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Functional Changes in Genetically Dysmyelinated Spinal Cord Axons of Shiverer Mice: Role of Juxtaparanodal Kv1 Family K⁺ Channels

¹Division of Cell and Molecular Biology, Toronto Western Research Institute, Krembil Neuroscience Center, Toronto Western Hospital, University Health Network, Toronto; and ²Division of Neurosurgery, Department of Surgery and ³Institute of Medical Sciences, University of Toronto, Ontario, Canada

Submitted 29 August 2005; accepted in final form 28 November 2005

	ABSTRACT

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Axonal dysfunction after spinal cord injury (SCI) and other types of neurotrauma is associated with demyelination and exposure of juxtaparanodal K⁺ channels. In this study, sucrose gap electrophysiology using selective and nonselective K⁺ channel blockers, confocal immunohistochemistry, and Western blotting were used to study the role of Kv1.1 and Kv1.2 K⁺ channel subunits in dysmyelination-induced spinal cord axonal dysfunction in shiverer mice, which lack the gene encoding myelin basic protein (MBP) and exhibit incomplete myelin sheath formation on CNS axons. The shiverer spinal cord axons exhibited smaller amplitude of compound action potentials (CAPs), reduced conduction velocity, reduced excitability, and greater degree of high-frequency conduction failure. The "fast" K⁺ channel blocker 4-aminopyridine, the toxin DTX-I, which targets the Kv1.1 and Kv1.2, but not DTX- K, which has higher selectivity for Kv1.1, increased the amplitude and area of CAPs of shiverer mice spinal cord axons but had insignificant effects in wild-type mice. Confocal immunohistochemistry showed that, unlike wild-type mice, which have a precise juxtaparanodal localization of the Kv1.l and Kv1.2 K⁺ channel subunits, shiverer mouse axons displayed a dispersed distribution of these subunits along the internodes. In contrast, the Kv1.l and Kv1.2 subunits, Na⁺ channels remained highly localized to the nodal regions. Western blotting showed an increased expression of Kv 1.1 and 1.2 in the shiverer mouse spinal cord. These results provide evidence that the neurological deficits associated with myelin deficiency reflect the altered distribution and expression of the K⁺ channel subunits Kv1.l and Kv1.2 along the internodes of spinal cord axons associated with the biophysical consequences caused by alterations in the myelin sheaths.

	INTRODUCTION

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The structure of the myelin sheath contributes to the unique spatial segregation of axonal ion channels (Devaux and Scherer 2005). The characteristic localization of voltage-gated ion channels is crucial for the saltatory conduction in myelinated nerve fibers (Chiu and Ritchie 1981). At the mammalian node of Ranvier, there is a sharp segregation of voltage-dependent ion channels. The Na⁺ channels are clustered at a very high-density within the nodes, while the rapidly activating voltage-dependent K⁺ channels are located in the juxtaparanodal regions beneath compact myelin and do not normally contribute to the action potential. Demyelination of intact axons by disease or injury results in altered distribution of the nodal/paranodal ion channels and cell adhesion proteins, thus hampering the propagation of action potentials (Boyle et al. 2001; Coetzee et al. 1996; Nashmi and Fehlings 2001a,b; Poliak and Peles 2003).

Demyelination is an important factor contributing to the loss of axonal function and to the development of significant neurological deficits in many CNS disorders including multiple sclerosis, stroke, spinal cord injury, and other types of neurotrauma (Blight and Decrescito 1986; Fehlings and Nashmi 1996; McDonald et al. 2000; Nashmi and Fehlings 2001a,b). Demyelination is associated with abnormal conduction properties including prolonged refractory period, reduced conduction velocity, and conduction block at subphysiological temperatures (Blight and Decrescito 1986; Nashmi and Fehlings 2001b). Recently, the contribution of axonal potassium channels as a critical factor in conduction loss after spinal cord injury has been emphasized in both animal model (Blight 1989; Nashmi and Fehlings 2001b; Targ and Kocsis 1985) and human victims (Hansebout et al. 1993; Hayes et al. 1994). In fact, 4-aminopyridine (4-AP), a blocker of fast K⁺ channels, has been shown to effectively enhance action potential propagation after spinal cord trauma by blocking potassium channels in in vitro studies (Blight 1989; Nashmi et al. 2000; Shi et al. 1997) and in improving the neurological function of human patients (Hansebout et al. 1993; Hayes et al. 1994). However, much remains to be learned regarding the mechanisms of these effects. The juxtaparanodal Shaker K⁺ channels containing Kv1.1 and Kv1.2 subunits are normally isolated electrically by the overlying myelin structures but are uncovered during demyelination. Demyelinated/dysmyelinated axons are characterized by an exposure and altered distribution of these K⁺ channel subunits and display electrophysiological evidence of dysfunction (Jensen and Shi 2003; Nashmi et al. 2000). These studies have suggested an important role for the Shaker K⁺ channel subunits Kv1.1 and Kv1.2 in axonal conduction block.

A diverse group of spontaneous and engineered mouse mutants, characterized by defects of myelin formation, has been intensively studied from the morphological to molecular levels to understand the pathophysiology underlying the demyelination/dysmyelination and regeneration research. However, relatively little work has been performed in terms of the physiological characterization of central axons in these models. Homozygous shiverer mice (shi/shi) are autosomal recessive mutants characterized by an almost complete lack of CNS myelin (Dupouey et al. 1979; Molineaux et al. 1986; Roach et al. 1985). These mice begin to exhibit behavioral differences from normal mice at around 14 days after birth, the time of onset of extensive myelination in the CNS, and exhibit neurological dysfunction characterized with the onset of shivering, tonic seizures, and early death (Chernoff 1981; Kirschner and Ganser 1980). Recently, shiverer mice have been widely used as a noninjury model in remyelination studies using transplantation of myelin-producing oligodendrocytes, their precursors derived from neural stem cells and other cell types such as olfactory ensheathing glia, all of which showed newly produced myelin identified by myelin basic protein (MBP) immunoreactivity (McDonald et al. 2000; Vignais et al. 1993; Vitry et al. 2003). However, the formation of myelin by transplanted cells does not in itself ensure impulse conduction and needs experimental verification. Axonal impulse conduction after cell transplantation depends not only on myelination but also on deployment of adequate numbers and types of ion channels in newly formed or remyelinated axons. Moreover, incomplete remyelination might be expected to lead to conduction failure because of impedance mismatch at junctions between myelinated and nonmyelinated axonal membrane regions (Utzschneider et al. 1994).

The molecular basis for the shi/shi mutation is well characterized and can be mimicked by antisense transgenic technology (Katsuki et al. 1988), and shiverer mutants have been partially rescued by transgenic gene therapy (Readhead and Muggleton 1991; Readhead et al. 1987). To date, however, there has been no quantitative assessment of the electrophysiological properties of dysmyelinated spinal cord axons of shiverer mice, which is essential in the assessment of the effectiveness of various treatment strategies designed to prevent or reverse neurological deficits. The goal of this study was to characterize the physiological properties of spinal cord axons of genetically dysmyelinated shiverer mice by in vitro sucrose gap electrophysiology and to use pharmacological, confocal immunohistochemistry, and Western blotting approaches to examine the role of the Kv1.1 and Kv1.2 K⁺ channel subunits in mediating these physiological changes.

Our results indicate that the abnormal conduction properties of shiverer mouse axons can be partially enhanced by the K⁺ channel blockers 4-AP and DTX-I. Moreover, these physiological effects were strongly associated with an altered distribution and expression of the K⁺ channel subunits Kv1.1 and Kv1.2 in dysmyelinated spinal cord axons of shiverer mice.

	METHODS

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All experimental protocols of this study were approved by the Animal Care Committee of the University Health Network in accordance with the policies established in the Guide to the Care and Use of Experimental Animals prepared by the Canadian Council of Animal Care. All efforts were made to minimize animal suffering and the number of animals used in the study. The mice were procured from Jackson Laboratories, USA.

Electrophysiological recording

ISOLATION OF MICE SPINAL CORD. The isolation of whole segments of the thoracic spinal cord and the electrophysiological recording methods were similar to procedures described earlier in our laboratory for mature rat spinal cord (Agrawal and Fehlings 1996; Eftekharpour et al. 2005; Fehlings and Nashmi 1996, 1997). Briefly, 10-wk-old female wild-type and shiverer mice were killed using an overdose of pentobarbital sodium. The mice were decapitated, and the T₁ to T₁₂ segments of the thoracic region were exposed. The exposed cord was isolated in cold (4°C) oxygenated (95% O₂-5% CO₂) artificial cerebrospinal fluid (ACSF; see composition below). The spinal cord was cleared of the meninges, separated from the exiting nerve roots, and allowed to stabilize at room temperature for 1 h in oxygenated ACSF before recording.

Sucrose gap recording

The sucrose gap technique allows for the recording of monophasic compound action potential (CAP) responses that closely resemble the shape of action potentials recorded intra-axonally and has the advantage over intra-axonal recordings in allowing stable recordings from small axons (1–3 µm diam) over several hours. The sucrose gap chamber (Department of Pharmacy, University of Toronto, Toronto, Canada) was comprised of three compartments (Fehlings and Nashmi 1997; Nashmi and Fehlings 2001b). The two outer compartments were perfused with oxygenated (95% O₂-5% CO₂) ACSF and isotonic KCl solutions, and the oxygenated (with 100% O₂ for pH considerations) isotonic sucrose solution flowed down the 1-mm-wide central compartment. The spinal cord traversed all three compartments through openings in the walls that were sealed with a plastic applicator and silicone grease. A bipolar platinum wire stimulating electrode, insulated by nail polish except for the undersurface contacting the cord, was positioned on top of the dorsal column of the spinal cord segment in the ACSF compartment, 5 mm from the sucrose gap. The temperature of the solutions was maintained at 33°C.

A constant current supramaximal stimulus of 100-µs duration (1.5 times the intensity that elicited a maximal response) was applied at 0.1 Hz from the SIU-PSIV6 stimulus isolation unit of a Grass S88 stimulator. Two agar bridge electrodes (Ag/AgCl wires in 0.5% agar prepared on 3 M NaCl or KCl) were placed in, respectively, the ACSF and KCl compartments and connected to the headstage of the amplifier (Axoprobe 1-A, Axon Instruments). The signals were amplified (100 times in AC mode and 10 times in DC mode), digitized at 12-bit resolution to 1,024 points, and stored and analyzed on a PC type computer using pClamp software (version 6.0, Axon Instruments).

EXPERIMENTAL PROTOCOL AND ANALYSIS. A baseline recording of 100 sweeps at 0.1 Hz was obtained. The conduction velocity was determined as described in our previous studies (Fehlings and Nashmi 1997; Nashmi and Fehlings 2001a). The latency was measured as the time from stimulus artifact to the peak of the CAP, and the conduction velocities were calculated by dividing the distance between the stimulating electrode and gap by the latency. Stimulus-response relationships were performed by varying stimulus intensity from 1 to 16 mA and measuring the amplitude of corresponding graded CAPs. This protocol allowed for comparison of the relative thresholds of activation of the population of conducting fibers in shiverer mouse spinal cords with those from wild-type mice. To examine the ability of the spinal cord axons to conduct signals at high frequency, the cord was stimulated with 500-ms-long, 100-pulse stimulus trains of 50-, 100-, 150-, 200-, 250-, and 300-Hz pulse frequency. The amplitude of last CAP was expressed as percentage of first CAP in the train.

POTASSIUM CHANNEL BLOCKERS. Drugs were bath-applied in the ACSF compartment, and the changes in the amplitude and area of the CAPs were assessed. The area was measured from the beginning of the CAP to 10 ms after the peak. The effects of 4-AP, a wide-range blocker of fast K⁺ channels, and of the more specific dendrotoxins DTX-I and DTX- K, which were used in this study to assess the role of Kv1.1 and Kv1.2 in axonal dysfunction of shiverer mice, were examined. DTX-I binds to both Kv1.1 and Kv1.2 subunits (IC₅₀ 0.13 and 3.1 nM, respectively), whereas DTX-K has a high selectivity for Kv1.1 subunits (EC₅₀ = 0.6 nM). After 20 min of baseline recording, the spinal cord was perfused with DTX-K (100 nM) for 20 min. After washout of the drug for another 15–20 min, dendrotoxin I (100 nM) was perfused for 20 min. At the end of the experiment, 4 AP (200 µM) was perfused for 20 min after washout of the DTX-I.

SOLUTIONS AND DRUGS. The ACSF was composed of (in mM) 124 NaCl, 3 KCl, 1 Na₂HPO₄, 26 NaHCO₃, 1.5 MgCl₂, 1.5 CaCl₂, and 10 glucose. The composition of isotonic KCl solution was (in mM) 7 NaCl, 120 KCl, 1 Na₂HPO₄, 26 NaHCO₃, 1.5 MgCl₂, 1.5 CaCl₂, and 10 glucose. The isotonic sucrose solution contained 300 mM sucrose. A stock solution of 4-AP (Fluka, Buchs, Switzerland) was made by dissolving 94.12 µg in 1 ml distilled water (concentration of stock 1 mM, pH 7.4) and dissolved in ACSF solution to 200 µM. Stock solution of DTX-I (Alomone Labs, Jerusalem, Israel) was made by dissolving 0.14 mg of DTX-I in 1 ml distilled H₂O (concentration of stock 20 µM, pH 7.5). DTX-K (Alomone Labs) was made by dissolving 70 µg of DTX-K in 1 ml distilled H₂O (concentration of stock 10 µM, pH 7.5). DTX-containing solutions were recycled once during the 20-min perfusion.

Histology and fluorescence immunohistochemistry

TISSUE PREPARATION. Animals were deeply anesthetized with an overdose of pentobarbital sodium and perfused transcardially with 4% paraformaldehyde (PFA) in 0.1 M PBS, pH 7.4. The T₅ to T₈ segments of the thoracic cord of wild-type and shiverer mice were dissected out and postfixed in the same PFA-PBS solution plus 10% sucrose overnight at 4°C. The tissues were cryoprotected in 20% sucrose in PBS for 24–48 h at 4°C. The tissue was embedded in HistoPrep (Fisher Scientific) on dry ice. Cryostat sections (10 µm) were cut and mounted onto gelatin-subbed slides.

LUXOL FAST BLUE/HEMATOXYLIN AND EOSIN STAINING. The sections were stained with Luxol fast blue (LFB) and counterstained with hematoxylin and eosin (H&E). The slides were examined under a bright field microscope (Nikon E800) using a 3CCD camera and Image Pro plus software (MediaCybernetics).

IMMUNOHISTOCHEMISTRY The procedures were similar to those described in recent publications from our laboratory (Eftekharpour et al. 2005; Karimi-Abdolrezaee et al. 2004). All incubations were performed at room temperature unless otherwise noted. Cryostat sections (10 µm) were cut longitudinally, mounted onto gelatin-subbed slides, and fixed for 30 min with ice-cold 4% PFA in PBS. Sections were blocked in PBS containing 1% bovine serum albumin (BSA), 5% nonfat dry milk, and 0.3% Triton-X for 1 h, and incubated with the following primary antibodies: polyclonal Kv1.1 (1:100), Kv1.2 (1:100) antibody (Alomone Labs; both 1:100), and mouse anti-MBP (Sternberger Monoclonal; 1:1,000) for myelin basic protein in the same blocking solution at 4°C overnight. The slides were washed three times (10 min each) in 1% BSA in PBS and treated with fluorescent Alexa Fluor 488 goat-anti rabbit secondary antibody (Molecular Probes; 1:400) for 1 h. For double labeling, the sections were incubated with NF200 monoclonal antibody (Sigma; 1:500) or panNa⁺ monoclonal antibody (Sigma; 1:50) in the preceding blocking solution for 1 h, and treated with fluorescent Alexa Fluor 594 goat anti-mouse secondary antibody (Molecular Probes; 1:400). The sections were washed in PBS (3 times, 10 min each) and coverslipped with Mowiol (Hoechst, Germany). The specificity of staining was confirmed by attenuation of immunostaining both by omitting the primary antibody and by competing the primary antibody with its corresponding peptide before incubation. The images were taken from dorsal or lateral columns using an LSM Zeiss 510 laser confocal microscope. Confocal images were scanned as one z stack with normal X-Y-Z dimensions with a voxel size of (0.11 x 0.11 x 1 µm).

WESTERN BLOT ANALYSIS. Animals were deeply anesthetized by halothane (1–2%) and a 1:1 mixture of O₂-N₂O and were decapitated. Spinal cord tissues prepared from thoracic segments of wild-type and shiverer mice were placed in ice-cold ACSF and separated from the meninges and nerve roots. One centimeter of spinal cord from thoracic area between T₅ and T₈ was dissected and homogenized in a protein homogenization buffer composed of 50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 0.1% SDS, 100 µM leupeptin, 1 µM pepstatin, 10 µg/ml aprotinin, and 100 µM phenylmethylsulfonyl fluoride, and centrifuged for 15 min at 560g at 4°C. Twenty micrograms of protein as determined by the Lowry protein assay was electrophoresed in a 7.5% polyacrylamide gel and electroblotted onto nitrocellulose membranes. The membranes were blocked in a solution containing 5% nonfat milk in Tween Tris-buffered saline (TTBS; 0.05% Tween 20 in 20 mM Tris-HCl, 250 mM NaCl, pH 7.4) and incubated with primary polyclonal antibodies against Kv1.1 (1:50) or Kv1.2 (1:100) (Alomone Labs) in the same blocking solution for 2 h at room temperature. The membranes were treated with a goat anti-rabbit horseradish peroxidase–conjugated secondary antibody for 1 h. Immunoreactivity was visualized using the Amersham ECL kit (Amersham, Little Chalfont, Buckinghamshire, UK). The membranes were reprobed with monoclonal antibodies against -actin (Chemicon) and treated with a goat-anti mouse horseradish peroxidase–conjugated secondary antibody for 1 h. For protein quantification, the bands were determined to be within the linear range of the radiographic film, and optical densities were determined using Gel Pro analysis software (Media Cybernetics, Silver Spring, MD). Actin was used to control for equal protein loading. Controls for Western blotting included a competition assay with incubation of the primary antibody with its target peptide and subsequent incubation on the membranes.

Statistical analyses

Throughout the paper, all data are reported as means ± SE. Significant differences were assessed by repeated-measures ANOVA and t-test for parametric data or Mann-Whitney rank sum tests for nonparametric data, not meeting normality assumptions (SigmaStat; SPSS statistical package). Post hoc comparisons were performed using Tukey's HSD test. To compare the effects of drugs on the CAP amplitudes and areas before and after drug administration in wild-type and shiverer mice white matter, paired t-tests were performed.

	RESULTS

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Characterization of axonal dysfunction in genetically dysmyelinated spinal cord axons

We used sucrose gap recordings to record axonal CAPs from the isolated thoracic spinal cord segments of wild-type or shiverer mice. We assessed changes in axonal function by determining the amplitude, conduction velocity, threshold, and ability to follow high-frequency trains of stimuli.

CAP AMPLITUDE AND CONDUCTION VELOCITY. Axonal conduction in spinal cord white matter of shiverer and wild-type mice was tested by recording CAPs (Fig. 1, A and B) evoked at a low frequency of stimulation (0.1 Hz). The CAP amplitude and conduction velocity are summarized in Fig. 1, C and D. The CAP amplitude was significantly smaller (P < 0.001, Mann-Whitney rank sum test) in shiverer mouse axons (1.58 ± 0.086 mV, n = 6) compared with wild-type axons (3.77 ± 0.504 mV, n = 8). Similarly, the axonal conduction velocity was significantly slower (P = <0.001, t-test) in shiverer mice (3.63 ± 0.232 m/s, n = 8) compared with wild-type mice (5.66 ± 0.313 m/s, n = 6).

View larger version (14K): [in this window] [in a new window]   FIG. 1. Compound action potentials (CAPs) recorded by sucrose gap technique from the isolated spinal cords of wild-type (A) and shiverer mice (B) at 33°C. Bar graphs of peak CAP amplitudes (C) and conduction velocities (D) in wild-type and shiverer mice cords (means ± SE; *P < 0.05 compared with wild-type). Amplitude and conduction velocity were significantly lower in shiverer mice compared with wild-type. Calibration pulses at the beginning of traces in A and B: 5 mV, 1 ms.

View larger version (18K): [in this window] [in a new window]   FIG. 2. Relationship between stimulus intensity and CAP amplitude in wild-type and shiverer mouse spinal cords. A: absolute values (mV). B: normalized values (taking maximal amplitude as 100%). Rightward shift of stimulus-response curves in shiverer mice indicates a significant reduction (P < 0.05) in excitability of shiverer mouse axons compared with wild-type spinal cord axons.

View larger version (16K): [in this window] [in a new window]   FIG. 3. Frequency dependence of CAPs in wild-type and shiverer mice. Cords were stimulated with trains of 100 stimuli at 200 Hz (A) and with trains of 100 stimuli and varying frequencies (50, 100, 150, 200, 250, and 300 Hz) (B). A: changes of CAP amplitude during the 200-Hz train, expressed as a percentage of the 1st CAP amplitude. Note a faster and greater decay of CAPs in shiverer mice spinal cord axons compared with wild-type axons. Each point represents mean of measurements in 5 separate experiments. B: frequency dependence of fatigue of CAPs at different train frequencies in wild-type and shiverer mice spinal cord axons. "Fatigue ratio" was quantified as amplitude of last CAP in the train expressed as percentage of the 1st CAP. Dysmyelinated axons of shiverer mice showed significantly higher susceptibility to frequency-dependent axonal conduction failure compared with those from wild-type mice at all frequencies between 50 and 300 Hz. Data are expressed as means ± SE.

Administration of 4-AP caused a striking increase in the amplitude and area of the CAPs in shiverer (Fig. 4B) but not wild-type (Fig. 4A) spinal cord axons. Quantitative analysis revealed that 4-AP (200 µM) produced a significant (P < 0.05) reversible increase in the CAP amplitude and area in shiverer mouse spinal cord axons (28.17 ± 10.26 and 40.14 ± 14.45%, respectively, n = 7) but not in wild-type (–2.98 ± 5.13, P = 0.65, and 10.01 ± 8.31%, P = 0.43, respectively, n = 3) spinal cord axons (Fig. 4, C and D).

View larger version (32K): [in this window] [in a new window]   FIG. 4. Representative recordings and quantitative analysis of the effects of potassium channel blockers 4-aminopyridine (4-AP, 200 µM), DTX-I (100 nM), and DTX-K (100 nM) on CAPs of wild-type and shiverer mouse spinal cords. Pharmacological agents were applied for 20 min. Averaged superimposed traces (n = 10, taken at 30-s intervals) from a representative experiment, before and after administering the blocker are shown. Note that the K+ channel blockers had minimal effect on average recordings from wild-type spinal cord axons. In contrast, 4-AP and DTX-I, but not DTX-K, caused striking increase in CAP amplitude in shiverer spinal cord axons. A, B, E, F, I, and J: effect of 4-AP, DTX-I, and DTX-K in wild-type and shiverer mouse spinal cords. Bar graph (C, D, G, H, K, and L) shows quantitative analysis of the effect of amplitude and area of CAPs in wild-type and shiverer mouse spinal cords. Data are normalized against predrug baseline values (100%). *Statistically significant differences for each pairwise comparison (P < 0.05).

Morphological and confocal immunohistochemical examination of the spinal cord of shiverer mice

We confirmed the absence of myelination in shiverer mice using LFB/H&E staining of spinal cord cross-sections. As shown in Fig. 5, A and B, in contrast to wild-type mice with normal myelination as identified by blue staining with LFB, shiverer mice had no detectable compact myelin in the spinal cord white matter. Using confocal fluorescence immunohistochemistry, we also confirmed the lack of MBP expression in the spinal cords of shiverer (Fig. 5, F–H) compared with wild-type mice (Fig. 5, C–E).

View larger version (105K): [in this window] [in a new window]   FIG. 5. Absence of myelin and myelin basic protein (MBP) staining in the spinal cord of shiverer mice. A and B: Luxol fast blue H&E staining of cross-sections of the thoracic cord of wild-type and shiverer mice. Note the absence of myelin staining (blue in A) in the shiverer cord (B). C–H: fluorescence immunohistochemistry for MBP in longitudinal sections of the spinal cord of wild-type (C–E) and shiverer (F–H) mice. Double labeling for the nuclear stain DAPI was performed to show cellular profiles. Figure 6, F–H, also confirms lack of MBP expression in shiverer mice.

View larger version (110K): [in this window] [in a new window]   FIG. 6. Distribution of Kv1.1 and Kv1.2 K+ channel subunits on spinal cord white matter axons of wild-type and shiverer mice is shown using confocal immunohistochemistry on longitudinal sections of the spinal cord. Localization of Kv1.1 and Kv1.2 (green, arrowheads) on dorsal column axons (identified by NF200 immunoreactivity red, arrowheads) is depicted. In wild-type mice, both Kv1.1 (A–C) and Kv1.2 (G–I) are confined to juxtaparanodal regions of axons. In contrast, shiverer mice show a clearly dispersed pattern of Kv1.1 (D–F) and Kv1.2 (J–L) distribution along internodes axons.

View larger version (90K): [in this window] [in a new window]   FIG. 7. Relative localization of Na+ channels (panNav) and Kv1.1 channel subunits in spinal cord white matter axons of wild-type (A–C and G–I) and shiverer (D–F and J–L) mice. Confocal immunohistochemistry on longitudinal sections of the spinal cord shows nodal regions of axons identified by panNav staining for Na+ channels (red, arrowheads). In contrast to wild-type spinal cord axons, shiverer mice show a dispersed immunostaining pattern of Kv1.1 (green, arrowheads) along length of axons while Na+ channels remain clustered in nodal regions. Bottom 2 rows show higher magnification images.

View larger version (95K): [in this window] [in a new window]   FIG. 8. Relative localization of Na+ channels (panNav) and Kv1.2 channel subunits in spinal cord white matter axons of wild-type (A–C and G–I) and shiverer (D–F and J–L) mice. Similar to the pattern for Kv1.1 in Fig. 7, Kv 1.2 in shiverer mouse spinal cord axons show a dispersed distribution along internodes (in contrast to juxtaparanodal localized in wild-type), whereas Na+ channels remained clustered at nodes. Bottom 2 rows show higher magnification images (green, arrowheads).

Next, we examined whether the increase in the distribution extent of Kv1.1 and Kv1.2 subunits of K⁺ channel on shiverer spinal cord axons was caused only by the redistribution of preexisting Kv1.1 and Kv1.2 subunits on axons or whether it is accompanied by the synthesis of new subunit proteins. To address this issue, we performed Western blot analysis to examine the expression of Kv1.1 and Kv1.2 subunit proteins in the thoracic spinal cord of wild-type and shiverer mice. The expression of Kv1.1 and Kv1.2 proteins was examined using affinity-purified polyclonal antibodies. Western blots showed immunoreactive bands for both Kv1.1 and Kv1.2 proteins at the molecular weight of 80 kDa (Fig. 9, A and D), which was in agreement with the manufacturer's product analysis (Alomone Labs) and previous reports (Karimi-Abdolrezaee et al. 2004; Sobko et al. 1998) The specificity of Kv1.1 and Kv1.2 antibodies was confirmed by peptide competition reactions with preadsorption of the antibodies with their corresponding antigens (Fig. 9, B and E). We normalized the expression of Kv1.1 and Kv1.2 proteins to -actin protein to adjust for variations in protein loading. Our quantitative Western blot analysis on the spinal cord of wild-type and shiverer mice showed an increase in expression of both Kv1.1 and Kv1.2 proteins in the spinal cord of shiverer mice. The expression of Kv1.1 and Kv1.2 proteins was elevated 2.6- and 2-fold, respectively, in the shiverer spinal cord compared with the wild-type littermate mice (n = 4/group). Our statistical analysis revealed that Kv1.1 protein expression was significantly increased in shiverer spinal cord compared with wild-type (P = 0.023). Although the elevation in expression of Kv1.2 protein displayed a similar trend as observed with Kv1.1 protein expression, these increases did not reach statistical significance (P = 0.065; Fig. 9, D–F).

View larger version (45K): [in this window] [in a new window]   FIG. 9. Changes in expression of Kv1.1 and Kv1.2 proteins in the spinal cord of shiverer mice. Representative Western blots for Kv1.1 (A–C) and Kv1.2 (D–F) show changes in immunostaining intensity of bands from wild-type to shiverer spinal cord. Western blots for Kv1. Immunostaining for actin was used as a control for protein loading. Specificity of Kv1.1 and Kv1.2 antibodies were confirmed by preincubation of antibodies with their corresponding peptide antigen. The 1st lane shows regular probing of spinal cord homogenates with the anti-Kv1.1 or anti-Kv1.2 antibody. The next lane shows probing of spinal cord homogenates with the antibody, which was preincubated with the target antigen peptide. Elevated expression of Kv1.1 protein (A–C) was significantly detectable in the spinal cord of shiverer mice compared with wild-type littermates (n = 4/group, *P = 0.023, Student's t-test). Each column and bar represent mean ± SE. For Kv1.2, although protein expression showed an increase with the same trend as observed for Kv1.1, it did not reach statistical significance (P = 0.065, Student's t-test). Each column and bar represent mean and SE of 4 independent animals.

	DISCUSSION

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To identify the specific functional roles of Kv1.1 and Kv1.2 K⁺ channel subunits in axons, we tested the effects of not only 4-AP, a wide-range blocker of fast potassium channels used in earlier studies on optic nerves of shiverer mice (Rasband et al. 1999b), but also of more selective blockers of the Kv1.1 and Kv1.2 K⁺ channel subunits. Our work showed that pharmacological blockade of Kv1.1 and Kv1.2 with DTX-I caused an increase in the amplitude and area of CAPs recorded from shiverer mice but not from wild-type controls. In contrast, more selective blockade of the Kv1.1 K⁺ channel subunits with DTX-K showed a similar trend although the changes in CAP area and amplitude were not significant, suggesting perhaps that Kv 1.1 and 1.2 acts in concert to mediate the physiological changes seen in myelin deficient spinal cord axons.

Axonal dysfunction in genetically dysmyelinated spinal cord axons in shiverer mice

Myelin is essential for the rapid conduction of electrical potentials in the nervous system, and its loss leads to impairment of neural function (Devaux and Scherer 2005). Membrane-associated galactolipid, (Coetzee et al. 1996; Ishibashi et al. 2002; Marcus and Popko 2002; Marcus et al. 2002) proteins include proteolipid protein (PLP) and MBP (Arroyo et al. 2002; Mathis et al. 2001), which are important molecular determinant of development of myelin sheath and axo-glial organization in the CNS. The mutation of the genes encoding these molecules results in abnormal neural function in rodents (Bhat et al. 2001; Boyle et al. 2001; Chernoff 1981; Jenkins and Bennett 2002; Macklin et al. 1987; Mathis et al. 2001; Nadon et al. 1990; Readhead et al. 1987) and humans (Fehlings and Nashmi 1996; Hudson 1990). The shiverer mice have an uncompacted CNS myelin lacking the major dense line caused by the absence of MBP (Baumann and Pham 2001; Privat et al. 1979; Rosenbluth 1980). Our electrophysiological data showing that dysmyelinated shiverer mice spinal cord axons display significant axonal dysfunction are in agreement with earlier studies in the optic nerve of the same mutant (Rasband et al. 1999b). The CAP is the algebraic summation of action potentials fired by individual axons. Axons with different diameters have different conduction velocities (Erlanger 1937) and thresholds to fire action potentials in response to stimulation (BeMent and Ranck 1969; West and Wolstencroft 1983). The lower amplitude and conduction velocity of CAPs in shiverer mice compared with wild-type may be caused by a smaller number of axons that are able to generate action potentials or to changes in the axonal size and spatiotemporal distribution of Na⁺ and K⁺ channels affecting the axonal excitability. The smaller amplitude of CAPs of spinal cord white matter is consistent with earlier studies with acute and chronic injury of the spinal cord (Nashmi and Fehlings 2001a,b; Shi et al. 1997). In agreement with our findings in shiverer mice, previous studies have reported a dramatic reduction in the conduction velocity of contactin mutant (Boyle et al. 2001) and galactosyltransferease (CGT)-deficient mice (Coetzee et al. 1996). These studies suggest that disruption of the paranodal junction might increase the duration of transient capacitive current and decrease the specific resistance through the paranodal junction. This decrease may result in radial shunting of internodal transmembrane current back through the paranode, thereby reducing the flow of longitudinal axoplasmic current to excite the downstream node. Moreover, our stimulus-response data show a higher threshold of activation, i.e., less excitability of dysmyelinated axons of shiverer mice compared with those from wild-type mice, which may also affect the conduction properties of the axons. This may be in part caused by the lower "safety factor" of action potential propagation.

An important functional difference of dysmyelinated axons found in our experiments is the impaired ability to conduct action potentials at higher frequencies. Axonal conduction failure during high-frequency stimulation has also been reported in demyelinating lesions in rats (Honmou et al. 1996; Mediratta and Nicoll 1983; Nashmi and Fehlings 2001a,b). In this study, we tested axonal conduction across a wide range of stimulus frequencies between 50 and 300 Hz. These experiments have revealed a markedly reduced ability of shiverer mouse axons to follow stimulation frequencies as low as 50 Hz. With increasing frequency of stimulation, the shiverer mice showed a significantly higher degree of reduction of action potentials compared with wild-type mice. This finding may have important implications in the understanding and interpretation of myelin-associated functional deficits in neurological disorders and neurotrauma.

Localization and expression of Kv1.1 and Kv1.2 K⁺ channel subunits on the spinal cord axons of shiverer mice

In normal myelinated axons, the nodal membrane contains a high-density of voltage-gated Na⁺ channels, which are linked to the spectrin cytoskeleton by ankyrin_G. The potassium channel subunits Kv1.1 and Kv1.2 are specifically located in the juxtaparanodal domain, whereas the paranodal membrane expresses the contactin associated protein Caspr/contactin complex in a highly localized manner (Coetzee et al. 1996; Karimi-Abdolrezaee et al. 2004; Peles and Salzer 2000; Poliak et al. 2003; Rios et al. 2000). The glial cell adhesion molecule, neurofascin155, is also present in the paranodal axo-glia junction, where it interacts with axonal proteins in close association with Caspr1 (Tait et al. 2000). Our immunohistochemical data confirm this spatial arrangement in myelinated axons of the white matter of wild-type mice spinal cord. In contrast to the localization of Kv1.1 and Kv1.2 subunits along the axons of wild-type mice, shiverer mice spinal white matter axons exhibited a profoundly diffuse expression pattern of Kv1.1. and Kv1.2 along the axonal membrane, in agreement with previous observations (Wang et al. 1995). The aberrant localization of axonal Kv1.1 and Kv1.2 channels has been also reported for other dysmyelinated mutants, the myelin-deficient rats (Arroyo et al. 2002) and Long Evans Shaker (LES) rats (Eftekharpour et al. 2005), as well as after spinal cord injury in rats (Karimi-Abdolrezaee et al. 2004; Nashmi et al. 2000). Interestingly, there was no apparent change in the highly confined localization of pan-Na⁺ channels in shiverer mice compared with wild-type mice. This observation differs from those reported for Nav1.2 expressing Na⁺ channels in the optic nerve of shiverer mice that showed that Na⁺ channels, excluding Nav1.6, which was present in low amounts, were expressed throughout the axonal membrane (Boiko et al. 2001). Westenbroek et al. (1992) also reported that expression of sodium channels, which are preferentially localized in unmyelinated fiber tracts, was elevated; however, no difference was observed in the localization of sodium channels, which are mainly present in neuronal cell bodies. In another study, it was observed that distribution of axolemmal sodium channels depends on the conformation of paranodal axoglial interaction (Noebels et al. 1991; Rosenbluth et al. 2003). We did observe, however, that panNa⁺ channels appeared in more than one cluster at the presumed nodal area of shiverer mouse spinal cord axons. Similar observations of node-like clusters of panNa⁺ channels, which were more numerous than in the wild-type, were reported in the ventral funiculi of myelin-deficient rats (Arroyo et al. 2002).

Our quantitative Western blot analysis showed an increase in expression of both Kv1.1 and Kv1.2 proteins in the spinal cord of shiverer mice. Our results are in agreement with previous observations by Wang et al. (1995) on shiverer mice and showed that Kv1.1 and Kv1.2 mRNA was up-regulated in different regions of the CNS including the spinal cord. Our data suggest that increased expression of Kv1.1 and Kv1.2 proteins on shiverer spinal cord axons may also contribute to disperse localization of these subunits on shiverer axons.

The altered distribution and increased expression of axonal ion channels is an important factor in the abnormal conduction properties of dysmyelinated axons (Boyle et al. 2001; Rasband et al. 1998). While the precise mechanisms of these changes remain to be elucidated, our study provides unequivocal evidence that the expression and redistribution of Kv1.1 and Kv1.2 channel subunits contributes substantially to the abnormal conduction properties of dysmyelinated axons in shiverer mice.

Functional role of Kv1.1 and Kv1.2 potassium channel subunits in abnormal axonal function in shiverer mice

The fast K⁺ channels that are involved in the repolarization phase of action potentials in myelinated axons are sensitive to 4-AP, produce a rapid outward rectification, and are believed to be localized in the myelinated portion of the axon (Blight 1989). The action potentials recorded from dysmyelinated spinal cord axons show significantly higher sensitivity to wide range blocker of fast K⁺ channels, 4-AP, than normal axons, which is consistent with the exposure of those K⁺ channels normally hidden under the myelin sheath (Chiu and Ritchie 1980). In this study, we showed that 4-AP and DTX I, a specific blocker of Kv1.1 and Kv1.2 subunits, significantly increased the area and amplitude of the CAP response in shiverer mouse spinal cord axons while having no effects on myelinated axons of wild-type mice. These results are in agreement with the idea that dysmyelination unmasks the normally hidden potassium channels that are largely silent in myelinated axons. Moreover, the redistribution of these K⁺ channels along the internodes of dysmyelinated axons likely also plays a role in mediating these effects. A potassium current leakage through these or other types of channels is likely to cause a conduction deficit or block by shunting currents gated by Na⁺ channels (Fehlings and Nashmi 1996; Jensen and Shi 2003; Nashmi and Fehlings 2001a). Blockade of voltage dependent fast K⁺ channels is also likely to increase the safety factor of spike propagation along the axon (Blight 1989).

The use of fast K⁺ channel blockers is an important tool to dissect the role of specific K channel types and subunits in the function of normally myelinated and dysmyelinated axons. This study, using specific blockers of Kv1.1 and Kv1.2 subunits, provides evidence that, while the combined blockade of Kv1.1 and Kv1.2 (with DTX-I) improves the axonal function, the selective blockade of Kv1.1 only (using DTX-K) has no significant effect. This suggests that Kv1.1 and Kv1.2 subunits in concert play a major role in the axonal excitability and conductive properties. DTX-K, which has higher affinity for Kv1.1, is not sufficient to overcome the abnormal conduction properties associated with dysmyelinated spinal cord axons. Previous studies (Hopkins 1998; Tytgat et al. 1995) have reported that potassium channels subunits have the ability to coassemble with members of the same molecularly defined subfamilies into heterotetramers. Therefore blocking of Kv1.1/ Kv1.2 heteromers K⁺ channels is a better strategy to improve axonal conduction deficits of dysmyelinated spinal cord axons. This conclusion is in agreement with our recent findings in another dysmyelinated model, the LES rat spinal cord white matter axons, and with earlier studies on the changes of axonal function after spinal cord injury (Eftekharpour et al. 2005; Nashmi and Fehlings 2001b).

In conclusion, this study showed that axonal conduction deficits in the spinal cord white matter of shiverer mice are associated with alteration of the axonal distribution and expression of the Kv1.1 and Kv1.2 K⁺ channel subunits. These conduction deficits have been improved by blocking Kv1.1 and Kv1.2 K⁺ channels. These results clarify some of the functional deficits that occur because of disorders related to loss of myelin or myelin deficiency and serve to further clarify the functional alterations of spinal cord axons in the shiverer mouse, which has become an increasingly important animal model to study demyelinating disorders.

	GRANTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by an operating grant from the Canadian Institutes of Health Research (CIHR-MIT14459). M. G. Fehlings is partially supported by the Krembil Chair in Neural Repair and Regeneration, and S. Karimi-Abdolrezaee receives financial support from an Ontario Neurotrauma Foundation Postdoctoral Fellowship.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Address for reprint requests and other correspondence: M. G. Fehlings, Div. of Neurosurgery, Univ. of Toronto, Toronto Western Hospital, Univ. Health Network, Rm. 4W-449, 399 Bathurst St., Toronto, Ontario M5T 2S8, Canada (E-mail: michael.fehlings{at}uhn.on.ca)

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