paranodes between the labelled oligodendrocyte and some of the axons in the unit. The rarity of HRP transfer to axons suggests that it may be a transient or labile event. It is not clear whether oligodendrocyte to axon macromolecular exchange has real physiological and/or pathological significance. © Wiley-Liss, Inc.     

Immunocytochemical localization of 5′-nucleotidase in myelinated peripheral nerves from the rat

In sciatic and trigeminal nerves from the rat, 5′-nucleotidase immunostaining was observed on the surfaces of the myelinated fibers and in the membranes encircling the outermost loops of the myelin sheaths, the paranodal loops, and perhaps the inner loops, but neither in the compact myelin nor in the axoplasm. These results, which were consistent with previous biochemical data regarding sciatic nerve, suggest that the function of 5′-nucleotidase in myelinated fibers in the peripheral nervous system may be to promote diffusion of adenosine between the glial and neuronal compartments.     

Cytoplasmic membrane elaborations in oligodendrocytes during myelination of spinal motoneuron axons

The ultrastructure of paranodal oligodendroglial cytoplasm, which is located in proximity to the forming myelin sheath, was studied during maturation of spinal motoneuron axons in rat. At 8 days postnatal, the paranodal oligodendroglial loops contain a network of membrane-bound tubulovesicular elements. These membrane elaborations are most common in oligodendroglial loops attached to the outermost layers of the myelin sheath, i.e., paranodal loops closest to the nodal gap. The number of oligodendroglial cytoplasmic profiles per paranodal loop falls over the course of five to ten sequential paranodal loops, and these profiles are nearly absent in paranodal oligodendroglial cytoplasm located distant from the nodal gap. Oligodendrocytes in spinal cords of 14- and 20-day-old rats and of adult rats did not exhibit networks of tubulovesicular profiles. The appearance of these membrane organelles within oligodendroglial cytoplasm during myelin maturation suggests increased membrane turnover within paranodal cytoplasm located adjacent to the axon that is being myelinated. Membrane turnover within oligodendrocytes may reflect axonal modulation of glial function during myelination.     

Acute swelling of nodes of ranvier caused by venoms which slow inactivation of sodium channels

Summary The venoms of the spiderPhoneutria nigriventer and the scorpionsLeiurus quinquestriatus andCentruroides sculpturatus cause acute, transient swelling of axons at nodes of Ranvier. The changes in the morphology of the node and paranode were studied in the mouse. Venom was injected into the sciatic nerve by means of a glass micropipette. After survival times ranging from 15 min to 3 weeks the nerves were examined by light and electron microscopy. The increase in nodal axoplasmic volume led within an hour to disruption of neurofilaments and microtubules, swelling of the paranodes and displacement of the terminal loops of myelin away from the node. Axonal calibre recovered within a few hours, but restoration of nodal width took several days and seemed to be accomplished by elongation and remodelling of the paranodal myelin. Occasional internodes were interrupted by node-like discontinuities in the myelin sheath. These developed within a few hours and persisted for the duration of the study.     

Age-related molecular reorganization at the node of Ranvier

In myelinated axons, action potential conduction is dependent on the discrete clustering of ion channels at specialized regions of the axon, termed nodes of Ranvier. This organization is controlled, at least in part, by the adherence of myelin sheaths to the axolemma in the adjacent region of the paranode. Age-related disruption in the integrity of internodal myelin sheaths is well described and includes splitting of myelin sheaths, redundant myelin, and fluctuations in biochemical constituents of myelin. These changes have been proposed to contribute to age-related cognitive decline; in previous studies of monkeys, myelin changes correlate with cognitive performance. In the present study, we hypothesize that age-dependent myelin breakdown results in concomitant disruption at sites of axoglial contact, in particular at the paranode, and that this disruption alters the molecular organization in this region. In aged monkey and rat optic nerves, immunolabeling for voltage-dependent potassium channels of the Shaker family (Kv1.2), normally localizing in the adjacent juxtaparanode, were mislocalized to the paranode. Similarly, immunolabeling for the paranodal marker caspr reveals irregular caspr-labeled paranodal profiles, suggesting that there may be age-related changes in paranodal structure. Ultrastructural analysis of paranodal segments from optic nerve of aged monkeys shows that, in a subset of myelinated axons with thick sheaths, some paranodal loops fail to contact the axolemma. Thus, age-dependent myelin alterations affect axonal protein localization and may be detrimental to maintenance of axonal conduction.     

Nodal sodium channel domain integrity depends on the conformation of the paranodal junction,not on the presence of transverse bands

Our understanding of the role that axoglial interactions play in node of Ranvier formation and maintenance remains incomplete. Previous studies of CNS myelinated fibers of CGT-null mice showed abnormalities in the arrangement of paranodal myelin loops and absence of a conspicuous component of the paranodal junction, the ridge-like intercellular transverse bands. Axolemmal sodium channel domains were largely preserved at nodes of Ranvier but displayed some abnormalities in form. Using a combination of freeze-fracture and immunocytochemical methods, we have found additional evidence documenting abnormalities in the size, shape, and location of axolemmal sodium channel clusters in CGT-null mice as well as evidence that these nodal abnormalities are complementary to the organization of paranodal myelin loops, despite the absence of transverse bands. We conclude that the differentiated form of the nodal axolemma and the distribution of axolemmal sodium channels depend on the conformation of paranodal axoglial contacts but not on the presence of transverse bands at the sites of contact.     

Cation-binding sites in trigeminal ganglia and maxillary nerve: unusual reactivity of perikarya, stem axons and satellite cells

We have used the cupric/ferrocyanide reaction to study cation-binding in trigeminal ganglia and maxillary nerve of adult rats. Unmyelinated axons did not react, whereas myelinated axons were stained at nodal, paranodal or cleft sites. At 'nodal' sites, metallic deposits were found in the axoplasm, along the axolemma, and at the extracellular interfaces of the paranodal myelin. At 'paranodal' sites, particles were concentrated in the paranodal axoplasm and in the intracellular spaces of the myelin loops. Most maxillary axons examined at successive sites had all nodal or all paranodal staining, but 13 of 51 had a mixture. In trigeminal ganglia there was no staining of perineurial sheath, endoneurial cells or mast cells. Satellite cells and their basal laminae were prominently stained, with those around small neurons more reactive than those of large neurons. Patches of neuronal membrane on cell bodies were stained, more often for small than large neurons. The axon hillock and proximal stem axon were not stained in some cases, but approximately half the neurons had staining of perikaryal cytoplasm at the axon hillock or a dense asymmetric band in the proximal stem axon. Strong intraaxonal staining was found at the junction between unmyelinated proximal and myelinated distal stem axon. In distal stem axons, staining was found at the first myelin segment and at each successively thicker myelin segment; staining was mostly weak and paranodal, with intensity proportional to myelin thickness. The T-junction between stem and main myelinated axon had nodal or paranodal patterns; unmyelinated T-junctions were not stained. The varied cation-binding patterns in trigeminal ganglia show unusual properties of satellite cells and important differences between stem and main axons. The results that the cell membrane of axon hillock and proximal stem regions of many sensory large and small neurons may have numerous sodium channels and could affect signal propagation.     

Paranodal pathology in Tangier disease with remitting-relapsing multifocal neuropathy.

Pathological studies of a sural nerve biopsy in a man with Tangier disease presenting as a remitting-relapsing multifocal neuropathy showed abnormalities in the paranodal regions, including lipid deposition (65%) and redundant myelin foldings, with various degrees of myelin splitting and vesiculation (43%) forming small tomacula and abnormal myelin terminal loops (4%). The internodal regions were normal in the majority of myelinated fibres. Abnormal lipid storage was also present in the Schwann cells of the majority of unmyelinated fibres (67%). The evidence suggests that the noncompacted myelin region of the paranode is a preferential site for lipid storage in the myelinated Schwann cell, and that the space-occupying effects of the cholesterol esters leads to paranodal malfunction and tomacula formation as the pathological basis for the multifocal relapsing-remitting clinical course.     

IgM deposits at nodes of Ranvier in a patient with amyotrophic lateral sclerosis, anti-GM1 antibodies, and multifocal motor conduction block

We studied a patient with amyotrophic lateral sclerosis, multifocal motor conduction block, and IgM anti-GM1 antibodies. A sural nerve biopsy demonstrated deposits of IgM at nodes of Ranvier by direct immunofluorescence. The deposits were granular and located in the nodal gap between adjacent myelin internodes, and in some instances, they extended along the surface of the paranodal myelin sheath. When injected into rat sciatic nerve, the serum IgM bound to the nodes of Ranvier, and the binding activity was removed by preincubation with GM1. These observations suggest that anti-GM1 antibodies may have caused motor dysfunction by binding to the nodal and paranodal regions of peripheral nerve.     

Ultrastructural concomitants of anoxic injury and early post-anoxic recovery in rat optic nerve

To study the effects of anoxia on CNS white matter, we examined the ultrastructure of axons and glial cells in a white matter tract, the rat optic nerve, that was subjected to a standardized anoxic insult in vitro. Previous electrophysiological studies showed that in this model, action potential conduction is rapidly abolished by anoxia, and conduction is restored after reoxygenation in about 30% of axons following a 60-min anoxic period. The present study examined the ultrastructural correlates of anoxic injury and early post-anoxic recovery in this model. Optic nerves examined immediately following 60 min of anoxia displayed numerous large, apparently empty zones located within myelin sheaths adjacent to the axon. The myelin remained compact and retained its periodicity. In some regions, the extracellular space was enlarged. There was mitochondrial swelling with loss of normal cristae. There was also loss of microtubules and, to a smaller degree, of neurofilaments in large-diameter axons. Some nodes of Ranvier in anoxic optic nerves displayed detachment of terminal oligodendroglial loops or retraction of the myelin from the node; the presence of tongue-like processes, extending from nearby cells under the detached myelin loops, suggested a possible role of cell-mediated damage to the paranodal myelin. Bundles of dense astrocyte processes were present, and there was vesicular degeneration of perinodal astrocyte processes. In optic nerves that had been permitted to recover for 60 min in oxygenated Ringers following the anoxic period, empty zones were only rarely observed within myelin sheaths and, when present, were smaller than in optic nerves immediately following 60 min of anoxia. The axoplasm of large fibers continued to show loss of microtubules and neurofilaments, as well as mitochondrial swelling. Myelin appeared normal, and only rare paranodal oligodendroglial processes remained unattached from the axon membrane. These results provide support for the idea that, during anoxia, myelinated axons are damaged with significant injury to cytoskeletal elements, probably due to an influx of calcium. The ultrastructural results, together with our earlier observations on the physiological correlates of anoxia and re-oxygenation, suggest that the development of intramyelinic spaces or damage to paranodes lead to conduction block in the anoxic optic nerve. These results also suggest that repair of these structural abnormalities may provide a morphological basis for the early recovery of conduction that occurs after re-oxygenation.     

Nodal and paranodal structural changes in mouse and rat optic nerve during Wallerian degeneration

Ultrastructural changes in nodal and paranodal regions of myelinated mouse and rat optic nerve fibers were followed between 4 h and 28 days during the course of Wallerian degeneration. In the mouse, axoplasmic changes, including accumulation of organelles and segregation of microtubules, were detectable 4 h after transection, and progressed to a maximum level on day 4, at which time many axons were markedly swollen. Dense axoplasm was seen as early as 16 h and was a common feature of degenerating axoplasm at later times. Paranodal changes, which first appeared as early as 16 h after injury, included detachment of terminal loops of myelin from the axolemma, disconnection of terminal loops from compact myelin lamellae and broadening of terminal loops, or separation of the loops from each other, resulting in paranodal elongation. In freeze-fracture replicas, the E-face of the axolemma showed the normal particle distribution as late as days 3-5. By day 8, however, the nodal particles were patchy and the overall nodal particle density was reduced to approximately half normal. Some normal-looking fibers were present at all stages examined, but their number had declined to about half the total population on day 5 and to less than 10% on day 11. In the rat, the overall sequence of events and time course were comparable to those in the mouse. Thus, the morphological changes found follow approximately the same sequence as that described previously in frog nerves, but progress more rapidly in the mouse and rat.     

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.     

Effects of IDPN-induced axonal swellings on conduction in motor nerve fibers

Paranodal demyelination produces a reduction of conduction velocity and conduction block. The relative proportions of these changes appear to vary among different demyelinating disorders. In this study we have examined the effects on conduction of paranodal demyelination produced by giant axonal swellings. The axonal swellings were induced in rats by administration of beta, beta'-iminodipropionitrile (IDPN). In this experimental model synchronous axonal swellings occur in the proximal region of virtually every alpha-motorneuron without evidence of segmental demyelination or fiber loss. Conduction across the motor neuron was evaluated by two methods: a monosynaptic reflex pathway and intracellular recording from single motor neurons. Increases in the delay across the central region of the monosynaptic reflex pathway began between 2 and 4 days after toxin administration. Intracellular studies confirmed that the slowing occurred across the proximal regions of the motor axons; more distal regions of the motor axons were unaffected. The substantial reduction in conduction velocity over the swollen segment occurs with only moderate evidence of conduction block, as assayed by a reduction in the H-reflex/M-response amplitude ratio. Parallel morphological studies showed that in the enlarged fibers the myelin terminal loops maintained contact with the axon but were displaced from the paranodal region into the internode. The appearance of this "passive" paranodal demyelination correlated closely with the increase in conduction delay. We suggest that the contact maintained by the displaced myelin terminal loops with the axolemma allows saltatory conduction to continue, and explains the paucity of conduction block in this model despite the prominent conduction slowing.     

Rapid alterations of the axon membrane in antibody-mediated demyelination

Alterations of nodal and paranodal axolemma of the rat sciatic nerve were investigated in antigalactocerebroside serum-induced demyelination. A ferric ion-ferrocyanide (FeFCN) stain that appears to stain the regions with a high sodium channel density in nerve fibers was applied. When acute conduction block was initiated 20 to 180 minutes after the antiserum injection, myelin terminal loops began to be detached from the paranodal axolemma and reaction product of FeFCN stain originally localized at the nodes decreased in density and extended to the paranodal axolemma. By the time that complete conduction block was established, 5 hours after the injection, FeFCN stain was barely detectable around the nodal area. The loss of staining was associated with detachment and vesiculovacuolar degeneration of the paranodal myelin. This rapid deterioration and disappearance of normal cytochemical characteristics of the axolemma in the presence of only modest paranodal demyelination could be a morphological correlate of the loss of excitability of the axon membrane.     

Ultrastructural identification of HRP-injected oligodendrocytes in the intact rat optic nerve

Glial cells in the rat optic nerve were visualized by intracellular injections of horseradish peroxidase (HRP). A novel class of cell was encountered that was presumed to be an oligodendrocyte on the basis of arguments related to its light microscopic appearance after intracellular staining (Butt and Ransom, Glia 1989;2:470-475). These cells had 10-20 parallel processes 200-300 microns long that were oriented exclusively along the long axis of the optic nerve; the parallel processes were connected to the cell body by thin branches 15-30 microns long. To determine if these HRP-filled cells were oligodendrocytes, they were examined ultrastructurally; all cells examined in this way were unequivocally found to be myelin-producing oligodendrocytes. The oligodendrocytes contained intracellular organelles that were characteristic of this cell type, including abundant Golgi profiles and microtubules. In addition, HRP was found to fill the inner and outer tongue processes of myelin sheaths and the paranodal loops at nodes of Ranvier, proving that the entire cytoplasmic border surrounding the myelin sheath rapidly communicates by intracellular diffusion with the cell body. This electron microscopic study demonstrates that oligodendrocytes in the rat optic nerve can be positively identified by their distinctive light microscopic appearance after intracellular dye injection, and provides light microscopic criteria for establishing the number, distribution, and dimensions of the myelin segments provided by individual oligodendrocytes.     

Periaxin mutations cause a broad spectrum of demyelinating neuropathies

Previous studies have demonstrated that apparent loss-of-function mutations in the periaxin gene cause autosomal recessive Dejerine-Sottas neuropathy or severe demyelinating Charcot-Marie-Tooth disease. In this report, we extend the associated phenotypes with the identification of two additional families with novel periaxin gene mutations (C715X and R82fsX96) and provide detailed neuropathology. Each patient had marked sensory involvement; two siblings with a homozygous C715X mutation had much worse sensory impairment than motor impairment. Despite early disease onset, these siblings with the C715X mutation had relatively slow disease progression and adult motor impairment typical of classic demyelinating Charcot-Marie-Tooth neuropathy. In contrast, a patient with the homozygous R82fsX96 mutation had a disease course consistent with Dejerine-Sottas neuropathy. The neuropathology of patients in both families was remarkable for demyelination, onion bulb and occasional tomacula formation with focal myelin thickening, abnormalities of the paranodal myelin loops, and focal absence of paranodal septate-like junctions between the terminal loops and axon. Our study indicates a prominent sensory neuropathy resulting from periaxin gene mutations and suggests a role for the carboxyl terminal domain of the periaxin protein.     

The formation of paranodal spirals at the ends of CNS myelin sheaths requires the planar polarity protein Vangl2

During axonal ensheathment, noncompact myelin channels formed at lateral edges of the myelinating process become arranged into tight paranodal spirals that resemble loops when cut in cross section. These adhere to the axon, concentrating voltage-dependent sodium channels at nodes of Ranvier and patterning the surrounding axon into distinct molecular domains. The signals responsible for forming and maintaining the complex structure of paranodal myelin are poorly understood. Here, we test the hypothesis that the planar cell polarity determinant Vangl2 organizes paranodal myelin. We show that Vangl2 is concentrated at paranodes and that, following conditional knockout of Vangl2 in oligodendrocytes, the paranodal spiral loosens, accompanied by disruption to the microtubule cytoskeleton and mislocalization of autotypic adhesion molecules between loops within the spiral. Adhesion of the spiral to the axon is unaffected. This results in disruptions to axonal patterning at nodes of Ranvier, paranodal axon diameter and conduction velocity. When taken together with our previous work showing that loss of the apico-basal polarity protein Scribble has the opposite phenotype—loss of axonal adhesion but no effect on loop–loop autotypic adhesion—our results identify a novel mechanism by which polarity proteins control the shape of nodes of Ranvier and regulate conduction in the CNS.     

Demyelination of Sternarchus electrocyte fibers by injection of diphtheria toxin

Diphtheria toxin was injected into the electric organ of the gymnotid fish, Sternarchus albifrons. After 10 days, there was extensive demeylination of electrocyte fibers in the area of injection. Electron microscopy showed that paranodal loops of myelin do not separately cleanly from the axon, and remnants of the myelin loops may persist after demyelination of the internodes is nearly complete. The dense cytoplasmic undercoating of the nodal axolemma may disappear before the paranodal junctions are completely gone. Observations of demyelination of internodes between the elaborate, inexcitable nodes suggest that the presence of myelin may not be necessary for the maintenance of structural differentiation of this region of the axolemma. Use of diphtheria toxin to demyelinate Sternarchus electrocytes may provide a useful system for experimental neuropathological studies.