Hyperosmolar -mannitol reverses the increased membrane excitability and the nodal swelling caused by Caribbean ciguatoxin-1 in single frog myelinated axons

The effects of hyperosmolar -mannitol were studied on single frog myelinated nerve fibres previously poisoned with Caribbean ciguatoxin-1 (C-CTX-1), a new toxin isolated from the pelagic fish Caranx latus inhabiting the Caribbean region. In current-clamped myelinated axons, C-CTX-1 (50–120 nM) caused spontaneous and repetitive action potential discharges after a short delay. In addition, the toxin produced a marked swelling of nodes of Ranvier of myelinated axons that reached a steady state within about 90 min, as revealed by using confocal laser scanning microscopy. The increased excitability and the nodal swelling caused by C-CTX-1 were prevented or reversed by an external hyperosmotic solution containing 100 mM -mannitol. Moreover, the C-CTX-1-induced nodal swelling was completely prevented by the blockade of voltage-sensitive sodium channels by tetrodotoxin (TTX). It is suggested that C-CTX-1, by increasing nerve membrane excitability, enhances Na⁺ entry into nodes of Ranvier through TTX-sensitive sodium channels, which directly or indirectly disturb the osmotic equilibrium between intra- and extra-axonal media resulting in an influx of water that was responsible for the long-lasting nodal swelling. The fact, that hyperosmolar -mannitol either reversed or prevented the neurocellular actions of C-CTX-1, is of particular interest since it provides the rational basis for its use to treat the neurological symptoms of ciguatera fish poisoning in the Caribbean area.     

Neurotoxins targetting receptor site 5 of voltage-dependent sodium channels increase the nodal volume of myelinated axons

The effects of a C57 type ciguatoxin (CTX-3C) and two types of brevetoxins (PbTx-1 and PbTx-3), known to bind to receptor site 5 of the neuronal voltage-dependent Na+ channel-protein, were studied on the morphology of living frog myelinated axons using confocal laser scanning microscopy. During the action of CTX-3C, PbTx-1, and PbTx-3 (10-50 nM), a marked swelling of nodes of Ranvier was observed without apparent modification of internodal parts of axons. In all cases, toxin-induced nodal swelling attained a steady-state within 75-100 min that was well maintained during an additional 90-115 min. The nodal swelling was reversed by an external hyperosmotic solution containing 100 mM D-mannitol and could be completely prevented by blocking voltage-dependent Na+ channels with 1 microM tetrodotoxin. It is suggested that CTX-3C, PbTx-1, and PbTx-3 by activating Na+ channels cause a continuous Na+ entry into axons, increasing internal Na+ concentration. Such an increase directly or indirectly disturbs the osmotic equilibrium between intra- and extra-axonal media, resulting in an influx of water, which is responsible for the long-lasting nodal swelling. Similar results were previously reported with two C60 type ciguatoxins (CTX-1B and CTX-4B). Thus, it is concluded that the four types of toxins targetting receptor site 5 of neuronal voltage-dependent Na+ channels, not only enhance nerve membrane excitability but also, on a long-term basis, cause a marked increase in the axonal volume.     

A new conotoxin isolated from Conus consors venom acting selectively on axons and motor nerve terminals through a Na+-dependent mechanism

A novel conotoxin was isolated and characterized from the venom of the fish-hunting marine snail Conus consors. The peptide was identified by screening chromatography fractions of the crude venom that produced a marked contraction and extension of the caudal and dorsal fins in fish, and noticeable spontaneous contractions of isolated frog neuromuscular preparations. The peptide, named CcTX, had 30 amino acids and the following scaffold: X11CCX7CX2CXCX3C. At the frog neuromuscular junction, CcTx at nanomolar concentrations selectively increased nerve terminal excitability so that a single nerve stimulation triggered trains of repetitive or spontaneous synaptic potentials and action potentials. In contrast, CcTx had no noticeable effect on muscle excitability even at concentrations 100 x higher than those that affected motor nerve terminals, as revealed by direct muscle stimulation. In addition, CcTx increased miniature endplate potential (MEPP) frequency in a Ca2+-free medium supplemented with ethylene glycol-bis-(beta-aminoethyl ether)-N,N,N', N'-tetraacetic acid (EGTA). Blockade of voltage-dependent sodium channels with tetrodotoxin (TTX) either prevented or suppressed the increase of MEPP frequency induced by the toxin. CcTx also produced a TTX-sensitive depolarization of the nodal membrane in single myelinated axons giving rise, in some cases, to repetitive and/or spontaneous action potential discharges. In addition, CcTx increased the nodal volume of myelinated axons, as determined using confocal laser scanning microscopy. This increase was reversed by external hyperosmolar solutions and was prevented by pretreatment of axons with TTX. It is suggested that CcTx, by specifically activating neuronal voltage-gated sodium channels at the resting membrane potential, produced Na+ entry into nerve terminals and axons without directly affecting skeletal muscle fibres. CcTx belongs to a novel family of conotoxins that targets neuronal voltage-gated sodium channels.     

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.     

Specific staining of the axon membrane at nodes of Ranvier with ferric ion and ferrocyanide

Ferric ion and ferrocyanide were used as stains for light and electron microscopy of peripheral nerves. In rat sciatic nerves, it was found that ferric ion preferentially binds to the cytoplasmic surface of the axon membrane at nodes of Ranvier but not at internodal regions. In myelinated axons in the electric organ of the gymnotid fish, Sternarchus albifrons, the small excitable nodes are similarly stained, but the larger inexcitable nodes are not stained by ferric ion. Staining of the inner surface of the nodal membrane appears to be related to a structural specialization of this membrane, rather than accessibility to stain. Our data thus show a chemical differentiation of the inner surface of the axon membrane between nodes and internodes in normal peripheral nerve fibers and between the inner surface of the axon membrane at active nodes, inactive nodes, and the internodes in the Sternarchus electrocyte axons.     

Axoplasmic transport of thioredoxin and thioredoxin reductase in rat sciatic nerve

Thioredoxin and thioredoxin reductase were localized immunohistochemically in the rat sciatic nerve by immunofluorescence using specific rabbit antisera. Both proteins showed strong immunoreactivity in the cytoplasm of Schwann cells and at the nodes of Ranvier. The axoplasm of myelinated axons also showed a low, evenly distributed immunoreactivity for both proteins. A single or double crush of the nerve caused accumulation of immunoreactivity in dilatated axons both proximally and distally to the crush for up to 8 h. Local cooling of the nerve or subepineural injection of either colchicine or vinblastine prevented the accumulation indicating a role of microtubules. The results showed that thioredoxin and thioredoxin reductase are synthesized in nerve cell bodies and rapidly transported in axons both in anterograde and retrograde directions.     

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.     

Nodes of Ranvier in skin biopsies of patients with diabetes mellitus

Paranodal demyelination has been discussed as a potential mechanism of nerve fiber damage in diabetic neuropathy (DNP). Studies on human tissue are limited, as nerve biopsies are invasive and only rarely performed in patients with confirmed DNP. Skin biopsy has recently been suggested as a tool to analyze paranodal and nodal changes of myelinated fibers. We analyzed the myelinated fibers of skin biopsies of 35 patients with DNP, 17 patients with diabetes mellitus (DM) without neuropathy, and 30 normal controls. Immunofluorescence of skin sections with antibodies against Caspr, neurofascin, sodium channels, and myelin basic protein was performed to assess paranodal/nodal architecture, segmental demyelination, and myelinated nerve fibers. Staining with antibodies against protein gene product 9.5 was used to quantify unmyelinated nerve fibers. There was an increase of elongated Ranvier nodes and a dispersion of neurofascin at the distal leg in patients with DM with and without neuropathy and at the finger in patients with DNP. An increased dispersion of Caspr was only found in biopsies of the finger in patients with DNP. Skin biopsy may be an appropriate tool to analyze nodes of Ranvier in patients with DM. Structural nodal changes are detectable in DNP and even in diabetic patients without neuropathy.     

Role of a Contactin multi-molecular complex secreted by oligodendrocytes in nodal protein clustering in the CNS

The fast and reliable propagation of action potentials along myelinated fibers relies on the clustering of voltage-gated sodium channels at nodes of Ranvier. Axo-glial communication is required for assembly of nodal proteins in the central nervous system, yet the underlying mechanisms remain poorly understood. Oligodendrocytes are known to support node of Ranvier assembly through paranodal junction formation. In addition, the formation of early nodal protein clusters (or prenodes) along axons prior to myelination has been reported, and can be induced by oligodendrocyte conditioned medium (OCM). Our recent work on cultured hippocampal neurons showed that OCM-induced prenodes are associated with an increased conduction velocity (Freeman et al., 2015). We here unravel the nature of the oligodendroglial secreted factors. Mass spectrometry analysis of OCM identified several candidate proteins (i.e., Contactin-1, ChL1, NrCAM, Noelin2, RPTP/Phosphacan, and Tenascin-R). We show that Contactin-1 combined with RPTP/Phosphacan or Tenascin-R induces clusters of nodal proteins along hippocampal GABAergic axons. Furthermore, Contactin-1-immunodepleted OCM or OCM from Cntn1-null mice display significantly reduced clustering activity, that is restored by addition of soluble Contactin-1. Altogether, our results identify Contactin-1 secreted by oligodendrocytes as a novel factor that may influence early steps of nodal sodium channel cluster formation along specific axon populations.     

Paranodal axoglial junction is required for the maintenance of the Nav1.6-type sodium channel in the node of Ranvier in the optic nerves but not in peripheral nerve fibers in the sulfatide-deficient mice

In myelinated axons, voltage-gated sodium channels specifically cluster at the nodes of Ranvier, while voltage-gated potassium channels are located at the juxtaparanodes. These characteristic localizations are influenced by myelination. During development, Nav1.2 first appears in the predicted nodes during myelination, and Nav1.6 replaces it in the mature nodes. Such replacements may be important physiologically. We examined the influence of the paranodal junction on switching of sodium channel subunits using the sulfatide-deficient mouse. This mutant displayed disruption of paranodal axoglial junctions and altered nodal lengths and channel distributions. The initial switching of Nav1.2 to Nav1.6 occurred in the mutant optic nerves; however, the number of Nav1.2-positive clusters was significantly higher than in wild-type mice. Although no signs of demyelination were observed at least up to 36 weeks of age, sodium channel clusters decreased markedly with age. Interestingly, Nav1.2 stayed in some of the nodal regions, especially where the nodal lengths were elongated, while Nav1.6 tended to remain in the normal-length nodes. The results in the mutant optic nerves suggested that paranodal junction formation may be necessary for complete replacement of nodal Nav1.2 to Nav1.6 during development as well as maintenance of Nav1.6 clusters at the nodes. Such subtype abnormality was not observed in the sciatic nerve, where paranodal disruption was observed. Thus, the paranodal junction significantly influences the retention of Nav1.6 in the node, which is followed by disorganization of nodal structures. However, its importance may differ between the central and peripheral nervous system.     

Saltatory conduction precedes remyelination in axons demyelinated with lysophosphatidyl choline

The changing electrical and morphological properties of demyleinating and remyelinating nerve fibres have been studied in rat ventral roots after intrathecal injection of lysophosphatidyl choline (LPC). The spatial distribution of electrical excitability within the lesion has been studied in undissected single fibres using high-resolution longitudinal current analysis. The distribution of excitability has been correlated with the ultrastructure of the fibres and with the distribution of the surrounding Schwann cells. Demyelinated axolemma was initially not excited, but conduction across demyelinated internodes appeared progressively from the 4th day after LPC injection. Conduction was never continuous, but proceeded via new foci of inward membrane current as early as 4 days after LPC injection, i.e. 3 days before the onset of remyelination. It is suggested that these foci (termed ?-nodes to distinguish them from the nodes of Ranvier distributed along myelinated nerve fibres) are precursors of nodes of Ranvier, and may indicate aggregates of sodium channels which form along the demyelinated axolemma prior to remyelination.     

Preferential conduction block of myelinated axons by nitric oxide

Conduction block by nitric oxide (NO) was examined in myelinated and unmyelinated axons from both the central nervous system and peripheral nervous system. In rat vagus nerves, mouse optic nerves at P12–P23, adult and developing mouse sciatic nerves, and mouse spinal cords, myelinated fibers were preferentially blocked reversibly by concentrations of NO similar to those encountered in inflammatory lesions. The possibility that these differences between myelinated and unmyelinated axons are due to the normal developmental substitution of Na⁺ channel subtype Na_v1.6 for Na_v1.2 at nodes of Ranvier was tested by repeating experiments on mice null for Na_v1.6. Results were unchanged in this mutant. In shiverer optic nerve, which has only scattered regions with nodes of Ranvier, only the fastest component of the compound action potential was reduced. NO was compared with three other methods of blocking conduction: low Na⁺, high K⁺, and tetrodotoxin (TTX). In each of these three cases, unmyelinated axons lost conduction simultaneously with myelinated fibers. From changes in conduction velocity in myelinated axons as they were blocked, it was ascertained that NO acted most similarly to TTX. It was concluded that NO likely interacts with axonal Na⁺ channels through an intermediate that is associated with myelin. © 2016 Wiley Periodicals, Inc.     

Axonal bifurcation in the dorsal root ganglion of the cat: a light and electron microscopic study

Bifurcation of the axon of unipolar neurons in the dorsal root ganglion of the cat has been studied by light and electron microscopy. Osmic acid, Golgi and silver preparations have revealed two types of bifurcation: (1) of myelinated fibers characterized by constriction at the nodal region with peripheral and central branches of equal diameter; (2) of small fibers characterized by a broad triangular expansion at the junctional region with a much thicker peripheral as compared to the central branch. These differences in bifurcation of unmyelinated and myelinated axons can be related to the velocity of conduction in the peripheral nerve and dorsal roots. The ultrastructure of the nodal region at the bifurcation resembles the node of Ranvier of a peripheral nerve fiber. The node of Ranvier at the bifurcation consists of three cells of Schwann and adjacent cells are closely apposed and interdigitated. Neurofilaments and microtubules are prominent structures within the axoplasm at the nodal regions. They group into two streams as the unipolar process bifurcates, entering the peripheral and central branch respectively. At the junctional region within the axoplasm, numerous mitochondria and scattered multivesicular bodies are always observed.     

Early terminal and nodal sprouting of motor axons after botulinum toxin

Axonal sprouting in distal motor axons was studied in an attempt to answer two questions: (a) is the cell body required for early axonal sprouting?, and (b) do nodal, as well as terminal, axonal sprouts arise after muscle inactivity not caused by nerve injury? Botulinum toxin (BT) was used to induce axonal sprouting without nerve trauma. Mice were injected in the right calf with a sublethal dose of BT and the soleus muscle examined ultrastructurally at times varying from 3 h to 5 days post-injection. Terminal axonal sprouts were seen 2 days after injection, and based on the time taken for BT to act and the growth rate of sprouts, axons were calculated to sprout within 24 h of muscle inactivity. This short time suggests that early axonal regrowth is initiated and controlled at the distal axon. Sprouts were seen arising from the intramuscular nodes of Ranvier from 2 days after BT injection. Unlike the terminal sprouts which elongated over time, the nodal sprouts remained short and confined by the basal lamina overlying the node, probably because without structural denervation there were no empty perineural sheaths to act as pathways to the motor endplates. The finding of terminal and nodal sprouts after botulinum toxin supports the hypothesis that muscle inactivity gives rise to a single growth factor for both terminal and nodal sprouting.     

Localization of the tetrodotoxin-resistant sodium channel NaN in nociceptors

Tetrodotoxin-resistant sodium currents contribute to the somal and axonal sodium currents of small diameter primary sensory neurons, many of which are nociceptive. NaN is a recently described tetrodotoxin-resistant sodium channel expressed preferentially in IB4-labeled dorsal root ganglion (DRG) neurons. We employed an antibody raised to a NaN specific peptide to show that NaN is preferentially localized along axons of IB4-positive unmyelinated fibers in the sciatic nerve and in axon terminals in the cornea. NaN immunoreactivity was also found at some nodes of Ranvier of thinly myelinated axons of the sciatic nerve, where it was juxtaposed to Kv1.2 potassium channel immunoreactivity. This distribution of NaN is consistent with a role for NaN sodium channels in nociceptive transmission.     

Ultrastructural and cytochemical observations in a case of dominantly inherited hypertrophic (Charcot-Marie-Tooth) neuropathy

Ultrastructural and cytochemical studies were carried out on the sural nerve of a 6 1/2 year old girl with dominantly inherited hypertrophic (Charcot-Marie-Tooth) neuropathy. Electron microscopy revealed a paucity of myelinated fibers, with inappropriately thin myelin sheaths and onion-bulb formations associated with those fibers that were myelinated. In some cases the nodal axolemma was folded so as to form irregular excrescences. At other nodes, the non-myelinated gap was enlarged. Following staining with ferric ion and ferrocyanide, dense precipitates were observed on the cytoplasmic surface of the axolemma at some nodes of Ranvier, as in normal peripheral axons. At other nodes, staining was attenuated or absent. The latter result is similar to our findings in the dy/dy dystrophic mouse. These results are consistent with the hypothesis that, in dominantly inherited hypertrophic neuropathy, there are abnormalities of structure of the axolemma, in addition to an abnormality of the myelin sheath.     

The Kv7 potassium channel activator flupirtine affects clinical excitability parameters of myelinated axons in isolated rat sural nerve

Flupirtine is an activator of Kv7 (KCNQ/M) potassium channels that has found clinical use as an analgesic with muscle relaxant properties. Kv7 potassium channels are expressed in axonal membranes and pharmacological activation of these channels may restore abnormal nerve excitability. We have examined the effect of flupirtine on the electrical excitability of myelinated axons in isolated segments of rat sural nerve. Axonal excitability was studied in vitro with the same parameters used by clinical neurophysiologists to assess peripheral nerve excitability in situ . Application of flupirtine in low micromolar concentrations resulted in an increase in threshold current, a reduction of refractoriness and an increase in post-spike superexcitability. These effects are consistent with an increase in Kv7 conductance and membrane hyperpolarization. Flupirtine also enhanced and prolonged the late, long-lasting period of axonal subexcitability that follows a short burst of action potentials. This effect was blocked by XE 991 (10 µM), an antagonist of Kv7 channels. In summary, flupirtine affects measures of excitability that are altered in the myelinated axons of patients with peripheral nerve disorders. This indicates that neuropathies with abnormal nerve excitability parameters corresponding to those affected by flupirtine may benefit from activation of axonal Kv7 potassium channels.     

Mild traumatic brain injury in the mouse induces axotomy primarily within the axon initial segment

Traumatic axonal injury (TAI) is a consistent component of traumatic brain injury (TBI), and is associated with much of its morbidity. Increasingly, it has also been recognized as a major pathology of mild TBI (mTBI). In terms of its pathogenesis, numerous studies have investigated the susceptibility of the nodes of Ranvier, the paranode and internodal regions to TAI. The nodes of Ranvier, with their unique composition and concentration of ion channels, have been suggested as the primary site of injury, initiating a cascade of abnormalities in the related paranodal and internodal domains that lead to local axonal swellings and detachment. No investigation, however, has determined the effect of TAI upon the axon initial segment (AIS), a segment critical to regulating polarity and excitability. The current study sought to identify the susceptibility of these different axon domains to TAI within the neocortex, where each axonal domain could be simultaneously assessed. Utilizing a mouse model of mTBI, a temporal and spatial heterogeneity of axonal injury was found within the neocortical gray matter. Although axonal swellings were found in all domains along myelinated neocortical axons, the majority of TAI occurred within the AIS, which progressed without overt structural disruption of the AIS itself. The finding of primary AIS involvement has important implications regarding neuronal polarity and the fate of axotomized processes, while also raising therapeutic implications, as the mechanisms underlying such axonal injury in the AIS may be distinct from those described for nodal/paranodal injury.     

Increased numbers of sodium channels form along demyelinated axons

Sodium channels, which are largely localized to the nodes of Ranvier in myelinated axons, appear to form new distributions along demyelinated axons. In this study a sensitive radioimmunoassay (RIA) was used to examine the changes in the total number of sodium channels that occur in nerves experimentally demyelinated in vivo with doxorubicin (adriamycin). The results clearly illustrate the development of an increased number of sodium channels during demyelination, suggesting that this process is associated with the formation of new sodium channels.     

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.