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

Molecular and Cellular Neuroscience 39 (2008) 180–192

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Molecular and Cellular Neuroscience j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y m c n e

Nav1.1 is predominantly expressed in nodes of Ranvier and axon initial segments Amandine Duflocq, Barbara Le Bras, Erika Bullier, François Couraud ⁎, Marc Davenne UPMC Univ Paris 06, UMR 7101, F-75005, Paris, France CNRS, UMR 7101, F-75005, Paris, France

a r t i c l e

i n f o

Article history: Received 30 April 2008 Revised 11 June 2008 Accepted 13 June 2008 Available online 24 June 2008

a b s t r a c t 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. © 2008 Elsevier Inc. All rights reserved.

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 ⁎ Corresponding author. UPMC Univ Paris 06, CNRS, UMR 7101, 7, quai St Bernard, Case 2, 75252 Paris Cedex 05, France. Fax: +33 1 44 27 25 08. E-mail address: [email protected] (F. Couraud). 1044-7431/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2008.06.008

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, 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 somatodendritic 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

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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. Results 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 distributed throughout the grey matter and whose diameter was similar to that of axons (Fig. 1A, yellow arrows). Before characterizing the nature of these segments, we assessed the specificity of the immunolabeling. We therefore used a monoclonal antibody directed against an unrelated epitope located at the Nav1.1 cytoplasmic C-terminus. The immunostaining obtained with the monoclonal antibody was totally identical to that obtained with the polyclonal antibody (Figs. 1A–C: yellow arrows) and when the polyclonal antibody was pre-incubated with the Nav1.1 peptide used for its generation no labeling above background level was observed (Fig. 1D). Furthermore all segments labeled with the Nav1.1 polyclonal antibody were also labeled with a PanNav antibody (Figs. 1E–G: yellow arrows), which specifically recognizes all vertebrate Nav α subunit isoforms (Rasband et al., 1999). Finally, on western blots from adult mouse spinal cord protein extracts, both Nav1.1 polyclonal (Fig. 1H) and monoclonal (data not shown) antibodies recognized a unique band of the expected molecular mass for Nav1.1 (around 260 kDa) (Gong et al., 1999) that was lost when the polyclonal antibody was pre-incubated with its antigenic peptide (Fig. 1H). Altogether these results strongly support that the monoclonal and polyclonal antibodies both specifically recognize

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Nav1.1. Noteworthy, the absence of staining observed with the same polyclonal antibody on tissues from homozygous Nav1.1 knock-out (Yu et al., 2006) and knock-in mice (Ogiwara et al., 2007) further supports the antibody specificity for Nav1.1 rather than reflects the absence of an unspecific antigen downstream from Nav1.1. Nav1.1 is predominantly expressed in nodes of Ranvier in the adult spinal cord We then characterized the nature of Nav1.1+ segments observed in the adult lumbar spinal cord. The panNav antibody is known to only label regions of high Nav expression density, ie nodes of Ranvier and AISs in the adult CNS. The fact that all Nav1.1+ segments were stained with the panNav antibody (Figs. 1E–G: yellow arrows) and that these segments had the same size as panNav+ segments not expressing Nav1.1 (Figs. 1F, G: green arrows) suggested that Nav1.1+ segments correspond to either of these structures. The Nav1.1+ segments being 2–3 μm long, shorter than the average 30 μm long AIS (Fig. 4), we tested the possibility that Nav1.1+ segments correspond to nodes of Ranvier. We used an antibody against ankyrin G, a specific marker of AISs and nodes of Ranvier (Kordeli et al., 1995) together with an antibody against paranodin (or Caspr), a specific marker of paranodes flanking every node (Menegoz et al., 1997). We found that 100% of Nav1.1-labeled segments (n = 275) co-expressed ankyrin G and were flanked by paranodin expression on both sides (69%, Figs. 2A–F: pink and green arrows) or only one side (31%, Figs. 2A–F: pink and green arrowheads). The latter systematically corresponded to Nav1.1+ segments oriented more perpendicularly to the image plane and whose second putative paranode was not contained within the tissue section (in contrast with the classical image of nodes of Ranvier, flanked by both paranodes, observed in the teased sciatic nerve or the optic nerve, where nodes have a more homogeneous orientation along the nerve). We therefore concluded that all Nav1.1+ segments observed in the spinal cord were nodes of Ranvier. This result provides the first demonstration that Nav1.1 sodium channels are expressed in nodes of Ranvier in the adult CNS. Heterogeneity of CNS adult nodes of Ranvier: a novel population of nodes expressing Nav1.1 but not Nav1.6 Nodes of Ranvier in the adult CNS were so far considered to be a quasi-homogeneous population in terms of Nav expression,

Fig. 1. Anti-Nav1.1 antibodies both specifically label short segments in the adult spinal cord. (A–C) The anti-Nav1.1 polyclonal (A) and monoclonal (B) antibodies identically (yellow arrows) labeled numerous short segments in the lumbar spinal cord grey matter (merge in C). (D) The polyclonal antibody staining was abolished by pre-incubating the antibody with its antigenic peptide. (E–G) All segments labeled with the anti-Nav1.1 polyclonal antibody (E) were also labeled with the panNav antibody (F, G, yellow arrows), but some panNav+ segments were not labeled by the anti-Nav1.1 antibody (G, green arrows). (H) In western blots of adult spinal cord protein extracts the polyclonal antibody recognized a unique band of 260 kDa, not detected after incubating the antibody with its antigenic peptide. Scale bar, 5 μm.

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expressing a unique Nav, Nav1.6 (Caldwell et al., 2000), except some nodes of retinal ganglion cells that express Nav1.2 (Boiko et al., 2001). Our finding that Nav1.1 was expressed in adult nodes of Ranvier prompted us to analyze whether Nav1.1+ nodes could be a separate population of nodes or the same as the ones expressing Nav1.6. We used triple immunolabelings of ankyrin G, Nav1.1 and Nav1.6 in order to address this issue. We first analyzed the ventral region of the adult spinal cord grey matter (Fig. 3U) and identified three types of ankyrin G+ nodes (Fig. 3A): nodes expressing Nav1.1 only (Nav1.1+ only, red arrows), nodes expressing Nav1.1 and Nav1.6 together (Nav1.1+/Nav1.6+, yellow arrows) and nodes expressing Nav1.6 only (Nav1.6+ only, green arrows; Figs. 3A–D). We also found these three populations of nodes in the dorsal region of the spinal cord (Fig. 3U). We quantified, for both the ventral and dorsal regions of the grey matter analyzed, the proportion of each population of nodes with respect to the total number of nodes analyzed. In the ventral region 43.8% of nodes were Nav1.1+ only, 22% were Nav1.1+/Nav1.6+ and 34.2% were Nav1.6+ only (Fig. 3E; n = 569). In the dorsal region, each of the three populations had a very similar proportion: 38.3% of nodes were Nav1.1+ only, 25.7% were Nav1.1+/Nav1.6+ nodes and 36% were Nav1.6+ only nodes (Fig. 3E; n = 1008). Each of the three nodal populations was thus similarly represented in both grey matter ventral and dorsal regions (Fig. 3E), suggesting that none of them is restricted to a particular type of axon or to parts of axons predominantly found in the spinal cord dorsal or ventral grey matter. Analysis from both regions also demonstrated that the novel population of nodes

uncovered, expressing Nav1.1 only and not Nav1.6, represents in the spinal cord grey matter a very large fraction of the total number of nodes (43.8% ventrally and 38.3% dorsally, Fig. 3E). On the contrary, in the spinal cord white matter, the proportion of nodes expressing Nav1.1, in particular that of Nav1.1+ only nodes, appeared drastically reduced (Figs. 3F–J). Two regions were examined in the white matter, one dorsal and one ventral (Fig. 3U). In the white matter ventral region, 10.7% of nodes were Nav1.1+ only, 29.5% were Nav1.1+/Nav1.6+ and 59.8% were Nav1.6+ only (Fig. 3J, n = 960). In the white matter dorsal region, 9.6% of nodes were Nav1.1+ only, 37.5% were Nav1.1+/Nav1.6+ and 52.8% were Nav1.6+ only (Fig. 3J, n = 358). The different proportions of nodes observed for each of the three populations between the dorsal and ventral region of the white matter may be associated to different unidentified tracts originating from particular neuronal types. In the white matter, the contribution of Nav1.1+ nodes, in particular that of Nav1.1+ only nodes, to the total number of nodes is thus much smaller than in the grey matter. Noteworthy, in both the spinal cord grey and white matter, the presence of nodes from each of the three populations did not seem to correlate with axon or node diameter. PNS adult nodes of Ranvier in the spinal cord ventral and dorsal roots do not express Nav1.1 We next investigated whether this novel population of adult nodes found in the CNS could also be found in the PNS, where all nodes have

Fig. 2. Nav1.1 is expressed in nodes of Ranvier in the adult spinal cord. (A–E) Double immunostaining of Nav1.1 and paranodin (A) and triple immunostaining of Nav1.1 (B), ankyrin G (C) and paranodin (D) (merge in E) in the adult lumbar spinal cord grey matter showed that all Nav1.1+ segments were ankyrin G+ and flanked by paranodin expression. The majority of Nav1.1+/ankyrin G+ segments (pink arrows) were flanked by paranodin expression on both sides (green arrows). The remaining Nav1.1+/ankyrin G+ segments (pink arrowhead) were contiguous to paranodin expression on one side only (green arrowhead) (E) due to tissue sectioning. (F) From a total of n = 275 Nav1.1+ segments identified in the spinal cord grey matter, 69% were flanked by paranodin expression on both sides and 31% by paranodin expression on one side only. Results are presented as mean ± SEM of three independent experiments. Scale bar, 5 μm.

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been considered so far to express a unique Nav, Nav1.6 (Caldwell et al., 2000), except for some nodes of thinly myelinated axons in the sciatic nerve that were found to express Nav1.9 (Fjell et al., 2000). We analyzed the lumbar spinal cord dorsal and ventral roots, composed respectively of peripheral sensory and motor axons of the sciatic

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nerve. By triple immunostaining of ankyrin G, Nav1.1 and Nav1.6, nodes of Ranvier could be analyzed in terms of Nav1.1 and/or Nav1.6 expression (Figs. 3K–T). In both ventral and dorsal roots no expression of Nav1.1 could be detected in nodes of Ranvier: from a total of n = 28 and n = 33 ankyrin G+ nodes of Ranvier analyzed in ventral and dorsal

Fig. 3. Three populations of nodes of Ranvier were identified in the adult spinal cord: expressing Nav1.1, Nav1.6 or both. (A–D, F–I, K–N, P–S) Triple immunostaining of ankyrin G (A, F, K, P), Nav1.1 (B, G, L, Q) and Nav1.6 (C, H, M, R) (merge in D, I, N, S) in the adult lumbar spinal cord grey matter (A–D), white matter (F–I), ventral root (K–N) and dorsal root (P–S). Three populations of nodes were identified: expressing Nav1.1 only (red arrows), Nav1.6 only (green arrows) or both (yellow arrows). (E, J, O, T) Six sub-regions were analyzed (represented in U): ventral and dorsal grey matter (E), ventral and dorsal white matter (J), ventral root (O) and dorsal root (T). For each of these sub-regions, at least three image acquisitions of identical x, y, z size were taken from sections treated in different experiments. The percentage of each population of nodes was measured within each acquisition. The mean and SEM obtained from all acquisitions of an identical sub-region were plotted (E, J, O, T). The total number of nodes analyzed was respectively: n = 569 and n = 1008 in the ventral and dorsal grey matter (E); n = 960 and n = 358 in the ventral and dorsal white matter (J); n = 28 (O) and n = 33 (T) in the ventral and dorsal root. (U) Schematic representation of a spinal cord coronal section displaying the location of the six sub-regions analyzed in E (ventral, EV; dorsal, ED), J (ventral, JV; dorsal, JD), O and T. Scale bar, 5 μm.

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roots respectively, 100% in both cases were Nav1.6+ only (Figs. 3K–O and P–T, green arrows). Thus, from the three populations of nodes identified in the spinal cord (Nav1.1+ only, Nav1.1+/Nav1.6+ and Nav1.6+

only), the peripheral sensory and motor axons analyzed in ventral and dorsal roots of the lumbar spinal cord appeared to carry a unique population of nodes, Nav1.6+ only nodes.

Fig. 4. Nav1.1 is concentrated in the proximal region of AISs of adult spinal cord neurons, including of most motor neurons. (A–H) Double immunostaining of ankyrin G (A) and Nav1.1 (B) (merge in C) and triple immunostaining of ankyrin G (D), Nav1.1 (E) and Nav1.6 (F) (merge in H) in the adult lumbar spinal cord grey matter showing that Nav1.1 is expressed in the AIS. Nav1.1 expression displayed a proximo-distal gradient, with a higher expression level in the AIS region proximal to the soma (brackets in B, C, E, G, H). The Nav1.1 gradient was found to be complementary to Nav1.6 expression. (I) Two populations of AISs were observed: expressing Nav1.6 alone or with Nav1.1. The mean percentage of each population and SEM were obtained from a total of n = 50 and n = 60 AISs analyzed from image acquisitions taken respectively in the ventral and dorsal grey matter in five independent experiments. (J–O) Triple immunostaining of peripherin (J), ankyrin G (M) and Nav1.1 (N) (merge in O) combined with Dapi staining (K) (J and K merged in L) in the ventral grey matter of adult lumbar spinal cord. Most peripherin-labeled motor neurons (blue arrow) expressed Nav1.1 in their ankyrin G+ AIS (J, M–O). In the higher magnification inserts, brackets indicate the AIS proximal region with higher Nav1.1 expression level (J, M–O). (P) The mean percentage of each population of AISs and SEM were obtained from a total of n = 35 AISs of peripherinlabeled motor neurons, analyzed from image acquisitions taken in the ventral grey matter in three independent experiments. Scale bar, 5 μm (A–H), 25 μm (J–O), 10 μm (inserts in J, M–O).

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Nav1.1 is also expressed in the AIS proximal region of adult spinal cord neurons including of most motor neurons The expression of Nav1.1 found in adult nodes of Ranvier suggested that Nav1.1 might also be expressed in other axonal compartments of high Nav channel density, such as AISs. We used ankyrin G immunostaining to label all AISs (Fig. 4A) and analyzed whether Nav1.1 was expressed at these AISs using either the monoclonal or polyclonal anti-Nav1.1 antibody. Numerous Nav1.1-expressing AISs could be found throughout the adult lumbar spinal cord grey matter with both antibodies (Figs. 4A–C). The Nav1.1 immunolabeling at the AIS was however very dim compared to that of ankyrin G (Fig. 4A); its signal intensity on the image (Figs. 4B, C) was therefore enhanced in order to track trace amounts of labeling. Noteworthy, in all cases Nav1.1 expression level was not homogeneous along the AIS: it displayed a proximo-distal gradient, being highest in an AIS region proximal to the soma and progressively decreasing towards the AIS distal end (Fig. 4B). Despite this gradient, Nav1.1 expression was however not completely excluded from the distal AIS: in most cases Nav1.1 immunolabeling could still be detected, although with a very weak intensity (Fig. 4B). Interestingly, the expression level of the

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sodium channel anchoring protein, ankyrin G, was rather uniform along the AIS (Fig. 4A) as was the labeling with a panNav antibody, which recognizes all Nav α subunit isoforms (Rasband et al., 1999) (Supplemental Fig. 1). This suggested that in the distal part of Nav1.1+ AISs, where Nav1.1 was almost absent, another Nav α subunit would be expressed. We thus analyzed the expression of Nav1.1 in parallel with that of Nav1.6, considered to be the major Nav isoform expressed at the AIS of most adult CNS neurons (Jenkins and Bennett, 2001; Boiko et al., 2003). We used triple immunolabeling of ankyrin G, Nav1.1 and Nav1.6 to assess the relative expression level and distribution of each protein. All AISs analyzed in the lumbar spinal cord grey matter (n = 110) expressed Nav1.6. We found Nav1.1 expression in 67.9% of AISs ventrally (n = 50, Figs. 4D–H) and 69.9% dorsally (n = 60) (Fig. 4I), suggesting that Nav1.1 expression at the AIS is not restricted to a neuronal population found predominantly or only in the dorsal or ventral spinal cord, like motor neurons. Hence, in contrast with nodes of Ranvier, no AISs were found expressing only Nav1.1, without Nav1.6. While Nav1.6 expression was uniform along all AISs expressing only Nav1.6 (data not shown), in Nav1.1+/Nav1.6+ AISs Nav1.6 expression displayed a distal-to-proximal gradient, being highest in

Fig. 5. Developmental expression of Nav1.1 in AISs and nodes of Ranvier in the spinal cord. (A–P) Triple immunostaining of ankyrin G (A, E, I, M), Nav1.1 (B, F, J, N) and Nav1.6 (C, G, K, O) in the grey matter at P0 (A–D), P7 (E–H), P14 (I–L) and P35 (M–P) (merge for each stage in D, H, L, P). Nav1.1 and Nav1.6 expression appears in the AIS respectively at P7 and P0; both appear in nodes of Ranvier at P14. At P14 the three populations of nodes identified in the adult can already be observed: Nav1.1+ only (red arrows), Nav1.1+/Nav1.6+ (yellow arrows) and Nav1.6+ only (green arrows). At P14, Nav1.1 expression in the AIS already displayed a proximo-distal gradient complementary to Nav1.6 expression (I–L, bracket showing the AIS proximal domain where Nav1.1 expression is stronger). Scale bar, 5 μm.

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confirmed in separate experiments (data not shown) by colocalization of peripherin expression with the more general motor neuron marker, ChAT (Houser et al., 1983), whose immunostaining was however not compatible with the low tissue fixation conditions required for Nav1.1 immunolabeling. We found Nav1.1-expressing AISs (Nav1.1+/Nav1.6+) in 79.8% of motor neurons (Figs. 4J–P). The remaining 20.2% of motor neurons had an AIS expressing only Nav1.6 (n = 35; Fig. 4P). In motor neurons with a Nav1.1+/Nav1.6+ AIS, Nav1.1 expression displayed a proximo-distal gradient revealing within the AIS a proximal region of higher Nav1.1 expression level (Figs. 4M–O: bracket in the higher magnification inserts). The fact that not all motor neurons express Nav1.1 at their AIS revealed an interesting heterogeneity among motor neurons in terms of AIS molecular composition. Developmental expression of Nav1.1 in AISs and nodes of Ranvier

Fig. 6. Quantitative analysis of Nav1.1 developmental expression in AISs and nodes of Ranvier in the spinal cord. (A) The percentage of each of the four populations of AISs observed (Nav1.1+ only, Nav1.1+/Nav1.6+, Nav1.6+ only and ankyrin G+ only) was measured at P0 (from a total of n = 57 AISs, defined as ankyrin G+), P7 (n = 127), P14 (n = 15), P21 (n = 44), P29 (n = 51), P35 (n = 59), P42 (n = 16) and P90 (n = 110). The AISs analyzed were pooled from several image acquisitions taken in the lumbar spinal cord grey matter. The mean and SEM values were obtained from three independent experiments, except for P90 (ten experiments). (B) The number of nodes of Ranvier for each of the three populations identified in the adult spinal cord (Nav1.1+ only, Nav1.1+/ Nav1.6+ and Nav1.6+ only) was counted in three image acquisitions (from different experiments) of identical x, y, z size in the same ventral region of the spinal cord at each stage: P0, P7, P14, P21, P29, P35, P42 and P90. The mean per FOV and SEM values were thus obtained from three independent experiments. (C) Western blotting analysis of Nav1.1 expression level from total spinal cord protein extracts from P0 to P90.

the distal AIS (Fig. 4F). Both Nav1.1 and Nav1.6 gradients seemed to be complementary (Figs. 4D–H). This resembles what was observed in adult retinal ganglion cells (Van Wart et al., 2007). These complementary gradients could thus explain the rather uniform panNav immunostaining observed along Nav1.1+ AISs (Supplemental Fig. 1). Noteworthy, for both Nav1.1 and Nav1.6 the change from a region of high expression level to a region of low expression level appeared mostly progressive, suggesting that the AIS is not sharply divided into two subcompartments segregating Nav1.1 and Nav1.6 expression. Given the large fraction of AISs found to express Nav1.1 in the ventral spinal cord (67.9%), we investigated whether motor neurons, whose cell bodies are concentrated in the ventral spinal cord, have Nav1.1+ AISs. Motor neurons were labeled with a peripherin antibody (Greene, 1989), which specifically labels motor neurons in the spinal cord ventral horns (Figs. 4J–L, blue arrow). This was

We investigated whether this expression of Nav1.1 in spinal cord AISs and nodes of Ranvier was the result of a late compartmentalization mechanism, possibly restricted to adult stages, or whether it was already occurring at early postnatal stages and even as soon as Nav1.1 channels started to be expressed. We therefore analyzed the time course of Nav1.1 expression in parallel with ankyrin G and Nav1.6 expression, from birth (P0) to adult stage (P90) in the (presumptive) ventral grey matter of the lumbar spinal cord. At P0, nodes of Ranvier are not yet present in the spinal cord and at this stage Nav channels (as revealed by panNav immunostaining) are found only in ankyrin G-labeled AISs (data not shown). At P0, from a total of n = 57 ankyrin G-labeled AISs analyzed, Nav1.6 expression is found in 14.1% of them, but Nav1.1 expression could not yet be detected (Figs. 5A–D (green brackets) and 6A). The vast majority (85.9%) of AISs at P0 thus express neither Nav1.6 nor Nav1.1 and most likely still express Nav1.2, as in retinal ganglion cells (Boiko et al., 2003). At P7 however, trace amounts of Nav1.1 immunostaining could be detected. Nav1.1 expression was observed in 9.3% of AISs without the concomitant expression of Nav1.6 and in 21.4% of AISs together with Nav1.6 expression (from a total of n = 127 AISs, Figs. 5E–H (red and yellow brackets respectively) and 6A). In addition 7.3% of AISs were found to express Nav1.6 without Nav1.1 and 62% still expressed

Table 1 Adult brain regions containing Nav1.1 expression in AISs and/or nodes of Ranvier Region

Olfactory bulb Cerebral cortex Hippocampal region CA1 CA2 CA3 Dentate gyrus Fimbria Corpus callosum Thalamus Ventral tegmental area Central gray Superior colliculus Inferior colliculus Occulomotor nucleus Dorsal raphe nucleus Pons Medulla Cerebellum Purkinje cells White matter Granular layer Deep cerebellar nuclei

Nav1.1 expression AIS

Nodes of Ranvier

+ +

+ +

+ + + + − − + + + + + + + + +

+ + + + − − + + + + + + + + +

+ − − +

+ + +

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ankyrin G alone without Nav1.1 and Nav1.6 (data not shown; Fig. 6A). Hence, from the earliest stage of Nav1.1 expression in the lumbar spinal cord, it is found in the AIS, either alone or together with Nav1.6 expression. At this stage Nav1.1 expression in the AIS was too dim (as was Nav1.6 expression) to identify, as in adult AISs, a proximo-distal gradient complementary to Nav1.6 expression. At P14, the first ankyrin G-labeled nodes of Ranvier were observed in the lumbar spinal cord. In contrast with AISs, the majority of nodes were found to express Nav1.1 only (a mean of 26.3 per field of view (FOV) analyzed, Figs. 5I–L (red arrows) and 6B). However, the two other populations of nodes that we identified in the adult were also found at this earliest stage of Nav α subunits nodal expression, although in different proportions: we found a mean of 9.3 Nav1.1+/ Nav1.6+ nodes and a mean of 2.7 Nav1.6+ only nodes per FOV (Figs. 5I– L (yellow and green arrows respectively) and 6B). Thus at P14 very few nodes were found to express Nav1.6 only, suggesting that in the earliest nodes of Ranvier, in contrast with the earliest AISs, Nav1.1 may be more prone than Nav1.6 to be expressed alone. In terms of AISs at P14, an important increase is observed in the proportions of Nav1.6+

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only (34.1%, data not shown) and of Nav1.1+/Nav1.6+ AISs (63.6% from a total of n = 15 AISs, Figs. 5I–L and 6A) at the expense of ankyrin G+ AISs not expressing Nav1.1 and Nav1.6, which disappeared. The proportion of Nav1.1+ only AISs decreased at P14 (2.3%). Interestingly, at P14 Nav1.1 expression in Nav1.1+/Nav1.6+ AISs already appeared displaying a proximo-distal gradient, complementary to Nav1.6 expression (Figs. 5J–L: yellow brackets showing the AIS proximal region of higher Nav1.1 expression) as observed in adult AISs (Fig. 4). From P14 to P42, the number of nodes of Ranvier from each of the three populations increased continuously, except for Nav1.1+/Nav1.6+ nodes, whose number remained quite constant after P29 (Fig. 6B, 5M– P and Supplemental Fig. 2 for triple ankyrin G, Nav1.1 and Nav1.6 immunostainings of AISs and nodes at P21, P29, P42 and P90). The contribution of Nav1.6+ only nodes to the total number of nodes from the three populations, which was very low at P14 (6.9%: a mean of 2.6 from a mean total of n = 38.3 nodes per FOV, Fig. 6B), increased continuously until P42 (31.4%: a mean of 92.7 from a mean total of n = 294.7 nodes per FOV, Fig. 6B) mostly at the expense of Nav1.1+ only nodes, whose contribution dropped from 68.7% at P14 to 50.6% at

Fig. 7. Nav1.1 is expressed in AISs and nodes of Ranvier in numerous brain areas. (A–I) Double immunostaining of ankyrin G (A, D, G) and Nav1.1 (B, E, H) (merge in C, F, I) showing Nav1.1 expression in the proximal part of AISs (red brackets) in a Purkinje cell (A–C), in the polymorphic cell layer of the dentate gyrus (D–F) and in the thalamus (G–I). (J–Q) Triple immunostaining of ankyrin G (J, N), Nav1.1 (K, O) and paranodin (L, P) (merge in M, Q) in the adult cerebral cortex (J–M) and the superior colliculus (N–Q) showing Nav1.1 expression in ankyrin G+ nodes of Ranvier (pink arrows) flanked by paranodin expression (green arrows).

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P42 (Fig. 6B). This result suggested that Nav1.6 channels which were not very prone to be expressed alone in early nodes of Ranvier at P14 progressively increased their ability to be expressed alone in nodes at later postnatal stages. Concerning AISs, the proportion of each of the two remaining populations, Nav1.1+/Nav1.6+ and Nav1.6+ only AISs, remained quite constant (Figs. 6A, 5M–P and Supplemental Fig. 2) and Nav1.1+ only AISs disappeared after P14. Throughout these stages Nav1.1 expression at AISs displayed a proximo-distal gradient (yellow brackets) and appeared complementary to Nav1.6 expression (Figs. 5M–P and Supplemental Fig. 2). From P42 to P90 (adult stage), a significant decrease in the mean total number of nodes per FOV (from n = 294.7 to n = 197.1) was observed, mostly due to a reduced number of Nav1.1+ only nodes (from a mean of 149 to 79.1 per FOV; Fig. 6B and Supplemental Fig. 2). The developmental expression level of Nav1.1 protein has been analyzed from P1 to adult stage in the brain (Gong et al., 1999; Ogiwara et al., 2007) but not in the spinal cord. We thus undertook the analysis of Nav1.1 protein level from P0 to P90 by western blotting from total spinal cord protein extracts with the anti-Nav1.1 polyclonal antibody. At P0, Nav1.1 proteins could not be detected (Fig. 6C). Detection started at P7 (Fig. 6C), confirming that the earliest immunolocalization found for Nav1.1 in the AIS at P7 (Fig. 5F) corresponds to the earliest traces of Nav1.1 protein expression detected in the spinal cord. The amount of spinal cord Nav1.1 proteins gradually increased after P7 (Fig. 6C), reflecting the widespread expression of Nav1.1 in nodes of Ranvier and AISs throughout the spinal cord from P7 to adult stage (Fig. 5A and Supplemental Fig. 2). Nav1.1 is also expressed in AISs and nodes of Ranvier in multiple adult brain regions We investigated whether the predominant expression of Nav1.1 in AISs and nodes of Ranvier that we found in the lumbar spinal cord could also be observed, in the adult, in other regions of the spinal cord and in the brain. We used triple immunolabeling of Nav1.1, ankyrin G and paranodin. First, other segments of the spinal cord (cervical, thoracic and sacral) as well as the medulla and the pons were analyzed and in each region Nav1.1 expression was observed in numerous AISs and nodes of Ranvier in the grey matter (Table 1) like in the spinal cord lumbar region (Figs. 2 and 4). The Nav1.1-expressing segments were identified as AISs or nodes of Ranvier based on ankyrin G colocalization and respectively the characteristic morphology of AISs or the concomitant expression of paranodin (in the paranodes) flanking Nav1.1 and ankyrin G colocalization at the node. In the adult brain, we found multiple areas where Nav1.1 expression was also predominantly expressed in AISs and/or nodes of Ranvier (Table 1). Noteworthy, as in the spinal cord, expression of Nav1.1 at the AIS was found to display, a proximo-distal gradient, complementary to Nav1.6 expression (data not shown). In the cerebellum, a low level of Nav1.1 expression at the proximal AIS was found in a vast majority of Purkinje cells (Figs. 7A–C) and in deep nuclei, but not in the granular layer and the white matter. Nav1.1 expression was also found in nodes of Ranvier in deep nuclei, in the granular layer and in rare nodes of Ranvier in the white matter. In the hippocampal region, Nav1.1 expression was found in a few AISs and nodes of Ranvier throughout the hippocampus (in stratum oriens, radiatum and lacunosum-moleculare as well as in the pyramidal cell layer of the CA1, CA2 and CA3 regions). Nav1.1 expression was also found in a few AISs and nodes of Ranvier in the polymorphic (Figs. 7D–F), molecular and granule cell layer of the dentate gyrus (Table 1). Given their orientation and morphology, the Nav1.1+ AISs found in the pyramidal and granule cell layers most likely corresponded to interneurons, except in the CA3 area where very few Nav1.1+ AISs seemed to belong to pyramidal cells.

Finally both Nav1.1+ AISs and nodes of Ranvier were observed in the cerebral cortex (Figs. 7J–M), the thalamus (where numerous Nav1.1+ AISs were observed, Figs. 7G–I), the superior (Figs. 7N–Q) and inferior colliculi, the oculomotor and dorsal raphe nuclei, the pons, the medulla, the central gray, the ventral tegmental area and the olfactory bulb (Nav1.1+ AISs were found in a few mitral and granule cells as well as in the periglomerular area; Nav1.1+ nodes of Ranvier were found in the internal plexiform and granule cell layers as well as in the lateral olfactory tract) (Table 1). In contrast with results from P14–P16 mice (Ogiwara et al., 2007), we did not find, in the adult, Nav1.1 expression in nodes of Ranvier or AISs in the fimbria nor in the corpus callosum (Table 1). 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 not found in PNS sensory and motor axons from spinal cord dorsal and ventral roots. Unlike nodes of Ranvier, only two populations of AISs were found in the spinal cord: expressing Nav1.6 only, or both Nav1.6 and Nav1.1. We found that 80% of motor neurons expressed Nav1.1 in their AIS. In the AIS, Nav1.1 displayed a proximo-distal gradient, being more concentrated in the region proximal to the soma, concomitant with a complementary distal-to-proximal gradient of Nav1.6 expression, revealing two AIS subcompartments of higher Nav1.1 and Nav1.6 expression respectively. Both expression of Nav1.1 in AISs and nodes of Ranvier appeared postnatally in the spinal cord, respectively as soon as Nav1.1 expression started (P7) and nodes were formed (P14). Finally, we found such an expression of Nav1.1 in AISs and nodes of Ranvier throughout the antero-posterior extent of the spinal cord, as well as in multiple brain regions. Specific and predominant immunolocalization of Nav1.1 in axonal instead of somato-dendritic domains This novel adult distribution of Nav1.1 found in nodes of Ranvier, as well as its adult distribution found in AISs, similar to the one observed in retinal ganglion cells (Van Wart et al., 2007) contrasts with earlier descriptions of Nav1.1 expression in the somato-dendritic compartment (Westenbroek et al., 1989; Gong et al., 1999; Whitaker et al., 2001). This discrepancy raises the question of immunostaining specificity. The Nav1.1 peptide sequences used to raise the antiNav1.1 polyclonal and monoclonal antibodies both display a minimal similarity with other Nav α subunits and unrelated proteins. The specificity of the anti-Nav1.1 monoclonal antibody had been tested with respect to Nav1.2 and Nav1.6 by ELISA as well as by immunofluorescence of both transfected COS cells and retinal tissue (Van Wart et al., 2005). The Nav1.1 immunostaining we first obtained in the spinal cord with this antibody may still have reflected the expression of another Nav α subunit or that of an unrelated protein. A similar cross-reactivity of the anti-Nav1.1 polyclonal antibody may also have given rise to the immunostaining observed in the spinal cord. In order to discard such unspecific immunolabelings, the concomitant use of the two anti-Nav1.1 antibodies, directed against two unrelated peptides from the Nav1.1 protein and poorly conserved among Nav α subunits, was determinant. The fact that both antibodies gave an identical immunostaining in the spinal cord together with the additional control experiments we performed (Fig. 1) helped us confirm the immunolabeling specificity of each anti-Nav1.1 antibody. We could therefore confidently conclude that the immunostaining

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observed with both antibodies in nodes of Ranvier and AISs indeed reflects the presence of Nav1.1 proteins. One possible reason that could explain the discrepancy with the somato-dendritic expression described in earlier studies (Westenbroek et al., 1989; Gong et al., 1999; Whitaker et al., 2001) is the tissue preparation. The distribution of Nav1.1 described here was observed with a very light paraformaldehyde tissue fixation protocol that seems to differ significantly from the protocols used in these earlier studies. Interestingly, the recent report that also described Nav1.1 expression in adult AISs, in retinal ganglion cells, used a light tissue fixation protocol as well (Van Wart et al., 2007). Noteworthy, Nav1.1 antigenicity seemed to be particularly sensitive to fixation: slightly stronger fixations with increased paraformaldehyde concentration, intracardiac perfusion volume and/or increased duration of postperfusion immersion fixation all abolished Nav1.1 immunolabeling in nodes of Ranvier and AISs (our unpublished results). Aldehyde fixatives introduce intra- and inter-molecular bridges which are useful to stabilize protein conformation and allow epitope recognition yet they may compromise antibody accessibility to some epitopes. Interestingly a similar fixative-sensitivity was reported for KCNQ2 and KCNQ3: a protocol with low or no tissue fixation revealed expression of these potassium channels in AISs and nodes of Ranvier (Devaux et al., 2004; Pan et al., 2006), differing from what had been observed with strong fixation protocols (Cooper et al., 2001). In contrast with what was observed in retinal ganglion cells (Van Wart et al., 2007), we were not able to detect, with a light fixation protocol, any Nav1.1 somato-dendritic immunostaining in the spinal cord, nor in the brain regions analyzed. However, it could be that Nav1.1 channels are expressed in the somato-dendritic compartment at a low density, much lower than in AISs (and nodes of Ranvier), as was shown in cortical pyramidal neurons (Kole et al., 2008), which could explain why we have not detected it. Localization of Nav1.1 to specific nodes of Ranvier and AIS proximal subcompartments This study demonstrates for the first time that Nav1.1 is expressed in adult nodes of Ranvier throughout the CNS. Eventhough Nav1.6 is considered as the major Nav α subunit expressed at CNS and PNS nodes of Ranvier (Caldwell et al., 2000; Krzemien et al., 2000; Tzoumaka et al., 2000), previous studies have shown that other Nav α subunits can also be expressed in adult nodes of Ranvier in restricted subsets of neurons: Nav1.2 in retinal ganglion cells (Boiko et al., 2001) and Nav1.9 in thinly myelinated axons of the sciatic nerve (Fjell et al., 2000). At P21, Nav1.8 and Nav1.2 expression in nodes of Ranvier of a few small axons in spinal cord white matter funiculi was mentioned (Arroyo et al., 2002). And at P14–P16, Nav1.1 expression was found in nodes of Ranvier in cerebellar white matter and deep nuclei as well as in the fimbria and corpus callosum (Ogiwara et al., 2007). Similarly for potassium channels, whereas KNCQ2 was found to be expressed in all CNS and PNS nodes (Devaux et al., 2004), Kv3.1b and KCNQ3 have both been observed in subsets of nodes of Ranvier only (Devaux et al., 2003, 2004; Pan et al., 2006). Together with these observations our results suggest that throughout the CNS and PNS multiple populations of nodes of Ranvier exist in terms of sodium (and potassium) channel expression. In addition, unlike other Nav α subunits, we found that Nav1.1 was expressed (either alone or together with Nav1.6) in a very large proportion of nodes of Ranvier (and probably in a large proportion of neurons) throughout the spinal cord, almost as large (in the grey matter) as the proportion of nodes expressing Nav1.6. This result suggests that Nav1.1 should also be considered as a major nodal sodium channel, at least in the spinal cord. Two hypotheses could explain the existence of the three populations of nodes of Ranvier we observed (Nav1.1+, Nav1.6+ or Nav1.1+/ Nav1.6+): it could reflect either different nodes present along individual axons or different axons each harboring a single type of

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node. The three populations were found throughout the spinal cord grey matter with no apparent regionalization and thus could not be associated with a particular type of axon or axonal domain predominantly found in the ventral and dorsal regions analyzed. In contrast, the proportion of nodes expressing Nav1.1 was much lower in the spinal cord white matter. Similarly, Nav1.1 was found to be expressed in nodes of Ranvier in several brain regions, but not in the corpus callosum and only in rare nodes in the cerebellum white matter. In addition, Nav1.1 was never found to be expressed in PNS nodes of Ranvier analyzed in the spinal cord ventral and dorsal roots. We thus cannot favor so far any of the two hypotheses that could explain the heterogeneity of nodes. It is however tempting to speculate, given the low proportion of Nav1.1+ nodes in white matter regions, that the nodal expression of Nav1.1 is predominantly found in axonal regions close to the soma, or as a corollary in short axons. Similarly, while Nav1.6 was considered to be the major Nav α subunit expressed at AISs in the adult CNS (Jenkins and Bennett, 2001), we found that Nav1.1 is expressed at the AIS in many brain regions and in the majority of spinal cord neurons, including 80% of motor neurons. Previous studies have shown that other Nav α subunits may also be expressed in AISs: Nav1.2 was shown to be expressed in postnatal but also adult retinal ganglion cell AISs (Boiko et al., 2003). It was also, like Nav1.8, mentioned to be expressed in a few AISs in the spinal cord grey matter at P21 (Arroyo et al., 2002). Finally, Nav1.1 expression at AISs has also been observed in the adult in retinal ganglion cells and 4% of neurons in the hippocampal area CA3 (Van Wart et al., 2007) as well as at P14–P16 in Purkinje cells and in parvalbumin-expressing interneurons of the neocortex and hippocampus (Ogiwara et al., 2007). In addition, we observed that AISs display an extra level of heterogeneity by expressing, in a complementary fashion, different Nav α subunits in distinct subcompartments: Nav1.1 in the proximal AIS and Nav1.6 in the distal AIS, as was observed in retinal ganglion cell AISs (Van Wart et al., 2007). Interestingly, AISs have also been shown to display a heterogeneous distribution of specific potassium channel isoforms: Kv1.2, KCNQ2 and KCNQ3, all mostly localized to the distal AIS subcompartment. (Inda et al., 2006; Van Wart et al., 2007; Devaux et al., 2004; Pan et al., 2006). Together with these observations, our results suggest that throughout the CNS AISs have a heterogeneous and complex molecular architecture, composed of distinct subcompartments expressing different combinations of sodium and potassium channels and thus endowed with very heterogeneous excitability properties. Mechanisms must exist to allow in a given neuron the selective expression of Nav1.1, versus other Nav α subunits, in specific axonal domains, such as AISs and nodes of Ranvier and in particular in a subpopulation of them or a specific AIS subcompartment. The compartmentalized expression of Nav α subunits along the neuronal membrane can be achieved by distinct mechanisms such as directed delivery, selective retention and/or transcytosis (Sampo et al., 2003; Wisco et al., 2003; Fache et al., 2004). The cytoplasmic loop between domains II and III of Nav1.2 was found to contain two determinants conferring axonal expression. One is responsible for selective endocytosis from somato-dendritic domains (Fache et al., 2004). The other one, composed of a nine amino-acid motif, highly conserved among Nav α subunits, allows binding to ankyrin G and tethers sodium channels at the AIS and possibly also at nodes of Ranvier (Garrido et al., 2003; Lemaillet et al., 2003; Fache et al., 2004). This motif could thus explain the presence of Nav1.1 in both AISs and nodes of Ranvier. An additional ankyrin G interaction site identified in the intracellular loop between domains I and II of Nav1.2 (Bouzidi et al., 2002) and highly conserved in Nav1.1 could also explain tethering of Nav1.1 to AISs and nodes of Ranvier. However, given the ubiquitous expression of ankyrin G in all nodes of Ranvier and AISs, other mechanisms that remain to be identified must exist to explain the differential distribution of Nav1.1 and Nav1.6 in these compartments. Nav1.1 and Nav1.6 may possess additional motifs responsible for their

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selective delivery or retention in specific nodal or AIS compartments. In addition, binding partners must exist that would be expressed in these specific compartments and would display isoform-dependent differences in binding affinity. The gradient-like change of Nav1.1 and Nav1.6 expression levels occurring in AISs in opposite directions and in a complementary fashion suggests however that, at least in AISs, both compete for a limited and constant number of the same anchoring sites, allowing the total amount of Nav channels expressed along the AIS to be uniform (as suggested by the panNav labeling). In such a case only one binding partner could be sufficient to explain the differential localization of Nav1.1 and Nav1.6. A gradient-like distribution of such a binding partner in specific AISs or its expression in specific nodes of Ranvier could enable for instance the preferential binding of Nav1.1 to anchoring sites, and allow it to outcompete the otherwise dominant Nav1.6. Such a competition scenario is supported by the fact that in Nav1.6 mutant mice Nav1.1 is expressed throughout the AIS of retinal ganglion cells and Purkinje cells (Van Wart and Matthews, 2006), cells where, in the presence of Nav1.6 in wild-type mice, Nav1.1 is normally confined to the proximal AIS only (Van Wart et al., 2007; the present study). Nav α subunits interact or are colocalized with several proteins at AISs and nodes of Ranvier, such as NrCAM, NF-186, βIV-spectrin and Nav β subunits, that could fulfill the role of such a binding partner and modulate the anchoring ability of Nav α subunits (Hedstrom and Rasband, 2006). However, none of these proteins has been shown so far to be differentially expressed in nodal and AIS compartments. The fact that Nav1.1 was only found in CNS but not in PNS nodes of Ranvier suggests that the extracellular environment may play a role, consistent with the fact that in PNS nodes aggregation of Nav channels is governed by an outside-to-inside mechanism (Dzhashiashvili et al., 2007). However it is not clear whether the same mechanisms are responsible for Nav channel aggregation at CNS nodes of Ranvier (Hedstrom and Rasband, 2006). Moreover this difference between the extracellular environment of CNS and PNS nodes of Ranvier is not sufficient to explain Nav1.1 expression in specific CNS nodes. Still in line with an extracellular influence, paranodal axoglial junctions, flanking nodes of Ranvier, were shown to regulate the identity and localization of sodium and potassium channels in CNS nodes (Hedstrom and Rasband, 2006). In addition oligodendrocyte-derived factors were also shown to influence the expression and localization of Nav channels at CNS nodes (Kaplan et al., 1997, 2001). However, whether paranodal junctions or oligodendrocyte-derived factors again differ along axons and could cause the differential distribution of Nav α subunits in CNS nodes of Ranvier remains to be shown. Functional significance of Nav1.1 expression in nodes of Ranvier and AISs The heterogeneous composition of AISs and nodes of Ranvier in terms of sodium channel expression suggests the existence of neurons with different action potential generation and propagation properties. How then would the presence of Nav1.1 in AISs and/or nodes of Ranvier modulate these properties? Analysis of Nav1.1 knock-out and knock-in mice have shown that the presence of Nav1.1 is necessary to maintain but not initiate sustained spiking in GABAergic interneurons or Purkinje cells: mutant cells rapidly decreased action potential frequency, number and amplitude in spiking trains (Yu et al., 2006; Ogiwara et al., 2007; Kalume et al., 2007). Expression of Nav1.1 in AISs or nodes of Ranvier may thus correlate with neurons that need to respectively generate or faithfully propagate sustained firing. Interestingly, we found Nav1.1 expression at the AIS of a vast majority of motor neurons, suggesting that it endows them with distinct firing properties. It will be of interest to analyze Nav1.1 function in these motor neurons and determine whether it plays the same role in the maintenance of sustained firing as it does in GABAergic interneurons and Purkinje cells and whether it could be due to Nav1.1 contributing to persistent sodium currents as observed in Purkinje cells (Kalume et

al., 2007). Analysis of Nav1.1 function in motor neurons might also give insights into some of the motor deficits observed in Nav1.1 knockout and knock-in mice (Yu et al., 2006; Ogiwara et al., 2007; Kalume et al., 2007) as well as in some epileptic syndromes associated with Nav1.1 mutations, such as severe myoclonic epilepsy in infancy (Dravet et al., 2005). As mentioned above, AISs appear to have a complex and heterogeneous molecular architecture in terms of sodium and potassium channels, which are differentially distributed in AIS microdomains. The subdivision of AISs into such subcompartments expressing various combinations of sodium and potassium channels is expected to endow neurons with excitability properties that differ between but also along AISs and this likely influences the generation and propagation of action potentials. The functional impact of Nav1.1 expression in proximal AIS microdomains will thus need to be evaluated taking into account the molecular architecture of its subcellular environment. It is likely that Nav1.1 fulfills different roles depending not only on the cell type where it is expressed but also on its precise distribution and molecular environment. Further investigations may thus be needed to unravel its role in epileptogenesis. Experimental methods 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 were used: anti-Nav1.1 rabbit polyclonal (AB5204, Millipore) and mouse monoclonal (clone K74/71, NeuroMab) antibodies raised respectively against amino acids 465-481 and 1929-2009 of rat Nav1.1; anti-Nav1.6 rabbit polyclonal (ASC009, Alomone) and mouse monoclonal (clone K87A/10, NeuroMab) antibodies raised respectively against amino acids 1042-1061 and 459-476 of rat Nav1.6; anti-panNav mouse monoclonal antibody (S8809, clone K58/35, Sigma) raised against a conserved sequence of the intracellular III–IV loop present in all vertebrate α subunit isoforms (Rasband et al., 1999), anti-ChAT goat polyclonal antibody (AB144P, Millipore), anti-peripherin mouse monoclonal antibody (MAB1527, clone 8G2, Millipore) and anti-ankyrin G rabbit polyclonal antibody, directed against residues 1633–1650 of human ankyrin G (Bouzidi et al., 2002). We also generated an anti-ankyrin G guinea pig polyclonal antibody directed against the same residues (1633–1650) of human ankyrin G. This antibody was used instead of the rabbit anti-ankyrin G antibody in triple immunostaining experiments already involving a rabbit (anti-Nav1.1 or anti-Nav1.6) antibody. The anti-paranodin (Caspr) rabbit polyclonal antibody was a generous gift from Dr. J.-A. Girault (University Pierre and Marie Curie, Paris); Menegoz et al., 1997). Alexa 488- or Alexa 594conjugated secondary antibodies (Invitrogen) were used to detect rabbit polyclonal or mouse monoclonal primary antibodies. Cy5conjugated secondary antibodies (Jackson Immuno Research) were used to detect guinea pig polyclonal primary antibodies. Immunohistochemistry Adult mice were deeply anesthetized and fixed by intracardiac perfusion with 5 ml of 1% paraformaldehyde (PFA) freshly prepared

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in phosphate-buffered saline (PBS, pH 7.4) at 4 °C. Spinal cords with their dorsal and ventral roots as well as brains were immediately dissected out (brains were sagittally hemisected), further immersion-fixed for 1 h in 1% PFA at 4 °C, rinsed in PBS and cryoprotected by overnight immersion in 20% sucrose in PBS at 4 °C. Postnatal spinal cords before postnatal day 29 (P29) were freshly dissected out, immersion-fixed for 1 h in 0.25% PFA, rinsed in PBS and cryoprotected overnight in 20% sucrose in PBS at 4 °C. Adult and postnatal spinal cords and adult brains were then frozen in OCT medium (Tissue Tec) and 10 μm cryosections (coronal sections for spinal cords and sagittal sections for brains) were collected onto slides. Dorsal and ventral roots from the spinal cord L3–L6 segments, containing the sciatic nerve sensory and motor axons respectively, were sectioned in a longitudinal plane relative to spinal roots. Slides were thawed at room temperature (RT) and washed 3 × 10 min in tris-buffered saline (TBS). Slides were incubated for 1 h in a blocking solution (10% goat serum in TBS) with 0.4% triton X100 and overnight at 4 °C in a sealed humidified chamber with the primary antibodies, diluted in the blocking solution with 0.2% triton X-100. Slides were then washed 3 × 10 min in TBS and incubated for 2 h with the secondary antibodies, diluted in the blocking solution with 0.2% triton X-100. After washing 3 × 10 min in TBS, slides were dried out and mounted in Mowiol medium (Calbiochem). When primary antibodies generated in the mouse were used, prior to the primary antibody incubation step, slides were incubated for 3 h in goat anti-mouse F(ab)’2 (Jackson Immuno Research), diluted in the blocking solution with 0.2% triton X-100 and washed 3 × 10 min in TBS. This supplementary step reduced non-specific fixation of antimouse secondary antibodies onto mouse tissues. In order to test the immunolabeling specificity of the anti-Nav1.1 polyclonal antibody on spinal cord tissue, cryosections adjacent to experimental sections were incubated with the anti-Nav1.1 polyclonal antibody preincubated with a large molar excess of the Nav1.1 peptide used for its generation. Images were acquired using a fluorescence microscope equipped with an Apotome module (Zeiss, Axiovert 200M) and processed with the NIH ImageJ software (Bethesda, MD). Each figure corresponds to a projection image from a stack of optical sections. Western blotting analysis OF1 mice were sacrificed by rapid decapitation and their spinal cord was freshly dissected out. Spinal cords were minced and homogenized in ice-cold homogenization buffer (0.32 M sucrose, 5 mM TrisHCl, pH 7.4, 2 mM EDTA and 1× protease inhibitor mixture, consisting of 0.5 mM APMSF with leupeptin, aprotinin, and pepstatin A at 1 μg/ml each). The homogenate was centrifuged at 750 ×g for 10 min. The supernatant was next centrifuged at 100,000 ×g for 1 h. The pellet was resuspended in solubilisation buffer (5 mM TrisHCl, pH 7.4, 1 mM EDTA and 1× protease inhibitor mixture) and the protein concentration was determined using the Bradford method (Perbio). Proteins were incubated for 5 min at 50 °C in a denaturing Laemmli buffer (50 mM TrisHCl, pH 6.8, 2% SDS and 10% glycerol) with 0.1 M DTT. For each sample, 50 μg of proteins was separated on a 3.5%–7.5% polyacrylamide-SDS gel, transferred onto nitrocellulose membrane and immunoblotted with the anti-Nav1.1 rabbit polyclonal or mouse monoclonal antibody. HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (Jackson ImmunoResearch) were used for detection in combination with the ECL Plus reagent (Amersham). A chemoluminescent sensitive film (BioMax Light Film, Kodak) was exposed to the membrane. Acknowledgments We are grateful to Lynda Demmou for her valuable participation in preliminary experiments and to Drs. Yoheved Berwald-Netter and

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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 (AFM) and the Fondation pour la Recherche Médicale (FRM). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.mcn.2008.06.008. References Arroyo, E.J., Xu, T., Grinspan, J., Lambert, S., Levinson, S.R., Brophy, P.J., Peles, E., Scherer, S.S., 2002. Genetic dysmyelination alters the molecular architecture of the nodal region. J. Neurosci. 22, 1726–1737. Beckh, S., Noda, M., Lubbert, H., Numa, S., 1989. Differential regulation of three sodium channel messenger RNAs in the rat central nervous system during development. EMBO J. 8, 3611–3616. 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