Peroxidase activity at nodes of Ranvier in some commonly used laboratory animals after intramuscular administration of horseradish peroxidase

During retrograde transport of intramuscularly administrated horseradish peroxidase (HRP) nodal axon segments of rabbit, rat, and mouse peripheral nerve fibers became HRP-positive and showed a striking proximodistal segregation of certain axoplasmic organelles. HRP-positive bodies and HRP-negative vesiculotubular membrane profiles were seen just distal and proximal to the nodal midlevel, respectively. This appearance was similar to that found earlier in HRP-transporting feline muscle nerve fibers. Some mechanisms that might explain the segregated appearance are briefly discussed.     

Nav1.1 is predominantly expressed in nodes of Ranvier and axon initial segments

Aggregation of voltage-gated sodium (Nav) channels in the axon initial segment (AIS) and nodes of Ranvier is essential for the generation and propagation of action potentials. From the three Nav channel isoforms (Nav1.1, Nav1.2 and Nav1.6) expressed in the adult CNS, Nav1.1 appears to play an important function since numerous mutations in its coding sequence cause epileptic syndromes. Yet, its distribution is still controversial. Here we demonstrate for the first time that in the adult CNS Nav1.1 is expressed in nodes of Ranvier throughout the mouse spinal cord and in many brain regions. We identified three populations of nodes: expressing Nav1.1, Nav1.6 or both. We also found Nav1.1 expression concentrated in a proximal AIS subcompartment in spinal cord neurons including 80% of motor neurons and in multiple brain areas. This novel distribution suggests that Nav1.1 is involved in the control of action potential generation and propagation.     

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.     

An ankyrinG-binding motif is necessary and sufficient for targeting Nav1.6 sodium channels to axon initial segments and nodes of Ranvier

Neurons are highly polarized cells with functionally distinct axonal and somatodendritic compartments. Voltage-gated sodium channels Na(v)1.2 and Na(v)1.6 are highly enriched at axon initial segments (AISs) and nodes of Ranvier, where they are necessary for generation and propagation of action potentials. Previous studies using reporter proteins in unmyelinated cultured neurons suggest that an ankyrinG-binding motif within intracellular loop 2 (L2) of sodium channels is sufficient for targeting these channels to the AIS, but mechanisms of channel targeting to nodes remain poorly understood. Using a CD4-Na(v)1.2/L2 reporter protein in rat dorsal root ganglion neuron-Schwann cell myelinating cocultures, we show that the ankyrinG-binding motif is sufficient for protein targeting to nodes of Ranvier. However, reporter proteins cannot capture the complexity of full-length channels. To determine how native, full-length sodium channels are clustered in axons, and to show the feasibility of studying these channels in vivo, we constructed fluorescently tagged and functional mouse Na(v)1.6 channels for in vivo analysis using in utero brain electroporation. We show here that wild-type tagged-Na(v)1.6 channels are efficiently clustered at nodes and AISs in vivo. Furthermore, we show that mutation of a single invariant glutamic acid residue (E1100) within the ankyrinG-binding motif blocked Na(v)1.6 targeting in neurons both in vitro and in vivo. Additionally, we show that caseine kinase phosphorylation sites within this motif, while not essential for targeting, can modulate clustering at the AIS. Thus, the ankyrinG-binding motif is both necessary and sufficient for the clustering of sodium channels at nodes of Ranvier and the AIS.     

A morphological study of the axons and recurrent axon collaterals of cat sciatic alpha-motoneurons after intracellular staining with horseradish peroxidase.

Utilizing the centrifugal neuronal transport of intracellularly injected horseradish peroxidase (HRP), we have performed a light microscopic (LM) investigation of the intramedullary parts of the axons and axon collaterals of sciatic alpha-motoneurons in the adult cat. The intramedullary parts of the alpha-motor axons had comparatively short internodes (down to 75 microns) and were thinner than reported in earlier studies on the ventral root. Positive correlations were obtained when relating nodal diameters (2.8-7.8 micron) or the mean diameters of the motor axons in the white matter (4.4-9.0 micron) to the diameters of the initial axonal segments (2.3-4.9 micron). Eighty percent of the motor axons gave off one to five collaterals. There was no correlation between the numbers of collaterals and the lengths of the parent motor axons in the gray matter. The branching patterns of the axon collaterals showed considerable variation and the number of end branches from a single collateral ranged between 1 and 39. The rostro-caudal distribution of the collateral end branches was arranged symmetrically within a narrow space (+/- 300 micron) around the origins of the first order collaterals. Outbulgings of the motor axon collaterals, interpreted as synaptic terminals, were found along (59%) or at the ends (41%) of the collateral branches, and were located 200-700 micron away from the origin of the first order collateral. No characteristic LM feature of the outbulgings was distinguished.     

Reassembly of the axon initial segment and nodes of Ranvier in regenerated axons of the central nervous system

<正>Myelinated axons of the peripheral and central nervous system(PNSCNS)are divided into molecularly distinct excitable domains,including the axon initial segment(AIS)and nodes of Ranvier.The AIS is composed of a dense network of cytoskeletal proteins,cell adhesion molecules,and voltage gated ion channels and is located at the proximal most region of the axon(Kole     

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.     

An ultrastructural study of cat lumbosacral gamma-motoneurons after retrograde labelling with horseradish peroxidase

Twelve retrogradely horseradish peroxidase (HRP)-labelled triceps surae motoneurons of gamma size (mean cell body diameter less than 38 micron) were studied ultrastructurally. The contours of the cell bodies, as observed in the transverse midnucleolus plane, were elongated to rounded. The axons identified all originated from the cell body. The mean diameter of the stem dendrites was 4.5 micron. A substantial part of the cell membrane was covered by glial extensions. The boutons and synaptic contacts apposing the gamma-motoneurons could be classified into two categories on the basis of the type of synaptic vesicles: S-type boutons with spherical synaptic vesicles and F-type boutons with flattened vesicles. In each neuron, the values for mean length and mean area of apposition, percentage synaptic covering, and packing density of S-type, F-type, and S+F-type boutons were estimated on the cell body and in two dendritic compartments. In comparison with alpha-motoneurons and Renshaw cells, the cell bodies of the gamma-motoneurons were covered by smaller and strikingly fewer boutons of both the S- and F-types. The values for percentage synaptic covering and packing density of boutons on the proximal dendrites were also lower for gamma-motoneurons than for both alpha-motoneurons and Renshaw cells, although the differences were less pronounced than on the cell body. No boutons of the C-, M-, and T-types described for alpha-motoneurons were found on the gamma-motoneurons.     

CNP is required for maintenance of axon-glia interactions at nodes of Ranvier in the CNS

Axoglial interactions underlie the clustering of ion channels and of cell adhesion molecules, regulate gene expression, and control cell survival. We report that Cnp1-null mice, lacking expression of the myelin protein cyclic nucleotide phosphodiesterase (CNP), have disrupted axoglial interactions in the central nervous system (CNS). Nodal sodium channels (Nav) and paranodal adhesion proteins (Caspr) are initially clustered normally, but become progressively disorganized with age. These changes are characterized by mislocalized Caspr immunostaining, combined with a decrease of clustered Na+ channels, and occur before axonal degeneration and microglial invasion, both prominent in older Cnp1-null mice. We suggest that CNP is a glial protein required for maintaining the integrity of paranodes and that disrupted axoglial signaling at this site underlies progressive axonal degeneration, observed later in the CNS of Cnp1-null mice.     

Cells expressing the NG2 antigen contact nodes of Ranvier in adult CNS white matter

The NG2 antibody, which recognises an integral membrane chondroitin sulphate, labels a significant population of cells in adult CNS white matter tracts of the rat optic nerve and anterior medullary velum (AMV). Adult NG2+ cells are highly complex with multiple branching processes and we show by EM immunocytochemistry that they extend perinodal processes, which contact nodes of Ranvier. NG2+ cells do not react to conventional immunohistochemical markers for adult glia and so we reservedly term them NG2P cells. In vitro, NG2 labels oligodendrocyte-type-2 astrocyte (O-2A) progenitors that can give rise to oligodendrocytes or type-2 astrocytes, depending on the culture medium. Thus, it is possible that NG2P cells may be derived from the same stem cells as oligodendrocytes. Interestingly, NG2+ cells identified previously in adult CNS displayed phenotypic characteristics of O-2Aadult progenitors and it is possible that, like them, NG2P cells might retain the capacity of generating oligodendrocytes in the adult CNS. This may be an important role of NG2P cells in demyelinating diseases such as multiple sclerosis. It is significant therefore that the perinodal processes of NG2P cells contact the only sites of exposed axolemma in myelinated axons, so that NG2P cells are ideally situated to detect and respond to changes in axonal function during demyelination. A further implication of our finding is that NG2P cells may perform functions at nodes of Ranvier previously attributed to perinodal astrocytes, including the clustering and maintenance of sodium channels in the axon membrane at nodes, during development and following demyelination.     

Cellular and extracellular components at nodes of Ranvier in rat white matter

Rat CNS nodes of Ranvier were investigated by electron microscopy and immunohistochemistry. Nodes along thin callosal axons possess tiny node gaps containing few or no astrocytic processes. Nodes along thick spinal axons exhibit spatious node gaps containing relatively few irregularly arranged astrocytic processes. Antibodies against HNK-1, chondroitin sulfate, tenascin or NSP-4 do not label small nodes but stain large nodes. We conclude that rat CNS fibers do not exhibit a strict relation between nodal complexity and fiber size comparable to that found in rat PNS fibers.     

Cytochemical characteristics of the axon membrane at nodes of Ranvier in resting, tetrodotoxin-blocked, and electrically stimulated peripheral nerves of the rat

To test whether intraaxonal ferric ion-ferrocyanide staining at nodes of Ranvier is influenced by functional state of the nodal membrane, the tibial nerves of Sprague-Dawley rats were subjected to one of the following experimental procedures prior to fixation and staining: General anesthesia to induce nerves at rest, tetrodotoxin blocking of nerve activity, and high-frequency (100 Hz) electrical stimulation. In all cases most but not all nodes were stained. No statistically significant differences were found either in the percentage of total stained nodes or in the percentage of stained nodes from large- and small-diameter fibers among the three conditions tested. An explanation is offered to account for the apparent discrepancy between these results and those from other studies involving related cytochemical markers of the nodal apparatus.     

Differential expression of proteoglycans at central and peripheral nodes of Ranvier

The nodes of Ranvier are regularly spaced gaps between myelin sheaths that are markedly enriched in voltage-gated sodium channels and associated proteins. Myelinating glia play a key role in promoting node formation, although the requisite glial signals remain poorly understood. In this study, we have examined the expression of glial proteoglycans in the peripheral and central nodes. We report that the heparan sulfate proteoglycan, syndecan-3, becomes highly enriched with PNS node formation; its ligand, collagen V, is also concentrated at the PNS nodes and at lower levels along the abaxonal membrane. The V1 isoform of versican, a chondroitin sulfate proteoglycan, is also present in the nodal gap. By contrast, CNS nodes are enriched in versican isoform V2, but not syndecan-3. We have examined the molecular composition of the PNS nodes in syndecan-3 knockout mice. Nodal components are normally expressed in mice deficient in syndecan-3, suggesting that it has a nonessential role in the organization of nodes in the adult. These results indicate that the molecular composition and extracellular environment of the PNS and CNS nodes of Ranvier are significantly distinct.     

Axon initial segments of the granule cell in the rat dentate gyrus: synaptic contacts on bundles of axon initial segments

In Golgi and electron microscopic studies, dentate granule cell axon initial segments were revealed to be fasciculated in the granule cell layer and to receive many synapses. Combined Golgi-EM studies indicated that the presynaptic elements of these synapses might be beaded string-like axon collaterals of some type of basket cell.     

Polyribosomes associated with synaptic specializations on axon initial segments: localization of protein-synthetic machinery at inhibitory synapses

Previous studies have revealed a selective association between polyribosomes and axospinous synapses in a variety of brain regions. The present study evaluates whether polyribosomes are also associated with the symmetrical and presumably inhibitory synaptic connections found on the initial segment of axons of some neurons in the CNS. The initial segments of pyramidal neurons in the sensorimotor cortex of the monkey and of granule cells in the hippocampus of the rat were examined. The initial segments of these cell types are contacted by GABAergic terminals that form symmetrical synaptic connections. In the present study, these initial segments were found to contain polyribosomes that tended to be selectively localized beneath the synaptic specializations. Both the synaptic connections and the polyribosomes were localized to the initial segment; after the point at which the axon became myelinated, neither synapses nor polyribosomes were found. The association between polyribosomes and synapses was also suggested by the position of the polyribosomes with respect to the synapse. In each cell type, the majority of the polyribosomes that were present in the initial segments appeared to be localized preferentially beneath synaptic sites, although some polyribosomes were also present in the core of the axon. These data reveal that polyribosomes are not peculiar to spine synapses, but rather are ubiquitous components of the subsynaptic region of many types of synapses. We propose that neurons may regulate their innervation by positioning protein-synthetic machinery (and appropriate mRNA molecules) at particular locations in order to construct particular types of synapses at defined positions on the postsynaptic cells' receptive surface.     

Reduced axon sprouting after treatment that diminishes microglia accumulation at lesions in the leech CNS

The role of mammalian microglia in central nervous system (CNS) repair is controversial. Microglia accumulate at lesions where they act as immune cells and phagocytize debris, and they may secrete neurotrophins, but they also produce molecules that can be cytotoxic, like nitric oxide (NO). To determine the importance of microglial accumulation at lesions on growth of severed CNS axons in the leech (Hirudo medicinalis), in which axon and synapse regeneration are notably successful even when isolated in tissue culture medium, microglial migration to lesions was reduced. Pressure (P) sensory neurons were injected with biocytin to reveal the extent of their sprouting 24 hours after lesioning. To reduce microglia accumulation at lesions, cords were treated for 3.5 hours with 3 mM ATP or 2 mM N(omega)-nitro-L-arginine methyl ester (L-NAME) or 50 microM Reactive blue-2 (RB2) beginning 30 minutes before injury. Lesioned controls were either not treated with drug or treated 3 hours later with one of the drugs, after the migration and subsequent accumulation of most microglia had occurred, but before the onset of axon sprouting, for a total of seven separate conditions. There was a significant reduction in total sprout lengths compared with controls when microglial accumulation was reduced. The results suggest that microglial cells are necessary for the usual sprouting of injured axons.     

Postnatal development of synaptic structure proteins in pyramidal neuron axon initial segments in monkey prefrontal cortex

In the primate prefrontal cortex (PFC), the functional maturation of the synaptic connections of certain classes of γ‐aminobutyric acid (GABA) neurons is very complex. For example, the levels of both pre‐ and postsynaptic proteins that regulate GABA neurotransmission from the chandelier class of cortical interneurons to the axon initial segment (AIS) of pyramidal neurons undergo marked changes during both the perinatal period and adolescence in the monkey PFC. In order to understand the potential molecular mechanisms associated with these developmental refinements, we quantified the relative densities, laminar distributions, and lengths of pyramidal neuron AIS immunoreactive for ankyrin‐G, βIV spectrin, or gephyrin, three proteins involved in regulating synapse structure and receptor localization, in the PFC of rhesus monkeys ranging in age from birth through adulthood. Ankyrin‐G‐ and βIV spectrin‐labeled AIS declined in density and length during the first 6 postnatal months, but then remained stable through adolescence and into adulthood. In contrast, the density of gephyrin‐labeled AIS was stable until approximately 15 months of age and then markedly declined during adolescence. Thus, molecular determinants of the structural features that define GABA inputs to pyramidal neuron AIS in monkey PFC undergo distinct developmental trajectories with different types of changes occurring during the perinatal period and adolescence. In concert with previous data, these findings reveal a two‐phase developmental process of GABAergic synaptic stability and GABA neurotransmission at chandelier cell inputs to pyramidal neurons that likely contributes to the protracted maturation of behaviors mediated by primate PFC circuitry. J. Comp. Neurol. 514:353–367, 2009. © 2009 Wiley‐Liss, Inc.     

Expression of IP3 receptor isoforms at the nodes of Ranvier in rat sciatic nerve

Inositol 1,4,5-trisphosphate receptors (IP3R) are modulated by the second messenger IP3, which induces intracellular calcium release. Using immunohistochemical techniques, we show that the three isoforms are expressed in sciatic nerve. IP3R1 and IP3R2 are mainly present in the nucleus of Schwann cells. IP3R1 is also expressed in Schmidt-Lanterman incisures. IP3R3 is primarily localized at very high levels in nonmyelinating Schwann cells. Interestingly, the three isoforms are expressed at the nodes of Ranvier. IP3R1 is clustered at the node of Ranvier, in a distribution that is similar to the Nav1.6 sodium channels in the sciatic nerve. IP3R3 is present in the paranodal regions of the nodes. IP3R2 is concentrated in the vicinity of the node, and the outer Schwann cell cytoplasm similar to the Kv1.5 potassium channel.     

Characterization of the distinctive neurofilament subunits of the soma and axon initial segments in the squid stellate ganglion

The stellate ganglion, which gives rise to the giant axons of the squid, was dissected into two parts, one containing primarily cell bodies and the other axon initial segments. A neurofilament protein-enriched extract of each was prepared and compared biochemically and immunochemically with an axoplasmic neurofilament preparation and with the glial sheath that surrounds the axons. Both parts of the ganglion lacked the 220 kDa subunit of axoplasmic neurofilaments (NFs). However, they did contain a protein of about 190 kDa that reacted with the Pruss anti-intermediate filament antibody (aIFA; Pruss et al.: Cell 27:419-428, 1981), but not with a phosphorylation-dependent NF antibody (Cohen et al.: J Neurosci 7: 2056-2074, 1987). Dephosphorylation of the axoplasmic NF220 yielded a product that comigrated on two-dimensional (2D) gel electrophoresis with the 190 kDa ganglion protein, suggesting that the latter represented the incompletely phosphorylated precursor of NF220. The major low molecular weight aIFA-reactive species in the ganglion preparations was a polypeptide of about 65 kDa. A relatively small quantity of that polypeptide was also found in axoplasm and it comigrated in 2D gels with an aIFA-reactive polypeptide from the glial sheath. These results indicate that the site of modification of the 190 kDa NF precursor to the 220 kDa axonal form is probably at the point where the axon initial segments leave the ganglion, which is several mm distal to its site of synthesis in the cell body. Furthermore, the filament network of the axoplasm and possibly the cell bodies includes a glial-like intermediate filament protein in addition to the NF protein subunits.     

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

Summary Antibodies to GM1 or Gal(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(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(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.Supported by center grants from the Muscular Dystrophy Association and NINCDS (NS11766) to Columbia University. F.P. Thomas and S. A. Sadiq were fellows of the Muscular Dystrophy Association and the Charles A. Dana Foundation; F.P.Thomas is a fellow of the German Cancer Research Center (DKFZ)