Immuno-ultrastructural localization of sodium channels at nodes of Ranvier and perinodal astrocytes in rat optic nerve

Immuno-electron microscopic localization of sodium channels at nodes of Ranvier within adult optic nerve was demonstrated with polyclonal antibody 7493. The 7493 antisera, which is directed against purified sodium channels from rat brain, recognizes a 260 kDa protein in immunoblots of the crude glycoprotein fraction from adult rat optic nerve. Intense immunoreactivity with 7493 antisera was observed at nodes of Ranvier. Axon membrane at the node was densely stained, whereas paranodal and internodal axon membrane did not exhibit immunoreactivity. The axoplasm beneath the nodal membrane displayed variable immunostaining. Neither terminal paranodal oligodendroglial loops nor oligodendrocyte plasmalemma were immunoreactive with 7493 antisera. However, perinodal astrocyte processes exhibited intense immunoreactivity with the anti-sodium channel antisera. Optic nerves incubated with pre-immune sera, or with 7493 antisera that had been pre-adsorbed with purified sodium channel protein, displayed no immunoreactivity. These results demonstrate localization of sodium channels at high density at mammalian nodes of Ranvier and in some perinodal astrocyte processes. The latter observation offers support for an active role for perinodal astrocyte processes in the aggregation of sodium channels within the axon membrane at the node of Ranvier.     

Nr-CAM and neurofascin interactions regulate ankyrin G and sodium channel clustering at the node of Ranvier.

Voltage-dependent sodium (Na(+)) channels are highly concentrated at nodes of Ranvier in myelinated axons and play a key role in promoting rapid and efficient conduction of action potentials by saltatory conduction. The molecular mechanisms that direct their localization to the node are not well understood but are believed to involve contact-dependent signals from myelinating Schwann cells and interactions of Na(+) channels with the cytoskeletal protein, ankyrin G. Two cell adhesion molecules (CAMs) expressed at the axon surface, Nr-CAM and neurofascin, are also linked to ankyrin G and accumulate at early stages of node formation, suggesting that they mediate contact-dependent Schwann cell signals to initiate node development. To examine the potential role of Nr-CAM in this process, we treated myelinating cocultures of DRG (dorsal root ganglion) neurons and Schwann cells with an Nr-CAM-Fc (Nr-Fc) fusion protein. Nr-Fc had no effect on initial axon-Schwann cell interactions, including Schwann cell proliferation, or on the extent of myelination, but it strikingly and specifically inhibited Na(+) channel and ankyrin G accumulation at the node. Nr-Fc bound directly to neurons and clustered and coprecipitated neurofascin expressed on axons. These results provide the first evidence that neurofascin plays a major role in the formation of nodes, possibly via interactions with Nr-CAM.     

alphaII-spectrin is essential for assembly of the nodes of Ranvier in myelinated axons

Saltatory conduction in myelinated axons requires organization of the nodes of Ranvier, where voltage-gated sodium channels are prominently localized [1]. Previous results indicate that alphaII-spectrin, a component of the cortical cytoskeleton [2], is enriched at the paranodes [3, 4], which flank the node of Ranvier, but alphaII-spectrin's function has not been investigated. Starting with a genetic screen in zebrafish, we discovered in alphaII-spectrin (alphaII-spn) a mutation that disrupts nodal sodium-channel clusters in myelinated axons of the PNS and CNS. In alphaII-spn mutants, the nodal sodium-channel clusters are reduced in number and disrupted at early stages. Analysis of chimeric animals indicated that alphaII-spn functions autonomously in neurons. Ultrastructural studies show that myelin forms in the posterior lateral line nerve and in the ventral spinal cord in alphaII-spn mutants and that the node is abnormally long; these findings indicate that alphaII-spn is required for the assembly of a mature node of the correct length. We find that alphaII-spectrin is enriched in nodes and paranodes at early stages and that the nodal expression diminishes as nodes mature. Our results provide functional evidence that alphaII-spectrin in the axonal cytoskeleton is essential for stabilizing nascent sodium-channel clusters and assembling the mature node of Ranvier.     

Sulfated acid mucopolysaccharides in the cortical granules of eggs. Effects of quaternary ammonium salts on fertilization

Sulfated acid mucopolysaccharides have been shown to be constituents of cortical granules in sea urchin and vertebrate eggs. These observations were made possible by retaining soluble acid mucopolysaccharides in situ within the eggs by precipitation during fixation with cetyltrimethyl-ammonium bromide, a quaternary ammonium salt. The sulfated mucopolysaccharides were then identified by staining with Astrablau at pH 0.2 and also by reaction with sodium rhodizonate. Staining reaction with Alcian blue at pH 2.5 showed that carboxylated mucopolysaccharides may also be present in cortical granules. The natural ionic environment of these eggs would favor the formation of very stable complexes between sulfated mucopolysaccharides and quaternary ammonium salts. Brief exposure of unfertilized sea urchin eggs to several quaternary ammonium compounds produced a residual adverse effect on subsequent fertilization in terms of increased vulnerability to polyspermy and reduced fertilizability. These results suggest that sulfated acid mucopolysaccharides participate in the function of the cortical granules and the establishment of the block to polyspermy at fertilization, and possibly in other cellular secretory processes.     

Ultrastructure of the nodes of Ranvier and their surrounding structures in the central nervous system

Summary The nodes of Ranvier and their surrounding structures have been studied by means of serial and single ultrathin sections of the frog's optic tract and diencephalon.The following aspects of the ultrastructure of the myelin sheath at the nodes are described: (a) the site and manner of termination of the compact myelin and glia-satellite cytoplasm; (b) the reflection of the glia-satellite cytoplasm at the node; (c) the formation of compact myelin during development; (d) the involvement of the glia-satellite cell in the metabolism and impulse conduction of the fiber.The nodes are surrounded by a considerable extension of the extracellular space — the perinodal extracellular space or perinodal matrix. The ground substance of the perinodal matrix consists of ill defined granules arranged in patches or in fibrillar or vacuolar manner. Microvilli-like protuberances of the glia and nervous processes emerge into the perinodal matrix. The shape and the volume of the perinodal extracellular space is determined using a reconstruction method from a series of sections through the node in diencephalon.In the diencephalon nerve endings show close contact with the node and its surroundings. These nerve endings contain synaptic vesicles, mitochondria and small opaque particles. Small opaque particles, up to the present not recognized as components of synapses, have been observed in a number of nerve endings in the diencephalon. The possibility is considered that such nervous configurations at the node could be involved in subliminal interactions between different neurons.Based on the ultrastructural data the concept of nodal apparatus is introduced as a working hypothesis. The nodal apparatus consists of the node, terminal compartments of the glia-satellite cell, the perinodal matrix, and the surrounding glia and nervous structures, which may be involved in the nodal activities. The structural pattern of such a nodal apparatus may vary in different parts of the central nervous system indicating the possibility of variation in the functioning of the corresponding nodes.This investigation was supported by a grant from the Medical Research Council, Canada (MA-1247).The author is very much indebted to Mrs. H. Rushforth for excellent technical assistance, to Mr. H. R. A. Meiborg, Groningen, for very skilful printing of the photographs and to Mr. Hoekstra, Groningen, who made the drawing of Fig. 8.     

Perlecan is recruited by dystroglycan to nodes of Ranvier and binds the clustering molecule gliomedin

Fast neural conduction requires accumulation of Na⁺ channels at nodes of Ranvier. Dedicated adhesion molecules on myelinating cells and axons govern node organization. Among those, specific laminins and dystroglycan complexes contribute to Na⁺ channel clustering at peripheral nodes by unknown mechanisms. We show that in addition to facing the basal lamina, dystroglycan is found near the nodal matrix around axons, binds matrix components, and participates in initial events of nodogenesis. We identify the dystroglycan-ligand perlecan as a novel nodal component and show that dystroglycan is required for the selective accumulation of perlecan at nodes. Perlecan binds the clustering molecule gliomedin and enhances clustering of node of Ranvier components. These data show that proteoglycans have specific roles in peripheral nodes and indicate that peripheral and central axons use similar strategies but different molecules to form nodes of Ranvier. Further, our data indicate that dystroglycan binds free matrix that is not organized in a basal lamina.     

Differential expression and functions of neuronal and glial neurofascin isoforms and splice variants during PNS development

The cell adhesion molecule neurofascin (NF) has a major neuronal isoform (NF186) containing a mucin-like domain followed by a fifth fibronectin type III repeat while these domains are absent from glial NF155. Neuronal NF isoforms lacking one or both of these domains are expressed transiently in embryonic dorsal root ganglia (DRG). These two domains are co-expressed in mature NF186, which peaks in expression prior to birth and then persists almost exclusively at nodes of Ranvier on myelinated axons. In contrast, glial NF155 is only detected postnatally with the onset of myelination. All these forms of NF bound homophilically and to Schwann cells but only the mature NF186 isoform inhibits cell adhesion, and this activity may be important in formation of the node of Ranvier. Schwann cells deficient in NF155 myelinated DRG axons in a delayed manner and they showed significantly decreased clustering of both NF and Caspr in regions where paranodes normally form. The combined results suggest that NF186 is expressed prenatally on DRG neurons and it may modulate their adhesive interactions with Schwann cells, which express NF155 postnatally and require it for development of axon-glial paranodal junctions.     

First node of Ranvier facilitates high-frequency burst encoding

In central neurons the first node of Ranvier is located at the first axonal branchpoint, ～ 100 μm from the axon initial segment where synaptic inputs are integrated and converted into action potentials (APs). Whether the first node contributes to this signal transformation is not well understood. Here it was found that in neocortical layer 5 axons, the first branchpoint is required for intrinsic high-frequency (≥ 100 Hz) AP bursts. Furthermore, block of nodal Na(+) channels or axotomy of the first node in intrinsically bursting neurons depolarized the somatic AP voltage threshold (～ 5 mV) and eliminated APs selectively within a high-frequency cluster in response to steady currents or simulated synaptic inputs. These results indicate that nodal persistent Na(+) current exerts an anterograde influence on AP initiation in the axon initial segment, revealing a computational role of the first node of Ranvier beyond conduction of the propagating AP.     

Molecular composition of the node of Ranvier: identification of ankyrin- binding cell adhesion molecules neurofascin (mucin+/third FNIII domain- ) and NrCAM at nodal axon segments

Neurofascin, NrCAM, L1, and NgCAM are a family of Ig/FNIII cell adhesion molecules that share ankyrin-binding activity in their cytoplasmic domains, and are candidates to form membrane-spanning complexes with members of the ankyrin family of spectrin-binding proteins in a variety of cellular contexts in the nervous system. Specialized forms of ankyrin, 270 kD and/or 480 kD ankyrinG are components of the membrane undercoat of axons at the node of Ranvier. This paper focuses on definition of the isoforms of ankyrin-binding cell adhesion molecules localized with ankyrinG at the nodal axon segment. The exon usage of two major forms of neurofascin was determined by isolation of full-length cDNAs and used to prepare isoform-specific antibodies. An isoform of neurofascin containing a mucin-like domain and lacking the third FNIII domain was concentrated at axon initial segments and colocalized at nodes of Ranvier with ankyrinG and the voltage-dependent sodium channel. An alternative form of neurofascin lacking the mucin-like domain and containing the third FNIII domain was present in unmyelinated axons. The antibody initially raised against neurofascin was used to screen a rat brain cDNA expression library. In addition to neurofascin, this screen yielded a clone with 80% sequence identity to NrCAM from chicken. The sequences of two full-length cDNAs are presented. NrCAM is most closely related to neurofascin among the other members of the L1/neurofascin/NgCAM family, with over 70% identity between cytoplasmic domains. NrCAM, visualized with antibodies specific for the ecto-domain, also was found to be coexpressed with neurofascin at nodes of Ranvier and at axon initial segments. This is the first characterization of defined neuronal cell adhesion molecules localized to axonal membranes at the node of Ranvier of myelinated axons.     

Axonal domains: role for paranodal junction in node of Ranvier assembly

A new study shows that communication between axons and glia at the paranodal junction can orchestrate the formation of the node of Ranvier.     

The Reactions of Isolated Mucopolysaccharides to Several Histochemical Tests

The reaction to three histochemical tests of preparations of hyaluronic acid, chondroitin sulfate, heparin, the acidic mucopolysaccharides from cornea, gastric mucin, and dentine, and also of the neutral mucopolysaccharide from gastric mucin was studied. To 1% aqueous solutions of the acid mucopolysaccharides, equal volumes of 1% casein solution were added; drops of the resulting solutions were placed on slides and dried at 37 °C. The films were then fixed in acetic-alcohol (1:9). The technics employed were the periodic acid-Schiff (PAS) test, the metachromatic reaction and the Hale test. The relative acidity of the preparations was demonstrated by staining in dilute aqueous methylene blue at pH 3-6. With the exception of the preparation from dentine, the acid mucopolysaccharides stained only weakly with PAS; the neutral mucopolysaccharide stained strongly. It is concluded, therefore, that the use of the PAS technic for the histochemical demonstration of acid mucopolysaccharides is misleading, for many important members of this class of tissue component do not react appreciably. On the other hand, metachromasia was shown by all the acidic compounds studied, and the intensity of staining was approximately correlated with the acidity of the preparations. The Hale method was found to be nonspecific.     

Early events in node of Ranvier formation during myelination and remyelination in the PNS

Action potential conduction velocity increases dramatically during early development as axons become myelinated. Integral to this process is the clustering of voltage-gated Na(+) (Nav) channels at regularly spaced gaps in the myelin sheath called nodes of Ranvier. We show here that some aspects of peripheral node of Ranvier formation are distinct from node formation in the CNS. For example, at CNS nodes, Nav1.2 channels are detected first, but are then replaced by Nav1.6. Similarly, during remyelination in the CNS, Nav1.2 channels are detected at newly forming nodes. By contrast, the earliest Nav-channel clusters detected during developmental myelination in the PNS have Nav1.6. Further, during PNS remyelination, Nav1.6 is detected at new nodes. Finally, we show that accumulation of the cell adhesion molecule neurofascin always precedes Nav channel clustering in the PNS. In most cases axonal neurofascin (NF-186) accumulates first, but occasionally paranodal neurofascin is detected first. We suggest there is heterogeneity in the events leading to Nav channel clustering, indicating that multiple mechanisms might contribute to node of Ranvier formation in the PNS.     

Histochemical localization of galactose-containing glycoconjugate at peripheral nodes of Ranvier in the rat

Lectin-horseradish peroxidase conjugates were used to study glycoconjugates in paraffin sections of dorsal roots of the rat spinal cord. Griffonia simplicifolia-B4 isolectin (GSA I-B4) and peanut agglutinin (PNA) stained strongly the nodes of Ranvier, localizing, respectively, terminal alpha- and beta-D-galactose. Sialidase digestion did not increase staining with PNA at the node of Ranvier, suggesting the presence of a neutral glycoconjugate. Staining of the nodal but not the internodal axolemma was observed with PNA. The outer surface of the myelin sheath in axons of the dorsal root stained strongly with GSA I-B4 but only weakly with PNA, demonstrating an abundance of terminal alpha-galactose. PNA staining was enhanced in this site by sialidase digestion, showing terminal sialic acid-beta-galactose dimers. The presence of sialic acid here was further evidenced by labeling of these membranes with the lectin derived from the slug, Limax flavus (LFA). Affinity for a high iron diamine-Alcian blue (pH 2.5) sequence demonstrated, in addition, the presence of sulfate esters in glycoconjugates on the outer myelin membrane. GSA I-B4 imparted strong reactivity to nonmyelinated fibers in the dorsal root and the spinal nerve. The present findings appear to reflect several localizations of biochemically described nervous system glycoproteins containing O-glycosidically linked side chains terminated by alpha- and beta-D-galactose.     

Molecular specialization of astrocyte processes at nodes of Ranvier in rat optic nerve

The HNK-1 and L2 monoclonal antibodies are thought to recognize identical or closely associated carbohydrate epitopes on a family of neural plasma membrane glycoproteins, including myelin-associated glycoprotein, the neural cell adhesion molecule, and the L1 and J1 glycoproteins, all of which have been postulated to play a part in mediating cell-cell interactions in the nervous system. We have used these two antibodies in immunofluorescence and immunogold-electron microscopic studies of semithin and ultrathin frozen sections of adult rat optic nerve, respectively, and we show that they bind mainly to astrocyte processes around nodes of Ranvier. Most other elements of the nerve, including astrocyte cell bodies and large astrocytic processes, are not labeled by the antibodies. To our knowledge, this is the first demonstration that perinodal astrocyte processes are biochemically specialized. We provide evidence that one of the HNK-1+/L2+ molecules concentrated around perinodal astrocyte processes is the J1 glycoprotein; our findings, taken together with previously reported observations, suggest that the other known HNK-1+/L2+ molecules are not concentrated on these processes. Since anti-J1 antibodies previously have been shown to inhibit neuron to astrocyte adhesion in vitro, we hypothesize that J1 may play an important part in the axon-glial interactions that presumably are involved in the assembly and/or maintenance of nodes of Ranvier.     

Acid protease activity during germination of microcysts of the cellular slime mold Polysphondylium pallidum.

Extracts of dormant microcysts of Polysphondylium pallidum demonstrate pH optima for the hydrolysis of casein at 3.5 and 6.0. During germination the intracellular pH 6.0 caseinolytic specific activity does not change significantly. The pH 6.0 protease is also active on azo-albumin, revealing the same developmental pattern with this substrate. Both acid protease activities are excreted during the germination process. Addition of purified nonspecific protease to cultures speeds up germination, suggesting that the excreted protease may play a role in removal of the microcyst wall. When cycloheximide is added to cultures, complete germination (emergence) is stopped whereas the pH 6.0 protease activity still accumulates to between 50 and 60% of the maximum control activity. Although this suggests that post-translational controls might mediate the accumulation of a portion of the pH 6.0 protease increase, mixing and dilution experiments with cell extracts do not reveal the differential presence of soluble activators or inhibitors of this activity at different developmental stages. The presence of tightly bound enzyme-inhibitor complexes for protease B in dormant microcysts has not been ruled out and is currently under study.     

pH and temperature resolve the kinetics of two pools of calcium bound to the sarcoplasmic reticulum Ca2+ -ATPase

Calcium bound to the sarcoplasmic reticulum Ca2+ -ATPase was removed by chelating free calcium ion with EGTA. The kinetic calcium binding reaction to the calcium-unbound ATPase was studied by varying the pH (6.0-8.8) and temperature (0-20 degrees C) at a saturating concentration of 50-100 microM [Ca2+]. At pH 6.0 and 0 degrees C, calcium sites of the enzyme at a rate of t1/2 approximately 10 s. By increasing the pH from 6.0 to 8.8, about half of the total calcium sites were converted from a slow binding state to a rapid binding state (less than 2s). The maximum level was reached at about pH 7.4, and the midpoint of the conversion was observed at about pH 6.7. On the other hand, the slow binding reaction to the other sites was not significantly affected by the pH increase. At pH 7.0 and 20 degrees C, about 90% of the total calcium sites rapidly (less than 2s) bound calcium. The present results suggest that pH and temperature resolve the kinetics of two pools of calcium bound to the Ca2+-ATPase.     

Different metal-binding properties of the two sites of human transferrin.

Transferrin, the serum serum iron-transport protein which can bind two metal ions at physiologic pH, binds just one Fe3+, VO2+, or Cr3+ ion at pH 6.0. Fe3+ and VO2+ appear to be bound at the same site, designated A, based on electron paramagnetic resonance (EPR) spectra of VO2+-transferrin and (Fe3+)1(VO2+)1-transferrin. The EPR spectra of (Cr3+)1(VO2+)1-transferrin and of (Cr3+), (FE3+)1-transferrin indicate that that Cr3+ is bound to site B at pH 6.0. Transferrin was labeled at site A with 59Fe at pH 6.0 and at site B with 55Fe at pH 7.5. When the pH of the resulting preparation was lowered to 6.3 and the dissociated iron was separated by gel filtration, about ten times as much 55Fe as 59Fe was lost. The same EPR and isotopic-labeling experiments showed that Fe3+ added to transferrin at pH 7.5 binds to site A with about 90% selectivity.     

Initiation of sodium channel clustering at the node of Ranvier in the mouse optic nerve

Ion fluxes in mammalian myelinated axons are restricted to the nodes of Ranvier, where, in particular, voltage-gated Na⁺ channels are clustered at a high density. The node of Ranvier is separated from the internode by two distinct domains of the axolemma, the paranode and the juxtaparanode. Each axonal domain is characterized by the presence of a specific protein complex. Although oligodendrocytes and/or myelin membranes are believed to play some instructive roles in the organization of axonal domains, the mechanisms leading to their localized distribution are not well understood. In this paper we focused on the involvement of myelin sheaths in this domain organization and examined the distribution of axonal components in the optic nerves of wild type, hypomyelinating jimpy mice and demyelinating PLP transgenic mice. The results showed that the clustering of Na⁺ channels does not require junction-like structures to be formed between the glial processes and axons, but requires mature oligodendrocytes to be present in close vicinity.     

Receptor-mediated vitellogenin binding to chicken oocytes.

The specific binding of vitellogenin to chicken oocyte membranes was characterized. This major hen serum phospholipoglycoprotein and one of its lower-molecular-weight components, phosvitin, bound to oocyte membranes with KD values of approx. 6 x 10-7 M. The optimum pH for binding was 6.0, the same as the pH of yolk contents. Phosvitin and vitellogenin compete with each other for binding; other proteins tested do not compete to the same degree. Phosvitin, which contains 10% phosphate by weight, appears to be the polypeptide recognized by the receptor. RNA failed to compete with either vitellogenin or phosvitin for binding, suggesting that the binding specificity may require more than polymeric phosphate. The binding was tissue-specific in that phosvitin and vitellogenin bound to oocyte surfaces (at both pH 6.0 and 7.5), but not to chicken erythrocytes (at either pH).