An isoform of ankyrin is localized at nodes of Ranvier in myelinated axons of central and peripheral nerves.

Two variants of ankyrin have been distinguished in rat brain tissue using antibodies: a broadly distributed isoform (ankyrinB) that represents the major form of ankyrin in brain and another isoform with a restricted distribution (ankyrinR) that shares epitopes with erythrocyte ankyrin. The ankyrinR isoform was localized by immunofluorescence in cryosections of rat spinal cord gray matter and myelinated central and peripheral nerves to: (a) perikarya and initial axonal segments of neuron cells, (b) nodes of Ranvier of myelinated nerve with no detectable labeling in other areas of the myelinated axons, and (c) the axolemma of unmyelinated axons. Immunogold EM on ultrathin cryosections of myelinated nerve showed that ankyrinR was localized on the cytoplasmic face of the axolemma and was restricted to the nodal and, in some cases, paranodal area. The major isoform of ankyrin in brain (ankyrinB) displayed a broad distribution on glial and neuronal cells of the gray matter and a mainly glial distribution in central myelinated axons with no significant labeling on the axolemma. These results show that (a) ankyrin isoforms display a differential distribution on glial and neuronal cells of the nervous tissue; (b) an isoform of ankyrin codistributes with the voltage-dependent sodium channel in both myelinated and unmyelinated nerve fibers. Ankyrin interacts in vitro with the voltage-dependent sodium channel (Srinivasan, Y., L. Elmer, J. Davis, V. Bennett, and K. Angelides. 1988. Nature (Lond.). 333:177-180). A specific interaction of an isoform of ankyrin with the sodium channel thus may play an important role in the morphogenesis and/or maintenance of the node of Ranvier.

Real-time imaging of axonally transported subresolution organelles in vertebrate myelinated axons.

A procedure is described for the real-time imaging of organelles of sub-resolution dimensions that undergo rapid axonal transport in myelinated axons. The procedure uses commercially available processors to enhance images that are obtained with a video microscope. Image detail is enhanced by spatial high-pass filtering. Small moving organelles are detected by subtracting a dynamically updated image of stationary cellular detail from one that contains the same stationary features plus images of the moving organelles.

Detection and differentiation of glycoconjugates in various cell types by lectin histochemistry.

Knowledge of chemical structure of glycoconjugates (GCs) at precise loci has increased through histochemical use of a battery of horseradish peroxidase-conjugated lectins, each possessing affinity for a specific terminal sugar or internal sugar linkage. Lectin histochemistry has shown tremendous variability among GCs in different histologic sites, reflecting known chemical diversity of these substances. GCs differ in structure among various cell types in an animal and differ at a given histologic site between species or between individuals in outbred but not inbred species. Lectin conjugates react with and detect GCs not otherwise demonstrable histochemically and, because of low concentration in tissue, not identified biochemically. Lectin-HRP conjugates have visualized unique GC with terminal GalNAc in primordial germ cells of rat embryos, with terminal Gal in epithelial basal cells of rodents and nodes of Ranvier in rats and with terminal GalNAc in a cell population in the thymus, Peyer's patches and intestinal lamina propria of some but not other mice.

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.

Filipin-sterol complexes at nodes of Ranvier.

Using the filipin-sterol technique, regional heterogeneity in the axonal and Schwann cell plasma membranes was investigated at the node of Ranvier and paranodes. Filipin-sterol complexes were abundant at the nodal axolemma but infrequent throughout the paranodal axolemma. The paranodal Schwann cell plasma membrane was rich in complexes which extended over the nodal Schwann cell microvilli. There were no regional differences in filipin labelling of the nodal-paranodal Schwann cell plasma membrane in relation to features such as paranodal cytoplasmic columns or mesaxonal furrows. However, the paranodes of adjacent Schwann cells were sometimes markedly different from each other in the amount of filipin labelling. The extent to which filipin labelling is indicative of cholesterol membrane content is discussed and the findings are related to current concepts of distribution, mobility and interaction of protein and lipid in biomembranes, with particular reference to the nodal axolemma.

The localization of sodium and calcium to schwann cell paranodal loops at nodes of Ranvier and of calcium to compact myelin.

High-voltage electron microscopy (HVEM) has been used to determine the distribution of cationic precipitates in myelinated axons resulting from the application of two cytochemical techniques: a direct osmium pyroantimonate treatment for precipitating Na+, Ca2+ and Mg2+; and a 5 mM Ca2+ inclusion procedure (Oschman & Wall) for imparting electron density to Ca2+ binding sites. Electron probe wavelength spectroscopy was then used on semi-thick tissue sections to identify the species of ions present in the following regions: Schwann cell paranodal loops, axoplasm at the node, compact myelin and extracellular matrix. With these combined procedures we were able to localize elevated concentrations of both Na+ and Ca2+ to cytoplasmic compartments of the Schwann cell paranodal loops, as well as to detect the presence of Ca2+ at elevated levels in compact myelin. The involvement of the Schwann cell paranodal loops in providing a source and/or sink for Na+ involved in impulse conduction is suggested by these results, and the significance of such a role is discussed. A role for Ca2+ in the formation and stabilization of myelin lamellae is also suggested.

[Association of brain glycoprotein (NSA 3) with neuronal membranes and with the nodes of Ranvier]. / Association de la glycoprotéine cérébrale (ANS 3) à la membrane du neurone et aux étranglements de Ranvier.

The localisation of brain glycoprotein NSA 3 was studied by means of indirect immunofluorescence on alcohol fixed, paraffin embedded sections of Rat brain. These techniques allowed the localisation of NSA 3 to the membrane of some (about 10%) of the neurons. In the white matter, the patterns were in agreement with the localisation of the Ranvier nodes. The nodes of Ranvier were also stained in peripheral nerves.

Localization of thiamine in cholinergic nerve terminals: a histofluorescence study in the electric organ of Torpedo.

Thiamine (vitamin B1) and its phosphoric acid esters were found at a surprisingly high level in the electric organ of the fish Torpedo marmorata. The localization of the vitamin was investigated by a sensitive reaction in which the thiamine molecule was converted into a blue fluorescent thiochrome by treatment with an alkaline potassium ferricyanide solution. Whereas only slight fluorescence occurred in the electroplaque cells, a strong fluorescent reaction took place in their nerve supply. Intense fluorescence was observed not only in the myelinated nerve fibres but also at the nodes of Ranvier; moreover, preterminal branches and nerve endings, which are non-myelinated, were also strongly stained. The emission spectrum of this fluorescent material was found to be nearly identical to that of standard thiochrome. These findings substantiate the hypothesis that vitamin B1 plays an important role in acetylcholine metabolism and release.