Department of Neurology and Paralyzed Veterans Association/Eastern Paralyzed Veterans Association Neuroscience Research Center, Yale Medical School, 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, M.,
T. R. Cummins,
W. N. Hormuzdiar, and
S. G. Waxman.
-SNS Produces the Slow TTX-Resistant Sodium Current in Large
Cutaneous Afferent DRG Neurons.
J. Neurophysiol. 84: 710-718, 2000.
In this study, we used sensory
neuron specific (SNS) sodium channel gene knockout (
/
) mice to ask
whether SNS sodium channel produces the slow Na+
current ("slow") in large (>40 µm diam) cutaneous afferent
dorsal root ganglion (DRG) neurons. SNS wild-type (+/+) mice were used as controls. Retrograde Fluoro-Gold labeling permitted the definitive identification of cutaneous afferent neurons. Prepulse inactivation was
used to separate the fast and slow Na+ currents.
Fifty-two percent of the large cutaneous afferent neurons isolated from
SNS (+/+) mice expressed only fast-inactivating Na+ currents ("fast"), and 48% expressed
both fast and slow Na+ currents. The fast and
slow current densities were 0.90 ± 0.12 and 0.39 ± 0.16 nA/pF, respectively. Fast Na+ currents were
blocked completely by 300 nM tetrodotoxin (TTX), while slow
Na+ currents were resistant to 300 nM TTX,
confirming that the slow Na+ currents observed in
large cutaneous DRG neurons are TTX-resistant (TTX-R). Slow
Na+ currents could not be detected in large
cutaneous afferent neurons from SNS (
/
) mice; these cells expressed
only fast Na+ current, and it was blocked by 300 nM TTX. The fast Na+ current density in SNS
(
/
) neurons was 1.47 ± 0.14 nA/pF, approximately 60% higher
than the current density observed in SNS (+/+) mice (P < 0.02). A low-voltage-activated TTX-R Na+
current ("persistent") observed in small C-type neurons is not present in large cutaneous afferent neurons from either SNS (+/+) or
SNS (
/
) mice. These results show that the slow TTX-R
Na+ current in large cutaneous afferent DRG is
produced by the SNS sodium channel.
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INTRODUCTION |
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Different repertoires of Na+
currents appear to endow different functional classes of dorsal root
ganglion (DRG) neurons with distinct encoding and/or transducing
properties. Thus it is important to establish the molecular identities
of the channels responsible for each of the Na+
currents in various classes of DRG neuron. It is now well established that, in addition to fast-activating and -inactivating ("fast") tetrodotoxin (TTX)-sensitive (TTX-S) Na+ current,
DRG neurons express slow ("slow") TTX-resistant (TTX-R) Na+ currents (Caffrey et al. 1992;
Elliott and Elliott 1993
; Kostyuk et al.
1981
; Roy and Narahashi 1992
; Scholz et
al. 1998
). More recently, a novel persistent TTX-R
Na+ current has been demonstrated in small C-type
DRG neurons (Cummins et al. 1999
; Dib-Hajj et al.
1999a
). To date, 11 distinct sodium channel
-subunits,
encoded by different genes, have been cloned (Plummer and
Meisler 1999
). Five of the sodium channel
-subunits are
expressed in DRG neurons (Black et al. 1996
). These
include two distinct TTX-R sodium channel
-subunits that are
preferentially expressed in DRG neurons, the first termed sensory
neuron specific (SNS) (Akopian et al. 1996
) or PN3
(Sangameswaran et al. 1996
) and the second termed NaN
(Dib-Hajj et al. 1998
) or SNS2 (Tate et al.
1998
). SNS encodes a slow TTX-R Na+
current when expressed in oocytes (Akopian et al. 1996
;
Sangameswaran et al. 1996
). Development of a transgenic
mouse model, in which the SNS gene has been knocked out, has made it
possible to establish definitively that the SNS channel produces the
slow TTX-R Na+ current in one of the normal host
cells in which it is expressed, i.e., in small C-type DRG neurons
(Akopian et al. 1999
). Cummins et al.
(1999)
have demonstrated the presence of the persistent TTX-R
Na+ current, presumably encoded by NaN channels,
in C-type DRG neurons in SNS knockout mice.
To date, studies on the physiological role of specific
TTX-R sodium channel isoforms (SNS and NaN) have focused on small
C-type DRG neurons. However, it is well established that a slow TTX-R Na+ current is also present in some subsets of
large DRG neurons. In particular, a slow TTX-R
Na+ current is seen in large cutaneous afferent
DRG neurons, although it is not present in muscle afferent neurons
(Honmou et al. 1994). While a one-to-one correspondence
with a specific channel isoform has been accomplished for the slow and
persistent TTX-R Na+ currents in small C-type DRG
neurons (Akopian et al. 1999
; Cummins et al.
1999
), the molecular identity of the channel producing the slow
TTX-R Na+ current in large cutaneous afferent
neurons has not yet been established.
In this study, we used SNS knockout (/
) mice to determine the
identity of the channel that is responsible for the slow TTX-R Na+ current in large cutaneous afferent neurons.
Retrograde labeling permitted us to definitively identify cutaneous
afferent neurons. Our results indicate that the slow TTX-R
Na+ current in large cutaneous neurons is
produced by
-SNS channels.
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METHODS |
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Animal care
A breeding pair of SNS (+/) mice (Akopian et al.
1999
) was generously provided by Prof. John Wood and a colony
of SNS (+/+) and SNS (
/
) animals were raised from this pair. Three
month-old SNS (+/+) and SNS (
/
) 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.
Animal care and surgical procedures followed a protocol approved by the
Animal Care and Use Committee of Yale University.
Fluoro-Gold labeling of cutaneous neurons
DRG cell bodies giving rise to cutaneous afferent fibers were
identified by retrograde labeling with Fluoro-Gold. Five to 10 µl of
4% solution of Fluoro-Gold dissolved in distilled water was injected
sub-cutaneously in the lateral plantar region of both legs 1 wk before
sacrifice for culture preparation (Honmou et al. 1994).
Culture of DRG neurons
The mice were exsanguinated under Ketamine/Xylazine anesthesia (38/5 mg/kg ip). Briefly, L4 and L5 lumbar DRG were freed from their connective sheaths in sterile calcium-free saline solution. The DRGs were then enzymatically digested for 15 min with collagenase A (1 mg/ml; Boehringer-Mannheim, Indianapolis, IN) in complete saline solution (CSS) containing 0.5 mM EDTA and 2 mg cysteine, and for 15 min with collagenase D (1 mg/ml; Boehringer-Mannheim, Indianapolis, IN) and papain (30 units/ml, Worthington Biochemical, Lakewood, NJ) in CSS containing 0.5 mM EDTA and 2 mg cysteine at 37°C. DRGs were carefully removed by pasteur pipette from the other cellular debri and placed in DRG culture medium (DMEM and F12 in a ratio of 1:1, 10% fetal calf serum, 100 U/ml penicillin and 0.1 mg/ml streptomycin) containing 1 mg/ml bovine serum albumin (Fraction V, Sigma Chemicals, St. Louis, MO) and 1 mg/ml trypsin inhibitor (Sigma Chemicals, St. Louis, MO). The DRGs were mechanically dispersed (5 strokes up and down) with a 1-ml pipette and plated on polyornithine and laminin-coated glass coverslips (100 µl suspension per coverslip). Neurons were placed in a 5% CO2-95% O2 incubator at 37°C and, 1 h after isolation, were fed with fresh culture medium. Na+ current properties in DRG neurons were investigated between 1 and 8 h after isolation. Short-term culture provided cells with truncated (<10 µm) axonal processes that can be voltage clamped readily and reliably, allowed the cells sufficient time to adhere to the glass coverslips, and were short enough to minimize changes in electrical properties that can occur in long-term cultures.
Electrophysiological recordings
Coverslips were mounted in a small flow-through chamber positioned on the stage of a Nikon Diaphot microscope (Nikon, Melville, NY) and were continuously perfused with the bath-external solution (see next paragraph) with a push-pull syringe pump (WPI, Saratoga, FL). Fluoro-Gold-labeled neurons were identified by fluorescence emission above 420 nm. A short arc mercury lamp was used as the excitation source, and it was defined by broadband (330-380 nm) pass filter (Nikon). Large (40-45 µm diam) Fluoro-Gold-labeled neurons were studied.
Cells were voltage clamped via the whole cell configuration of the
patch-clamp with an Axopatch-200B amplifier (Axon Instruments, Foster
City, CA) using standard techniques (Hamill et al.
1981). Micropipettes were pulled from borosilicate glasses
(Boralex) with a Flaming Brown micropipette puller (P80, Sutter
Instrument, Novato, CA) and polished by placing them close to a glass
bead on a microforge (Narishige, Tokyo) to obtain electrode resistance ranging from 0.4 to 0.8 M
. To reduce the pipette capacitance, micropipettes were coated with a mixture prepared by adding
approximately three parts of finely shredded parafilm to one part each
of light and heavy mineral oil (Sigma, St. Louis, MO) and vigorously
stirring over heat for 30-60 min. The pipette solution contained (in
mM) 140 CsF, 1 EGTA, 10 NaCl, and 10 HEPES, pH 7.3, and was adjusted to
310 mosmol/l with glucose. The following bath solution was used (in
mM): 140 NaCl, 3 KCl, 1 MgCl2, 1 CaCl2, 0.1 CdCl2, and 20 HEPES, pH 7.3; it was adjusted to 320 mosmol/l with glucose. CdCl2 was used to block
Ca2+ currents. To eliminate the small residual
sustained outward current that was seen at very depolarizing pulses, in
some experiments we included 10 and 20 mM TEA in bath and pipette
solutions, respectively, and CsF concentration was decreased to 120 mM
in the pipette solution. Na+ current density did
not appear to be different in these experiments. The pipette potential
was zeroed before seal formation, and the voltages were not corrected
for liquid junction potential. Capacity transients were canceled, and
series resistance was compensated (>80%) as necessary. The leakage
current was digitally subtracted on-line using hyperpolarizing control
pulses, applied before the test pulse, of one-sixth test pulse
amplitude (-P/6 procedure). Access resistance was monitored throughout
the recording, and the cells were discarded if the resistance was
3
M
. Whole cell currents were filtered at 5 kHz and acquired at 50 kHz
by a computer using Clampex 8.01 software (Axon Instruments). Digidata
1200B interface (Axon Instruments) was used for A-D conversion, and the
data were stored on compact disk for analysis. For current density
measurements, membrane currents were normalized to membrane capacitance. Membrane capacitance was calculated as the integral of the
transient current in response to a brief hyperpolarizing pulse from
100 mV (holding potential) to
110 mV. The average of the membrane
capacitance of the DRG neurons used for electrophysiological experiments was 54.56 ± 2.54 pF (mean ± SE,
n = 21 from 5 different preparations) for SNS (+/+)
mice and 58.92 ± 2.15 pF (n = 45; P > 0.05) for SNS (
/
) mice.
Calculation of fast and slow Na+ current density using prepulse inactivation
Prepulse inactivation takes advantage of the differences in the
inactivation properties of the fast and slow Na+
currents (Cummins and Waxman 1997; McLean et al.
1988
; Roy and Narahashi 1992
).
Na+ currents were elicited by 20-ms test pulses
to
10 mV after 500 ms prepulses to potentials over the range of
130
to
10 mV. Fast Na+ currents were obtained by
subtracting the current obtained at approximately
50 mV prepulse,
which elicits only slow Na+ current, from the
current obtained with more hyperpolarizing prepulses that elicit both
fast and slow Na+ currents. Densities for fast
TTX-S Na+ currents were calculated from cells
that express fast Na+ currents and from cells
expressing both fast and slow Na+ currents.
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RESULTS |
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In addition to TTX-S fast Na+ currents, two
distinct TTX-R Na+ currents in DRG neurons can be
distinguished on the basis of their inactivation properties: slow TTX-R
and persistent TTX-R Na+ currents (Cummins
and Waxman 1997; Cummins et al. 1999
). In this study we asked the following: 1) whether the slow
Na+ currents are produced by SNS channels in
large cutaneous DRG neurons and 2) whether a persistent
Na+ current is present in these cells.
Identification of large cutaneous afferent DRG neurons by retrograde Fluoro-Gold labeling
DRG neurons isolated from the L4 and
L5 ganglia of SNS (+/+) and SNS (/
) mice
consisted of small (<25 µm apparent diameter), medium (25-40 µm)
and large DRG (>40 µm) cells. Of the overall population of DRG
neurons, approximately 50% were small in size, 40% were medium in
size, and 10% were large in size. The size and shape of these three
types of neurons were similar in SNS (+/+) and SNS (
/
) mice. Figure
1A shows the optical image of large (>40 µm) and medium (<40 µm) size DRG neurons isolated from a SNS (
/
) mouse DRG. Figure 1B shows the retrograde
labeling of one of these neurons following subcutaneous injection of
Fluoro-Gold, indicating that it is a cutaneous afferent neuron. Only
fluorescent neurons, which we could identify definitively as cutaneous
afferents, were used in this study.
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Both fast and slow Na+ currents are present in SNS (+/+) large cutaneous afferent neurons
Sodium currents were elicited from large cutaneous afferent
(40-45 µm diam) neurons with the whole cell patch-clamp technique by
test pulses from 100 to +40 mV. Two overall patterns of
Na+ current expression, one group of cells
expressing only fast Na+ currents (Fig.
2A; 11 of 21 neurons tested)
and the other group expressing fast and slow Na+
currents (Fig. 2B; 10 of 21 neurons tested), were seen in
these cells. The Na+ current traces illustrated
in Fig. 2A reach peak amplitude within 1 ms and inactivate
back to the baseline within ~5 ms, and therefore these are fast
Na+ currents. The Na+
current traces from another neuron, illustrated in Fig. 2B,
also reach the peak amplitude within 1 ms but take more than 20 ms to
inactivate back to the baseline, indicating the presence of slow
Na+ currents in addition to fast
Na+ currents. These results suggest that large
cutaneous afferent neurons isolated from SNS (+/+) mouse can express
either fast Na+ currents (Fig. 2A) or
co-express fast and slow Na+ currents (Fig.
2B). The percentage of cells expressing only fast Na+ current is ~52%; and co-expressing fast
and slow Na+ currents are ~48% (see below for
the distribution of fast and slow Na+ currents).
Large cutaneous afferent neurons expressing only slow Na+ currents were not observed in this study
(n = 21; see Table 1).
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The voltage dependence of steady-state inactivation of fast and slow
Na+ currents are markedly different, and this has
been used to separate fast and slow Na+ currents
in DRG neurons (Cummins and Waxman 1997; McLean
et al. 1988
; Roy and Narahashi 1992
). Recordings
showing steady-state inactivation of Na+ currents
expressing only fast Na+ current (11 neurons from
5 different preparations) or co-expressing fast and slow
Na+ currents (10 neurons from 5 different
preparations) are displayed in Fig. 3,
A and D, respectively. Steady-state inactivation
of Na+ currents was obtained using 40-ms test
pulses to
10 mV after 500-ms prepulses to potentials from
130 to
10 mV. All of the current traces illustrated in Fig. 3A
reach peak amplitude in <1 ms and inactivate completely to the
baseline within a few milliseconds. The activation and inactivation of
the fast Na+ currents of large cutaneous afferent
neurons are similar to the fast Na+ currents
present in small C-type DRG neurons (Cummins and Waxman 1997
; Renganathan et al. 2000
).
Na+ current traces illustrated in Fig.
3D reach peak amplitude in <1 ms, but inactivation is
incomplete and biphasic, indicating the presence of both fast and slow
Na+ currents in this neuron.
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Prepulse inactivation takes advantage of the differences in the
inactivation properties of the fast and slow Na+
currents (Cummins and Waxman 1997; McLean et al.
1988
; Roy and Narahashi 1992
), i.e., fast
Na+ currents inactivate completely at
approximately
50 mV, whereas the slow Na+
currents do not inactivate at approximately
50 mV (see Cummins and Waxman 1997
). In these studies, fast
Na+ currents were obtained by subtracting the
response with the prepulse that elicits only slow
Na+ currents, from the response with more
hyperpolarizing prepulses that elicit both fast and slow
Na+ currents. The process of separating fast and
slow Na+ currents by prepulse inactivation and
subtraction will be hereafter termed as prepulse subtraction. Prepulse
subtraction was used to separate the fast and slow
Na+ currents in large cutaneous afferent DRG
neurons: neurons that expressed only fast Na+
current in unsubtracted recordings showed fast current (Fig. 3B) and no slow Na+ current following
prepulse subtraction (Fig. 3C; 11 neurons from 5 different
preparations). In contrast, neurons that in unsubtracted records
co-expressed both fast and slow Na+ current
appeared to yield fast (Fig. 3E) and slow
Na+ currents (Fig. 3F) after prepulse
subtraction (10 neurons from 5 different preparations).
Consistent with earlier studies in C-type DRG neurons
(Cummins and Waxman 1997; Elliott and Elliott
1993
; Roy and Narahashi 1992
), the fast
Na+ currents in large cutaneous afferent neurons
were sensitive to nanomolar concentrations of TTX, whereas the slow
Na+ currents were resistant to 300 nM TTX (Fig.
4, n = 4). Prepulse inactivation and TTX subtraction give essentially the same results when
used to separate fast and slow Na+ currents (Fig.
3F and Fig. 4), as reported previously in C-type DRG neurons
(Cummins and Waxman 1997
).
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Distribution of fast and slow Na+ currents in SNS (+/+) large cutaneous afferent neurons
In whole cell recordings of 1-8 h cultured cutaneous DRG neurons from SNS (+/+) mice, varying amounts of fast and slow Na+ currents were observed (see below in current densities). One hundred percent of large cutaneous neurons expressed fast Na+ currents, with 48% of the neurons co-expressing fast and slow Na+ currents. We did not observe any large cutaneous afferent neurons expressing only slow Na+ current (21 neurons from 5 different preparations).
Only fast Na+ currents are present in SNS (/
) large
cutaneous afferent neurons
Figure 5 illustrates a family of
Na+ current traces elicited by depolarizing
voltage test pulses from 100 to +40 mV from a typical large cutaneous
afferent neuron isolated from SNS (
/
) mouse. The
Na+ current traces inactivate back to the
baseline completely within ~10 ms (see DISCUSSION for an
explanation of the slower inactivation kinetics), suggesting that only
fast Na+ current is present.
Na+ current steady-state inactivation from a
typical large cutaneous afferent neuron isolated from SNS (
/
) mouse
indicates the presence of only fast Na+ current
(Fig. 6A). When prepulse
inactivation was used to separate the fast and slow
Na+ currents, only fast Na+
current was observed (Fig. 6B), and no slow
Na+ current was seen (Fig. 6C, 45 neurons from 5 different preparations).
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The mid-point potential and the slope of the steady-state activation
curves for fast Na+ currents were 25.7 ± 4.5 mV and 6.8 ± 1.6 mV/e-fold in SNS (+/+) large
cutaneous sensory neurons (n = 11), and
27.6 ± 3.8 mV and 5.9 ± 1.2 mV/e-fold, in SNS (
/
) large
cutaneous sensory neurons (n = 20), respectively. The
mid-point potential and the slope of the steady-state inactivation
curves for fast Na+ currents were
79.62 ± 3.44 mV and
8.70 ± 2.39 mV/e-fold in SNS (+/+) large
cutaneous sensory neurons (n = 11), and
72.84 ± 2.23 and
9.25 ± 1.20 mV/e-fold in SNS (
/
) large
cutaneous sensory neurons (n = 45), respectively. These
values are not significantly different between the large cutaneous
sensory neurons of SNS (+/+) and SNS (
/
) mice (P > 0.05).
The fast Na+ currents in large cutaneous DRG
neurons were sensitive to nanomolar concentrations of TTX, and no slow
Na+ currents were seen in the presence of 300 nM
TTX (Fig. 7, n = 10). A
total of 45 large cutaneous afferent neurons isolated from 5 different
SNS (/
) mice were investigated for Na+
currents, and in all of them only fast TTX-S Na+
currents were seen. Slow Na+ currents could not
be detected either using prepulse inactivation (n = 45)
or in the presence of 300 nM TTX (n = 10), in large
cutaneous afferent neurons isolated from SNS (
/
) mice. These
results demonstrate that SNS produces the slow TTX-R
Na+ currents in large cutaneous neurons.
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Fast and slow Na+ current densities are both altered in
SNS (/
) mice
In these experiments the fast and slow
Na+ current amplitudes were measured in 140 mM
NaCl in bath solution, a concentration similar to that found in vivo
and used routinely to quantify Na+ current
density in DRG neurons (Cummins and Waxman 1997;
Renganathan et al. 2000
). The series resistance problem
usually associated with large amounts of Na+
current and large DRG neurons were minimized by using a low-resistance pipette (0.4-0.8 M
) and using 80-90% series resistance
compensation. In a small number of large cutaneous afferent DRG neurons
isolated from SNS (
/
) mice, the Na+ current
amplitude exceeded 200 nA, reaching the limits of the amplifier, and
these neurons were not included for the quantitation of
Na+ currents. Prepulse subtraction
(Cummins and Waxman 1997
; McLean et al.
1988
; Roy and Narahashi 1992
) was used to
separate and quantitate fast and slow Na+
currents expressed in each large cutaneous afferent neuron. The fast
and slow Na+ current amplitudes in large
cutaneous afferent neurons isolated from SNS (+/+) mice were 49.15 ± 5.14 nA and 18.82 ± 1.12 nA, respectively. The fast
Na+ current amplitudes in large cutaneous
afferent neurons from SNS (
/
) mice were 93.62 ± 8.64 nA
(P < 0.01); slow Na+ currents,
as noted above, could not be detected in cells from SNS (
/
) mice.
To compensate for differences in cell size, currents were normalized to
cell capacitance and expressed as current densities. The capacitance of
the large cutaneous neurons isolated from SNS (+/+) and SNS (
/
)
mice were 54.56 ± 2.54 pF and 58.92 ± 2.15 pF,
respectively. The fast and slow Na+ current
densities in large cutaneous neurons isolated from SNS (+/+) mice were
0.90 ± 0.12 and 0.39 ± 0.16 nA/pF, respectively. The fast
Na+ current density in large cutaneous afferent
DRG neurons isolated from SNS (
/
) mice, was 1.47 ± 0.14 nA/pF, approximately 60% higher than the current density observed in
SNS (+/+) mice (P < 0.02).
Persistent Na+ currents are absent in large cutaneous afferent DRG neurons
Two TTX-R Na+ channels have been cloned from
DRG neurons, SNS (Akopian et al. 1996;
Sangameswaran et al. 1996
) and NaN (Dib-Hajj et
al. 1998
; Tate et al. 1998
).
-SNS transcripts
produce a slow TTX-R Na+ current (Akopian
et al. 1996
), while NaN transcripts appear to produce a
persistent TTX-R Na+ currents in small C-type DRG
neurons (Cummins et al. 1999
). Persistent TTX-R
Na+ currents activate at low depolarizing
voltages, i.e.,
80 mV, whereas activation of fast and slow
Na+ currents occurs at approximately
40 mV.
Furthermore, at test potentials between
80 and
50 mV, the
persistent Na+ currents do not inactivate or very
slowly inactivate (Cummins et al. 1999
; Dib-Hajj
et al. 1999a
; Renganathan et al. 2000
). The
pulse protocols employed in the experiments shown in Figs. 2, 4, 5, and
7 would have been expected to elicit persistent TTX-R Na+ currents if there were any present in large
cutaneous afferent DRG neurons because the same protocols elicited both
the persistent and slow TTX-R Na+ currents in
small C-type DRG neuron (Fig.
8A, n = 4).
However, we were unable to elicit persistent TTX-R
Na+ currents in large cutaneous afferent neurons,
i.e., at test potentials between
80 to
50 mV. Figure 8,
C and D, juxtaposes Na+
current recordings from small (15 µm) and large cutaneous afferent neurons. As seen in this figure low depolarizing test pulses, i.e.,
from
80 to
50 mV, elicited a typical persistent TTX-R Na+ current in small C-type DRG neurons (Fig.
8C), but not in large cutaneous afferent DRG neurons (Fig.
8D, n = 45).
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DISCUSSION |
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Large cutaneous afferent DRG neurons exhibit different functional
properties compared with muscle afferent neurons and express a slow
TTX-R Na+ current that is not present in muscle
afferent neurons (Honmou et al. 1994). In this study we
demonstrate that the slow TTX-R Na+ current is
present in SNS (+/+) large cutaneous afferent neurons but absent in
transgenic SNS (
/
) large cutaneous afferent neurons identified by
retrograde Fluoro-Gold labeling. These results support the hypothesis
that
-SNS transcript encodes the slow TTX-R
Na+ current in large cutaneous DRG neurons. This
conclusion is strengthened by our findings that the slow
Na+ currents that are seen in SNS (+/+) large
cutaneous afferent neurons are, as predicted by the SNS sequence,
resistant to 300 nM TTX. Previous studies have demonstrated that,
although SNS mRNA and protein are most highly expressed in small C-type
DRG neurons, they are also present in large DRG neurons (Black
et al. 1996
; Dib-Hajj et al. 1996
;
Novakovic et al. 1998
; Sangameswaran et al.
1996
).
The size criteria used in the study are based on the assumption that
DRG neurons <25 µm diam tend to give rise to C-fiber afferent axons,
whereas neurons >40 µm diam tend to have low-threshold afferent
fibers. This assumption is supported by the effects of capsaicin on DRG
neurons from in vitro and in vivo mouse studies (Hiura and
Sakamoto 1987; Urban and Dray 1993
). Mouse DRG
neurons with C-fiber characteristics (conduction velocity <0.6 m/s;
broad action potential with shoulder on the descending slope) were
depolarized and generated action potentials when 100-700 nM capsaicin
was added to the bath solution for 30 s. However, capsaicin at
these concentrations did not affect the membrane potential of DRG
neurons with myelinated fibers (conduction velocity >2.0 m/s),
indicating that neurons <25 µm diam have properties of nociceptors
and neurons >40 µm tend to have low-threshold afferent fibers
(Urban and Dray 1993
). Capsaicin treatment of neonatal
mice decreased the number of small DRG neurons (8-25 µm) by
51-77%, whereas the number of large-sized neurons (25-40 µm)
decreased by 14-52%, suggesting that capsaicin affects small and
certain types of large neurons (Hiura and Sakamoto
1987
). Since a part of the 25-40 µm diam neuron population
was affected by capsaicin treatment, we focused on neurons >40 µm
diam with Fluoro-Gold labeling to unequivocally study large cutaneous
afferent neurons in this investigation.
The total (and fast) Na+ currents in SNS (/
)
large sensory neurons (Figs. 5 and 6) appear to take longer to return
to baseline than do the total and fast Na+
currents in SNS (+/+) large sensory neurons (Figs. 2 and 3). Analysis
of the fast Na+ current traces obtained from SNS
(
/
) or SNS (+/+) large cutaneous sensory neurons revealed that
~50% of these large cutaneous sensory neurons had current traces
that inactivated back to baseline within 5 ms, whereas the other 50%
of the large cutaneous sensory neurons had current traces that
inactivated back to baseline within 10 ms. The inactivation of the fast
Na+ currents (test potential, 0 mV) in
approximately one-half the SNS (+/+) large cutaneous sensory neurons
was best fitted with a single exponential function with the time
constant of 0.47 ± 0.20 ms. In the other half of the large
cutaneous sensory neurons, a second exponential function was
needed to best fit the inactivation phase of the fast
Na+ currents. The values for the time constant
for inactivation from the fit were
th1 = 0.69 ± 0.10 ms,
amplitude = 0.8 ± 0.2; th2 = 6.70 ± 0.86 ms, amplitude = 0.2 ± 0.1. A similar
pattern was observed in large cutaneous sensory neurons isolated from
SNS (
/
) mice. In 40% of the neurons, the inactivation of fast
Na+ currents could be fit with a single
exponential function (th = 0.53 ± 0.20 ms), and in the other 60% of the neurons the
inactivation of fast Na+ currents was best fitted
with two exponential functions. The time constants for inactivation
thus obtained were th1 = 0.65 ± 0.04 ms, amplitude = 0.83 ± 0.12;
th2 = 4.94 ± 0.34 ms,
amplitude = 0.17 ± 0.11. The inactivation time constants of
the fast Na+ currents are not significantly
different within the large cutaneous sensory neurons of SNS (+/+) and
SNS (
/
) mice or between them (P > 0.05). The
inactivation time constants obtained with a single exponential function
or the smaller time constants obtained with two exponential functions
from large cutaneous sensory neurons of SNS (+/+) and SNS (
/
) mice
are similar to the values reported earlier (Cummins and Waxman
1997
). We do not have a clear explanation for the observation
that fast Na+ currents in some of the large
cutaneous sensory neurons of SNS (+/+) or SNS (
/
) could be fit with
two exponential functions. It is possible that fast
Na+ currents in these neurons could originate
from two types of TTX-S fast Na+ channels. The
possibility that the fast Na+ currents in SNS
(
/
) neurons contain a different subtype of Na+ channel that is up-regulated or expressed de
novo, compared with fast Na+ currents in SNS
(+/+) neurons, is under investigation.
Recently, a novel persistent TTX-R Na+ current
has been described in small DRG neurons (Cummins et al.
1999). We found that neither SNS (+/+) nor SNS (
/
) large
cutaneous afferent DRG neurons express persistent TTX-R
Na+ currents. Previous studies (Dib-Hajj
et al. 1998
, 1999a
; Fjell et al.
1999
; Tate et al. 1998
) indicate that NaN mRNA
has a more restricted expression than SNS and is selectively expressed
in small DRG neurons, and immunocytochemical studies with NaN-specific antibodies (Fjell et al. 2000
) demonstrate NaN protein
in unmyelinated and small-diameter myelinated axons that arise from
small DRG neurons. Our results support the conclusion (Cummins
et al. 1999
) that NaN channels produce a persistent TTX-R
Na+ current.
Differentiating large DRG neurons into cutaneous and muscle afferents
by subcutaneous injection of Fluoro-Gold, Honmou et al.
(1994) demonstrated the presence of slow TTX-R
Na+ currents in large cutaneous afferent neurons.
Consistent with these observations in rat DRG neurons (Honmou et
al. 1994
), the present results show that SNS (+/+) large
cutaneous DRG neurons express two types of Na+
currents, a fast TTX-S Na+ current and slow TTX-R
Na+ current. In the earlier study, fast
Na+ current was observed together with slow
Na+ current in 40%, and slow
Na+ current alone was seen in ~60% of the
large cutaneous DRG neurons (Honmou et al. 1994
).
However, we observed fast Na+ current in all
large cutaneous afferent DRG neurons from mice (21 of 21 neurons) and
slow Na+ current together with fast
Na+ current in 48% of the neurons (10 of 21 neurons). These apparently contradictory observations may be reconciled
by differences between the two studies: 1) there may be
species difference between rat and mouse DRG neurons, 2) in
the earlier study the rat neurons were cultured for 1-2 days prior to
patch clamping, whereas, in this study, the neurons were studied
between 1 and 8 h after plating.
The deletion of SNS gene leads to the loss of slow TTX-R
Na+ current in large cutaneous DRG neurons (45 neurons of 45 neurons from 5 different preparations). The absence of
slow TTX-R Na+ current in SNS (/
) mice
suggests that other types of sodium channels are unlikely to produce
slow TTX-R Na+ current in large cutaneous
afferent DRG neurons. Associated with the loss of SNS, we observed an
increased expression of fast TTX-S Na+
currents in SNS (
/
) large cutaneous DRG neurons. This apparent up-regulation is similar to the increase observed in an earlier study
in SNS (
/
) DRG neurons that focused on small cells (Akopian et al. 1999
), where the fast TTX-S Na+
current density increased twofold. Akopian et al.
(1999)
, using semi-quantitative polymerase chain
reaction, could not detect changes in the less abundant TTX-S
-subunit transcripts; however, northern blots of the abundant PN1
channel transcript showed an increase in transcript expression of more
than 50%. Patch-clamp studies on heterologously expressed PN1 channels
have shown that they produce a fast TTX-sensitive current
(Cummins et al. 1998
). The increase of about 60% in
fast Na+ current density and the similar increase
in PN1 channel transcript in SNS (
/
) mice suggest that the increase
in fast Na+ current density may be due to
up-regulation of PN1 channel expression at the transcriptional level.
Interestingly, the increase in PN1 expression does not appear to be
part of a global or nonspecific up-regulation of sodium channel
expression. Within the normal nervous system, PN1 is expressed in small
and large diameter DRG neurons (Black et al. 1996
;
Toledo-Aral et al. 1997
), whereas the expression of NaN
is confined to small DRG neurons (Dib-Hajj et al. 1998
,
1999a
; Fjell et al. 1999
,
2000
; Tate et al. 1998
). The lack of
increased levels of NaN-type (persistent) Na+
currents in SNS (
/
) large cutaneous afferents, even though NaN and
SNS are encoded by genes that are located within a cluster on a single
chromosome (mouse chromosome 3) (Dib-Hajj et al. 1999b
), suggests that compensatory up-regulation of the
Na+ channel subtypes may be confined to channel
isoforms that are normally synthesized in these cells.
Slow depolarizations, first described in sensory axons by Kocsis
et al. (1983), have been associated with slow TTX-resistant Na+ channels (Honmou et al. 1994
)
and have been implicated in the generation of repetitive spike activity
in large cutaneous afferents. Activity of these slow channels appears
to contribute to abnormal bursting in injured sensory axons that can
contribute to paresthesias (Kocsis et al. 1986
). Here we
have identified SNS as the channel that produces the slow TTX-resistant
Na+ currents in large cutaneous afferents.
Together with earlier studies that have correlated other
Na+ channel isoforms in DRG neurons with specific
physiological signatures (Cummins et al. 1998
,
1999
) and with future studies on channel isoforms whose
physiological action in DRG are not yet understood, these results
should ultimately contribute to an understanding of the molecular basis
for electrogenesis in normal and injured spinal sensory neurons.
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ACKNOWLEDGMENTS |
---|
A breeding pair of SNS (+/) mice was kindly provided by Prof.
J. N. Wood, University College London. We thank Drs. S. D. Dib-Hajj and J. A. Black for help in genotyping and raising a colony of SNS (+/+) and SNS (
/
) mice from SNS (+/
) mice. We thank
B. Tuftness for help with the figures.
This work was supported in part by grants from the Medical Research Service and Rehabilitation Research Service, Department of Veterans Affairs, and the National Multiple Sclerosis Society. We also thank the Eastern Paralyzed Veterans Association and the Paralyzed Veterans of America for support.
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FOOTNOTES |
---|
Address for reprint requests: S. G. Waxman, Dept. of Neurology, LCI 707, Yale School of Medicine, 333 Cedar St., New Haven, CT 06510 (E-mail: stephen.waxman{at}yale.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 2 March 2000; accepted in final form 26 April 2000.
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REFERENCES |
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