Department of Neurology and Paralyzed Veterans of America/Eastern Paralyzed Veterans Association Neuroscience Research Center, Yale University School of Medicine, New Haven 06510; and Rehabilitation Research Center, Veterans Affairs Connecticut Healthcare Center, West Haven, Connecticut 06516
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ABSTRACT |
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Renganathan, Muthukrishnan,
Theodore R. Cummins, and
Stephen G. Waxman.
Contribution of Nav1.8 Sodium Channels to
Action Potential Electrogenesis in DRG Neurons.
J. Neurophysiol. 86: 629-640, 2001.
C-type dorsal root
ganglion (DRG) neurons can generate tetrodotoxin-resistant (TTX-R)
sodium-dependent action potentials. However, multiple sodium channels
are expressed in these neurons, and the molecular identity of the TTX-R
sodium channels that contribute to action potential production in these
neurons has not been established. In this study, we used current-clamp
recordings to compare action potential electrogenesis in
Nav1.8 (+/+) and (/
) small DRG neurons maintained for 2-8 h in vitro to examine the role of sodium channel Nav1.8 (
-SNS) in action potential
electrogenesis. Although there was no significant difference in resting
membrane potential, input resistance, current threshold, or voltage
threshold in Nav1.8 (+/+) and (
/
) DRG
neurons, there were significant differences in action potential
electrogenesis. Most Nav1.8 (+/+) neurons generate all-or-none action potentials, whereas most of
Nav1.8 (
/
) neurons produce smaller graded
responses. The peak of the response was significantly reduced in
Nav1.8 (
/
) neurons [31.5 ± 2.2 (SE) mV] compared with Nav1.8 (+/+)
neurons (55.0 ± 4.3 mV). The maximum rise slope was 84.7 ± 11.2 mV/ms in Nav1.8 (+/+) neurons, significantly
faster than in Nav1.8 (
/
) neurons where it
was 47.2 ± 1.3 mV/ms. Calculations based on the action potential overshoot in Nav1.8 (+/+) and (
/
) neurons,
following blockade of Ca2+ currents, indicate
that Nav1.8 contributes a substantial fraction (80-90%) of the inward membrane current that flows during the rising
phase of the action potential. We found that fast TTX-sensitive Na+ channels can produce all-or-none action
potentials in some Nav1.8 (
/
) neurons but,
presumably as a result of steady-state inactivation of these channels,
electrogenesis in Nav1.8 (
/
) neurons is more sensitive to membrane depolarization than in
Nav1.8 (+/+) neurons, and, in the absence of
Nav1.8, is attenuated with even modest depolarization. These observations indicate that
Nav1.8 contributes substantially to action
potential electrogenesis in C-type DRG neurons.
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INTRODUCTION |
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Dorsal root ganglia
(DRG) neurons, particularly small C-type DRG neurons, as well as other
sensory neurons such as trigeminal and nodose neurons, are unusual in
that they are capable of generating tetrodotoxin-resistant (TTX-R)
action potentials (Yoshida et al. 1978). Studies using
sharp microelectrodes have shown that, in some DRG neurons, these TTX-R
action potentials are sodium dependent (Matsuda et al.
1978
), suggesting that they might be produced by TTX-R
Na+ channels. More recently, patch-clamp studies
have demonstrated that DRG neurons are unique in expressing TTX-R as
well as classical fast TTX-sensitive (TTX-S), Na+
currents (Caffrey et al. 1992
; Cummins et al.
1999
; Elliott and Elliott 1993
; Kostyuk
et al. 1981
; Rizzo et al. 1994
; Roy and Narahashi 1992
). Electrophysiological studies have indicated
that one or more TTX-R sodium channels can support action potential conduction in the unmyelinated C fibers that arise from small DRG
neurons (Jeftinija 1994
; Quasthoff et al.
1995
)
It is now clear from studies at the molecular level that DRG neurons
express multiple sodium channel isotypes (Black et al. 1996). Two neuronal TTX-R sodium channels,
Nav1.8 (SNS) (Akopian et al. 1996
;
Sangameswaran et al. 1996
; see Goldin et al.
2001
, with respect to nomenclature) and
Nav1.9 (NaN) (Dib-Hajj et al. 1998
), both expressed in small DRG neurons, have been cloned; but the molecular identity of the sodium channel that produces TTX-R
sodium-dependent action potentials has thus far not been determined.
Behavioral observations in transgenic Nav1.8-null mice suggest a role of Nav1.8 in inflammatory
pain (Akopian et al. 1999
), and altered pain behavior
after administration of Nav1.8 antisense DNA is
consistent with a role in pain (Gold 1999
;
Porreca et al. 1999
). However, the role of
Nav1.8 in action potential electrogenesis has not
been examined. In this study, we compared small DRG neurons isolated
from transgenic Nav1.8 (+/+) and (
/
) mice
(Akopian et al. 1999
) to examine the contribution of
Nav1.8 to electrogenesis. Our results identify
Nav1.8 as a TTX-R sodium channel that contributes
significantly to the production of sodium-dependent action potentials
in C-type DRG neurons.
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METHODS |
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Animal care
A breeding pair of Nav1.8 (+/) [SNS
(+/
)] mice (Akopian et al. 1999
) was generously
provided by Prof. John Wood, and a colony of
Nav1.8 (+/+) and Nav1.8
(
/
) animals were raised from this pair. Three-month-old
Nav1.8 (+/+) and Nav1.8
(
/
) mice (25-g weight) from the colony were used in this study.
Animals were fed ad libitum and housed in a pathogen-free area at the
Veterans Affairs Medical Center, West Haven. All animals were
individually genotyped prior to death. Animal care and surgical
procedures followed a protocol approved by the Animal Care and Use
Committee of Yale University.
Culture of dorsal root ganglia (DRG) neurons
Mice were rendered unconscious by exposure to rising
concentrations of CO2 and decapitated. L4 and
L5 lumbar DRG were freed from their connective tissue
sheaths in sterile calcium-free saline solution. Cell cultures were
prepared as previously described (Renganathan et al.
2000b). Briefly, the L4 and L5 DRG
ganglia were harvested, treated with collagenase and papain, and
dissociated in DMEM and Ham's F12 medium supplemented with 10% fetal
bovine serum. The DRG neurons were plated on polyornithine and
laminin-coated glass coverslips. Neurons were placed in a 5%
CO2-95% O2 incubator at 37°C and, 1 h
after isolation, were fed with fresh culture medium. DRG neurons were
studied using patch clamp 2-8 h after isolation because at this time
in culture, neurons had not yet sprouted neurites and yielded a better
seal and a higher yield of high-quality recordings. Only C-type DRG
neurons (20-25 µm diam) were used in this study.
Electrophysiological recordings
Coverslips were mounted in a series 20 recording/perfusion
chamber (Warner Instruments, Hamden, CT) positioned on the stage of a
Nikon Diaphot microscope (Nikon) and were continuously perfused with
the bath-external solution (see following text) with a push-pull syringe pump (WPI, Saratoga, FL). Cells were current-clamped via the
whole cell configuration of the patch clamp with an Axopatch-200B amplifier (Axon Instruments, Foster City, CA) using standard
techniques. Micropipettes were pulled from borosilicate glasses
(Boralex) with a Flaming Brown micropipette puller (P80, Sutter
Instrument, Novato, CA) and polished on a microforge (Narishige, Tokyo)
to obtain electrode resistances ranging from 1 to 2.5 M. The pipette solution contained (in mM) 140 KCl, 0.5 EGTA, 5 MgATP, and 5 HEPES, pH
7.3 with KOH. To determine the contribution of
Na+ currents produced by
Nav1.8 to peak action potential amplitude, 100 µM CdCl2 was used to block
Ca2+ currents, and in some experiments, 5 mM
Na-HEPES was used instead of HEPES to elevate the intracellular
Na+ concentration to 5 mM. The following bath
solution was used (in mM): 140 NaCl, 3 KCl, 1 MgCl2, 1 CaCl2, and 10 HEPES, pH 7.3. The pipette potential was zeroed before seal formation.
Liquid junction potentials were
4 mV and were not corrected. Whole
cell membrane voltage changes were filtered at 5 kHz and acquired at 20 kHz by a computer using pCLAMP 8.1 software (Axon Instruments). A
Digidata 1200B interface (Axon Instruments) was used for A-D conversion, and the data were stored on compact disk and analyzed using
pCLAMP 8.1 software (Axon Instruments). Experiments were carried out at
a room temperature of 22-25°C.
As shown in Fig. 4, action potential characteristics were
affected by resting potential. Therefore only cells with a stable resting potential up to 45 mV were used in this study. Action potentials were detected in 100% of cells in response to
suprathreshold current injections. Input resistance was calculated by
recording voltage changes by injection of
10-pA hyperpolarizing
currents. Action potential threshold was measured at the beginning of
the sharp upward rise of the depolarizing phase of the action
potential. Current threshold was determined by a series of depolarizing
currents from 0 to 250 pA in 10-pA step increments. The waveform
characteristics of the action potentials recorded from
Nav1.8 (+/+) and (
/
) small DRG neurons, i.e.,
maximum rise slope, maximum fall slope, rise time, decay time, action
potential duration, were determined using Clampfit 8.1 software
(Axon Instruments). Repetitive firing of action potentials was measured
by recording voltage changes in response to sustained (1 s) injection
of depolarizing currents.
Statistics
All results are expressed as means ± SE. Statistical significance was evaluated using Student's t-test. P < 0.05 was considered significant.
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RESULTS |
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The principal aim of this study was to investigate the role of
Nav1.8 channels in action potential
electrogenesis. Because the slow TTX-R Na+
currents produced by Nav1.8
Na+ channels are present in >90% of wild-type
small C-type DRG neurons (Cummins and Waxman 1997;
Cummins et al. 1999
) but are absent in C-type neurons
from Nav1.8 (
/
) mice (Akopian et al.
1999
; Cummins et al. 1999
), we compared C-type
DRG neurons isolated from Nav1.8 (+/+) wild-type
and (
/
) null mice in this study.
C-type DRG neurons can generate TTX-R action potentials
Nav1.8 (+/+) C-type DRG neurons
generated action potentials with long duration (mean duration 3.87 ± 0.29 ms measured at 0 mV) and peaking at about +55 mV (Fig.
1A). Digital differentiation revealed two peaks in the falling phase of somatic action potential (Fig. 1A, inset), indicating the presence of
inflection in most cells studied. The long action potential duration
and inflection have been associated with small neurons within intact
DRGs (Harper and Lawson 1985; Villiere and
McLachlan 1996
; Waddell and Lawson 1990
), and
their presence suggests that isolation did not alter fundamental
electrophysiological features of the sensory neurons examined in our
study. Consistent with earlier sharp microelectrode recordings in mouse
DRG neurons (Matsuda et al. 1978
), action potentials
were still evoked in four of five small neurons when 250 nM TTX was
added to the bath solution, and the peak action potential amplitude did
not decrease on addition of TTX (Fig. 1B). The inflection in
the falling phase was still observed in the presence of TTX (Fig.
1B, inset), demonstrating that inflections can
occur in the absence of TTX-S Na+ currents.
Further, repetitive firing of action potentials was observed in the
presence of 250 nM TTX (Fig. 1C). These results demonstrate
the presence of TTX-R action potentials in C-type neurons.
|
Resting potential, input resistance, action potential threshold,
and current threshold are unchanged in Nav1.8 (/
)
DRG neurons
The mean resting membrane potential was 52.9 ± 1.2 mV
(n = 32) in Nav1.8 (+/+) C-type
neurons and
53.7 ± 1.5 mV (n = 42) in
Nav1.8 (
/
) DRG neurons (P > 0.05, Student's t-test, Fig. 2A). The input resistance for
Nav1.8 (+/+) DRG neurons was 521.3 ± 43.6 M
(n = 32) and for Nav1.8
(
/
) DRG neurons was 596.9 ± 37.8 M
(n = 42; Fig. 2B). No spontaneous firing of action potentials was
observed either in Nav1.8 (+/+) or
Nav1.8 (
/
) DRG neurons at resting membrane
potential. On injection of depolarizing current, all
Nav1.8 (+/+) and (
/
) neurons generated
overshooting responses. Action potential threshold for
Nav1.8 (+/+) and (
/
) DRG neurons was
26.9 ± 1.5 mV (n = 32) and
26.8 ± 1.7 mV (n = 42), respectively (Fig. 2C). The
current threshold required to elicit action potentials for
Nav1.8 (+/+) and (
/
) neurons was 68.2 ± 11.9 pA (n = 32) and 65.0 ± 19.8 pA
(n = 42), respectively (Fig. 2D).
|
Action potential characteristics are different in
Nav1.8 (/
) DRG neurons
Action potentials elicited from Nav1.8 (+/+)
and (/
) C-type neurons peaked at +55.0 ± 4.3 mV
(n = 32 from 7 different preparations) and +31.5 ± 2.2 mV (n = 42 from 7 different preparations),
respectively (Fig. 2E). The decrease in action potential
amplitude in Nav1.8 (
/
) C-type DRG neuron is
statistically significant (P < 0.0001). These results
demonstrate that TTX-R Na+ currents produced by
Nav1.8 Na+ channels
contribute to action potential overshoot in small C-type DRG neurons.
The action potential characteristics of Nav1.8
(+/+) and (/
) neurons are summarized in Table
1. The rate of depolarization is 80%
faster in Nav1.8 (+/+) neurons than
Nav1.8 (
/
) neurons, which is reflected in a
smaller 10-90% rise time for Nav1.8 (+/+) neurons. These results suggest that a current component that
contributes to the rate of depolarization is missing in
Nav1.8 (
/
) neurons. Other parameters,
including rate of repolarization, decay time, and half-width duration
in Nav1.8 (+/+) and (
/
) neurons, did not show
statistically significant differences, although half-width was 13%
larger in Nav1.8 (
/
) neurons. Because action
potential amplitude is smaller in Nav1.8 (
/
)
neurons, we also determined the action potential duration at 0 mV and
found no significant difference between Nav1.8
(+/+) and (
/
) neurons.
|
Most Nav1.8 (+/+) neurons produce all-or-none overshooting action potentials
Most Nav1.8 (+/+) small DRG neurons evoked
all-or-none action potentials (Fig.
3A). That is, on injection of
depolarizing current, these neurons displayed action potentials with
amplitude of ~115 mV (55-60 mV), and on further depolarization,
the action amplitude did not increase. This pattern of all-or-none
action potential was observed in 84% (27 of 32) of the DRG neurons.
Mean resting potential in this subgroup of neurons was
55.5 ± 0.9 mV. The action potential amplitude in these neurons peaked at +59.2 ± 3.9 mV. In contrast, in 5 of 32 small DRG neurons,
depolarization evoked smaller graded responses (Fig. 3B),
which tended to peak at an average potential of +26.7 ± 0.9 mV.
With graded depolarization, these neurons initially generated responses
with a peak close to 0 mV, which increased to about +30 mV on further
depolarization. These graded responses were observed in neurons with
resting potentials of
48.2 ± 1.2 mV, where most of the TTX-S
fast Na+ channels in these neurons are inactive
(Cummins and Waxman 1997
; Renganathan et al.
2000a
) and only a few channels are able to participate in the
generation of action potentials. Figure
4, A and B, shows a
cell that initially exhibited a depolarized resting potential, close to
30 mV, and small graded responses to depolarizing stimuli immediately
after whole cell configuration was established. After 4 min, resting
potential shifted to
60 mV and large, overshooting (+68 mV) action
potentials were seen. These observations suggest that in some
Nav1.8 (+/+) neurons, TTX-S fast
Na+ channels can produce all-or-none action
potentials but, presumably as a result of steady-state inactivation of
these channels, electrogenesis in these neurons is more sensitive to
membrane depolarization.
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|
Most Nav1.8 (/
) neurons produce small graded
responses
Thirty-two of 42 small DRG neurons isolated from
Nav1.8 (/
) null mice produced small graded
responses in response to depolarizing stimuli when studied immediately
after establishing whole cell configuration (Fig. 3C),
unlike the pattern of all-or-none action potentials observed in most
Nav1.8 (+/+) neurons. The mean resting potential
in this majority of the neurons was
49.5 ± 0.9 mV. The graded
responses in Nav1.8 (
/
) neurons peaked at a
maximum of +24.6 ± 1.4 mV. These results demonstrate that in the
absence of slow TTX-R Na+ currents produced by
Nav1.8, the ability of most
Nav1.8 (
/
) neurons to generate overshooting
all-or-none action potentials is impaired.
In 10 of 42 small Nav1.8 (/
) DRG neurons
studied immediately after establishing whole cell configuration,
depolarizing stimuli evoked all-or-none action potentials that peaked
at 45.9 ± 1.2 mV (Fig. 3D), significantly higher than
observed in the other 32 Nav1.8 (
/
) neurons
(P < 0.0001). The larger action potential amplitude
and all-or-none behavior in this population of
Nav1.8 (
/
) neurons may reflect their
relatively hyperpolarized resting potentials (
65.1 ± 0.9 mV for
this subgroup of neurons) compared with the other 32 neurons, which as
noted in the preceding text, had resting potential amplitude of
49.5 ± 0.9 mV. At the more hyperpolarized resting potential,
~50% of the fast TTX-S Na+ channels would be
expected to be available for activation (see Cummins and Waxman
1997
) and therefore able to contribute to the all-or-none
action potential overshoot. We tested this hypothesis in a
Nav1.8 (
/
) neuron whose resting potential was
70 mV immediately after establishing whole cell configuration. At
this resting potential (Fig. 4C), this cell generated
all-or-none action potentials with a peak at +55 mV. When we
depolarized the cell and held it at a resting potential of
50 mV, the
cell generated smaller graded responses (Fig. 4D).
TTX-S Na+ channels produce all-or-none action
potentials in Nav1.8 (/
) neurons
To determine whether TTX-S Na+ channels
produce all-or-none action potentials in Nav1.8
(/
) neurons, Nav1.8 (
/
) neurons that
generated all-or-none action potentials were exposed to 250 nM TTX.
Figure 5 shows recordings from a
representative Nav1.8 (
/
) neuron and
illustrates action potential generation in the absence of TTX (Fig.
5A) and the failure of action potential electrogenesis in
the presence of 250 nM TTX (Fig. 5B). In the absence of TTX, the neuron produced action potentials with peak at +60 mV, and on
addition of TTX, the neuron produced subthreshold membrane depolarizations but did not generate action potentials on depolarizing current injection. Similar results were obtained in 3/3
Nav1.8 (
/
) neurons exposed to 250 nM TTX.
These results indicate that TTX-S Na+ channels
can produce all-or-none action potentials in
Nav1.8 (
/
) neurons.
|
Inflected responses can occur in the absence of Nav1.8 Na+ channels
Action potentials in C-type DRG neurons have been shown to have a
shoulder (inflection) on the falling phase (Gold et al. 1996a; Harper and Lawson 1985
; Villiere
and McLachlan 1996
; Waddell and Lawson 1990
).
C-cell action potentials have been suggested to be TTX resistant
(Villiere and McLachlan 1996
), while all those without
inflections have been reported to be TTX sensitive (Villiere and
McLachlan 1996
; Waddell and Lawson 1990
).
Axotomized cutaneous neurons display a reduction in TTX-R
Na+ current and an increase in TTX-S
Na+ current with a reduction in the number of
neurons that had inflections on the falling phase of the action
potential (Oyelese et al. 1997
). Taken together, these
results might be interpreted as suggesting that the presence of
Nav1.8 Na+ currents endows
DRG neurons with inflections on the falling phase of the action
potential. However, we found that most (37 of 42) Nav1.8 (
/
) neurons displayed an inflection or
hump on the falling phase, and only a few (5 of 42) neurons had simple,
uninflected falling phase of the action potential. A broad plateau or
hump on the falling phase was observed in 7 of 42 Nav1.8 (
/
) neurons (Fig.
6), while less pronounced inflections in
the falling phase was observed in 30 of 42 Nav1.8
(
/
) neurons (Fig. 8, A and B). These results
demonstrate that Nav1.8 is not required for an
inflection in the falling phase of the somatic action potential.
|
Ca2+ channels can contribute to inflection of the action potential in Nav1.8 (+/+) neurons
A majority of Nav1.8 (+/+) neurons (24/29) showed inflections on the falling phase of the action potential. To determine whether Ca2+ channels contribute to inflection on the falling phase of the action potential, Nav1.8 (+/+) neurons were depolarized to evoke action potentials in the absence and presence of 100 µM Cd2+, a concentration that effectively blocks Ca2+ currents in these neurons. Figure 7A illustrates an action potential evoked from a typical Nav1.8 (+/+) neuron in the absence of 100 µM Cd2+. Its derivative, which indicates the corresponding rate of depolarization, is given in Fig. 7B. The presence of two peaks on the falling phase of the derivative is due to the presence of inflection on the repolarization phase of action potential. The same neuron, when exposed to 100 µM Cd2+, generates action potentials with similar peak amplitude (Fig. 7C), but the falling phase depicts only one peak (Fig. 7D). Similar results were obtained in 4/5 neurons studied. These results demonstrate that the inflection on the falling phase of the action potential can be produced by calcium currents in Nav1.8 (+/+) neurons. In one neuron, Cd2+ did not eliminate the inflection on the falling phase, suggesting that inflection can occur in the absence of calcium currents in some DRG neurons. Peak action potential amplitude was not significantly decreased in Nav1.8 (+/+) neurons by exposure to Cd2+. The action potential in the absence and presence of Cd2+ peaked at 59.6 ± 1.8 and 56.7 ± 4.3 mV, respectively (P < 0.05). The rate of depolarization, repolarization and half-width duration were not significantly different in the presence of Cd2+ in Nav1.8 (+/+) neurons (Table 2).
|
|
Ca2+ channels produce overshooting action potentials in
some Nav1.8 (/
) neurons
Responses were elicited from Nav1.8 (/
)
neurons (n = 10) in the absence and presence of 100 µM Cd2+ to determine whether
Ca2+ channels contribute to action potential
electrogenesis. Action potentials elicited from a
Nav1.8 (
/
) neuron in the absence of
Cd2+ are shown in Fig.
8A. On exposure to 100 µM
Cd2+, the same neuron elicited graded responses
with smaller amplitude and no clear threshold (Fig. 8B). In
8/10 neurons studied, Cd2+ had this effect. The
response in the absence and presence of Cd2+
peaked at 27.5 ± 2.5 and 5.3 ± 3.2 mV (n = 8, P < 0.0001), respectively. The rate of
depolarization and repolarization, but not the half-width duration,
were significantly reduced in the presence Cd2+
in SNS (
/
) neurons (Table 2). These results suggest that
Ca2+ currents can contribute significantly to
electrogenesis in Nav1.8 (
/
) neurons.
|
Temporal patterns of action potential generation are different in
Nav1.8 (/
) and Nav1.8 (+/+) neurons
Although injection of sustained depolarization currents evoked
repetitive firing in ~40% of Nav1.8 (+/+) and
(/
) neurons, the pattern of firing was different. Repetitive firing
from a typical Nav1.8 (+/+) neuron is shown in
Fig. 9, A and B,
while firing of a representative Nav1.8 (
/
)
neuron is shown in Fig. 9, C and D. The current
protocols that elicited these responses are illustrated in Fig. 9,
E and F. The Nav1.8 (+/+)
neuron generated repetitive firing of action potentials at a frequency
of 9 Hz (Fig. 9A) on injection of 75-pA current for 1 s. With a stronger depolarizing current injection (150 pA), the same
neuron evoked action potentials at a higher frequency (14 Hz; Fig.
9B). Repetitive firing of Nav1.8
(
/
) neurons was not as robust as in Nav1.8 (+/+) neurons; the number of spikes was smaller, and they either occurred in bursts (Fig. 9C) or the spike train aborted
after firing a few spikes (Fig. 9D). In addition, the spike
amplitudes were smaller and the later spikes tended to fall off in
amplitude in Nav1.8 (
/
) neurons. In
Nav1.8 (+/+) and (
/
) neurons, the peak
amplitude for the first spike was 63.4 ± 1.3 mV
(n = 8) and 22.5 ± 3.6 mV (n = 13, P < 0.003), respectively. The peak amplitude for
the fourth spike in Nav1.8 (+/+) and (
/
)
neurons was 59.1 ± 0.9 and 0.6 ± 3.1 mV, respectively
(P < 0.00001). The action potential amplitude for the
fourth spike displayed a small but statistically significant reduction
(4%) compared with the first spike in Nav1.8
(+/+) neurons, but the decrease was much more dramatic (30%) in
Nav1.8 (
/
) neurons. These results suggest that Nav1.8 contributes to secure repetitive
firing in DRG neurons.
|
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DISCUSSION |
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In this study, we compared action potentials generated by small
DRG neurons from Nav1.8 (+/+) and (/
) mice to
investigate the role of Nav1.8 channels in action
potential electrogenesis in these neurons. Our experiments did not
reveal significant differences in resting membrane potential, input
resistance, current threshold, or voltage threshold in
Nav1.8 (+/+) and (
/
) DRG neurons. However, there were major differences in action potential electrogenesis between
Nav1.8 (+/+) and (
/
) DRG neurons. The peak
action potential response was significantly reduced and the rate of
depolarization was substantially slower in Nav1.8
(
/
) neurons compared with Nav1.8 (+/+)
neurons. We also observed different temporal patterns of spike
generation in Nav1.8 (+/+) and (
/
) neurons
with robust and continuous firing in response to sustained
depolarization in Nav1.8 (+/+) neurons while
frequency-related failure was observed in Nav1.8
(
/
) neurons. These results indicate that
Nav1.8 TTX-R Na+ channels
can play an important role in generating and maintaining the action
potential in C-type DRG neurons in contrast to
Nav1.9 TTX-R Na+ channels,
which are thought to contribute to modulating resting potential
(Herzog et al. 2001
).
Nav1.8 mRNA (Dib-Hajj et al. 1996)
and protein (Sleeper et al. 2000
) as well as the slowly
inactivating TTX-R Na+ currents attributable to
Nav1.8 (Cummins and Waxman 1997
)
are all downregulated in DRG neurons following axotomy within the sciatic nerve. The absence of significant differences in resting membrane potential and input resistance in Nav1.8
(+/+) and (
/
) neurons is similar to the results obtained in DRG
neurons isolated from control uninjured and axotomized neurons
(Zhang et al. 1997
). The absence of difference in
somatic action potential threshold observed between
Nav1.8 (+/+) and (
/
) DRG neurons, however, is
different from the results obtained in axotomized neurons (Zhang et al. 1997
, 1999
), where a reduction in action potential
threshold was seen. In electrophysiological studies that measured
dorsal root compound action potentials in response to sciatic nerve
stimulation, Akopian et al. (1999)
observed a small
(~20% for maximal compound action potential) decrease in C-fiber
threshold in Nav1.8 (
/
) compared with
Nav1.8 (+/+) mice. The disparity between this
result and the findings in the present study may reflect a difference in channel expression in neuronal somata compared with axons. Previous
studies have also demonstrated an upregulation of mRNA for
Nav1.7 that encodes a TTX-S
Na+ channel (Akopian et al. 1999
)
and TTX-S fast Na+ currents (Akopian et
al. 1999
; Renganathan et al. 2000b
) in
Nav1.8 (
/
) neurons. These compensatory
changes may maintain or even lower the threshold of these neurons.
We found that TTX-S Na+ channels can produce
all-or-none action potentials in some Nav1.8
(/
) neurons. The Nav1.8 (
/
) neurons that
generated all-or-none action potentials had a more hyperpolarized resting potential than the neurons that generated only graded responses. This indicates that electrogenesis in
Nav1.8 (
/
) neurons is more sensitive to
resting potential than is electrogenesis in
Nav1.8 (+/+) neurons. As noted in the following
text, this may be due to the relatively hyperpolarized voltage
dependence of steady-state inactivation of the TTX-S channels that are
expressed in DRG neurons (see e.g., Cummins and Waxman
1997
; Cummins et al. 1998
).
Nav1.8 (/
) and action potential inflection
We observed that Nav1.8 (/
) neurons
generate action potentials with inflections on the falling phase. Thus
Nav1.8 is not required for these inflections.
Na+ channels other than
Nav1.8 (Black et al. 1996
), or
Ca2+ channels (Baccei and Kocsis
2000
) and K+ channels (Gold et al.
1996b
) in small DRG neurons may in theory contribute to these
inflections. We found that in some neurons, Ca2+
channels can contribute to inflections in the action potential. While
inflections in the action potential have been used to classify DRG
neurons into distinct groups (Gold et al. 1996b
;
Harper and Lawson 1985
; Villiere and McLachlan
1996
; Waddell and Lawson 1990
), our results
indicate that the presence of inflections per se cannot be attributed
to the activity of any single channel isotype.
Different action potential characteristics in Nav1.8
(/
) DRG neurons
Action potentials in Nav1.8 (/
) neurons
displayed a significantly lower peak, (+31.5 ± 2.2 mV) compared
with Nav1.8 (+/+) neurons (+55.0 ± 4.3 mV;
P < 0.0001). One explanation for this is that
Nav1.8 channels in Nav1.8
(+/+) cells are open at the peak of the action potential, adding to the
total PNa. A slower rate of membrane
potential depolarization in Nav1.8 (
/
)
neurons (Table 1) further suggests that Nav1.8
channels contribute to the magnitude of the action potential in
Nav1.8 (+/+) neurons.
Peak action potential amplitude determined in the absence of
Ca2+ channel activity, i.e., in the presence of
100 µM Cd2+, can be used to estimate the ratio
of PNa (Na+
channel permeability) in Nav1.8 (/
) and (+/+)
neurons, i.e., the ratio of PNa(
/
)
to PNa(+/+). According to the
Goldman-Hodgkin-Katz equation
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Under our experimental conditions, the reversal potentials for
K+ and Na+ ions are 98.0
and +85.0 mV, respectively. The peak action potential overshoots in
Nav1.8 (+/+) and (
/
) neurons in the presence
of Cd2+ are 56.7 ± 4.3 mV
(n = 8) and 5.3 ± 3.2 mV (n = 8),
respectively. These values yield
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The present results indicate that Nav1.8
participates in action potential electrogenesis in the cell bodies of
small DRG neurons. Although we have no direct evidence concerning the
role of Nav1.8 within the axons of these neurons,
immunocytochemical studies demonstrate the presence of
Nav1.8 within the superficial laminae of the
spinal cord where the central projections of the C-type neurons
terminate (Novakovic et al. 1998). Scroggs and
Fox (1992)
used action potential waveforms as stimuli and found
that activation of N-type calcium channels is highly dependent on
action potential shape and amplitude. The effects of nerve injury on
Nav1.8 expression in the axon terminals of DRG
neurons have not been studied, but it is known that axotomy produces
long-lasting decreases in expression of Nav1.8
mRNA within DRG neurons (Dib-Hajj et al. 1996
). We have demonstrated (Fig. 3 and Table 1) that in C-type DRG neurons that lack
Nav1.8, amplitudes of action potentials are
decreased. Moreover action potential amplitude in
Nav1.8 (
/
) neurons is especially sensitive to
resting potential (Fig. 4), consistent with the voltage dependence of
steady-state inactivation of the TTX-S Na+
channels in DRG neurons (Cummins and Waxman 1997
;
Cummins et al. 1998
). This raises the question of
whether action potential waveform at the central terminals of C-type
DRG neurons is altered with a resultant change in the efficacy of
transmission to second-order sensory neurons as a result of reduced
Nav1.8 transcription after nerve injury.
In addition, we observed different temporal patterns of action
potential generation in Nav1.8 (+/+) and (/
)
neurons. Nav1.8 Na+
channels exhibit rapid recovery from inactivation (Cummins and Waxman 1997
; Elliott and Elliott 1993
); this
appears to be due, at least in part, to the presence of a specific
tripeptide insertion in the D4S3-S4 linker (Dib-Hajj et al.
1997
). This rapid recovery from inactivation, coupled with the
relatively depolarized voltage dependence of steady-state inactivation
observed for Nav1.8 Na+
channels (Akopian et al. 1996
), might allow neurons that
express Nav1.8 channels to sustain repetitive
firing at depolarized membrane potentials. Upregulation of
Nav1.8 expression has been observed in cerebellar
Purkinje cells in two experimental models of multiple sclerosis
(Black et al. 1999
, 2000
) and in multiple sclerosis (Black et al. 2000
). Although the physiological
concomitants of abnormal Nav1.8 expression in
Purkinje cells have not yet been directly studied, Black et al.
(2000)
speculated that the abnormal expression of
Nav1.8 could alter the pattern of electrogenesis, thereby contributing to clinical abnormalities such as cerebellar ataxia. We observed different patterns of action potential
electrogenesis, in response to the same stimulus, in
Nav1.8 (+/+) and (
/
) DRG neurons. While
compensatory changes in the expression of other channels in the
Nav1.8 (
/
) neurons (Akopian et al.
1999
) may also contribute to these differences, the altered
pattern of action potential generation in Nav1.8
(+/+) as compared with Nav1.8 (
/
) DRG neurons
is consistent with the idea that Nav1.8
expression can modify the pattern of firing in neurons where it is expressed.
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ACKNOWLEDGMENTS |
---|
A breeding pair of Nav1.8 (+/) [SNS (+/
)] mice
was kindly provided by Prof. J. N. Wood, University College
London. We thank Drs. Joel Black and Sulayman Dib-Hajj for establishing
and genotyping the colony of Nav1.8 (
/
) mice and B. Toftness for help with figures.
This work was supported in part by grants from the Medical Research Service and Rehabilitation Research Service, Department of Veterans Affairs and from the National Multiple Sclerosis Society. We also thank the Eastern Paralyzed Veterans Association and the Paralyzed Veterans of America for support, including a grant which supports the Yale-London collaboration.
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FOOTNOTES |
---|
Address for reprint requests: S. G. Waxman, Dept. of Neurology, LCI 707, Yale Medical School, 333 Cedar St., New Haven, CT 06510 (E-mail: stephen.waxman{at}yale.edu).
Received 8 January 2001; accepted in final form 23 April 2001.
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REFERENCES |
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