Immunocytochemical and electrophysiological techniques were used to localize TTX-sensitive sodium channels (NaChs) over the soma-dendritic axis of basilar and nonbasilar pyramidal cells of the electrosensory lateral line lobe (ELL) of weakly electric fish (Apteronotus leptorhynchus). Dense NaCh-like immunolabel was detected on the membranes of basilar and nonbasilar pyramidal cell somata. Punctate regions of immunolabel (approximately 15 microns) were separated by nonlabeled expanses of membrane over the entire extent of basal dendrites. Similar punctate immunolabel was observed over the apical dendrites, and frequently on membranes of afferent parallel fiber boutons in the distal apical dendritic region. Intracellular recordings from pyramidal cell somata or proximal apical dendrites (75–200 microns) were obtained using an in vitro ELL slice preparation. TTX- sensitive potentials were identified by focal pressure ejection of TTX. Somatic recordings demonstrated both TTX-sensitive fast spike discharge and a slow prepotential; similar but lower amplitude potentials were recorded in apical dendrites. Dendritic spikes were composed of at least two active components triggered by a fast prepotential (FPP) generated by the somatic spike. TTX-sensitive spikes propagated in a retrograde fashion over at least the proximal 200 microns of the apical dendrites, as determined by the conduction of an antidromic population spike and focal TTX ejections. Somatic spikes were followed by a depolarizing afterpotential (DAP) that was similar in duration and refractory period to that of proximal dendritic spikes. During repetitive spike discharge, the DAP could increase in amplitude and attain somatic spike threshold, generating a high-frequency spike doublet and a subsequent hyperpolarization that terminated spike discharge. Repetition of this process gave rise to an oscillatory burst discharge (2–6 spikes/burst) with a frequency of 40–80 Hz. Both the DAP and oscillatory discharge were selectively blocked by TTX ejections restricted to the proximal apical dendritic region. The present study demonstrates an immunolocalization of NaChs over somatic and dendritic membranes of a vertebrate sensory neuron that correlates with the distribution of TTX-sensitive potentials. The interaction of somatic and dendritic action potentials is further shown to underlie an oscillatory discharge believed to be important in electrosensory processing.

Polyclonal antibodies were raised against a well conserved region of the vertebrate Na+ channel and were affinity purified for use in immunocytochemistry. Focal demyelination of rat sciatic axons was initiated by an intraneural injection of lysolecithin and Na+ channel clustering was followed at several stages of myelin removal and repair. At 1 week post-injection axons contained long, fully demyelinated regions. Na+ channel clusters appeared only at heminodes forming the borders of these zones, and at widely spaced isolated sites that may represent former nodes of Ranvier. Over the next few days proliferating Schwann cells adhered to axons and began to extend processes. Clusters of Na+ channels appeared at the edges of these structures. As the Schwann cells elongated, the clusters seemed to move with them, since they remained at edges and the distance between aggregates increased. Clusters associated with different Schwann cells ultimately approached each other and appeared to fuse. Na+ channels then coalesced further at these sites, forming new nodes of Ranvier in regions that previously were internodal. If Schwann cell proliferation were blocked by mitomycin, no new clusters of Na+ channels appeared within internodes. Under these conditions, heminodal clusters remained visible at 1 week postinjection, but by 2 weeks they were no longer detectable, suggesting that proliferating Schwann cells are required for their maintenance. Clusters at normal nodes of Ranvier remained. It is concluded that Na+ channel aggregation and mobility in demyelinated nerve fibers is controlled by adhering Schwann cells, resulting in the formation of stable new nodes of Ranvier during remyelination.

Painful neuromas from 16 patients were examined using site-specific antisodium channel antibodies employed in immunocytochemical and radioimmunoassay methods. Normal sural nerves from six of these patients served as controls. Immunocytochemistry showed abnormal segmental accumulation of sodium channels within many axons in the neuromas. Dens immunolocalization was especially apparent within the axonal tips. Radioimmunoassay confirmed a significantly greater density of sodium channels in the neuromas as compared with the sural nerves. Thus, sodium channel accumulate abnormally within the axons of neuromas in humans. This alteration of the sodium channels may underlie the generation of axonal hyperexcitability and the resulting abnormal sensory phenomena (pain and paresthesias), which frequently occur after peripheral nerve injury.

Voltage-gated sodium channels are largely localized to the nodes of Ranvier in myelinated axons, providing a physiological basis for saltatory conduction. What happens to these channels in demyelinated axons is not known with certainty. Experimentally demyelinated axons were examined by using a well-characterized polyclonal antibody directed against sodium channels. Immunocytochemical and radioimmunoassay data were consistent with the distribution of an increased number of sodium channels along segments of previously internodal axon. These findings affirm the plasticity of sodium channels in demyelinated axolemma and may be relevant to understanding how axons recover conduction after demyelination.The sodium channel is a transmembrane protein that mediates the voltage-dependent sodium permeability of electrically excitable membranes. The presence of sodium channels is of obvious importance for the generation and propagation of action potentials along axolemma. In normal myelinated axons sodium channels are largely localized to the nodes of Ranvier. The most recent electrophysiological and biochemical studies demonstrate a sodium channel density of several thousand channels per gm2 at the nodes of Ranvier compared with a density of <25 per gm2 in internodal segments (1-5). In contrast to these observations in myelinated axons, little is known regarding the distribution of sodium channels in demyelinated axons. Such information is important because the resumption of axonal conduction appears the basis for recovery in many demyelinating diseases (6-8). The present report describes the use of an antibody directed against sodium channels to study their distribution along peripheral nerve axons. The immunocytochemical and radioimmunoassay data that follow indicate that the distribution of sodium channels changes along experimentally demyelinated axons. MATERIALS AND METHODSPreparation of Antibody. Tetrodotoxin is a compound that binds with high affinity and specificity to sodium channels. Using well-established methods, we purified the tetrodotoxin-binding protein (TTXR) of sodium channels from the electric organ of the eel, Electrophorus electricus (9). This large polypeptide alone appears to contain the entire sodium channel apparatus in Electrophorus (10, 11).Polyclonal antibodies to TTXR (anti-TJXR) were raised in rabbits, and their specificity was demonstrated by described radioimmunoassay and immunoprecipitation methods (4,12). Although some of these antibodies are species specific, others recognize sodium channels from various fish (T.E.F., unpublished data; 13). In particular, the antisera strongly interact with sodium channels in peripheral nerves of Carassius auratus (goldfish).Production of Demyelinative Lesions. tTo whom reprint requests should be addressed. 6777The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

In the interest of continuing structure-function studies, highly purified sodium channel preparations from the eel electroplax were incorporated into planar lipid bilayers in the presence of veratridine. This lipoglycoprotein originates from muscle-derived tissue and consists of a single polypeptide. In this study it is shown to have properties analogous to sodium channels from another muscle tissue (Garber, S. S., and C. Miller. 1987. J0urna/ of General Physiology. 89:459-480), which have an additional protein subunit. However, significant qualitative and quantitative differences were noted. Comparison of veratridine-modified with batrachotoxin-modified eel sodium channels revealed common properties. Tetrodotoxin blocked the channels in a voltage-dependent manner indistinguishable from that found for batrachotoxin-modified channels. Veratridine-modified channels exhibited a range of single-channel conductance and subconductance states. The selectivity of the veratridine-modified sodium channels for sodium vs. potassium ranged from 6-8 in reversal potential measurements, while conductance ratios ranged from 12-15. This is similar to BTX-modified eel channels, though the latter show a predominant single-channel conductance twice as large. In contrast to batrachotoxin-modified channels, the fractional open times of these channels had a shallow voltage dependence which, however, was similar to that of the slow interaction between veratridine and sodium channels in voltage-clamped biological membranes. Implications for sodium channel structure are discussed.