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The Journal of Neuroscience, July 15, 1998, 18(14):5234-5239
The Sodium Channel Scn8a Is the Major Contributor to
the Postnatal Developmental Increase of Sodium Current Density in
Spinal Motoneurons
Kelly D.
García1,
Leslie K.
Sprunger2,
Miriam
H.
Meisler2, and
Kurt G.
Beam1
1 Department of Anatomy and Neurobiology, Colorado
State University, Fort Collins, Colorado 80523-1670, and
2 Department of Human Genetics, University of Michigan, Ann
Arbor, Michigan 48109-0618
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ABSTRACT |
Sodium currents were recorded from motoneurons that were isolated
from mice at postnatal days 0-8 (P0-P8) and maintained in culture for
12-24 hr. Motoneurons from normal mice exhibited a more than threefold
increase in peak sodium current density from P0 to P8. For mice lacking
a functional Scn8a sodium channel gene, motoneuronal
sodium current density was comparable at P0 to that of normal mice but
failed to increase from P0 to P8. The absence of Scn8a
sodium channels is associated with the phenotype "motor end plate
disease," which is characterized by a progressive neuromuscular failure and is fatal by 3-4 postnatal weeks. Thus, it appears that the
development and function of mature motoneurons depends on the postnatal
induction of Scn8a expression.
Key words:
motoneurons; sodium channels; Scn8a; postnatal
development; motor end plate disease; neuromuscular system; mouse
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INTRODUCTION |
Motoneuronal electrical activity
plays a critical role in the prenatal and postnatal development of the
neuromuscular system (for review, see Grinnell, 1995 ). For example,
individual muscle fibers are multiply innervated at birth but are
singly innervated in the adult. Elimination of the supernumerary
synapses occurs during the first 2 postnatal weeks and is governed by
neuromuscular activity, which is obviously influenced by the ion
channels expressed in the motoneurons. Previous work has shown that the
expression of calcium currents in motoneurons changes during the period
of initial synapse elimination (Mynlieff and Beam, 1992b). Sodium current density has been shown to increase as a function of the length
of time that embryonic mouse motoneurons are maintained in
vitro (MacDermott and Westbrook, 1986). However, the in
vivo postnatal development of motoneuronal sodium currents has not been characterized. Here, we have measured sodium currents in identified motoneurons that were obtained from mice of varying postnatal ages and maintained overnight in primary culture.
In addition to cells from normal mice, we examined motoneurons from
mice with motor end plate disease (med), a genetic defect that causes progressive neuromuscular failure. The gene altered by the
med mutation was recently identified (Burgess et al., 1995) as the voltage-gated sodium channel Scn8a that is expressed
in brain and spinal cord (Schaller et al., 1995). Four independent mutations of Scn8a have been characterized at the molecular
level: a transgene-induced intragenic deletion (Burgess et al., 1995), two mutations that affect splicing (Kohrman et al., 1996a), and one
missense mutation (Kohrman et al., 1996b). Mice homozygous for the
transgene-induced mutation lack functional Scn8a channels and display altered cerebellar function and progressive neuromuscular weakness, which begins ~10 d after birth and results in death within
3-4 weeks (Burgess et al., 1995). Analysis of homozygous transgenic
mice demonstrated that Scn8a channels play an important role
in spontaneous and repetitive firing of cerebellar Purkinje cells
(Raman et al., 1997). Mice homozygous for the missense mutation display
profound cerebellar dysfunction without neuromuscular weakness and
survive to adulthood (Dick et al., 1986; Harris and Pollard, 1986).
Early studies of mice homozygous for the splicing mutations
demonstrated progressive loss of evoked neurotransmitter release at the
neuromuscular junction (Duchen and Stefani, 1971; Weinstein, 1980;
Harris and Pollard, 1986), indicating that the Scn8a channel
is essential for motoneuron function. Scn8a transcripts have
been detected in motoneurons of the rat by in situ
hybridization (Schaller et al., 1995; Felts et al., 1997). However, no
previous studies have evaluated the quantitative contribution of
Scn8a to motoneuronal sodium current.
We found that peak sodium current density in motoneurons from normal
mice increased more than threefold between postnatal day 0 (P0) and P8.
At P0, motoneurons from mice homozygous for a disrupted
Scn8a gene had a sodium current density that differed little
from that of normal P0 motoneurons. However, sodium current density
failed to increase between P0 and P8 in motoneurons lacking an
operational Scn8a gene. Thus, the postnatal increase in
sodium current expression that occurs in normal motoneurons depends on an intact Scn8a gene and appears to be essential for the
development and function of mature motoneurons.
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MATERIALS AND METHODS |
Animals. The medtg mutation arose
by nontargeted insertion of a transgene into a chromosome derived from
strain C3 Hr/HeJ mice and has been maintained by crossing with strain
C57BL/6J since 1992 (Kohrman et al., 1995). C57BL/6J mice were obtained
from the Jackson Laboratory (Bar Harbor, ME). Heterozygous transgenic mice (genotype tg/+) were crossed to produce litters
containing +/+, tg/+, and tg/tg mice. See Figure
1 for control studies using strain 129/ReJ mice (Jackson).
Motoneuron cultures. The procedures used for culturing
motoneurons were similar to those described previously (Mynlieff and Beam 1992a,b; García and Beam, 1996). Neonatal mouse pups were anesthetized and injected in all four limbs with a suspension of DiI
(Molecular Probes, Eugene, OR) (2.5 mg/ml, 20% ethanol, 80% rodent
Ringer's solution with 0.1% bovine serum albumin). The pups were
returned to their mothers for 9-11 hr to allow the dye to label
motoneuronal cell bodies (Honig and Hume, 1986). The pups were then
reanesthetized and decapitated, and the spinal cords were removed in
oxygenated rodent Ringer's solution (in mM: 146 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 11 glucose, and 10 HEPES, pH 7.4). The spinal cord was cleaned of meninges
and any adhering dorsal root ganglia, cut into small pieces (<1
mm3), and placed in 0.5 ml of a 0.1% type XI
trypsin and 0.01% DNase I (both from Sigma, St. Louis, MO) solution in
PIPES-buffered saline (in mM: 120 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 25 glucose, and
20 piperazine-N,N'-bis[2-ethanesulfonic acid], pH 7.0).
After 15-20 min of incubation at 35°C, the tissue was rinsed with
neural basal medium containing B27 supplement (Life Technologies, Grand Island, NY), 100 µg/ml streptomycin, and 60 µg/ml penicillin, and
triturated with a fire-polished pipette. The cells were plated onto 35 mm poly-L-lysine-coated (4-15 kDa; 1 mg/ml in 0.15 M boric acid, pH 8.4) dishes, maintained overnight in a
humidified atmosphere of 95% air and 5% C02 at 37°C,
and recorded from on the following morning.
Ionic currents. The majority of recordings (>75%) were
obtained from motoneurons identified on the basis of DiI staining
(Honig and Hume, 1986), with the remainder from nonlabeled cells
identified as motoneurons on the basis of morphological criteria (Smith
et al., 1986; Milligan et al., 1994; Mynlieff and Beam, 1994). Current densities in nonlabeled cells were not significantly different from
those in labeled cells (see Results). Membrane currents were recorded
at room temperature (20°C) with whole-cell patch-clamp configuration
(Hamill et al., 1981) using a DAGAN 3900 patch-clamp amplifier (Dagan,
Minneapolis, MN) equipped with a 3911 whole-cell expander (Dagan). The
currents were electronically filtered at 1 kHz (8-pole Bessel filter)
and then sampled and stored with a digital computer (Indec Systems,
Capitola, CA). Linear components of leak and capacitive currents were
removed from test currents by digital subtraction of scaled control
currents elicited by 20 mV hyperpolarizations from the holding
potential ( 80 mV). To normalize for differences in membrane area,
current densities were calculated as the ratio of measured current to
linear cell capacitance. Data are given as mean ± SEM. Data were
tested for significant differences (p < 0.05)
by an unpaired Student's t test.
Patch electrodes were made from soda lime glass, had resistances of
4-5 M , and were coated with wax to reduce capacitance. The pipette
filling solution contained (in mM): 140 Cs-aspartate, 5 MgCl2, 10 Cs2EGTA, and 10 HEPES, pH 7.4. For measurement of sodium currents, the bath contained (in
mM): 35 NaCl, 115 TEA-Cl, 2 CaCl2, 1 MgCl2, and 10 HEPES, pH 7.4. For measurement of
calcium currents, the external solution contained (in mM):
10 CaCl2, 145 TEA-Cl, 0.0005 TTX, and 10 HEPES, pH
7.4.
Ribonuclease protection assay. Spinal cords from wild-type
(+/+) mice at P0 (n = 6), P7 (n = 5),
and adult (n = 3) were pooled, and total RNA was
prepared using the Trizol reagent (Life Technologies). Ribonuclease
protection assays were performed as described previously, using a 511 bp [ -32P]UTP-radiolabeled riboprobe that contained a
381 bp coding fragment (Burgess et al., 1995).
Determination of genotype. Genotype was determined either
from muscle samples obtained at the time of spinal cord dissection or
from toe clips obtained at P3 or P4. The samples were frozen on dry ice
and stored at 80°C until further processing. Genomic DNA was
prepared from the muscle samples by digestion with proteinase K,
phenol-chloroform extraction and ethanol precipitation, and prepared
from the toe clips by a modification of the method of Pomp and Murray
(1991). Specifically, these samples were digested for 2 hr at 55°C in
250 µl of PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.01% gelatin, and 1.5 mM
MgCl2) containing 0.45% Tween 20, 0.45% NP-40, and
0.2 µg/µl proteinase K. Five-microliter aliquots were incubated
under mineral oil for 10 min at 95°C to inactivate proteinase K and
amplified in 25 µl PCR using the multiplexed PCR protocol described
below.
PCR reactions contained 50-100 ng of genomic DNA. Two genotype assays
were used. One was a previously described assay involving separate
amplification of the transgene and the wild-type Scn8a allele (Raman et al., 1997). In the new multiplex assay, a single aliquot of DNA from each mouse was amplified with two compatible primer
pairs: primer pair 1 (forward, GAGGG AGGGC TGAGG GTTTG AAGTC; reverse,
CCATG GTGTC TGTTT GAGGT TGCTAG) amplifies a 244 bp fragment from the
transgene (Kohrman et al., 1995); and primer pair 2 (forward, GAAGT
GAAAC CTTTA GAGGA GCTGT ATGAG; reverse, GGAAT TCCTT CTGGA AGTCG CCGTT
CCTGT GAATG TCC) amplifies a 103 bp fragment of exon 14 from the
wild-type Scn8a that is missing in the transgenic allele
(Raman et al., 1997). After denaturation for 3 min at 94°C, PCRs were
performed for 35 cycles with annealing for 45 sec at 65°C,
followed by 45 sec at 72°C and 45 sec at 94°C.
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RESULTS |
Postnatal increase in sodium current density in
normal motoneurons
Figure 1A
illustrates representative sodium currents in motoneurons isolated from
P0, P4, and P8 mice and cultured overnight. It is evident that the P4
currents are larger than the P0 currents, and that the P8 currents are
substantially larger still. This postnatal increase in sodium current
is also apparent in the I-V relationships obtained by
averaging data obtained from many cells (Fig. 1B).
Measured at a test potential of 10 mV, peak current density increased
almost fivefold between P0 and P7-P8 (Fig. 1C). As can be
seen in the raw currents, the high-resistance pipettes necessary to
record from motoneurons often caused a loss of voltage control for
potentials in the negative slope region of the I-V curve.
This was true, although the currents were measured with a reduced
extracellular sodium concentration (35 mM). Thus, it is
difficult to determine whether there were developmental changes in
either voltage dependence or kinetics.
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Figure 1.
Postnatal increase of sodium current density in
normal mouse motoneurons. A, Representative sodium
currents in motoneurons isolated from normal mice at P0, P4, and P8 and
cultured overnight. Here (and in Fig. 4), the illustrated currents were
elicited by step depolarizations ranging from 40 to +10 mV at 10 mV
intervals, extracellular sodium concentration was 35 mM,
and the holding potential was 80 mV. B, Average
current-voltage relationships for motoneurons obtained from normal
mice at P0-P1 (n = 45), P4 (n = 7), and P7-P8 (n = 12). Error bars indicate ± SEM. C, Peak sodium current density as a function of
postnatal age. The data point plotted midway between P7 and P8 was
obtained from P7 and P8 motoneurons (n = 28, 17, 16, 9, 8, and 12 at P0, P1, P2, P3, P4, and P7-P8,
respectively).
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Scn8a transcripts in neonatal spinal cord
Previous analysis with a specific ribonuclease protection assay
demonstrated that the Scn8a transcript is present in the
spinal cord of normal adult mice but absent from the spinal cord of
homozygous transgenic mice lacking a functional Scn8a gene
(Burgess et al., 1995). To determine whether the Scn8a
transcript is present during early postnatal development of the normal
spinal cord, RNA was prepared from spinal cords of mice at P0 and P7.
The Scn8a transcript was readily detected at both P0 and P7
(Fig. 2), consistent with the hypothesis
that Scn8a channels contribute to the postnatal increase in
motoneuronal sodium current illustrated in Figure 1.
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Figure 2.
Expression of Scn8a in mouse spinal
cord. Twenty-microgram aliquots of total RNA were assayed by
ribonuclease protection using a 511 bp Scn8a specific
riboprobe. The predicted size of the protected fragment is 381 bp.
Left panel, All samples were run on the same gel, which
was dried and exposed to film for 1.5 hr (probe) or 5 hr (samples plus
probe). Right panel, The concentration and integrity of
the RNA samples were compared by staining with ethidium bromide after
electrophoresis of 1 µg aliquots on a 1.0% agarose gel.
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Identification of mice with a disrupted Scn8a gene
To evaluate the contribution of Scn8a to the postnatal
increase in motoneuronal sodium current, we examined cells from
homozygous medtg mice in which both copies of the
Scn8a gene are inactivated by transgene (tg)
insertion. Homozygous mice were obtained from crosses between
tg/+ heterozygotes. To determine genotypes of the offspring, genomic DNA from each animal was analyzed by PCR with two primer pairs:
one for amplification of the transgene and one for amplification of the
wild-type Scn8a gene. Genotypes are inferred from the
presence or absence of transgenic and wild-type allele products of 244 and 103 bp, respectively (Fig. 3).
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Figure 3.
Determination of Scn8a genotypes by
PCR. Genomic DNA was prepared from offspring of the cross
(tg/+ × tg/+) and amplified using two
pairs of primers, as described in Materials and Methods. Amplification
of the wild-type Scn8a gene generated a 103 bp product;
amplification of the transgene generated a 244 bp product. The inferred
genotypes, based on presence or absence of each product, are shown
above each lane. A minor 280 bp product was occasionally
amplified from wild-type DNA by the transgene primers
(arrowhead). The positions of molecular weight markers
(1 kb ladder; BRL, Bethesda, MD) are shown at the left
in base pairs.
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Sodium currents in motoneurons from
medtg mice
To determine whether the absence of a functional Scn8a
gene affected motoneuronal sodium currents, cells were isolated from P0-P8 mouse pups obtained from tg/+ crosses. For each pup,
a sample of tissue was frozen at the time of spinal cord dissection.
Mouse genotype was determined by PCR analysis only after currents had been recorded and analyzed. This, together with the fact that P0-P8
medtg mice do not exhibit any phenotypic signs of
altered neuromuscular function, ensured that the currents were recorded
under blind conditions.
As shown by both raw currents (Fig.
4A) and average
I-V relationships (Fig.
4B-D), sodium currents were nearly normal
in motoneurons from P0-P3 medtg mice but very much
reduced in cells from P7-P8 medtg mice. Of 11 P7
and P8 medtg motoneurons analyzed, 5 had little or
no sodium current (peak density of  15 pA/pF) like the one illustrated
in Figure 4A, 4 had modest sodium currents (peak
density of ~45 pA/pF), and 2 had currents (peak densities of 91 and
142 pA/pF) almost as large as the mean for normal cells. This
variability may reflect the biological heterogeneity of the motoneuron
pool, because the neuromuscular weakness characteristic of motor end
plate disease is not uniform from muscle to muscle (Duchen, 1970;
Duchen and Stefani, 1971).
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Figure 4.
Lack of postnatal increase in sodium current
density in medtg motoneurons. A,
Representative sodium currents in motoneurons isolated from
medtg mice at P0, P3, and P7. Note that the
currents from the P7 motoneuron are displayed at a 10-fold higher gain.
B, C, D, Average
current-voltage relationships for wild-type and
medtg motoneurons at P0-P1, P2-P3, and P7-P8.
In B, C, and D,
n = 4, 7, and 11 for the wild-type motoneurons, and
n = 6, 9, and 11 for the
medtg motoneurons, respectively. Each of the
data points is based on currents measured in at least two separate
motoneuron cultures.
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For the P7 medtg motoneuron illustrated in Figure
4A, the sodium current was so small that it was
partially obscured by another current, with an amplitude and voltage
dependence suggestive of calcium current (the bath contained 2 mM calcium). Similar presumptive calcium currents were seen
in other P7-P8 medtg cells with very small sodium
currents, indicating that the small sodium currents were not
attributable to a generalized membrane pathology. We also recorded
currents from a few medtg cells under conditions
that isolate calcium currents (sodium-free external solutions with 10 mM calcium and 0.5 µM tetrodotoxin). Currents
recorded from medtg cells under these conditions
(Fig. 5) were qualitatively similar to
previously described calcium currents in normal motoneurons (Mynlieff
and Beam, 1992a,b; García and Beam, 1996).
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Figure 5.
Calcium currents in motoneurons from a P0
(top traces) or a P6 (bottom traces)
medtg mouse. Test pulses were to potentials of
20 to +20 mV, in 10 mV increments. Peak calcium current densities at
+10 mV were as follows: P0, 3.6 and 5.4 pA/pF; P6, 10.9 and 9.9 pA/pF.
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Figure 6 compares normalized peak sodium
currents (Vtest = 10 mV) in motoneurons from
medtg mice with those from their heterozygous and
homozygous normal littermates. The sodium currents from heterozygous
motoneurons were not significantly different (p > 0.05) from those of normal motoneurons at any of the three age
groups examined (P0-P1, P2-P3, and P7-P8). Moreover, sodium currents
in medtg motoneurons were not significantly
different from those of heterozygous or homozygous cells at P0-P1
(p > 0.05). However, average peak sodium
current density in medtg motoneurons was slightly
reduced at P2-P3 and substantially diminished at P7-P8 (for both age
groups, p < 0.05 for medtg cells
compared with either heterozygous or homozygous cells). Especially at
P7-P8, when the yield of viable cells from the dissociation procedure
was ~10-fold lower than in younger animals, recordings were made not
only from DiI-labeled cells but also from unlabeled cells identified
morphologically as motoneurons (Fig. 6, circles and
triangles, respectively). For none of the three genotypes of
P7-P8 cells was there a significant difference
(p > 0.05) in sodium current density between
labeled and unlabeled cells.
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Figure 6.
Peak sodium current density as a function of
postnatal age in motoneurons from P0-P1, P2-P3, and P7-P8 animals
that were wild-type (shaded symbols), heterozygous
(filled symbols), or medtg
(open symbols). The numbers of cells for the three age
groups were 4, 7, and 11 (wild-type), 4, 7, and 10 (heterozygous), and
6, 9, and 11 (medtg), respectively. Cells
identified as motoneurons by DiI labeling are indicated with
circles, and cells not identified by DiI labeling are
indicated by triangles.
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DISCUSSION |
We have found that sodium current density increases severalfold in
motoneurons isolated from normal mice of increasing postnatal age
(P0-P8). We also found that the transcript for the Scn8a
sodium channel is present in the spinal cord of normal neonatal mice. In motoneurons from medtg mice, which lack a
functional Scn8a gene, we did not observe the postnatal
increase in average sodium current density. These results are
compatible with the hypothesis that postnatal upregulation of
Scn8a expression is responsible for much of the increase in sodium current density in normal motoneurons. As previously observed for cerebellar Purkinje cells (Raman et al., 1997), the current density
in motoneurons from heterozygous null mice did not differ from the
normal wild-type level (Fig. 6). Thus, the loss of one functional
allele does not compromise the number of channels in the membrane.
Because acutely isolated motoneurons have fairly extensive processes
and are thus poorly suited for voltage-clamp measurements, we used
motoneurons isolated and cultured overnight. Cultured motoneuronal
somata are likely to express ion channels that would have been targeted
to other cellular structures (dendrites, axon, and axon terminal)
in vivo. This has been shown to be the case for cultured
neurons from the squid stellate ganglion; sodium channels, which are
only found in the axon in vivo, appear in the cell bodies
after the neurons have been axotomized and placed in culture (Gilly and
Brismar, 1989). Our measurements on cultured motoneurons do not allow
us to say which cellular structure(s) would have been the destination
for the sodium channels that produced the observed postnatal increase
in current density. An obvious possibility is that these sodium
channels would have been targeted to the axon. Consistent with this
possibility, the time frame of sodium current upregulation found in the
present study corresponds to that reported previously for increased
numbers of sodium channels at nodes of Ranvier in rat peripheral nerve
(Vabnick et al., 1996).
Our knowledge remains quite fragmentary as to the identity and
subcellular distribution of sodium channel isoforms in motoneurons. Based on in situ hybridization, the Scn8a mRNA is
expressed at high levels in adult rat motoneurons (Schaller et al.,
1995), and an RNase protection assay indicates that the transcript is also present in the spinal cord of P0 and P7 mice (Fig. 3). Thus, it
seems reasonable to attribute the postnatal increase we have observed
in motoneuronal sodium current density to an increase in
Scn8a transcript within motoneurons, particularly because
the increase does not occur in animals lacking a functional
Scn8a gene. However, it is also possible that
Scn8a activity influences the expression of other sodium
channel genes in motoneurons.
Northern blot analysis has revealed that the rat spinal cord also
contains mRNAs for the RI, RII, and RIII sodium channels, with levels
of RII and RIII being relatively high at birth and subsequently
declining, and levels of RI being low at birth and increasing
postnatally (Beckh et al., 1989). If motoneurons follow the same
general pattern as the spinal cord as a whole, then RII and/or RIII
channels might account for much of the sodium current present in P0
motoneurons of both normal and medtg mice. Moreover,
RI channels have been shown by immunohistochemistry to be present in
adult rat motoneurons (Westenbroek et al., 1989) and may contribute to
the postnatal increase of sodium current density described here.
It is not entirely clear why the absence of Scn8a sodium
channels causes both the cerebellar abnormalities and progressive neuromuscular weakness characteristic of motor end plate disease (Duchen et al., 1967; Duchen, 1970; Duchen and Stefani, 1971). That
both cerebellum and motoneurons are affected is consistent with the
evidence from in situ hybridization that indicates that Scn8a transcript is widely distributed in the CNS, including
motoneurons and cerebellar granule and Purkinje cells (Schaller et al.,
1995; Felts et al., 1997). Interestingly, the neuromuscular weakness in
mice lacking Scn8a is more severe in the proximal
musculature (Duchen, 1970), and the extent of neuromuscular failure
varies considerably from muscle to muscle within a limb (Duchen and
Stefani, 1971; Harris and Pollard, 1986). Thus, the dysfunction in
these mice shows heterogeneity within the motoneuron population.
Consistent with such heterogeneity, we found that some motoneurons from
medtg mice had a sodium current density that was
only slightly subnormal, whereas most had small currents.
Neuromuscular failure in motor end plate disease may be explained if
Scn8a is required for propagation of action potentials along
motor axons or for excitation-secretion coupling at motor nerve
terminals. Duchen and Stefani (1971) demonstrated that stimulating the
biceps nerve in affected mice elicited little or no twitch, although
the response to direct electrical stimulation of the muscle was normal.
Using microelectrode penetrations of the endoneurium, Duchen and
Stefani (1971) were able to record extracellular action potentials from
intramuscular branches of the nerve and thus concluded that peripheral
nerves remained capable of generating and propagating action
potentials, at least down to the intramuscular branches. Intracellular
recordings from end plate regions of paralyzed muscles have revealed
that raising the extracellular potassium from 5 to 20 mM
causes an acceleration of spontaneous neurotransmitter release in
mutant mice similar to that in neuromuscular preparations from normal
mice (Duchen and Stefani, 1971; Harris and Pollard, 1985). This
suggests that the coupling between depolarization and transmitter
release is normal, consistent with our observation of normal calcium
currents in medtg motoneurons.
Because muscle nerves contain sensory fibers in addition to motor axons
(cf. Boyd and Davey, 1968), our observation of reduced sodium currents
in medtg motoneurons may be reconcilable with the
observation of Duchen and Stefani (1971) that propagated action
potentials are detected by subendoneurial recordings. Specifically, it
may be that these action potentials were antidromically propagated via
sensory fibers from the site of stimulation (the cut end of the biceps
nerve) to the site of recording (intramuscular branches of the nerve). Another possible way to reconcile our results and those of Duchen and
Stefani is to postulate that in motor end plate disease, action potential generation in motor nerves is compromised, but only at sites
distal to the major intramuscular branches. In this regard, it is
interesting that extracellular recordings reveal the presence of sodium
channels in the unmyelinated terminals of motor axons (Mallart and
Brigant, 1982; Konishi, 1985). Immunohistochemical studies to determine
whether Scn8a sodium channels are present in nodes of
Ranvier and/or presynaptic terminals will be invaluable for
understanding the function of these channels in normal motoneurons and
the reason that their absence produces motor end plate disease.
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FOOTNOTES |
Received May 8, 1987; revised April 28, 1998; accepted May 4, 1998.
This research was supported by National Institutes of Health Grants
NS34509 (M.H.M.), HL02972 (L.K.S.), and NS24444 (K.G.B.) and a Muscular
Dystrophy Association grant to Jesús García. We thank
Jesús García for assistance in performing some of the measurements of sodium currents.
Correspondence should be addressed to Kurt Beam, Department of Anatomy
and Neurobiology, Colorado State University, Fort Collins, CO
80523-1670.
Dr. García's present address: Department of Physiology and
Biophysics, University of Illinois at Chicago, 900 South Ashland Avenue, Chicago, IL 60607.
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REFERENCES |
-
Beckh S,
Noda M,
Lubbert H,
Numa S
(1989)
Differential regulation of three sodium channel messenger RNAs in the rat central nervous system during development.
EMBO J
8:3611-3616[Web of Science][Medline].
-
Boyd IA,
Davey MR
(1968)
In: Composition of peripheral nerves. Edinburgh: Livingstone.
-
Burgess DL,
Kohrman DC,
Galt J,
Plummer NW,
Jones JM,
Spear B,
Meisler MH
(1995)
Mutation of a new sodium channel gene, Scn8a, in the mouse mutant "motor endplate disease."
Nat Genet
10:461-465[Web of Science][Medline].
-
Dick DJ,
Boakes RJ,
Candy JM,
Harris JB,
Cullen MJ
(1986)
Cerebellar structure and function in the murine mutant "jolting."
J Neurol Sci
76:255-267[Web of Science][Medline].
-
Duchen LW
(1970)
Hereditary motor end-plate disease in the mouse: light and electron microscopic studies.
J Neurol Neurosurg Psychiatry
33:238-250[Free Full Text].
-
Duchen LW,
Stefani E
(1971)
Electrophysiological studies of neuromuscular transmission in hereditary "motor end-plate disease" of the mouse.
J Physiol (Lond)
212:535-548[Abstract/Free Full Text].
-
Duchen LW,
Searle AG,
Strich SJ
(1967)
An hereditary motor end-plate disease in the mouse.
J Physiol (Lond)
189:4-6.
-
Felts PA,
Yokoyama S,
Dib-Hajj S,
Black JA,
Waxman SG
(1997)
Sodium channel -subunit mRNAs I, II, III, NaG, Na6 and hNE (PNL): different expression patterns in developing rat nervous system.
Mol Brain Res
45:71-82[Medline].
-
García KD,
Beam KG
(1996)
Reduction of calcium currents by Lambert-Eaton syndrome sera: motoneurons are preferentially affected and L-type currents are spared.
J Neurosci
16:4903-4913[Abstract/Free Full Text].
-
Gilly WF,
Brismar T
(1989)
Properties of appropriately and inappropriately expressed sodium channels in squid axon and its somata.
J Neurosci
9:1363-1374.
-
Grinnell AD
(1995)
Dynamics of nerve-muscle interaction in developing and mature neuromuscular junctions.
Physiol Rev
75:789-834[Abstract/Free Full Text].
-
Hamill OP,
Marty A,
Neher E,
Sakmann B,
Sigworth FJ
(1981)
Improved patch-clamp techniques for high resolution current recording from cells and cell free membrane patches.
Pflügers Arch
391:85-100[Web of Science][Medline].
-
Harris JB,
Pollard SL
(1985)
Reversal of paralysis in nerve-muscle preparations isolated from animals with hereditary motor endplate disease.
Br J Pharmacol
84:6-8[Web of Science][Medline].
-
Harris JB,
Pollard SL
(1986)
Neuromuscular transmission in the murine mutants "motor end-plate disease" and "jolting."
J Neurol Sci
76:239-253[Web of Science][Medline].
-
Honig MG,
Hume RI
(1986)
Fluorescent carbocyanine dyes allow living neurons of identified origin to be studies in long term cultures.
J Cell Biol
103:171-187[Abstract/Free Full Text].
-
Kohrman DC,
Plummer NW,
Schuster T,
Jones JM,
Jang W,
Burgess DL,
Galt J,
Spear BT,
Meisler MH
(1995)
Insertional mutation of the motor endplate disease (med) locus on mouse chromosome 15.
Genomics
26:171-177[Web of Science][Medline].
-
Kohrman DC,
Harris JB,
Meisler MH
(1996a)
Mutation detection in the med and medJ alleles of the sodium channel Scn8a: unusual patterns of exon skipping are influenced by a minor class AT-AC intron.
J Biol Chem
271:17576-17581[Abstract/Free Full Text].
-
Kohrman DC,
Smith MR,
Goldin AL,
Harris JB,
Meisler MH
(1996b)
A missense mutation in the sodium channel gene Scn8a is responsible for cerebellar ataxia in the mouse mutant jolting.
J Neurosci
16:5993-5999[Abstract/Free Full Text].
-
Konishi T
(1985)
Electrical excitability of motor nerve terminals in the mouse.
J Physiol (Lond)
366:411-421[Abstract/Free Full Text].
-
MacDermott AB,
Westbrook GL
(1986)
Early development of voltage-dependent sodium currents in cultured mouse spinal cord neurons.
Dev Biol
113:317-326[Web of Science][Medline].
-
Mallart A,
Brigant J-L
(1982)
Electrical activity at motor nerve terminals of the mouse.
J Physiol (Paris)
78:407-411[Medline].
-
Milligan CE,
Oppenheim RW,
Schwartz LM
(1994)
Motoneurons deprived of trophic support in vitro require new gene expression to undergo programmed cell death.
J Neurobiol
25:1005-1016[Web of Science][Medline].
-
Mynlieff M,
Beam KG
(1992a)
Characterization of voltage-dependent calcium currents in mouse motoneurons.
J Neurophysiol
68:85-92[Abstract/Free Full Text].
-
Mynlieff M,
Beam KG
(1992b)
Developmental expression of voltage-dependent calcium currents in identified mouse motoneurons.
Dev Biol
152:407-410[Web of Science][Medline].
-
Mynlieff M,
Beam KG
(1994)
Adenosine acting at an A1 receptor decreases N-type calcium current in mouse motoneurons.
J Neurosci
14:3628-3634[Abstract].
-
Pomp D,
Murray JD
(1991)
Single day detection of transgenic mice by PCR of toe-clips.
Mouse Genome
89:279.
-
Raman IM,
Sprunger LK,
Meisler MH,
Bean BP
(1997)
Altered sub-threshold sodium currents and disrupted firing patterns in Purkinje neurons of Scn8a mutant mice.
Neuron
19:881-891[Web of Science][Medline].
-
Schaller KL,
Krzemien DM,
Yarowsky PJ,
Krueger BK,
Caldwell JH
(1995)
A novel, abundant sodium channel expressed in neurons and glia.
J Neurosci
15:3231-3242[Abstract].
-
Smith RG,
Vaca K,
McManaman J,
Appel SH
(1986)
Selective effects of skeletal muscle extract fractions on motoneuron development in vitro.
J Neurosci
6:439-447[Abstract].
-
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].
-
Weinstein SP
(1980)
A comparative electrophysiological analysis study of motor end-plate diseased skeletal muscle in the mouse.
J Physiol (Lond)
307:453-464[Abstract/Free Full Text].
-
Westenbroek RE,
Merrick D,
Catterall WA
(1989)
Differential subcellular localization of the RI and RII Na+ channel subtypes in central neurons.
Neuron
3:695-704[Web of Science][Medline].
Copyright © 1998 Society for Neuroscience 0270-6474/98/18145234-06$05.00/0
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Abstract
Introduction
