Ranvier In Peripheral Nerves Research Articles

Glial M6B stabilizes the axonal membrane at peripheral nodes of Ranvier.

Glycoprotein M6B and the closely related proteolipid protein regulate oligodendrocyte myelination in the central nervous system, but their role in the peripheral nervous system is less clear. Here we report that M6B is located at nodes of Ranvier in peripheral nerves where it stabilizes the nodal axolemma. We show that M6B is co-localized and associates with gliomedin at Schwann cell microvilli that are attached to the nodes. Developmental analysis of sciatic nerves, as well as of myelinating Schwann cells/dorsal root ganglion neurons cultures, revealed that M6B is already present at heminodes, which are considered the precursors of mature nodes of Ranvier. However, in contrast to gliomedin, which accumulates at heminodes with or prior to Na+ channels, we often detected Na+ channel clusters at heminodes without any associated M6B, indicating that it is not required for initial channel clustering. Consistently, nodal cell adhesion molecules (NF186, NrCAM), ion channels (Nav1.2 and Kv7.2), cytoskeletal proteins (AnkG and βIV spectrin), and microvilli components (pERM, syndecan3, gliomedin), are all present at both heminodes and mature nodes of Ranvier in Gpm6b null mice. Using transmission electron microscopy, we show that the absence of M6B results in progressive appearance of nodal protrusions of the nodal axolemma, that are often accompanied by the presence of enlarged mitochondria. Our results reveal that M6B is a Schwann cell microvilli component that preserves the structural integrity of peripheral nodes of Ranvier.

Electron tomographic analysis of cytoskeletal cross-bridges in the paranodal region of the node of Ranvier in peripheral nerves

The node of Ranvier is a site for ionic conductances along myelinated nerves and governs the saltatory transmission of action potentials. Defects in the cross-bridging and spacing of the cytoskeleton are a prominent pathological feature in diseases of the peripheral nerve. Electron tomography was used to examine cytoskeletal-cytoskeletal, membrane-cytoskeletal, and heterologous cell connections in the paranodal region of the node of Ranvier in peripheral nerves. Focal attachment of cytoskeletal filaments to each other and to the axolemma and paranodal membranes of the Schwann cell via narrow cross-bridges was visualized in both neuronal and glial cytoplasm. A subset of intermediate filaments associates with the cytoplasmic surfaces of supramolecular complexes of transmembrane structures that are presumed to include known and unknown junctional proteins. Mitochondria were linked to both microtubules and neurofilaments in the axoplasm and to neighboring smooth endoplasmic reticulum by narrow cross-bridges. Tubular cisternae in the glial cytoplasm were also linked to the paranodal glial cytoplasmic loop juxtanodal membrane by short cross-bridges. In the extracellular matrix between axon and Schwann cell, junctional bridges formed long cylinders linking the two membranes. Interactions between cytoskeleton, membranes, and extracellular matrix associations in the paranodal region are likely critical not only for scaffolding, but also for intracellular and extracellular communication.

Immunolocalization of sodium channel isoform NaCh6 in the nervous system

Sodium channel 6 (NaCh6) is the α-subunit of a voltage-gated sodium channel expressed in the rat nervous system. The mRNA for this isoform has been shown to be expressed in both neuronal and glial cells by in situ hybridization. To examine localization of NaCh6 protein, polyclonal antibodies specific for NaCh6 were generated against peptides from two cytoplasmic domains and a fusion protein from an extracellular domain. Affinity-purified antibodies were used to localize NaCh6 in the brain, spinal cord, peripheral nervous system, and neuromuscular junction. There was widespread labeling of neurons in the brain and spinal cord. NaCh6 was present in both sensory and motor pathways. Radial glial cells in the cerebellum were intensely labeled for both GFAP and NaCh6. At the subcellular level, NaCh6 is found in axons, dendrites, and the cell body. Motor neurons and primary sensory neurons in dorsal root ganglia had strong cytoplasmic and axonal staining. Nodes of Ranvier in peripheral nerve and in the spinal cord were also intensely labeled. Motor neuron axons near the neuromuscular junction were labeled up to, but not including, terminal boutons. Dendrites of pyramidal cells in the cortex, hippocampus, and cerebellum were labeled. NaCh6 is the first NaCh subtype to be localized either at the node of Ranvier or to a dendrite. We conclude that NaCh6 is widely distributed in the central and peripheral nervous systems and is likely to be important for the electrical properties of the axon and dendrite. J. Comp. Neurol. 420:70–83, 2000. © 2000 Wiley-Liss, Inc.

Immunolocalization of sodium channel isoform NaCh6 in the nervous system

Sodium channel 6 (NaCh6) is the alpha-subunit of a voltage-gated sodium channel expressed in the rat nervous system. The mRNA for this isoform has been shown to be expressed in both neuronal and glial cells by in situ hybridization. To examine localization of NaCh6 protein, polyclonal antibodies specific for NaCh6 were generated against peptides from two cytoplasmic domains and a fusion protein from an extracellular domain. Affinity-purified antibodies were used to localize NaCh6 in the brain, spinal cord, peripheral nervous system, and neuromuscular junction. There was widespread labeling of neurons in the brain and spinal cord. NaCh6 was present in both sensory and motor pathways. Radial glial cells in the cerebellum were intensely labeled for both GFAP and NaCh6. At the subcellular level, NaCh6 is found in axons, dendrites, and the cell body. Motor neurons and primary sensory neurons in dorsal root ganglia had strong cytoplasmic and axonal staining. Nodes of Ranvier in peripheral nerve and in the spinal cord were also intensely labeled. Motor neuron axons near the neuromuscular junction were labeled up to, but not including, terminal boutons. Dendrites of pyramidal cells in the cortex, hippocampus, and cerebellum were labeled. NaCh6 is the first NaCh subtype to be localized either at the node of Ranvier or to a dendrite. We conclude that NaCh6 is widely distributed in the central and peripheral nervous systems and is likely to be important for the electrical properties of the axon and dendrite.

The distribution of ganglioside-like moieties in peripheral nerves.

GM1 ganglioside has been implicated as a target of immune attack in some diseases of the peripheral nervous system. Anti-GM1 ganglioside antibodies are associated with certain acquired immune-mediated neuropathies. It is not clear how anti-GM1 antibodies cause nerve dysfunction and injury; however, sodium and/or potassium ion channel dysfunction at the node of Ranvier has been implicated. To gain insight into the pathogenesis of these neuropathies, we examined the distribution of GM1 ganglioside and Gal(beta1-3)GalNAc moieties in nerve fibres and their relationship to voltage-gated sodium and potassium (Kv1.1, 1.5) channels at the nodes of Ranvier in peripheral nerves from human, rat and dystrophic mice. Gal(beta1-3)GalNAc moieties were localized via the binding of cholera toxin and peanut agglutinin. As a control for the specificity of these findings, we compared the distribution of GM1 moieties to that of the ganglioside GT1b. Our study provides definitive evidence for the presence of Gal(beta1-3)GalNAc bearing moieties on the axolemmal surface of mature myelinated fibres and on Schwann cells. Gal(beta1-3)GalNAc binding sites did not have an obligatory co-localization with voltage-gated sodium channels or the potassium ion channels Kv1.1 and Kv1.5 and are thus not likely carried by these ion channels. In contrast with Gal(beta1-3)GalNAc, GT1b-like moieties are restricted to the axolemma.

Identification of Gal(β1–3)GalNAc bearing glycoproteins at the nodes of ranvier in peripheral nerve

A subset of human anti-GM1 ganglioside antibodies cross-reacts with Gal(beta 1-3)GalNAc bearing glycoproteins in peripheral nerve and spinal cord. The same oligosaccharide determinant is recognized by the lectin peanut agglutinin (PNA) which binds at the nodes of Ranvier in intact peripheral nerve. The Gal(beta 1-3)GalNAc bearing glycoproteins were isolated using PNA lectin affinity chromatography followed by separation on Western blot, and the proteins were subjected to partial amino acid sequence analysis. Two major PNA binding glycoproteins were identified in peripheral nerve and spinal cord; one had an approximate molecular weight of 120 kD and had sequence homology to the oligodendrocyte-myelin glycoprotein (OMgp). The other migrated between 70 and 80 kD and had sequence homology to the hyaluronate binding domain of versican, which has been reported to share sequence homology with the 70 kD proteins hyaluronectin and the glial hyaluronic acid binding protein (GHAP). By immunocytochemistry, OMgp was localized to the paranodal region of myelin, and the protein homologous to the hyaluronate binding domain of versican was localized to the nodal gap in peripheral nerve. These PNA binding glycoproteins might be target antigens for autoantibodies in peripheral nerve.

Experimental autoimmune neuropathy with anti-GM1 antibodies and immunoglobulin deposits at the nodes of Ranvier

Antibodies to GM1 or Gal(beta 1-3)GalNAc are associated with motor or sensorimotor neuropathy and with motor neuron disease. To investigate the role of these antibodies in the neurological disorder, rabbits were immunized with GM1 or with Gal(beta 1-3)GalNAc-BSA, and studied serologically, electrophysiologically and pathologically. Development of antibodies to the immunizing antigens was associated with a fall in the ratio of the amplitudes of the compound muscle action potential evoked by proximal versus distal stimulation of the sciatic nerve. Pathological studies revealed mild axonal degeneration and immunoglobulin deposits at the nodes of Ranvier in peripheral nerve, resembling those reported in a patient with motor neuropathy, motor conduction block and anti-GM1 antibodies. These studies provide evidence that anti-GM1 or anti-Gal(beta 1-3)GalNAc antibodies cause conduction abnormalities and indicate that the antibodies may exert their effect, in part, by binding at the nodes of Ranvier in peripheral nerve.

Node of Ranvier formation along fibres regenerating through silicone tube implants: a freeze-fracture and thin-section electron microscopic study.

Thin-section and freeze-fracture electron microscopy have been used to examine the morphogenesis of the node of Ranvier in peripheral nerves regenerating through silicone tubes. A major question posed by this study is whether node formation in fibres regenerating across a gap recapitulates that occurring in normal development. Node formation occurs concurrently with myelination and follows a similar spatial gradient of progression from a proximal to distal direction along the regenerated nerve. Presumptive nodal sites appear prior to myelin formation and are identified as a prominent subaxolemmal density in thin sections and axonal particle patches in freeze-fracture. Following the appearance of presumptive nodes in regenerating fibres, dimeric particles are inserted into the axolemma adjacent to the node. These particles are in close apposition to the overlying Schwann cell terminal processes and with maturity adopt the same circumferential orientation seen in adult nodes. The nodal axolemma of regenerating fibres shows a characteristic increase in the prominence of its subaxolemmal densification and number of heterogeneously sized particles. Mature regenerated nodes demonstrate a complete annulus of nodal particles indistinguishable from control nodes. The results of the present study show that the nodal architecture of regenerating fibres is a faithful reconstruction of normal mature nodes, thus indicating that the morphological correlates associated with saltatory conduction at the node are present in regenerated nodes.

Unequal branch point filtering action in different types of dorsal root ganglion neurons of frogs

The influence of the intraganglionic branch point on impulse conduction in single neurons of frog dorsal root ganglia (DRG) has been determined by measuring the least interval at which it will conduct two action potentials into the dorsal root. At 21–23°C, branch points of myelinated fibers had long least conduction intervals and low safety factors for orthodromic impulse conduction compared to nodes of Ranvier in peripheral nerve. DRG neurons with broad somatic spikes with a shoulder on the falling phase and slowly conducting myelinated or non-myelinated axons had the longest least conduction intervals (lowest safery factors). DRG neurons with brief somatic spikes with little or no shoulder on the falling phase had short least conduction intervals (higher safety factor) regardless of their conduction velocity. The results indicate that certain DRG neurons have found a way to minimize branch point filtering action.

Evidence for inorganic phosphate binding at nodes of Ranvier in peripheral nerves

Ferric ion is bound to different sites at nodes of Ranvier, depending on how the nerves are prepared. By immersing fresh, unfixed nerves in phosphate buffer, cacodylate buffer, or physiological saline prior to staining with ferric ion and ferrocyanide, it can be shown that binding of ferric ion to the extracellular nodal gap substance requires pretreatment with inorganic phosphate. This implies that phosphate anions are bound to the gap substance where they may then promote precipitation of ferric ion. These results call for a re-evaluation of data that depend on ferric ion binding to nodes of Ranvier. They also open the possibility that affinity for anions in general, or phosphate in particular, may be a significant feature of extracellular molecules present at nodes.

Further studies of electronic influences on toxic interactions between aryl esters and tissue chemoreceptors

Selected, monosubstituted phenylacetate esters of 3-tropanol and 2-α-tropanol have been studied as acute toxins in mice and cats and as blocking agents in excitable nodes of Ranvier in peripheral nerve, in efforts to evaluate contributions of substituent electronegativity and ring isomerism in agonists to gross toxic interactions with chemoreceptors. Using monofluoro and mononitro substituted esters, it has been observed that inductive effects of the electronegative F and NO 2 groups are primarily operative in determining level of potency in the intact animal, and that increasing electronegativity beyond that afforded by the F group yields no further increments in toxicity. In the single node of Ranvier preparation, esters with an electron-supplying substituent (CH 3) on the aryl group afford greater blockade potency than those bearing relatively electronegative groups. With both intact animal and single node preparations, potency of esters as controlled by tropane ring isomerism decreases in the power sequence 3-tropanyl (transoid) > 3-tropanyl (ψ, cisoid) ≥ 2-α-tropanyl (transoid). These and earlier findings are interpreted in terms of possible models for ester-chemoreceptor triggering interactions, which involve charged loci on tissue receptors in complementary configuration to major centers of charge localization in the substituted phenylacetate esters.