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The Journal of Neuroscience, January 15, 1999, 19(2):747-758
Dynamic Potassium Channel Distributions during Axonal Development
Prevent Aberrant Firing Patterns
Ian
Vabnick1,
James S.
Trimmer3,
Thomas L.
Schwarz4,
S. Rock
Levinson5,
Dipesh
Risal1, and
Peter
Shrager1, 2
Departments of 1 Biochemistry and Biophysics and
2 Neurobiology and Anatomy, University of Rochester Medical
Center, Rochester, New York 14642, 3 Department of
Biochemistry and Cell Biology, State University of New York, Stony
Brook, New York 11794, 4 Department of Molecular and
Cellular Physiology, Beckman Center, Stanford University, Stanford,
California 94305, and 5 Department of Physiology,
University of Colorado, Denver, Colorado 80262
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ABSTRACT |
The distribution and function of Shaker-related
K+ channels were studied with immunofluorescence and
electrophysiology in sciatic nerves of developing rats. At nodes of
Ranvier, Na+ channel clustering occurred very early
(postnatal days 1-3). Although K+ channels
were not yet segregated at most of these sites, they were directly
involved in action potential generation, reducing duration, and the
refractory period. At ~1 week, K+ channel clusters
were first seen but were within the nodal gap and in paranodes, and
only later (weeks 2-4) were they shifted to juxtaparanodal regions.
K+ channel function was most dramatic during this
transition period, with block producing repetitive firing in response
to single stimuli. As K+ channels were increasingly
sequestered in juxtaparanodes, conduction became progressively
insensitive to K+ channel block. Over the first 3 weeks, K+ channel clustering was often asymmetric,
with channels exclusively in the distal paranode in ~40% of cases. A
computational model suggested a mechanism for the firing patterns
observed, and the results provide a role for K+
channels in the prevention of aberrant excitation as myelination proceeds during development.
Key words:
potassium channels; node of Ranvier; Schwann cell; myelin; axons; development
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INTRODUCTION |
The function of myelinated axons is
dependent on a very heterogeneous distribution of ion channels, as well
as a complex interaction of glial and axonal elements. In all known
adult myelinated fibers, Na+ channels are localized
at high density within the nodal gap and are responsible for the
upstroke of the action potential. In amphibian axons, voltage-dependent
K+ channels are likely to be colocalized with
Na+ channels, because their outward currents are
recorded from nodes under voltage clamp and contribute to the
repolarization of action potentials (Dodge and Frankenhaeuser, 1958 ;
Schmidt and Stampfli, 1966; Hille, 1967). In normal mammalian PNS
fibers, on the other hand, voltage-dependent K+
currents are absent from nodes (Chiu et al., 1979), and their role in
axons has thus been unclear. Chiu and Ritchie (1981, 1982) found that
after acute disruption of myelin with lysolecithin, delayed rectifier
K+ currents appeared, suggesting a paranodal or
internodal origin. It was later demonstrated that heteromultimers of
the Shaker K+ channel  subunits Kv1.1 and Kv1.2
and the cytoplasmic Kv 2 subunit, cluster in
juxtaparanodal zones, internodal regions just beyond the paranodes
(Wang et al., 1993; Mi et al., 1995; Rhodes et al., 1997). Both
Na+ channels and delayed rectifier
K+ channels are also present throughout the
internode but at low density (Chiu and Ritchie, 1982; Shrager, 1987,
1988, 1989). Smart et al. (1998) recently demonstrated that genetic
deletion of Kv1.1 resulted in hyperexcitability in CNS axons, as well
as changes in the falling phase and refractory period of sciatic nerve
action potentials. Finally, beyond their axonal segregation,
K+ channels are differentially distributed within
neuronal somata, dendrites, and terminals (Sheng et al., 1992,
1994).
These various features of axonal structure do not appear simultaneously
during development. Passive electrical properties change as glial cells
adhere and ensheathe fibers, ultimately forming compact myelin with
mature paranodes. Na+ channels initially cluster in
the axolemma adjacent to the edges of myelinating Schwann cell
processes. As Schwann cell processes grow longitudinally,
Na+ channel clusters are reorganized into nodes of
Ranvier (Dugandzija-Novakovic et al., 1995; Vabnick et al., 1996).
Conduction velocity increases by up to two orders of magnitude during
this time (Ziskind-Conheim, 1988; Vabnick and Shrager, 1998).
Despite these rather extensive alterations in structure, action
potential propagation must be continually reliable and stable. Voltage-dependent K+ channels can strongly influence
conduction, even causing complete block if they are expressed in
inappropriate locations. After demyelination, juxtaparanodal
K+ channels are no longer electrically isolated.
Application of the K+ channel blocking drug
4-aminopyridine (4-AP), which is without effect on normal fibers,
significantly increases excitability, a finding of considerable
clinical interest (Sherratt et al., 1980; Polman and Hartung, 1995;
Schwid et al., 1997; Rasband et al., 1998). However,
K+ channel block restores conduction in some
demyelinated fibers but induces repetitive firing in others (Burchiel
and Russell, 1985; Baker and Bostock, 1992), perhaps by enabling
reentry depolarization of paranodal regions (Chiu and Ritchie, 1981,
1984). It is thus important to understand the relationship between
excitability and axonal K+ channel organization. We
find here that during the early postnatal period K+
channels are essential for stable axonal conduction, and they are
extensively redistributed to serve this function.
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MATERIALS AND METHODS |
Primary antibodies. Polyclonal anti-Kv1.1 antibodies
were raised in rabbits against the C-terminal sequence
EDMNNSIAHYRQANIRTG. The peptide was synthesized at the Beckman Center
(Stanford University, Stanford, CA) and coupled to porcine
thyroglobulin. Immunization, serum collection, and purification were as
described previously (Mi et al., 1995). Mouse monoclonal anti-Kv1.1
antibody K20/78 was produced by immunization with the rat C-terminal
peptide CEEDMNNSIAHYRQANIRTG (Quality Controlled Biochemicals,
Hopkintown, MA) conjugated to either keyhole limpet hemocyanin (KLH) or
bovine serum albumin (Bekele-Arcuri et al., 1996). Labeling patterns
with the polyclonal and monoclonal anti-Kv1.1 antibodies were
identical, and the choice was dependent on the antibody type used for
double labeling. The mouse monoclonal anti-Kv1.2 antibody K14/16
(Bekele-Arcuri et al., 1996; Shi et al., 1996) was produced by
immunization with a glutathione-S-transferase (GST) fusion
protein containing amino acids 428-499 of rat Kv1.2. K14/16 binds to a
synthetic peptide corresponding to amino acids 463-480 of Kv1.2. The
mouse monoclonal anti-Kv2 antibody K17/70 (Bekele-Arcuri
et al., 1996; Rhodes et al., 1996) was produced by immunization with a
GST fusion protein containing the entire 367 amino acid rat
Kv2 polypeptide. K17/70 binds to a synthetic peptide
corresponding to amino acids 1-17 of Kv2. Hybridomas
were grown in Balb/c mice for production of ascites fluid as described
previously (Trimmer et al., 1985). Immunoglobulins were purified by
ammonium sulfate precipitation, followed by DEAE chromatography
(Trimmer et al., 1985). Monoclonal antibodies against myelin associated
glycoprotein (MAG) were a kind gift of Dr. Melitta Schachner (Zentrum
fur Molekulare Neurobiologie, Hamburg, Germany) and were prepared by
immunization with affinity-purified glycoproteins carrying the L2
epitope from chicken brain (Poltorak et al., 1987). The IgG antibody
was obtained after fusion of the mouse myeloma clone P3X63Ag8.653, with
spleen cells from immunized mice. The polyclonal
anti-Na+ channel antibody was raised against a
highly conserved segment (TEEQKKYYNAMKKLGSKK) located between domains
III and IV in the vertebrate Na+ channel subunit. The peptide, with a C-terminal cysteine, was synthesized at
the University of Colorado Medical School institutional facility
(Denver, CO) and was conjugated to maleimide-activated KLH. Antibodies
were affinity purified using the immunizing peptide coupled to a
Sulfolinked column (Pierce, Rockford, IL).
Immunocytochemistry. Procedures were similar to those used
in earlier studies on developing and remyelinating nerve (Vabnick et
al., 1996; Rasband et al., 1998). The animal was killed, and sciatic
nerves and/or dorsal and ventral roots were dissected, desheathed, and
dissociated in collagenase (3 mg/ml) for 10-20 min at room temperature
(RT). One to 3 mm sections of the tibial branch were teased and
attached to coverslips precoated with spots of Cell Tac (Collaborative
Research, Bedford, MA). The tissue was fixed in 4% paraformaldehyde,
pH 7.2, for 30 min at RT, washed in 0.05 M phosphate buffer
(PB), pH 7.4, and air dried. Alternatively, in some experiments animals
were deeply anesthetized and perfused with 4% paraformaldehyde in 0.1 M PB. The sciatic nerve was then dissected and post-fixed
in 4% paraformaldehyde for 4 hr at 4°C, rinsed in 0.1 M
PB for 10 min, incubated overnight in 20% sucrose, and again in 30%
sucrose overnight. The nerve was then frozen in OCT mounting medium
(Miller, Inc.) and cut in 30-µm-thick sections. These sections were
then spread on gelatin-coated slides, fixed again for 10 min in 4%
paraformaldehyde, rinsed in 0.05 M PB, and allowed to dry.
Both teased axons and cryosections were exposed to a solution
containing 45 ml of 0.1 M PB, 150 µl of Triton X-100, and
5 ml of goat serum (PBTGS) for 2 hr. All subsequent solutions used the
PBTGS mixture for dilutions or washing. Primary antibodies were applied
for 15 hr at RT. For rabbit polyclonal antisera, the secondary labeling
consisted of biotinylated goat anti-rabbit Fab2' (1:200, 1 hr), followed by Extravidin-fluorescein isothiocyanate (1:200, 1 hr). For monoclonal antibodies, the fluorescent ligand was goat
anti-mouse tetramethyl rhodamine isothiocyanate. In most experiments, axons were double labeled by applying one primary antibody
and its secondary antibody, followed by the second series. Preparations
were washed three times with PBTGS after each reagent. After
immunolabeling, coverslips were washed sequentially in PBTGS, 0.1 M PB, and 0.05 M PB for 5 min each. Tissue
preparations were air dried and mounted on slides for visualization
under a Nikon Microphot-SA fluorescence microscope fitted with a Dage
MTI (Michigan City, IN) SIT 68 camera. Images were captured and stored
in a computer for later analysis. Preabsorption of
Na+ and Kv1.1 antibodies with peptide antigens
eliminated immunolabel (Dugandzija-Novakovic et al., 1995; Rasband et
al., 1998).
We required criteria for designating K+ channel
immunoreactivity as nodal, paranodal, or juxtaparanodal. The nodal gap
was readily identified by either Na+ channel
labeling or MAG staining of paranodes. The point at which the paranodal
loops end and the juxtaparanodes begin cannot be determined at the
light level in immature fibers. We used published data to estimate
paranodal lengths of 4-8 µm in the adult and 2-4 µm in early
development (Allt, 1969; Tao-Cheng and Rosenbluth, 1982, 1983; Yamamoto
et al., 1996). K+ channel immunoreactivity was
considered paranodal if it was <5 µm from a node. In adult fibers,
the axon diameter increases sharply to the internodal value at the
border of the paranode, and label within this expanded zone (and >2
µm from the node) was thus considered to be juxtaparanodal. By the
end of the first postnatal week, MAG is expressed at elevated levels in
the terminal loops and thus demarcates the paranodes. Double labeling
with anti-MAG antibodies beyond postnatal day 6 (P6) confirmed the
validity of the above criteria and was also used in many experiments as
an additional determinant of localization. In cases in which
K+ channel immunoreactivity was present in two
regions, i.e., nodal/paranodal or paranodal/juxtaparanodal, the site
was included in both categories. Throughout the paper, the term
"site" refers to nodal regions, including the nodal gap, paranodes,
and juxtaparanodes.
Suction electrode recording. Rat sciatic nerves were
dissected and desheathed as above and placed in Locke's solution
containing (in mM): NaCl 154, KCl 5.6, CaCl2 2, D-glucose 5.6, and HEPES 10, pH 7.4. The floor of the nerve
chamber was constructed from glass coated on the outside surface with
indium tin oxide, allowing heating with an electric current. This
provided a highly uniform bath temperature without the need for
mechanical agitation. A thermistor and feedback electronics controlled
the temperature to ±0.5°C. Each end of the nerve was drawn into a
glass capillary electrode with a constricted orifice. Brief (50 µsec)
stimulating pulses were applied to the proximal (toward spinal cord)
electrode, and the compound action potential was recorded from the
distal end. With the stimulus parameters then set, the distal end was removed from its electrode and dissociated in collagenase (3 mg/ml) for
5-30 min until the tips of individual fibers could be visualized. This
enzymatic procedure has been shown not to affect compound action
potentials (Shrager, 1987). A single axon was then drawn into a broken
microelectrode (2-15 µm inner diameter) for a distance of
25-100 µm. After a stimulus, signals were recorded with an Axopatch
200A amplifier (Axon Instruments, Foster City, CA) in current-clamp mode (with steady-state current set to zero) and then
passed through an alternating current amplifier (Warner DP301). Amplitude corrections for much of the variation in seal resistance (Rseal) at the recording pipette
were made by the method of Stys et al. (1991), except for a few cases
in which Rseal was <1 M . The primary
information, however, is in the shape and timing of signals. Action
potentials were digitized and stored in a computer for later analysis.
The influence of K+ channels was tested by applying
one of several blocking drugs. Tetraethylammonium ion (TEA) (10 mM) or dendrotoxin I (DTX-I) (100 nM;
Calbiochem, San Diego, CA) were typically applied first, because
effects were readily reversed on washing. 4-AP (1 mM) was
then added. In a few experiments, washout of 4-AP was attempted, with
partial or complete reversal of effects over ~45 min. Responses after
application of these drugs were observed for at least 10 min to ensure
that the effect was maximal.
Computational model. Using the Neuron modeling computer
program (www.neuron.yale.edu/neuron) (Hines and Carnevale, 1997), we
designed an equivalent circuit for a P16 axon. The fiber
included 21 nodes and 20 internodes. Nodal, paranodal, juxtaparanodal, and internodal zones were defined as shown in the sketch within Table
1. Morphological and electrical
parameters were obtained from several published sources and are listed
in Table 1. The number of myelin lamellae covering paranodal segments
was decreased linearly toward the node, corresponding to the geometry
of the terminal loops. The length of the paranode was calculated by
multiplying the number of myelin layers by a terminal loop periodicity
of 0.15 µm (Rosenbluth, 1988). K+ channel
permeabilities were estimated by taking the ratio of peak
K+ conductance to peak Na+
conductance after paranodal retraction (Chiu and Ritchie, 1981) and
multiplying by the nodal Na+ channel permeability.
The ratio of internodal to maximal paranodal or juxtaparanodal
K+ channel permeability per unit area is close to
the value estimated by Roper and Schwartz (1989). To mimic the
maturation of the paranodal axoglial junctions (Tao-Cheng and
Rosenbluth, 1983; Yamamoto et al., 1996), we constricted the axoglial
gap linearly starting at 20 Å nearest the node and increasing to
90-120 Å at the paranodal/juxtaparanodal border (Table 1, sketch).
K+ channels were excluded from paranodal regions
with a gap of 20 Å, i.e., zones of mature junctions. The axon was
stimulated with a pulse of current (100 nA, 50 µsec) at the first
node of Ranvier. The last internode was placed inside a simulated
suction electrode with a Rseal of 10 M. The
extracellular voltage was calculated at the midpoint of this
internode.
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RESULTS |
K+ channel localization during early
postnatal development
At birth, rat sciatic axons had almost no myelin, and neither
Na+ nor K+ channels were detected
by immunocytochemistry. As will be shown later, conduction in these
nerves is slow and is likely to be mediated by channels diffusely
distributed below immunodetectable levels. Over the first few days,
myelination proceeded rapidly. Clusters of Na+
channels were readily detected at gaps between Schwann cell processes, but Kv1.2 and Kv1.1 immunoreactivity was absent in 90% of these sites
(Fig.
1A,B,
respectively). The Na+ channel clusters at this
stage were either binary (Fig. 1A) or uniform and
broad (Fig. 1B), typical of early node formation
(Vabnick et al., 1996). By P6-P9, nodal Na+ channel
clusters were more focal, but in 80% of these cases, K+ channels remained undetectable (Fig.
1C). However, K+ channel immunoreactivity
was seen in the other 20% of fibers at this stage, usually within
paranodes (Fig. 1D). Thus, Na+ and
K+ channels differ both temporally and spatially in
clustering during early development. The segregation of
Na+ channels precedes that of K+
channels and advances to a secondary stage before the density of Kv1.1
or Kv1.2 channels is detectable.
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Figure 1.
The distribution of K+ channels
in developing axons. All preparations were double stained with
antibodies to K+ (red) and
Na+ (A-I, green)
channels or MAG (J, green).
A, B, Lack of
K+ channel immunoreactivity (A,
Kv1.2; B, Kv1.1) at P3. Na+ channel
clusters were either binary (A) or broad
(B). C, Focal
Na+ channel cluster with no detectable Kv1.1
channels at P7. D, Diffuse Kv1.1 channel
immunoreactivity within paranodes, adjacent to Na+
channel clusters (P9). E, Asymmetric Kv1.2 localization
predominantly in the distal paranode at P13. F,
Juxtaparanodal Kv1.1 immunoreactivity at P61. Note sharp increase in
axonal diameter in this region. G, H,
K+ channel clustering within the nodal gap. Two
axons double labeled for Kv1.2 (G) and
Na+ (H) channels. In
the bottom fiber, a node (arrowhead) is
characterized by colocalization of these channels. The distal paranode
at this site is also immunopositive for Kv1.2, although separated from
the nodal cluster by a small gap. In the top axon, only
Na+ channel immunoreactivity is strong.
I, Organization of Kv1.2 channels in transverse bands
resembling helices at P21. J, Another site with
K+ channels (Kv1.1) in transverse bands double
labeled for MAG (green). The partial overlap
(yellow) indicates that these channels are both
paranodal and juxtaparanodal (P14). Scale bars, 10 µm.
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By the second postnatal week, >50% of sites had intense Kv1.1 and
Kv1.2 immunoreactivity within paranodal regions. However, almost half
of the K+ channel-immunopositive locations were
labeled asymmetrically. Notably, at these sites, the distal paranode
was invariably the one with K+ channels. Figure
1E illustrates this organization for Kv1.2 at P13.
This pattern was transient, and by the end of the third postnatal week,
K+ channel labeling had the symmetry characteristic
of adult fibers. (As will be shown shortly, Kv1.1 and Kv1.2 were
colocalized at all stages, and they are thus discussed interchangeably
here.) K+ channel immunofluorescence shifted
gradually toward the internode over the next several weeks. At this
stage of development, there was a mixed population of
Kv1.1-immunopositive sites in which at some locations labeling extended
across both paranodes and juxtaparanodes, whereas at others, channels
were exclusively juxtaparanodal. Eventually, the majority of sites
contained K+ channels segregated only within the
juxtaparanodal zones, as can be seen in Figure 1F at P61.
Between P6 and P8, Kv1.1 and Kv1.2 immunofluorescence was present
within the nodal gap at ~20% of sites, although typically at low intensity. Figure 1, G and H, illustrates
nodes of Ranvier in two axons at P7, labeled with antibodies against
Kv1.2 (G) and Na+
(H) channels. It can be seen that one of these sites
(arrowheads) included a Kv1.2 cluster (G)
that colocalized with Na+ channels
(H). The distal paranode at this site
(G) had diffuse K+ channel
labeling that appeared to be separated from the nodal cluster by a
small gap. In the second node of Ranvier seen in Figure 1, G
and H, Kv1.2 label was not detected, and as noted above,
this was true in the majority of cases. Nodal Kv1.1 and Kv1.2
distributions disappeared rapidly and were virtually absent by the end
of the first postnatal week.
At early stages, K+ channels were frequently found
organized in several transverse bands or spirals. Figure
1I illustrates one such region, in a P21 axon. MAG is
expressed by Schwann cells after they reach an overlapping (~1.5
wraps) ensheathement, indicating a commitment to myelination (Martini
and Schachner, 1986). MAG is initially rather uniformly distributed
over the Schwann cell surface, but as myelination proceeds, this
molecule is increasingly sequestered within terminal loops and other
cytoplasm-containing regions. MAG labeling thus helps in defining the
extent of the paranodes. In Figure 1J, the region of
overlap of Kv1.1 with MAG (yellow) shows that the K+
channel bands were within these latter zones, associated with the
paranodal termini furthest from the node. In this example K+ channel immunoreactivity extended beyond the
MAG-positive zone into the juxtaparanode. The banded-spiral pattern of
Kv1.1 and Kv1.2 channels was reminiscent of the regularly spaced
terminal Schwann cell loops (Rosenbluth, 1988). However, the width of
the K+ channel bands was typically ~0.4 µm, and
the periodicity was ~1.2 µm. Mature terminal loops are 0.1-0.2
µm in width and are usually tightly packed (Rosenbluth, 1988). On the
other hand, lakes of intramembranous particles up to 0.4 µm in width
have been seen in freeze-fracture of paranodal regions in ~40% of
spinal roots (Fields et al., 1986). At later stages of development, as K+ channels became increasingly sequestered at
juxtaparanodal zones, there was minimal overlap between
K+ channels and MAG, and the banded appearance
became very rare.
Kv1.1 and Kv1.2 subtypes were colocalized in most axons at all stages
of development studied. Figure 2,
A and B, illustrates a node of Ranvier at P13
with overlapping clusters of Kv1.2 and Kv1.1 subunits, respectively, at
the distal paranode. A node at P40 at which the juxtaparanodal regions
were immunopositive for both channels is shown in Figure 2,
C and D. Even at the earliest times of detection
of K+ channel clustering (P6-P9), Kv1.1 and Kv1.2
were almost invariably coexpressed in paranodal regions. The
K+ channel subunit Kv2 promotes
surface expression and colocalizes with Shaker-type subunits in rat
brain juxtaparanodal regions (Shi et al., 1996; Rhodes et al., 1997).
This association is present in developing sciatic nodes of Ranvier
also. Figure 2, E and F, illustrates a node of
Ranvier at P13, with asymmetric but overlapping paranodal staining of
Kv2 (E) and Kv1.1 (F). When both
antigens tested (Kv1.1/Kv1.2 or Kv1.1/Kv2) were
immunopositive at a particular region, the overlap was typically very
strong, as in the above examples. At a small number of sites, however,
fluorescence from one of the two antibodies tested was absent. It could
not be determined whether these locations truly lacked the tested
subunit or whether there was a technical failure of the antibody
labeling. In remyelinating axons, ~15% of sites were positive for
Kv1.1 and negative for Kv1.2 (Rasband et al., 1998), but in the
developing fibers the immunonegative subunit varied.
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Figure 2.
Colocalization of K+ channel
subunits at developing paranodes and juxtaparanodes. A,
A node with an asymmetric distribution of Kv1.2 subunits at P13.
B, Colocalization of Kv1.1 at the same site as in
A. C, D, Juxtaparanodal
immunoreactivity of Kv1.2 (C) and Kv1.1
(D) at P40. E, Kv2
subunits at the distal side of a node at P13. F, The
site in E double labeled for Kv1.1 subunits. Scale
bars, 10 µm.
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The frequency of occurrence of several K+ channel
patterns is plotted in Figure
3A. Because Kv1.1 and Kv1.2
immunofluorescence was the same at all stages examined, only results
for the former are shown. The classification included (1) sites with no
detectable Kv1.1 signal ( ); (2) nodes at which Kv1.1 and
Na+ channel immunofluorescence overlapped, with or
without additional paranodal label ( ); (3) paranodal regions,
defined as Kv1.1 label adjacent to a node but not present within it
( ); and (4) juxtaparanodal zones, defined by Kv1.1 immunoreactivity
>2 µm from nodal Na+ channels ( ). Despite our
attempts at defining criteria for classification, it was not possible
to distinguish between paranodal and juxtaparanodal label in many
cases. During P23-P61, a significant percentage of sites had
K+ channel immunoreactivity that appeared to extend
through both regions, and these were counted in both groups (see
Materials and Methods). Most features of these curves were measurable
with accuracy; the major uncertainty is in the timing of the initial appearance of juxtaparanodal channels, which may be shifted earlier. Figure 3A shows that at birth Kv1.1 was undetectable.
The earliest appearance of this channel in clusters was within the
nodal gap, and that configuration represented ~20% of the sites
examined by P6. However, this localization was highly transient, and
the percentage of sites with nodal K+ channels
dropped nearly to zero after P9. Approximately 90% of these sites had
associated paranodal immunofluorescence. The rate of appearance of
paranodal channels rose rapidly during the second postnatal week,
reaching a maximum frequency of ~75%. The percentage of sites with
juxtaparanodal Kv1.1 clusters increased steadily over weeks 2-4, and
this became the predominant adult form. The behavior of the paranodal
and juxtaparanodal data suggest that sites with the former localization
progress to the latter. The question of whether K+
channels begin within the nodal gap at all sites is addressed in
Discussion. Up to the maximum age examined, ~10% of sites remained immunonegative for Kv1.1 or Kv1.2. We found that during development K+ channel clusters were not detected in axons <2
µm in diameter.
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Figure 3.
The variation in K+ channel
distribution and influence on electrical signals during development.
A, Kv1.1 patterns. Sites were tallied by comparing loci
of K+ and Na+ channel
immunoreactivity and placed in the following categories: sites with
undetectable Kv1.1 (); Kv1.1 colocalized with nodal
Na+ channel clusters (); paranodal Kv1.1 (label
adjacent to but not overlapping Na+ channel) ();
and juxtaparanodal Kv1.1 immunofluorescence, >2 µm from a node
(). B, Sensitivity to block as a measure of
K+ channel function at different developmental
stages. Changes in single fiber waveforms at RT on introduction of 4-AP
were classified as the following: monophasic (); repetitive firing
(); and biphasic as in the absence of drug (). Results were
grouped as noted on the abscissa. Recordings were made from a total of
97 fibers, with the numbers per group indicated below the abscissa.
Because the number of fibers tested in each individual experiment was
small (often only 1), error bars for percentages could not be
calculated. C, Similar to B but at
37°C. D, Calculated levels of different
K+ channel distributions for a simple kinetic
scheme: none  nodal paranodal juxtaparanodal. The rate
constants for the three irreversible steps used in this fit were 0.15, 0.70, and 0.04 d 1, respectively. In this plot, the
four categories are mutually exclusive. If we allowed for sites with
either nodal/paranodal or paranodal/juxtaparanodal overlapping
immunoreactivity, then the paranodal curve would be effectively higher
in amplitude, and the fit to the data in A would be
improved.
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The frequency of occurrence of sites with K+
channels detected only at the distal paranode during development is
plotted in Figure 4, . Also shown is
the frequency of symmetrical labeling (). The data suggest that at
about half of the developing nodal regions K+
channels cluster preferentially at the distal paranode during the
second postnatal week. However, this situation is transient, and over
the succeeding week paranodes become symmetrically labeled at all such
sites. A possible functional significance of the asymmetric expression
is explored later using a computational model. During development,
dorsal and ventral spinal roots respond differently to pharmacological
block of K+ channels (Bowe et al., 1985). However,
we found no significant differences in Kv1.1/Kv1.2 organization among
different roots (data not shown; examined at P8, P12, and P19). In
contrast to the sciatic nerve, very few sites in spinal roots were
labeled preferentially at one paranode at any stage examined.
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Figure 4.
Asymmetrical distribution of K+
channels. The fraction of sites with Kv1.1 either symmetrically
distributed () or present exclusively in the distal paranode and
juxtaparanode () are plotted versus postnatal day.
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Single fiber recording as a test for K+
channel function
Longitudinal currents were measured from single axons at all
stages of postnatal development. Even small premyelinated fibers at P0
could be drawn into suction electrodes with minimal mechanical stress.
All-or-none action potentials were recorded before and after adding
blocking drugs as a test of K+ channel participation
in signal generation and propagation. Over the first week, during early
node formation and Na+ channel clustering, 4-AP had
small but significant effects, slowing the falling phase of action
potentials. This is evident in Figure 5
in which the biphasic undershoot present in the control trace (A) is absent after introduction of the drug
(B). This change in shape was observed in ~90% of
fibers from P0 through P8. This result indicates that although
Kv1.1/Kv1.2 subunits are not detectable by immunocytochemistry at this
stage, K+ channels (possibly including non-Shaker
types) are present in the axolemma at low density and function
in action potential generation. During the second and third postnatal
week, 4-AP induced more significant changes in the action potential
waveform. After a single stimulus, axons fired repetitively, as shown
at P20 in Figure 5D. The secondary responses were more
prominent at lower temperatures, and the exact pattern in each fiber
varied somewhat with successive stimuli. These multiple spikes were
never seen in the absence of 4-AP. Spiking waveforms after
K+ channel block at this stage of development have
been reported also by Kocsis et al. (1983). The bursting behavior
remained through P41, but by P62, 4-AP had only small effects, as may
be seen in Figure 5, E and F.
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Figure 5.
Sensitivity of propagating action potentials to
K+ channel block. Traces were derived
from single fibers drawn into suction electrodes. Records are shown
before (A, C, E) and after
(B, D, F)
introduction of 1 mM 4-AP in the bath. At P0, a typically
biphasic recording (A) became monophasic, with
loss of the negative phase indicating a slower repolarization in the
presence of the drug (B). At P20, 4-AP induced
repetitive firing in response to a single stimulus (C,
D). There is relatively little change in waveform at
P62, and the trace remained biphasic, indicative of
rapid repolarization (E, F).
Stimulus artifacts have been blanked. The time of stimulation is
indicated by the start of the gap in each record. All records are at
37°C.
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The electrical responses after exposure to 4-AP were divided in three
categories: monophasic, repetitive firing, and biphasic, as represented
by the traces in Figure 5, B, D,
and F, respectively. The frequency of occurrence of each of
these is plotted as a function of age in Figure 3, B (at RT)
and C (at 37°C). These results are now compared with those
for K+ channel clustering. Monophasic recordings
() represent over 90% of axons tested over the first postnatal week
but then fall off rapidly. This coincides closely with the curve for
axons with no detectable K+ channel clusters in
Figure 3A, . The spiking response was transient and
corresponds most closely to the localization of K+
channels in paranodes (Fig. 3A, ). Traces that remained
biphasic in 4-AP rose in frequency over the period studied and thus
corresponded most closely to the data for juxtaparanodal localization
in Figure 3A, . This comparison of electrophysiological
results with immunocytochemistry suggests that voltage-dependent
K+ channels play important roles in signaling during
early development.
The refractory period represents another important determinant of
axonal function. As a measure of this property, three stimuli at
equally spaced intervals were applied. The interstimulus interval was
progressively shortened until the second response was blocked and could
no longer be recovered, even with an increase in the stimulus
amplitude. This is illustrated for a P6 axon in Figure 6, A-D. In normal Locke's
solution, three stimuli at intervals of 2 msec result in three action
potentials (Fig. 6A). When the interval is reduced to
1 msec, the second action potential is missing (Fig.
6B). After exposure to 1 mM 4-AP, the
refractory period is much longer. At an interval of 8.5 msec, all
spikes are present (Fig. 6C), but at 8 msec, the fiber is
refractory (Fig. 6D). K+ channel
block thus increased the refractory period for this axon from <2 to 8 msec. The increase in refractory period with K+
channel block was strong over the first postnatal week and then declined, disappearing at ~6 weeks. This could not, however, be quantitated rigorously, because over weeks 3-5 repetitive firing in
the presence of 4-AP introduced uncertainties in the measurement of the
refractory period. Multiple spikes were often present in the first
response only, even when the primary action potential was generated in
response to all three stimuli (Fig. 6E).
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Figure 6.
Measurement of the refractory period. In each
record, three stimuli were applied at equal intervals
( t). The tic marks below each
sweep denote the times of stimulation.
A-D, Signals from a P6 fiber. A,
Locke's solution, t = 2 msec. B,
Locke's solution, t = 1 msec. C,
One millimolar 4-AP, t = 8.5 msec.
D, One millimolar 4-AP, t = 8 msec. E, P16 axon, 1 mM 4-AP,
t = 14 msec. Temperature for all
traces was 37°C.
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DTX-I has a more selective spectrum of targets than does 4-AP and
blocks mouse Kv1.1, Kv1.2, and Kv1.6 channels selectively with
IC50 values of 3.1, 0.13, and 9 nM,
respectively (Swanson et al., 1990; Hopkins et al., 1994). At P0 and
P1, four of six fibers tested were sensitive to DTX-I (100 nM). The records of Figure
7A came from one such axon and
suggest a significant and reversible slowing of the falling phase of
action potentials by this drug. Neither of two fibers at P6 and P7
exposed to 100 nM DTX-I were affected. Subsequent to
addition of DTX-I, all of these axons were found to be sensitive to
4-AP. By P6-P7, access of the peptide toxin to paranodal
channels may be restricted. TEA is a much more potent blocker of
homomultimeric Kv1.1 channels (IC50 value of 0.3 mM) than Kv1.2 channels (IC50 values of
107-560 mM) (Grissmer et al., 1994; Hopkins et al., 1994).
Heteromultimers are blocked with an IC50 value of ~10
mM (Hopkins et al., 1994). Only 3 of 24 fibers tested over
P0-P61 had a significant response to this drug when applied at a
concentration of 10 mM. In all other cases, the action
potential waveform was only minimally affected (Fig. 7B).
This result is consistent with the low sensitivity of internodal
delayed rectifier K+ currents to externally applied
TEA (Chiu and Ritchie, 1981; Shrager, 1987).
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Figure 7.
Response of early developing fibers to the
K+ channel blocking drugs DTX-I and TEA.
A, One hundred nanomolar DTX-I added to the bath
inverted the negative phase of the extracellular recording, suggesting
a widening or plateau in the action potential. The change was
reversible on washing (P0). B, Lack of response of a P1
axon to 10 mM TEA added to the Locke's solution. In this
experiment, the Rseal was inadequate for
normalization of amplitudes.
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Computational models of developing nerves
A computational model of a P16 axon was developed that reproduced
as many details of morphology and ion channel distribution as are known
and can be quantified (see Materials and Methods). This system was then
used to test the role of K+ channels in several
physiological responses. In Figure
8A, the sweep on the left was calculated for an axon with
paranodal/juxtaparanodal K+ channels distributed
symmetrically about each developing node. In the trace on
the right, all K+ channels were removed,
simulating a response in 4-AP. There were multiple depolarizations in
response to a single stimulus, comparable to records in Figure 5,
C and D. The sweeps in Figure
8A were calculated as extracellular potentials within
a suction electrode to allow a comparison with experimental results.
The model allowed a calculation of membrane potential as well, and this
is plotted in Figure 8B for a node just outside the
suction electrode, under the same conditions as in Figure
8A. With K+ channels removed
(right), there were two prominent peaks in the response. It
was of interest that the relatively small secondary spikes detected by
the extracellular electrode can represent large propagating signals
outside the pipette. The low density of internodal Na+ channels normally produced a current that was
delayed relative to the peak nodal Na+ current.
Removal of these internodal Na+ channels eliminated
the repetitive firing (data not shown). The calculations thus suggest
that the paranodal/juxtaparanodal K+ channels
normally act to suppress a reentry depolarization mediated by the
internodal Na+ channels in these immature
fibers.
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Figure 8.
Computed action potentials as a test of
K+ channel function. A,
Left, A calculated propagating action potential in a P16
axon as recorded by the simulated suction electrode.
Right, After removal of the paranodal/juxtaparanodal
K+ channel clusters. B, Computed
transmembrane potentials at a node outside the electrode for the same
conditions as in A. C, Calculated suction
electrode response to three stimuli applied at 7 msec intervals;
K+ channels blocked. D,
Left, Computed transmembrane voltage with either
proximal or distal paranode/juxtaparanode depleted of clustered
K+ channels. Right, After doubling
the K+ channel density on the populated side.
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The model also reproduced several aspects of links between
K+ channels and the frequency dependence of
developing fibers. In Figure 8C, three stimuli were applied
to a model axon with no paranodal/juxtaparanodal K+
channels, and extra spikes were present only in response to the first
stimulus, a result similar to that seen in Figure 6E
with 4-AP. The computed trace did, however, include a delay in the response to the second stimulus that was not seen in the experiment. Again, the internodal Na+ channels are implicated in
the repetitive firing, because inward current through these channels
was much reduced in the second and third responses. The internodal
channels remained inactivated, presumably because of long
charging and discharging times of the internodal axolemma (Barrett and
Barrett, 1982). It should be noted that the spatial separation of nodal
and internodal Na+ channels also had a significant
effect on bursting behavior. Decreasing the size of the juxtaparanode
or paranode resulted in the loss of the spiking response (data not
shown). Further, if the paranodal periaxonal space were reduced below
90 Å, as seen at mature paranodes, K+ channel block
no longer resulted in repetitive firing. There was also an increase in
the computed refractory period with block of K+
channels, from 4 to 6.5 msec. Finally, we tested possible consequences of an asymmetrical distribution of K+ channels. If
the density of channels in either paranode/juxtaparanode were reduced
to the internodal value, there was repetitive firing (Fig.
8D, left sweep). However, doubling the
channel density in the populated side restored a normal response (Fig.
8D, right sweep). Thus, the restriction of
K+ channels to just one side can lead to
instabilities, but this seems to be attributable primarily to the
reduced total number of K+ channels near a node and
not to the asymmetry in the distribution.
As noted earlier, at ~20% of sites, K+ channels
were seen within the nodal gap at very early stages. This was of
interest, because during the formation of new nodes of Ranvier in
remyelinating axons these channels clustered first at this location and
only later shifted into paranodal zones (Rasband et al., 1998). In developing axons, nodal K+ channel immunoreactivity
was relatively weak and was very transient, virtually disappearing by
P9. Further, the percentage of sites with this expression pattern was
significantly less than the 60% seen during remyelination.
Nonetheless, we tested the possibility that this was an obligatory
first step in K+ channel segregation. We considered
a simple model in which the sequence: none nodal paranodal juxtaparanodal, with reaction rates k1,
k2, and k3
respectively, represented a series of irreversible first order
reactions. The kinetic equations were solved and a best fit (by eye)
obtained with k1 = 0.15 d1,
k2 = 0.70 d1, and
k3 = 0.06 d1, i.e., the
transition nodal paranodal about five times faster than the initial
clustering at the node. Results are plotted in Figure 3D.
This scheme is clearly vastly oversimplified, but a comparison with the
data in Figure 3A demonstrates that the fact that the nodal
category never included >20% of the observed sites and was highly
transient does not rule it out as an essential step.
| |
DISCUSSION |
When a rat is born, axons in the peripheral nervous system are in
a very immature state. Schwann cells are associated with these fibers
but are not yet committed to myelination. Action potentials propagate
but at very slow velocity. Signals are blocked by TTX, indicating that
they are mediated by Na+ channels. However, the
latter are present at very low density because they are not detected by
an antibody that recognizes all known vertebrate voltage-dependent
Na+ channels. The Shaker group K+
channel subtypes Kv1.1 and Kv1.2 are similarly not detected by immunocytochemistry. Two months later, these axons are very different. Fibers >1 µm in diameter are typically wrapped by more than 35 lamellae of tightly compacted myelin. Na+ and
K+ channels are strongly segregated at high density
within the nodal gap and the juxtaparanodes, respectively, and at much
lower levels in the internodal axolemma (Shrager, 1989). Action
potential conduction has changed from a continuous to a saltatory mode
and has increased in velocity by one to two orders of magnitude. A
remarkable feature of this progression is that axons must continue to
function throughout this period, including all intermediate stages,
with high stability and reliability. This study suggests that the
changing distribution of K+ channels plays an
essential role in achieving this goal.
The clustering of K+ channels is temporally and
spatially distinct from that of Na+ channels.
Segregation of Na+ channels is seen as soon as
adherent Schwann cells reach the state of ensheathement (~1.5 wraps),
characterized by MAG expression (Vabnick et al., 1996). This begins as
early as P1 and is primarily complete by the end of the first postnatal
week. By P7, there are large numbers of presumptive nodes, each with a
focal cluster of Na+ channels centered in the nodal
gap. However, at this time, Kv1.1 and Kv1.2 channels are detected in
only a small fraction of these sites. K+ channel
clusters are associated with large numbers of nodes only after P10.
Further, in most of these sites K+ channel
immunoreactivity is detected first in paranodal regions, and only later
there is a progressive transition to the juxtaparanode. This maturation
is about half complete after 1 month, but continues even beyond 2 months, the latest developmental period studied here.
Over P2-P9, K+ channel immunoreactivity was present
within the nodal gap in a fraction of sites. Could this expression
represent an essential stage in K+ channel
clustering? Our calculations left this possibility open by
demonstrating that a simple kinetic scheme could describe the results.
One outcome argues against this sequence. In Figure 1G, a
small gap in immunoreactivity could be seen between nodal and paranodal
channels. If there is lateral diffusion from node to paranode then we
would expect to see a continuous gradient of fluorescence in this
region. On the other hand, it may be that, as for
Na+ channel clustering during development, the level
of expression at a particular locus is dependent on the relative rates
of synthesis-insertion versus glial differentiation. In fibers in
which the latter is relatively late, K+ channels may
appear first at the node. During remyelination, adult fibers have a
significant reserve of K+ channels in the internodal
axolemma (Shrager, 1988, 1989), and clustering at newly forming nodes
may thus be rapid. In remyelinating axons, nodal Kv1.1/Kv1.2 channels
strongly blocked conduction, as judged by improvement effected by 4-AP
(Rasband et al., 1998), and development may therefore proceed in a
manner designed to minimize the likelihood of this configuration.
The spatial and temporal changes in K+ channel
distribution that occur during development suggest that maturation of
the axoglial junctions may play an important role. These structures
link the terminal Schwann cell loops with the axolemma. They are formed by transverse bands consisting of parallel rows of intramembranous particles in glial and axonal membranes, some of which may bridge the
gap and interact to form bars of electron-dense intercellular material
(Schnapp et al., 1976; Wiley and Ellisman, 1980; Tao-Cheng and
Rosenbluth, 1983; Yamamoto et al., 1996). At these regions, the gap
between glial and axonal membranes is reduced to just 20-30 Å. The
formation of axoglial junctions in the PNS during development parallels
the progressive localization of K+ channels in the
paranodes and juxtaparanodes. At the time of initial Schwann cell
adhesion, junctions are not found (Tao-Cheng and Rosenbluth, 1983). At
approximately P10, axoglial junctions form preferentially at terminal
loops closest to the node. Over the next few weeks, they appear in
increasingly more of the paranode, and by P31 almost all terminal loops
are involved (Yamamoto et al., 1996). Of interest here is the fact that
this is remarkably similar to the time course of sequestration of
K+ channels in the juxtaparanode, as shown in Figure
3A. It is thus strongly suggestive of a mechanism in which
K+ channels are excluded from regions of axoglial
junctions and thereby directed to adjacent areas. A similar mechanism
was proposed by Rosenbluth (1988) based on numerous measurements of
intramembranous particle patches seen in freeze-fracture. Axoglial
junction formation is not, however, likely to account for initial
Na+ channel clustering, which occurs as early as P1,
nor can it be the sole mechanism of K+ channel
localization, because these channels are likewise absent from the nodal
gap in mature fibers.
Further evidence for the influence of paranodal maturation in
K+ channel clustering is provided by our
measurements of asymmetrical distributions. At 2-3 weeks of age, as
many as 42% of sites were characterized by paranodal
K+ channels confined to the distal side, but by P30
this value was decreased to ~10%. In ultrastructural studies of rat
sciatic nerves at P2-P12, up to two-thirds of nodes developed
asymmetrically, with one paranode much more differentiated than the
other (Allt, 1969; Tao-Cheng and Rosenbluth, 1983). (In these studies,
proximal vs distal sidedness was not determined.) In the adult,
paranodes are generally symmetric (Tao-Cheng and Rosenbluth, 1983). In
developing rat spinal roots (P0-P14), on the other hand, most nodes
developed with symmetry, and only occasional asymmetric sites were seen (Wiley-Livingston and Ellisman, 1980). Correspondingly, we found very
few examples of asymmetrical clustering of K+
channels in dorsal and ventral roots.
The role of K+ channels in
signal propagation
Our data indicate that the function of Kv1.1 and Kv1.2 channels
varies during development in parallel with the progression described
above for the anatomical localization of the channels and indeed with
changes in the overall architecture of the myelinating nerve. The
functional significance of these channels appears to move through three
distinct stages corresponding to the early axons in which paranodes
have not yet formed, transitional fibers with paranodes in the process
of formation, and adult nerves in which myelination is advanced and
paranodes are mature. During the early period (P0-P10),
K+ channels are directly involved in action
potential generation, speeding repolarization, and decreasing the
refractory period to allow trains at high frequency. During this time,
Kv1.1 and Kv1.2 immunoreactivity is not detected in most axons but is
present at nodal and paranodal zones in 10-20% of fibers. Because
almost all axons are sensitive to 4-AP, K+ channels
are likely to be present in all cases but primarily at densities too
low for detection with immunocytochemistry.
Perhaps the most striking role of K+ channels occurs
at 2-6 weeks of age, when they function to prevent bursting behavior
in response to a single stimulus. This period corresponds closely to
the presence of K+ channels within paranodes. Our
results suggest that it is specifically this intermediate level of
maturity that is susceptible to repetitive firing. If the longitudinal
resistance in the periaxonal space within the paranodes is low, as at
very early times, then the delay in "reflection" of the
depolarization from the next node is so short that the nodal
Na+ channels are inactivated and cannot fire again.
At later stages, as axoglial junctions develop and the longitudinal
resistance increases, internodal Na+ channels become
electrically isolated and cannot trigger a regenerative response at the
node. Simultaneously, K+ channels become
increasingly sequestered in the juxtaparanodes, reducing or eliminating
a direct involvement in action potential generation and rendering the
function of these channels at this stage unclear. Alterations in Kv1.1
in the PNS have been linked to episodic ataxia-myokymia (Browne et
al., 1994; Adelman et al., 1995; Smart et al., 1998).
K+ channels may also play an important protective
role during injury or disease. Although conduction block is the most
serious clinical consequence of demyelination, there are also
"positive" neurological symptoms, including pain and paresthesia,
that result from hyperexcitability. Foci of demyelination have been
shown to serve as origins of ectopic impulses or of multiple spikes
evoked from single stimuli (Howe et al., 1976; Smith and McDonald,
1980; Calvin et al., 1982). Particularly noteworthy is the finding that
at these sites K+ channel block exacerbates this
spontaneous discharge (Burchiel and Russell, 1985; Baker and Bostock,
1992), and therapeutic attempts to restore conduction must therefore be
balanced to avoid introducing hyperexcitability. Thus, during disease,
K+ channels may recapitulate their developmental
function to stabilize conduction while the passive electrical
properties of the axon are undergoing major transformations.
| |
FOOTNOTES |
Received Sept. 10, 1998; revised Oct. 29, 1998; accepted Oct. 29, 1998.
This work has been supported by National Institutes of Health Grants
NS17965, NS34383, and NS34375 and National Multiple Sclerosis Society
Grant RG-2687. We thank Dr. Michael Hines for modification of the
Neuron program and Matthew N. Rasband for comments on this manuscript.
Katia Kazarinova helped with model calculations. Ms. Ellen
Brunschweiger provided excellent technical assistance.
Correspondence should be addressed to Dr. Peter Shrager, Department of
Neurobiology and Anatomy, Box 603, Room 4-5428, University of
Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642.
| |
REFERENCES |
-
Adelman JP,
Bond CT,
Pessia M,
Maylie J
(1995)
Episodic ataxia results from voltage dependent potassium channels with altered functions.
Neuron
15:1449-1454[Web of Science][Medline].
-
Allt G
(1969)
Ultrastructural features of the immature peripheral nerve.
J Anat
105:283-293[Web of Science][Medline].
-
Baker M,
Bostock H
(1992)
Ectopic activity in demyelinated spinal root axons of the rat.
J Physiol (Lond)
451:539-552[Abstract/Free Full Text].
-
Barrett EF,
Barrett JN
(1982)
Intracellular recording from vertebrate myelinated axons: mechanism of the depolarizing afterpotential.
J Physiol (Lond)
323:117-144[Abstract/Free Full Text].
-
Bekele-Arcuri Z,
Matos MF,
Manganas MM,
Strassle BW,
Monaghan MM,
Rhodes KJ,
Trimmer JS
(1996)
Generation and characterization of subtype-specific monoclonal antibodies to K+ channel alpha and beta subunit polypeptides.
Neuropharmacology
35:851-865[Web of Science][Medline].
-
Bowe CM,
Kocsis JD,
Waxman SG
(1985)
Differences between mammalian ventral and dorsal spinal roots in response to blockade of potassium channels during maturation.
Proc R Soc Lond Ser B
224:355-366[Medline].
-
Browne DL,
Gancher ST,
Nutt JG,
Brunt ERP,
Smith EA,
Kramer P,
Litt M
(1994)
Episodic ataxia/myokymia syndrome is associated with point mutations in the human potassium channel gene, KCNA1.
Nat Genet
8:136-140[Web of Science][Medline].
-
Burchiel KJ,
Russell LC
(1985)
Effects of potassium channel-blocking agents on spontaneous discharges from neuromas in rats.
J Neurosurg
63:246-249[Web of Science][Medline].
-
Calvin WH,
Devor M,
Howe JF
(1982)
Can neuralgias arise from minor demyelination? Spontaneous firing, mechanosensitivity, and afterdischarge from conducting axons.
Exp Neurol
75:755-763[Web of Science][Medline].
-
Chiu SY,
Ritchie JM
(1981)
Evidence for the presence of potassium channels in the paranodal region of acutely demyelinated mammalian single nerve fibres.
J Physiol (Lond)
313:415-437[Abstract/Free Full Text].
-
Chiu SY,
Ritchie JM
(1982)
Evidence for the presence of potassium channels in the internode of frog myelinated nerve fibres.
J Physiol (Lond)
322:485-501[Abstract/Free Full Text].
-
Chiu SY,
Ritchie JM
(1984)
On the physiological role of internodal potassium channels and the security of conduction in myelinated nerve fibres.
Proc R Soc Lond Ser B
220:415-422[Medline].
-
Chiu SY,
Ritchie JM,
Rogart RB,
Stagg D
(1979)
A quantitative description of membrane currents in rabbit myelinated nerve.
J Physiol (Lond)
292:149-166[Abstract/Free Full Text].
-
Dodge F,
Frankenhaeuser B
(1958)
Membrane currents in isolated frog nerve fibre under voltage clamp conditions.
J Physiol (Lond)
143:76-90.
-
Dugandzija-Novakovic S,
Koszowski AG,
Levinson SR,
Shrager P
(1995)
Clustering of Na channels and node of Ranvier formation in remyelinating axons.
J Neurosci
15:492-502[Abstract].
-
Fields RD,
Black JA,
Bowe CM,
Kocsis JD,
Waxman SG
(1986)
Differences in intramembranous particle distribution in the paranodal axolemma are not associated with functional differences of dorsal and ventral roots.
Neurosci Lett
67:13-18[Web of Science][Medline].
-
Frankenhaeuser B,
Huxley AF
(1964)
The action potential in the myelinated nerve fibre of Xenopus laevis as computed on the basis of voltage clamp data.
J Physiol (Lond)
171:302-315.
-
Grissmer S,
Nguyen AN,
Aiyar J,
Hanson DC,
Mather RJ,
Gutman GA,
Karmilowicz MJ,
Auperin DD,
Chandy KG
(1994)
Pharmacological characterization of five cloned voltage-gated K+ channels, types Kv1.1, 1.2, 1.3, 1.5, and 3.1, stably expressed in mammalian cell lines.
Mol Pharmacol
45:1227-1234[Abstract].
-
Hille B
(1967)
The selective inhibition of delayed potassium currents in nerve by tetraethylammonium ion.
J Gen Physiol
50:1287-1302[Abstract/Free Full Text].
-
Hines M,
Carnevale NT
(1997)
The NEURON simulation.
Neural Comput
9:1179-1209[Web of Science][Medline].
-
Hopkins WF,
Allen ML,
Houamed KM,
Tempel BL
(1994)
Properties of voltage-gated K+ currents expressed in Xenopus oocytes by mKv1.1, mKv1.2 and their heteromultimers as revealed by mutagenesis of the dendrotoxin-binding site in mKv1.1.
Pflügers Arch
428:382-390[Web of Science][Medline].
-
Howe JF,
Calvin WH,
Loeser JD
(1976)
Impulses reflected from dorsal root ganglia and from focal nerve injuries.
Brain Res
116:139-144[Web of Science][Medline].
-
Kocsis JD,
Ruiz JA,
Waxman SG
(1983)
Maturation of mammalian myelinated fibers: changes in action-potential characteristics following 4-aminopyridine application.
J Neurophysiol
50:449-463[Free Full Text].
-
Martini R,
Schachner M
(1986)
Immunoelectron microscopic localization of neural cell adhesion molecules (L1, N-CAM, and MAG) and their shared carbohydrate epitope and myelin basic protein in developing sciatic nerve.
J Cell Biol
103:2439-2448[Abstract/Free Full Text].
-
Mi H,
Deerinck TJ,
Ellisman MH,
Schwarz TL
(1995)
Differential distribution of closely related potassium channels in rat Schwann cells.
J Neurosci
15:3761-3774[Abstract].
-
Milton RL,
Mathias RT,
Eisenberg RS
(1985)
Electrical properties of the myotendon region of frog twitch muscle fibers measured in the frequency domain.
Biophys J
48:253-267[Web of Science][Medline].
-
Polman CH,
Hartung HP
(1995)
The treatment of multiple sclerosis: current and future.
Curr Opin Neurol
8:200-209[Web of Science][Medline].
-
Poltorak M,
Sadoul R,
Keilhauer G,
Landa C,
Fahrig T,
Schachner M
(1987)
Myelin-associated glycoprotein, a member of the L2/HNK-1 family of neural cell adhesion molecules, is involved in neuron-oligodendrocyte and oligodendrocyte-oligodendrocyte interaction.
J Cell Biol
105:1893-1899[Abstract/Free Full Text].
-
Rasband MN,
Trimmer JS,
Schwarz TL,
Levinson SR,
Ellisman MH,
Schachner M,
Shrager P
(1998)
Potassium channel distribution, clustering, and function in remyelinating rat axons.
J Neurosci
18:36-47[Abstract/Free Full Text].
-
Rhodes KJ,
Monaghan MM,
Barrezueta NX,
Nawoschik S,
Bekele-Arcuri Z,
Matos MF,
Nakahira K,
Schechter LE,
Trimmer JS
(1996)
Voltage-gated K+ channel beta subunits: expression and distribution of Kv beta 1 and Kv beta 2 in adult rat brain.
J Neurosci
16:4846-4860[Abstract/Free Full Text].
-
Rhodes KJ,
Strassle BW,
Monaghan MM,
Bekelearcuri Z,
Matos MF,
Trimmer JS
(1997)
Association and colocalization of the Kv-beta-1 and Kv-beta-2 beta subunits with Kv1 alpha subunits in mammalian brain K+ channel complexes.
J Neurosci
17:8246-8258[Abstract/Free Full Text].
-
Roper J,
Schwarz JR
(1989)
Heterogeneous distribution of fast and slow potassium channels in myelinated rat nerve fibres.
J Physiol (Lond)
416:93-110[Abstract/Free Full Text].
-
Rosenbluth J
(1988)
Role of glial cells in the differentiation and function of myelinated axons.
Int J Dev Neurosci
6:3-24[Web of Science][Medline].
-
Schmidt H,
Stampfli R
(1966)
Die wirkung von tetraathylammoniumchlorid auf den einzelnen ranvierschen schnurring.
Pflügers Arch
287:311-325.
-
Schnapp B,
Peracchia C,
Mugnaini E
(1976)
The paranodal axo-glial junction in the central nervous system studied with thin sections and freeze-fracture.
Neuroscience
1:181-190[Web of Science][Medline].
-
Schwid SR,
Petrie MD,
McDermott MP,
Tierney DS,
Mason DH,
Goodman AD
(1997)
Quantitative assessment of sustained-release 4-aminopyridine for symptomatic treatment of multiple sclerosis.
Neurology
48:817-821[Abstract/Free Full Text].
-
Sheng M,
Tsaur ML,
Jan YN,
Jan LY
(1992)
Subcellular segregation of two A-type K+ channel proteins in rat central neurons.
Neuron
9:271-284[Web of Science][Medline].
-
Sheng M,
Tsaur ML,
Jan YN,
Jan LY
(1994)
Contrasting subcellular localization of the Kv1.2 K+ channel subunit in different neurons of rat brain.
J Neurosci
14:2408-2417[Abstract].
-
Sherratt RM,
Bostock H,
Sears TA
(1980)
Effects of 4-aminopyridine on normal and demyelinated mammalian nerve fibres.
Nature
283:570-572[Medline].
-
Shi G,
Nakahira K,
Hammond S,
Rhodes KJ,
Schechter LE,
Trimmer JS
(1996)
Beta subunits promote K+ channel surface expression through effects early in biosynthesis.
Neuron
16:843-852[Web of Science][Medline].
-
Shrager P
(1987)
The distribution of sodium and potassium channels in single demyelinated axons of the frog.
J Physiol (Lond)
392:587-602[Abstract/Free Full Text].
-
Shrager P
(1988)
Ionic channels and signal conduction in single remyelinating frog nerve fibres.
J Physiol (Lond)
404:695-712[Abstract/Free Full Text].
-
Shrager P
(1989)
Sodium channels in single demyelinated mammalian axons.
Brain Res
483:149-154[Web of Science][Medline].
-
Smart SL,
Lopantsev V,
Zhang CL,
Robbins CA,
Wang H,
Chiu SY,
Schwartzkroin PA,
Messing A,
Tempel BL
(1998)
Deletion of the Kv1.1 potassium channel causes epilepsy in mice.
Neuron
20:809-819[Web of Science][Medline].
-
Smith KJ,
McDonald WI
(1980)
Spontaneous and mechanically evoked activity due to central demyelinating lesion.
Nature
286:154-155[Medline].
-
Stampfli R
(1952)
Bau und funktion isolierter markhaltiger nervenfasern.
Ergeb Physiol Biol Chem Exp Pharmakol
47:70-165.
-
Strichartz GR,
Small RK,
Pfenninger KH
(1984)
Components of the plasma membrane of growing axons. III. Saxitoxin binding to sodium channels.
J Cell Biol
98:1444-1452[Abstract/Free Full Text].
-
Stys PK,
Ransom BR,
Waxman SG
(1991)
Compound action potential of nerve recorded by suction electrode: a theoretical and experimental analysis.
Brain Res
546:18-32[Web of Science][Medline].
-
Swanson R,
Marshall J,
Smith JS,
Williams JB,
Boyle MB,
Folander K,
Luneau CJ,
Antanavage J,
Oliva C,
Buhrow SA,
Bennett C,
Stein RB,
Kaczmarek LK
(1990)
Cloning and expression of cDNA and genomic clones encoding three delayed rectifier potassium channels in rat brain.
Neuron
4:929-939[Web of Science][Medline].
-
Tao-Cheng JH,
Rosenbluth J
(1982)
Development of nodal and paranodal membrane specializations in amphibian peripheral nerves.
Brain Res
255:577-594[Medline].
-
Tao-Cheng JH,
Rosenbluth J
(1983)
Axolemmal differentiation in myelinated fibers of rat peripheral nerves.
Brain Res
285:251-263[Medline].
-
Trimmer JS,
Trowbridge IS,
Vacquier VD
(1985)
Monoclonal antibody to a membrane glycoprotein inhibits acrosome reaction and associated Ca++ and H+ fluxes of sea urchin sperm.
Cell
40:697-703[Web of Science][Medline].
-
Vabnick I,
Shrager P
(1998)
Ion channel redistribution and function during development of the myelinated axon.
J Neurobiol
37:80-96[Web of Science][Medline].
-
Vabnick I,
Novakovic SD,
Levinson SR,
Schachner M,
Shrager P
(1996)
The clustering of axonal sodium channels during development of the peripheral nervous system.
J Neurosci
16:4914-4922[Abstract/Free Full Text].
-
Wang H,
Kunkel DD,
Martin TM,
Schwartzkroin PA,
Tempel BL
(1993)
Heteromultimeric K+ channels in terminal and juxtaparanodal regions of neurons.
Nature
365:75-79[Medline].
-
Webster HD
(1971)
The geometry of peripheral myelin sheaths during their formation and growth in rat sciatic nerves.
J Cell Biol
48:348-367[Abstract/Free Full Text].
-
Wiley CA,
Ellisman MH
(1980)
Rows of dimeric-particles within the axolemma and juxtaposed particles within glia, incorporated into a new model for the paranodal glial-axonal junction at the node of Ranvier.
J Cell Biol
84:261-280[Abstract/Free Full Text].
-
Wiley-Livingston C,
Ellisman MH
(1980)
Development of axonal membrane specializations defines nodes of Ranvier and precedes Schwann cell myelin elaboration.
Dev Biol
79:334-355[Web of Science][Medline].
-
Yamamoto K,
Merry AC,
Sima AA
(1996)
An orderly development of paranodal axoglial junctions and bracelets of Nageotte in the rat sural nerve.
Dev Brain Res
96:36-45[Medline].
-
Ziskind-Conheim L
(1988)
Physiological and morphological changes in developing peripheral nerves of rat embryos.
Dev Brain Res
42:15-28.
Copyright © 1999 Society for Neuroscience 0270-6474/99/192747-12$05.00/0
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|
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Abstract
Introduction
