Brain Advance Access originally published online on April 22, 2003
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Brain, Vol. 126, No. 7, 1552-1561,
July 2003
© 2003 Guarantors of Brain
doi: 10.1093/brain/awg153
Abnormal sodium channel distribution in optic nerve axons in a model of inflammatory demyelination
Department of Neurology and PVA/EPVA Center for Neuroscience Research, Yale University School of Medicine, New Haven and Rehabilitation Research Center, VA Connecticut Healthcare System, West Haven, CT, USA
Correspondence to: Stephen G. Waxman, MD, PhD, Department of Neurology LCI 707, Yale University School of Medicine, 333 Cedar Street, PO Box 208018, New Haven, CT 06520-8018, USA E-mail: stephen.waxman{at}yale.edu
Received January 7, 2003. Revised January 31, 2003. Accepted February 13, 2003.
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Summary |
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Myelinated fibres are characterized by the aggregation of Nav1.6 sodium channels within the axon membrane at nodes of Ranvier, where their presence supports saltatory conduction. In this study, we used immunocytochemical methods to study the organization of sodium channels along axons in experimental allergic encephalomyelitis (EAE), a model of multiple sclerosis. We studied axons within the optic nerve, a CNS tract commonly affected in multiple sclerosis, and their cell bodies of origin (retinal ganglion cells), using subtype-specific antibodies generated against sodium channel subtypes Nav1.1, Nav1.2, Nav1.3 and Nav1.6, which previously have been shown to be expressed by retinal ganglion cells. We demonstrate a significant switch from Nav1.6 to Nav1.2 expression in the optic nerve in EAE; there was a reduction in frequency of Nav1.6-positive nodes (84.5% Nav1.6-immunopositive nodes in control versus 32.9% in EAE) and increased frequency of Nav1.2-positive nodes (11.8% Nav1.2 immunopositive nodes in control versus 74.9% in EAE). Moreover, we observed a significant increase in the number of linear (presumably demyelinated) axonal profiles demonstrating extended diffuse immunostaining for Nav1.2 in EAE versus control optic nerves. These changes within the optic nerve are paralleled by decreased levels of Nav1.6 and increased Nav1.2 protein, together with increased levels of Nav1.2 mRNA, within retinal ganglion cells in EAE. Our findings of a loss of Nav1.6 and increased expression of Nav1.2 suggest that electrogenesis in EAE may revert to a stage similar to that observed in immature retinal ganglion cells in which Nav1.2 channels support conduction of action potentials along axons.
Keywords: sodium channels; demyelination; multiple sclerosis; visual pathways; nodes of Ranvier
Abbreviations: Caspr= contactin-associated protein; EAE = experimental allergic encephalomyelitis; MBP = myelin basic protein; MOG = myelin oligodendrocyte glycoprotein; RGC = retinal ganglion cell
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Introduction |
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In adult myelinated fibres, the axon membrane at nodes of Ranvier is highly specialized, and contains a high density of voltage-gated sodium channels (for reviews see Arroyo and Scherer, 2000
; Peles and Salzer, 2000). Although at least nine molecularly distinct subtypes of sodium channel are expressed within the nervous system, one particular channel subtype, Nav1.6, is predominant at nodes (Caldwell et al., 2000). The expression and distribution of sodium channels within myelinated axons demonstrate a developmental sequence of expression: Nav1.2 channels are found in the membrane of premyelinated axons and at immature nodes within CNS white matter, but there is a switch to Nav1.6 at mature nodes of Ranvier with completion of the myelin sheath (Boiko et al., 2001; Kaplan et al., 2001). Studies using pan-specific sodium channel antibodies (which do not distinguish between channel subtypes) have provided evidence for the reorganization of the nodal membrane, with a loss of nodal sodium channel clustering following PNS demyelination and reappearance with remyelination (Dugandzija-Novakovic et al., 1995; Novakovic et al., 1996, 1998). Boiko et al. (2001) demonstrated in Shiverer mice, a mutant model that is compact myelin deficient, a lack of Nav1.6 immunostaining and retention of Nav1.2 distributed along axons, and suggested that myelination dictates the targeting of Nav1.6 to nodes. Arroyo et al. (2002) demonstrated in mutant myelin-deficient (md) rats, which have a profound lack of CNS myelin, a marked disruption of the paranodal apparatus with displacement of neurofascin155, contactin and contactin-associated protein (Caspr), and aberrant localization of the Kv1.1 and Kv1.2 potassium channels, but no alteration in the expression of Nav1.2 or Nav1.6 sodium channel isoforms, at nodes of Ranvier.
In contrast to the studies with dysmyelinating mutant models, there have been no studies on the expression of specific sodium channel subtypes in demyelinated axons. In the present study, we used immunocytochemical methods with subtype-specific antibodies to examine the distribution of sodium channels along axons within the optic nerve, a tract that is commonly affected in multiple sclerosis, and their cell bodies of origin, the retinal ganglion cells (RGCs), in experimental allergic encephalomyelitis (EAE), an inflammatory demyelinating model of multiple sclerosis. Since sodium channels Nav1.1, Nav1.2, Nav1.3 and Nav1.6 have been shown previously to be expressed in these cells (Fjell et al., 1997), we utilized subtype-specific antibodies generated against these channels to examine their expression in EAE.
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Methods |
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Induction of EAE
Animal protocols followed guidelines established by the NIH and were approved by the Yale University Institutional Animal Care and Use Committee. Biozzi mice (Harlan, UK) aged 6–10 weeks were injected in the flank with 200 µl of emulsion composed of 300 µg of myelin oligodendrocyte glycoprotein (MOG) 35–55 peptide (rat origin, synthesized by the W. M. Keck Biotechnology Resource Center, Yale University) in incomplete Freund’s adjuvant (IFA; Sigma, St Louis, MO, USA) supplemented with 500 µg of Mycobacterium (8 : 1 ratio of tuberculosis and butyricum) (Difco, Detroit, MI, USA). The MOG injection, with Mycobacterium-supplemented IFA, was repeated in the alternate flank 1 week later. In addition, 500 ng of pertussis toxin in 200 µl of phosphate-buffered saline (PBS) was administered i.p to each mouse coincident with the first MOG injection and repeated 48 h later. Age- and sex-matched Biozzi mice served as controls.
All animals induced with MOG developed a relapsing–remitting clinical phenotype, with each animal having at least two relapses prior to sacrifice at 92–142 days post-injection (average length of disease of 111.6 ± 12 days). Staining of optic nerve sections for five animals demonstrated loss of myelin basic protein (MBP) immunoreactivity, suggesting the presence of demyelination, that extended throughout the length of the optic nerves. There was no apparent oedema within EAE optic nerves (Fig. 1). Optic nerves from these EAE mice (n = 5) were compared with optic nerves from aged-matched Biozzi controls (n = 5).
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Immunocytochemistry
Mice were anaesthetized with ketamine/xylazine (80/5 mg/kg, i.p.) and perfused with 4% paraformaldehyde in 0.14 M phosphate buffer (PBS). Tissue was post-fixed for 30 min in 4% paraformaldehyde in 0.14 M PBS, and cryoprotected overnight at 4°C in 30% sucrose in 0.14 M PBS. Sections were cut at 12 µm for retina. After flat embedding in rectangular moulds in OCT medium, optic nerves were sectioned longitudinally at 6 µm and desiccated overnight prior to processing. Retinal tissue sections were processed for immunocytochemistry as previously described (Black et al., 1999
). Briefly, sections were incubated in blocking solution [PBS containing 5% normal goat serum and 1% bovine serum albumin (BSA)] containing 0.1% Triton X-100 and 0.02% sodium azide at room temperature for 30 min, and then incubated with antibodies to Na+ channel 
-subunits Nav1.1 (residues 465–481; 1 : 100; Alomone, Jerusalem), Nav1.2 (residues 467–485; 1 : 100; Alomone), Nav1.3 (residues 511–524; 1 : 2000; Hains et al., 2002) and Nav1.6 (residues 1042–1061; 1 : 100; Alomone) overnight at 4°C. Retinal sections were washed in PBS and incubated with biotinylated goat anti-rabbit IgG (1 : 1000, Sigma) in blocking solution for 3 h, washed in PBS and then incubated in avidin–horseradish peroxidase (HRP; 1 : 1000; Sigma) in blocking solution for 3 h. Sections were washed in PBS and incubated in heavy metal-enhanced DAB (3,3' diaminobenzidine hydrochloride) solution (Pierce, Rockford, IL, USA) for 7 min.
Optic nerve sections were subjected to a similar protocol, but incubated simultaneously with monoclonal antibodies to Caspr (1 : 500; provided by M. Rasband; Rasband et al., 1999) or MBP (1 : 4000; Sternberger Monoclonals Inc., Lutherville, MD, USA) and polyclonal antibodies to Nav1.1 Nav1.2, Nav1.3 and Nav1.6. Sections were then washed in PBS and incubated with goat anti-rabbit IgG-Cy3 (1 : 2000, Amersham, Piscataway, NJ, USA) and goat anti-mouse IgG-Cy2 (1 : 1000, Amersham) secondary antibodies in blocking solution for 3 h, washed in PBS and mounted.
Control experiments which included the omission of primary or secondary antibodies showed no staining (data not shown).
In situ hybridization
Mice were subject to a similar surgical and perfusion protocol. Tissue was post-fixed and cryoprotected overnight at 4°C in 30% sucrose in 4% paraformaldehyde solution, and serial sections were cut onto slides and desiccated overnight. Sections from control and EAE were processed for in situ hybridization cytochemistry as previously described using isoform-specific riboprobes (Black et al., 1996). Sense riboprobes yielded no signals on in situ hybridization (data not shown).
Analysis of tissue sections
For analysis of optic nerve sections, multiple images from segments of optic nerve were taken by confocal microscopy with a NIKON Eclipse E600 microscope. Analysis was confined to images in which axons were sectioned longitudinally as evident from linear arrangement of paranodal Caspr immunostaining and nodes; the presence of linear (presumably demyelinated) axonal profiles with diffuse sodium channel immunoreactivity, running for >20–30 µm within the plane of single sections, further verifed the longitudinal orientation of sections from EAE optic nerves. For counting of nodal regions, a pre-designed grid (consisting of 12 quadrants each 25 µm x 33.3 µm) was superimposed on an image. To ensure random sampling that included the entire range of nodal morphologies in the optic nerve in EAE and controls, four quadrants were selected from each of three images (both quadrants and images were randomly selected) and analysed to sample a total field of 104 µm2 per animal. In view of the loss of MBP staining throughout the length of the optic nerves, it would be expected that very few axons sampled in EAE optic nerves were from non-demyelinated regions.
All nodes were scored within each of the selected quadrants and classified into one of two main categories. (i) Nodes: robust/well-defined Caspr staining on both sides of the nodal region (see examples in Fig. 2A and B; yellow arrowheads). (ii) Nodes with attenuated Caspr: ill-defined or attenuated Caspr staining on one or both sides of the node (see the example in Fig. 2C, inset). They were then scored as Nav1.6-immunopositive or -negative, or as Nav1.2-immunopositive or -negative, on the basis of the presence, or absence, of nodal red immunostaining signal. This method of analysis was used in preference to quantitative microdensitometry because: (i) as a result of the high density of sodium channels and non-linearity of signal intensity, microdensitometry would not have detected fluctuations of less than 
50% in density of channels at the node; and (ii) it provided a measure of abnormal nodal/paranodal morphology as well as channel immunofluorescence. A total of 622 and 527 nodes were examined in sections immunoreacted with Nav1.6 or Nav1.2 antibodies, respectively, in control optic nerve within a 5 x 104 µm2 area (104 µm2 from each of five animals); 736 and 641 nodes were examined in sections immunoreacted with Nav1.6 or Nav1.2 antibodies, respectively, in EAE optic nerve within a 5 x 104 µm2 area. To facilitate comparison, the data are presented in terms of the mean number of nodes or nodes with attenuated Caspr ± SEM per 104 µm2. A small proportion (10% of the total) of heminodes (Arroyo et al. 2002), with robust Caspr immunostaining flanking a nodal region on only one side, were identified but excluded from the data presented. Inclusion of these heminodes (data not presented) did not significantly alter the results. Due to substantial changes in EAE (see Results), it was not possible to blind the observer as to whether a section was from EAE or control optic nerve. To validate the scoring of Caspr, and of sodium channel immunopositivity, two observers independently evaluated 100 nodes each and differed by <9% of nodes with respect to robust/well-defined versus attenuated Caspr, and in 1% of nodes with respect to sodium channel immunopositivity versus immunonegativity.
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Within the optic nerve sections, we also identified extended regions of diffuse sodium channel immunoreactivity for either Nav1.2 or Nav1.6 along (presumably demyelinated) linear axonal profiles (see Fig. 2D, F and G; open arrows). As an index of the frequency of these profiles, we counted the number of axonal profiles that displayed diffuse regions of immunostaining to either Nav1.2 or Nav1.6, extending >8 µm length along the optic nerve (therefore excluding nodal clusters of immunostaining). To facilitate quantification, a target line (six 100 µm increments for a total length 600 µm) perpendicular to the axis of nerve fibres was overlaid on randomly selected images (see the example in Fig. 4B), and linear (>8 µm length) profiles with sodium channel immunostaining that intersected the target line were counted. The data presented represent the mean number of axonal profiles with diffuse Nav1.2 or Nav1.6 immuno staining ± SEM per 600 µm of target in both control and EAE optic nerves.
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For quantification of RGC neurons, images were captured using a Nikon Eclipse E800 light microscope. Quantitative microdensitometry of the immunostaining signal was performed using IPLab Scientific Image Processing soft ware (Scanalytics Inc., Fairfax, VA, USA; Craner et al., 2002
). Signal intensities were obtained by manual out lining of RGC neurons, using the IPLab integrated densitometry function to calculate mean signal intensities for the outlined areas. Background signal was subtracted from both EAE and control images. Statistical analysis was performed using the Student’s t test. Data are presented as the mean ± SEM. |
Results |
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Loss of nodal integrity indicated by attenuated Caspr immunostaining in EAE
Caspr was the first molecular component of the paranodal region to be identified (Einheber et al., 1997
; Menegoz et al., 1997) and is an integral constituent of paranodal junctions that are believed to participate in demarcation of ion channel domains at nodes of Ranvier (Dupree et al., 1999; Bhat et al., 2001). Therefore, we utilized Caspr immunolabelling to delineate nodal regions for subsequent analysis in the distribution of sodium channels. However, it was strikingly apparent that in all regions sampled from the optic nerves of EAE mice, there was a significant reduction in the number of nodes flanked by robust/well-defined Caspr staining, suggesting that the pathological process in EAE is not confined solely to a redistribution of sodium channels but also affects the paranodal junctions. Quantification of nodal density reveals that the mean number of nodes, bounded on both sides by robust/well-defined Caspr immunostaining, is reduced from 94.9 ± 6.4 per 104 µm2 in control optic nerve to 52.3 ± 3.8 per 104 µm2 (P < 0.005) in EAE (Fig. 3A, left panel). Concurrent with the reduction in the density of nodes with robust/well-defined Caspr immunostaining, there is an increase in the number of nodes with attenuated Caspr staining from 27 ± 1.5 per 104 µm2 in control optic nerve to 87.0 ± 9.0 per 104 µm2 in EAE optic nerve (P < 0.005) (Fig. 3A, right panel).
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Consistent with remyelination and development of shortened internodes, we observed a non-significant trend for an overall increase in the total number of nodes in EAE (139.3 ± 11.1 per 104 µm2) compared with control (121.9 ± 7.2 per 104 µm2) optic nerves.
Altered distribution of Nav1.6 and Nav1.2 at nodal regions in EAE
As previously reported (Caldwell et al., 2000; Arroyo et al., 2002), we confirm that Nav1.6 is the predominant sodium channel at nodes of Ranvier, with Nav1.6 immunostaining occurring at 84.5% (Table 1) of nodes delineated by robust Caspr labelling in control optic nerves (Fig. 3B, left panel). Nav1.2 immunostaining occurred at 11.8% (Table 1) of nodes (Fig. 3B, right panel). We observed no specific nodal staining for Nav1.1 or Nav1.3 sodium channels in either control or EAE optic nerves.
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In all of the regions sampled from the optic nerves of mice with EAE, we observed a significant reduction in the frequency of Nav1.6 immunopositivity at nodes with robust Caspr immunostaining, which fell to 32.9% (Table 1) (Fig. 3B, left panel) (P < 0.005). In contrast, we observed a significant shift to Nav1.2 expression at nodes in EAE, with 74.9% (Table 1) of nodes with robust Caspr demonstrating Nav1.2 in EAE optic nerves (Fig. 3B, right panel; see also yellow arrowheads in Fig. 2B, D and H).
We also examined Nav1.6 and Nav1.2 expression at nodes with attenuated Caspr staining. We observed a significant decrease in the proportion of nodes displaying Nav1.6 immunostaining in EAE, which fell to 30.6% (Table 1) compared with 52.8% (Table 1) in control (Fig. 3C, left panel) (P < 0.05). We observed an overall increase in the number of Nav1.2-immunopositive nodes with attenuated Caspr staining in EAE (31.6 ± 4.3 per 104 µm2) compared with control (12.0 ± 1.9 per104 µm2). However, as a result of the 3-fold increase in the number of nodes with attenuated Caspr staining in EAE optic nerves (see above), there was not an increase in the frequency of Nav1.2 immunopositivity (42.4%; Table 1) at nodes with attenuated Caspr staining in EAE optic nerves compared with that of Nav1.2 immunopositivity at nodes with attenuated Caspr staining (41.4%; Table 1) in control optic nerves (Fig. 3C, right panel).
Increased number of linear axon profiles with diffuse Nav1.2 and Nav1.6 immunostaining in EAE
We observed a significant increase in linear (presumably demyelinated) axonal profiles in which diffuse sodium channel immunostaining extended for >8 µm along the fibre axis in EAE. Consistent with previous reports indicating that virtually all axons in adult rodent optic nerves are myelinated (Forrester and Peters, 1967; Foster et al., 1982), we found only 2 ± 1.5 linear axonal profiles with diffuse Nav1.6 immunostaining and 4.2 ± 0.4 linear axonal profiles with diffuse Nav1.2 immunostaining that intersected a 600 µm target line in control tissue (Fig. 4). In contrast, in EAE optic nerves, there was a significant increase in the number of linear axonal profiles with diffuse Nav1.6 immunostaining (26.6 ± 7.7, P < 0.05) and linear axonal profiles with diffuse Nav1.2 immunostaining (75.8 ± 11.4, P < 0.005) intersecting 600 µm of target (Fig. 4). These extended diffuse regions of Nav1.6 and Nav1.2 immunostaining were associated in some cases with Caspr staining (e.g. Fig. 2D, open arrow). In some instances, sodium channel immunostaining extended for >30 µm (Fig. 2D and F, open arrow) along linear axon profiles.
Downregulation of Nav1.6 and upregulation of Nav1.2 protein within retinal ganglion neurons in EAE
To determine if the changes observed within optic nerve axons are accompanied by altered channel expression in the neuronal cell bodies that give rise to these fibres, we examined and quantified Nav1.6 and Nav1.2 immunostained signals within RGCs of control and EAE mice. We observed a significant downregulation of Nav1.6 signal intensity in RGCs of EAE mice, with a mean intensity of 45.8 ± 8.7 in EAE compared with 77.1 ± 2.5 in control mice (P < 0.05) (Figures 4 and 5).
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Consistent with the increased number and percentage of Nav1.2-immunopositive nodes with robust Caspr immunostaining in EAE, we found a significant increase in Nav1.2 signal intensity in the RGCs in EAE compared with control mice (EAE, 82.0 ± 3.0; control, 61.4 ± 5.6, P < 0.05) (Figures 4 and 5) and upregulation of in situ hybridization signal for Nav1.2 mRNA in RGCs in EAE (Fig. 6), consistent with increased transcription of this channel subtype. We also observed upregulation of Nav1.2 immunostaining within both the retinal nerve fibre layer (which contains the non-myelinated segments of ganglion cell axons, prior to their entrance into the largely myelinated optic nerve; Black et al., 1985
) and inner nuclear layer, which contains cell bodies of the retinal bipolar cells (Fig. 5). There was no significant change observed in Nav1.1 and Nav1.3 protein within the RGCs, which remained low as in control tissue.
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Discussion |
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In this study, we used subtype-specific antibodies to examine sodium channel distribution in the optic nerve, a tract in which essentially 100% of axons are myelinated within the normal CNS (Forrester and Peters, 1967
; Foster et al., 1982) and which is commonly affected in multiple sclerosis. We have demonstrated that in chronic relapsing EAE, an experimental model of multiple sclerosis, MBP immunoreactivity is decreased throughout the length of the optic nerve, indicating demyelination, and this loss of myelin is accompanied by a reduced number and proportion of nodes expressing Nav1.6. Moreover, we observed an increased number and proportion of nodes with robust Caspr immunostaining in EAE optic nerves that expressed Nav1.2. There was also an increase in the number of linear (presumably demyelinated) axon profiles displaying diffuse Nav1.6 or Nav1.2 immunostaining. These changes are paralleled by downregulation of Nav1.6 and upregulation of Nav1.2 within retinal ganglion neurons.
Nodes of Ranvier in normal white matter exhibit a complex molecular organization, including the aggregation of sodium channels at high densities in the axon membrane at the node (Ritchie and Rogart, 1977; Waxman, 1977) flanked by paranodal sepate-like axo-glial junctions containing the contactin–Caspr heterodimer (Einheber et al., 1997; Menegoz et al., 1997; Rios et al., 2000). The mechanisms that determine nodal specialization and clustering of sodium channels have not been fully delineated. Some evidence suggests that myelinating oligodendrocytes in the CNS (Kaplan et al., 2001) or Schwann cells in the PNS (Dugandzija-Novakovic et al., 1995; Tzoumaka et al., 1995) play a role (for a review see Vabnick and Shrager, 1998) and that nodal sodium channel clustering and segregation from potassium channels are dependent on axo-glial contact (Rasband et al., 1999) and Caspr (Bhat et al., 2001). However, it has also been suggested that sodium channel clustering can occur under some circumstances in which Schwann cells have ensheathed axons, but have not formed compact myelin or mature paranodal junctions (Waxman and Foster, 1980; Deerinck et al., 1997; Vabnick et al., 1997). A developmental switch has been observed, with early expression of Nav1.2 along pre-myelinated axons and initial development of Nav1.2 clusters, preceding the clustering of Nav1.6 along axons of RGCs in vitro (Kaplan et al., 2001) and in vivo (Boiko et al., 2001). The present results demonstrate a significant reduction in the number of nodes with robust Caspr staining that were Nav1.6 immunopositive in EAE. This was not offset by an increase in Nav1.6 immunostaining at nodes with attenuated Caspr immunolabelling, which may have been partially demyelinated. In contrast, we observed a significant increase in the number of nodes that displayed Nav1.2 immunostaining. Although we cannot distinguish whether these Nav1.2-positive nodes represent preserved (original) nodes at which there was a switch to Nav1.2 expression, or the emergence of new nodes expressing Nav1.2 along remyelinated axons, the upregulated expression of Nav1.2, in the context of reduced Nav1.6 expression, is consistent with the idea that different mechanisms are responsible for channel clustering of Nav1.6 versus Nav1.2 (Boiko et al., 2001; Kaplan et al., 2001). The relatively large number of nodes that we observed in EAE optic nerves (139.3 ± 11.1 per 104 µm2, compared with 121.9 ± 7.2 per 104 µm2 in controls) suggests that some nodes were formed as a result of remyelination, as has been observed in other EAE models (e.g. Prineas et al., 1969; Pender et al., 1989)
Similar to previous descriptions in models of genetic dysmyelination (Boiko et al., 2001) and in experimental allergic neuritis (Novakovic et al., 1998) and doxorubicin-induced demyelination (England et al., 1990), we observed diffuse sodium channel immunostaining that extended in a relatively non-focal manner along linear axon profiles. Our use of subtype-specific antibodies permitted us to identify Nav1.2 and Nav1.6 immunostaining that extended diffusely for at least 8 µm along linear axonal profiles, with a majority of these displaying Nav1.2 immunostaining. A similar pattern of expression of Nav1.2 immunostaining is observed along axon profiles during early development prior to myelination (Boiko et al., 2001) and in genetic dysmyelination (Boiko et al., 2001). Nav1.2 (Westenbroek et al., 1989; Gong et al., 1999; Whitaker et al., 2000) and Nav1.6 (Black et al., 2002) channels are known to be present along non-myelinated axons within the CNS, and Nav1.6 has been shown to contribute to action potential conduction along non-myelinated axons (Black et al., 2002). The relatively non-focal distribution of Nav1.2 and Nav1.6 channels may provide a substrate for continuous (Bostock and Sears, 1976, 1978) or saltatory (Smith et al., 1982) impulse conduction, both of which have been observed in demyelinated axons.
The acquisition of sodium channels by the demyelinated axonal membrane may be a result of altered transcription in RGCs or redistribution of channels from nodes of Ranvier. Rosenbluth (1976) first suggested that sodium channels might diffuse within the axon membrane after damage to the paranodal axo-glial junctions. Consistent with this hypothesis, Ishibashi et al. (2002) demonstrated that in cerebroside sulfotransferase-deficient mice, the numbers of sodium channel clusters were dramatically reduced with age but that protein levels detected with a pan-specific sodium channel antibody were not reduced. Our demonstration of a downregulation of Nav1.6 protein in the RGCs in EAE suggests that the increase in diffuse Nav1.6 immunostaining over extended regions that we observed in a small number of linear axon profiles may not be due to the production of new Nav1.6 channels within the neuronal cell body, but rather to a redistribution of existing Nav1.6 sodium channels from their previous sites at nodes of Ranvier. We also observed that an upregulation of Nav1.2 protein in the RGCs accompanies the increase in the number of Nav1.2-immunopositive nodes and of linear axon profiles diffusely immunostained for Nav1.2 in EAE. Consistent with increased transcription of Nav1.2 in EAE, we observed an upregulation of the level of Nav1.2 mRNA by in situ hybridization.
The functional importance of the altered Nav1.2 and Nav1.6 sodium channel distribution in EAE is not yet clear. Nav1.2 and Nav1.6 channels, expressed in Xenopus oocytes, produce currents with subtly different functional properties that appear to allow Nav1.6 channels to maintain higher firing rates (Zhou and Goldin, 2002). Moreover, Nav1.6 channels produce subthreshold persistent currents that contribute to repetitive firing in some neuronal cell types (Raman et al., 1997; Smith et al., 1998). These observations suggest that a loss of Nav1.6 may lead to perturbed generation of impulse trains within ganglion cells and/or impaired conduction from the retina through the optic nerve to the visual cortex.
Action potentials are conducted, albeit with slowed conduction velocities (Foster et al., 1982; Rasband et al., 1999), along CNS fibres prior to maturation of myelin. The (re)development of an immature pattern of expression of Nav1.2 channels in demyelinated axons may provide a mechanism for slowed conduction of at least low-frequency impulses along these fibres. Although the detailed time course of expression of Nav1.2 and Nav1.6 sodium channels in RGCs has not been delineated, it is known that there is a shift in sodium current density and kinetics during the developmental period (Schmid and Guenther, 1998) and these changes parallel the sequential expression of Nav1.2 and subsequently Nav1.6 sodium channels in optic nerve (Boiko et al., 2001). Our findings of a loss of Nav1.6 and increased expression of Nav1.2 thus suggest that action potential electrogenesis in EAE may revert to a stage similar to that observed (Skaliora et al., 1993; Wang et al., 1997; Rothe et al., 1999) in immature RGCs, which can generate single action potentials, but are less reliable in terms of sustained high-frequency firing.
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Acknowledgements |
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We wish to thank Dr Matthew Rasband, University of Connecticut Health Center, for the generous gift of Caspr antibody and for helpful comments, and Lynda Tyrrell and Bart Toftness for excellent technical assistance. This work was supported in part by grants from the National Multiple Sclerosis Society (RG-1912) and the Rehabilitation Research Service and Medical Research Service, Department of Veterans Affairs. The authors also thank the Paralyzed Veterans of America, the Eastern Paralyzed Veterans Association and the Nancy Davis Foundation for support. M.J.C. thanks Medical Director General, UK, for support.
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References |
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TopSummaryIntroductionMethodsResultsDiscussionReferences |
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Arroyo EJ, Scherer SS. On the molecular architecture of myelinated fibers. Histochem Cell Biol 2000; 113: 1–18.[CrossRef][Web of Science][Medline]
Arroyo EJ, Xu T, Grinspan J, et al. Genetic dysmyelination alters the molecular architecture of the nodal region. J Neurosci 2002; 22: 1726–37.
Bhat MA, Rios JC, Lu Y, et al. Axon–glia interactions and the domain organization of myelinated axons requires neurexin IV/Caspr/Paranodin. Neuron 2001; 30: 369–83.[CrossRef][Web of Science][Medline]
Black JA, Waxman SG, Hildebrand C. Axo-glial relations in the retina–optic nerve junction of the adult rat: freeze-fracture observations on axon membrane structure. J Neurocytol 1985; 14: 887–907.[CrossRef][Web of Science][Medline]
Black JA, Dib-Hajj SD, McNabola K, et al. Spinal sensory neurons express multiple sodium channel -subunit mRNAs. Brain Res Mol Brain Res 1996; 43: 117–31.[Medline]
Black JA, Renganathan M, Waxman SG. Sodium channel Nav1.6 is expressed along nonmyelinated axons and it contributes to conduction. Brain Res Mol Brain Res 2002; 105: 19–28.[Medline]
Boiko T, Rasband MN, Levinson SR, et al. Compact myelin dictates the differential targeting of two sodium channel isoforms in the same axon. Neuron 2001; 30: 91–104.[CrossRef][Web of Science][Medline]
Bostock H, Sears TA. Continuous conduction in demyelinated mammalian nerve fibers. Nature 1976; 263: 786–7.[CrossRef][Medline]
Bostock H, Sears TA.The internodal axon membrane: electrical excitability and continuous conduction in segmental demyelination. J Physiol 1978; 280: 273–301.
Caldwell JH, Schaller KL, Lasher RS, Peles E, Levinson SR. Sodium channel Na(v)1.6 is localized at nodes of Ranvier, dendrites, and synapses. Proc Natl Acad Sci USA 2000; 97: 5616–20.
Craner MJ, Klein JP, Renganathan M, Black JA, Waxman SG. Changes of sodium channel expression in experimental painful diabetic neuropathy. Ann Neurol 2002; 52: 786–92.[CrossRef][Web of Science][Medline]
Deerinck TJ, Levinson SR, Bennett GV, Ellisman MH. Clustering of voltage-sensitive sodium channels on axons is independent of direct Schwann cell contact in the dystrophic mouse. J Neurosci 1997; 17: 5080–8.
Dugandzija-Novakovic S, Koszowski AG, Levinson SR, Shrager P. Clustering of Na+ channels and node of Ranvier formation in remyelinating axons. J Neurosci 1995; 15: 492–503.[Abstract]
Dupree JL, Girault JA, Popko B. Axo-glial interactions regulate the localization of axonal paranodal proteins. J Cell Biol 1999; 147: 1145–52.
Einheber S, Zanazzi G, Ching W, et al. The axonal membrane protein Caspr, a homologue of neurexin IV, is a component of the septate-like paranodal junctions that assemble during myelination. J Cell Biol 1997; 139: 1495–506.
England JD, Gamboni F, Levinson SR, Finger TE. Changed distribution of sodium channels along demyelinated axons. Proc Natl Acad Sci USA 1990; 87: 6777–80.
Fjell J, Dib-Hajj S, Fried K, Black JA, Waxman SG. Differential expression of sodium channel genes in retinal ganglion cells. Brain Res Mol Brain Res 1997; 50: 197–204[Medline]
Forrester J, Peters A. Nerve fibres in optic nerve of rat. Nature 1967; 214: 245–7.[CrossRef][Medline]
Foster RE, Connors BW, Waxman SG. Rat optic nerve: electro physiological, pharmacological and anatomical studies during development. Brain Res 1982; 255: 371–86.[Medline]
Gong B, Rhodes KJ, Bekele-Arcuri Z, Trimmer JS. Type I and type II Na+ channel alpha-subunit polypeptides exhibit distinct spatial and temporal patterning, and association with auxiliary subunits in rat brain. J Comp Neurol 1999; 412: 342–52.[CrossRef][Web of Science][Medline]
Hains BC, Black JA, Waxman SG. Primary motor neurons fail to up-regulate voltage-gated sodium channel Na(v)1.3/brain type III following axotomy resulting from spinal cord injury. J Neurosci Res 2002; 70: 546–52.[CrossRef][Web of Science][Medline]
Ishibashi T, Dupree JL, Ikenaka K, et al. A myelin galactolipid, sulfatide, is essential for maintenance of ion channels on myelinated axon but not essential for initial cluster formation. J Neurosci 2002; 22: 6507–14.
Kaplan MR, Cho MH, Ullian EM, Isom LL, Levinson SR, Barres BA. Differential control of clustering of the sodium channels Na(v)1.2 and Na(v)1.6 at developing CNS nodes of Ranvier. Neuron 2001; 30: 105–19.[CrossRef][Web of Science][Medline]
Menegoz M, Gaspar P, Le Bert M, et al. Paranodin, a glycoprotein of neuronal paranodal membranes. Neuron 1997; 19: 319–31.[CrossRef][Web of Science][Medline]
Novakovic SD, Deerinck TJ, Levinson SR, Shrager P, Ellisman MH. Clusters of axonal Na+ channels adjacent to remyelinating Schwann cells. J Neurocytol 1996; 25: 403–12.[CrossRef][Web of Science][Medline]
Novakovic SD, Levinson SR, Schachner M, Shrager P. Disruption and reorganization of sodium channels in experimental allergic neuritis. Muscle Nerve 1998; 21: 1019–32.[CrossRef][Web of Science][Medline]
Peles E, Salzer JL. Molecular domains of myelinated axons. Curr Opin Neurobiol 2000; 10: 558–65.[CrossRef][Web of Science][Medline]
Pender MP. Recovery from acute experimental allergic encephalomyelitis in the Lewis rat. Early restoration of nerve conduction and repair by Schwann cells and oligodendrocytes. Brain 1989; 112: 393–416.
Prineas J, Raine CS, Wisniewski H. An ultrastructural study of experimental demyelination and remyelination. 3. Chronic experi mental allergic encephalomyelitis in the central nervous system. Lab Invest 1969; 21: 472–83.[Web of Science][Medline]
Raman IM, Sprunger LK, Meisler MH, Bean BP. Altered subthreshold sodium currents and disrupted firing patterns in Purkinje neurons of Scn8a mutant mice. Neuron 1997; 19: 881–91.[CrossRef][Web of Science][Medline]
Rasband MN, Peles E, Trimmer JS, Levinson SR, Lux SE, Shrager P. Dependence of nodal sodium channel clustering on paranodal axo-glial contact in the developing CNS. J Neurosci 1999; 19: 7516–28.
Rios JC, Melendez-Vasquez CV, Einheber S, et al. Contactin-associated protein (Caspr) and contactin form a complex that is targeted to the paranodal junctions during myelination. J Neurosci 2000; 20: 8354–64.
Ritchie JM, Rogart RB. Density of sodium channels in mammalian myelinated nerve fibers and nature of the axonal membrane under the myelin sheath. Proc Natl Acad Sci USA 1977; 74: 211–5.
Rosenbluth J. Intramembranous particle distribution at the node of Ranvier and adjacent axolemma in myelinated axons of the frog brain. J Neurocytol 1976; 5: 731–45.[CrossRef][Web of Science][Medline]
Rothe T, Juttner R, Bahring R, Grantyn R. Ion conductances related to development of repetitive firing in mouse retinal ganglion neurons in situ. J Neurobiol 1999; 38: 191–206.[CrossRef][Web of Science][Medline]
Schmid S, Guenther E. Alterations in channel density and kinetic properties of the sodium current in retinal ganglion cells of the rat during in vivo differentiation. Neuroscience 1998; 85: 249–58[CrossRef][Web of Science][Medline]
Skaliora I, Scobey RP, Chalupa LM. Prenatal development of excitability in cat retinal ganglion cells: action potentials and sodium currents. J Neurosci 1993; 13: 313–23.[Abstract]
Smith KJ, Bostock H, Hall SM. Saltatory conduction precedes remyelination in axons demyelinated with lysophosphatidyl choline. J Neurol Sci 1982; 54: 13–31.[CrossRef][Web of Science][Medline]
Smith MR, Smith RD, Plummer NW, Meisler MH, Goldin AL. Functional analysis of the mouse Scn8a sodium channel. J Neurosci 1998; 18: 6093–102.
Tzoumaka EE, Novakovic SD, Levinson SR, Shrager P. Na+ channel aggregation in remyelinating mouse sciatic axons following transection. Glia 1995; 15: 188–94.[CrossRef][Web of Science][Medline]
Vabnick I, Shrager P. Ion channel redistribution and function during development of the myelinated axon. J Neurobiol 1998; 37: 80–96.[CrossRef][Web of Science][Medline]
Vabnick I, Messing A, Chiu SY, et al. Sodium channel distribution in axons of hypomyelinated and MAG null mutant mice. J Neurosci Res 1997; 50: 321–36.[CrossRef][Web of Science][Medline]
Wang GY, Ratto G, Bisti S, Chalupa LM. Functional development of intrinsic properties in ganglion cells of the mammalian retina. J Neurophysiol 1997; 78: 2895–903.
Waxman SG. Conduction in myelinated, unmyelinated and demyelinated fibers. Arch Neurol 1977; 34: 585–590
Waxman SG, Foster RE. Development of the axon membrane during differentiation of myelinated fibres in spinal nerve roots. Proc R Soc Lond B Biol Sci 1980; 209: 441–6.[Medline]
Westenbroek RE, Merrick DK, Catterall WA. Differential subcellular localization of the RI and RII Na+ channel subtypes in central neurons. Neuron 1989; 3: 695–704.[CrossRef][Web of Science][Medline]
Whitaker WR, Clare JJ, Powell AJ, Chen YH, Faull RL, Emson PC. Distribution of voltage-gated sodium channel alpha-subunit and beta-subunit mRNAs in human hippocampal formation, cortex, and cerebellum. J Comp Neurol 2000; 422: 123–39.[CrossRef][Web of Science][Medline]
Zhou W, Goldin AL. Functional differences between the Nav1.6 and Nav1.2 sodium channels [abstract]. Soc Neurosci Abstr 2002; 32: Prog. No. 834.4
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