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

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Abstract

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.

Introduction

Nodes of Ranvier and axon initial segments (AISs) are characterized by a high density of voltage-gated sodium (Nav) channels, essential for the generation and propagation of action potentials. In both compartments, aggregation of Nav channels involves a macromolecular complex of interacting proteins, including the cytoskeletal adaptor protein ankyrin G (Kordeli et al., 1995), the actin-binding protein βIV-spectrin (Berghs et al., 2000) and the cell adhesion molecules neurofascin-186 (NF186) and NrCAM (Davis et al., 1996) as well as Nav channel auxiliary β subunits (Isom, 2002), which may all three provide extracellular interactions. Ankyrin G plays an essential role as a protein scaffold coordinating the expression of Nav channels at AISs and nodes of Ranvier (Zhou et al., 1998, Bennett and Lambert, 1999, Jenkins and Bennett, 2001, Dzhashiashvili et al., 2007, Hedstrom et al., 2007, Pan et al., 2006).

Nav channels are heteromultimeric protein complexes consisting of one large pore-forming α subunit and one or more small auxiliary β subunits (Yu and Catterall, 2003). Nine genes encode α subunits (Nav1.1 to Nav1.9) (Goldin et al., 2000). However from the nine α subunits, three (Nav1.1, Nav1.2 and Nav1.6) are expressed in the adult central nervous system (CNS) (Trimmer and Rhodes, 2004). Since their biophysical properties differ, the nature of Nav channels expressed at AISs and nodes of Ranvier is an important determinant endowing neurons with specific excitability and signaling properties (Catterall et al., 2005). Nodes of Ranvier and AISs in the adult CNS are considered to contain a unique Nav, Nav1.6 (Caldwell et al., 2000, Jenkins and Bennett, 2001), except in some retinal ganglion cells that also contain Nav1.2 in their nodes and/or AISs (Boiko et al., 2001, Boiko et al., 2003).

Nav1.1 appears to play an important function in the control of neuronal and network excitability: more than 200 mutations in its gene have been associated to human inherited epileptic syndromes, including the most severe form, severe myoclonic epilepsy in infancy (Ragsdale, 2008). Nav1.1 is known to be expressed in the CNS from the first postnatal week to adult stages (Gordon et al., 1987, Beckh et al., 1989, Brysch et al., 1991, Gong et al., 1999). It is generally described as having a somato-dendritic expression throughout the brain (Westenbroek et al., 1989, Gong et al., 1999, Whitaker et al., 2001) and was found to be expressed in somatas of spinal cord neurons, including of motor neurons (Westenbroek et al., 1989). However, Nav1.1 expression was also recently observed at the AIS of adult retinal ganglion cells and 4% of neurons in the hippocampal area CA3 (Van Wart et al., 2007). Nav1.1 expression was found to be confined to the proximal part of these AISs, spatially segregated from Nav1.6 channels (Van Wart et al., 2007). In P14–P16 mice, Nav1.1 expression has also been recently observed at the AIS of parvalbumin-expressing interneurons in the neocortex and hippocampus as well as of cerebellar Purkinje cells (Ogiwara et al., 2007). In addition, in these P14–P16 mice, Nav1.1 expression was also found in a few nodes of Ranvier in the cerebellar white matter and deep nuclei, as well as in the corpus callosum and fimbria. The distribution of Nav1.1 thus remains controversial, in particular in the adult where a quasi-unique cell population was shown to display a novel distribution of Nav1.1 in the AIS.

Given that Nav1.1 was known to be strongly expressed in caudal regions of the CNS including the spinal cord (Gordon et al., 1987, Beckh et al., 1989, Black et al., 1994), we investigated the distribution of Nav1.1 in the adult mouse spinal cord. We found a predominant Nav1.1 expression in numerous nodes of Ranvier throughout the spinal cord. We identified three populations of nodes: expressing Nav1.1, Nav1.6 or both. We also found Nav1.1 expression in numerous AISs throughout the spinal cord including in 80% of motor neurons. Expression of Nav1.1 in the AIS was found to display a proximo-distal gradient, complementary to Nav1.6 expression, thus defining two spatially segregated AIS subcompartments with different Nav composition. Finally, Nav1.1 expression in nodes of Ranvier and AISs could also be observed in multiple brain areas of adult mice.

Section snippets

Specificity of Nav1.1 immunostaining

In order to analyze the localization of Nav1.1 we used mouse tissues that were treated with a light paraformaldehyde fixation. We first analyzed the distribution of Nav1.1 by immunohistochemistry on adult mouse lumbar spinal cord sections with an anti-Nav1.1 polyclonal antibody raised against a peptide derived from the Nav1.1 cytoplasmic linker between transmembrane domains I and II. This antibody predominantly labeled a very large number of short segments (around 2 μm long) homogeneously

Discussion

This study provides the first demonstration that Nav1.1 is expressed in nodes of Ranvier in the adult CNS. Instead of the single population of nodes considered to exist in the adult, expressing a unique Nav, Nav1.6 (Caldwell et al., 2000), we uncovered three populations of nodes in the adult spinal cord: expressing Nav1.1 only, Nav1.6 only, or both. An unsuspected population of nodes was thus identified, that does not express Nav1.6 but only Nav1.1. This nodal expression of Nav1.1 was however

Animals

OF1 adult and postnatal mice (obtained from Charles River) were housed under standard laboratory conditions. All animal experiments were performed in compliance with European Community guiding principles on the care and use of animals (86/609/CEE, CE off. J. no. L358, 18 December 1986), the French decree no. 97/748 of October 19, 1987 (J Off République Française, 20 October 1987, pp. 12245–12248) and recommendations from the CNRS and University Pierre and Marie Curie.

Antibodies

The following antibodies

Acknowledgments

We are grateful to Lynda Demmou for her valuable participation in preliminary experiments and to Drs. Yoheved Berwald-Netter and Isabelle Dusart for their helpful discussions. We thank Drs. Gisèle Alcaraz (University Aix-Marseille II, Marseille) and Jean-Antoine Girault (University Pierre and Marie Curie, Paris) for generously providing respectively the anti-ankyrin G rabbit antibody and the anti-paranodin antibody. This work was supported by the Association Française contre les Myopathies

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