1Department of Anesthesia,
Strassman, A. M. and
S. A. Raymond.
Electrophysiological evidence for tetrodotoxin-resistant sodium
channels in mechanosensitive nerve endings of slowly conducting fibers
in the intracranial dura. A tetrodotoxin (TTX)-resistant sodium channel was recently identified that is expressed only in small
diameter neurons of peripheral sensory ganglia. The peripheral axons of
sensory neurons appear to lack this channel, but its presence has not
been investigated in peripheral nerve endings, the site of sensory
transduction in vivo. We investigated the effect of TTX on
mechanoresponsiveness in nerve endings of sensory neurons that
innervate the intracranial dura. Because the degree of TTX resistance
of axonal branches could potentially be affected by factors other than
channel subtype, the neurons were also tested for sensitivity to
lidocaine, which blocks both TTX-sensitive and TTX-resistant sodium
channels. Single-unit activity was recorded from dural afferent neurons
in the trigeminal ganglion of urethan-anesthetized rats. Response
thresholds to mechanical stimulation of the dura were determined with
von Frey monofilaments while exposing the dura to progressively
increasing concentrations of TTX or lidocaine. Neurons with slowly
conducting axons were relatively resistant to TTX. Application of 1 µM TTX produced complete suppression of mechanoresponsiveness in all
(11/11) fast A- Small-diameter peripheral sensory neurons exhibit
a distinctive type of voltage-gated sodium current that is resistant to blockade by tetrodotoxin (TTX) (Arbuckle and Docherty
1995 Because nociceptive neurons comprise the major population of small
sensory neurons, the TTX-resistant sodium current may be important in
the transmission of sensory signals evoked by painful stimuli. This
idea is supported by the observations that TTX resistance is specific
to nociceptive neurons (Ritter and Mendell 1992 No study has yet investigated the presence of TTX resistance in C-fiber
peripheral nerve endings, the site of sensory tranduction in vivo.
Therefore we examined TTX resistance and its relation to fiber
conduction velocity in mechanosensitive axonal branches of sensory
neurons that innervate the intracranial dura. The dura receives a
sensory innervation from A- Surgical preparation and recording
Urethan-anesthetized male rats (400-600 g) were placed in a
stereotaxic headholder. The right transverse sinus and the caudal part
of the superior sagittal sinus were exposed by craniotomy. The exposed
dura was bathed in a synthetic interstitial fluid (SIF) consisting of
135 mm NaCl, 5 mM KCl, 1 mM MgCl2, 5 mM CaCl2, 10 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid, and 10 mM glucose, pH 7.2. Tungsten microelectrodes were advanced into the trigeminal ganglion by a dorsal approach through the cerebral
cortex. Single-unit recordings were obtained from dural afferent
neurons in the trigeminal ganglion that were identified by their
constant latency response to single shock stimuli (0.5-ms pulses, 5 mA,
0.7 Hz) applied to the dura overlying the ipsilateral transverse sinus
through bipolar stimulating electrodes (Strassman et al.
1996 Identification of shortest latency site
Response thresholds and latencies to electrical stimuli were
mapped at multiple sites across the surface of the exposed dura. The
dural stimulation site associated with the shortest latency response
was identified. The shortest latency site was typically on the
transverse sinus at a distance of 3-6 mm from the midline, which
corresponds well with the position at which the tentorial nerve reaches
the dura from its course through the underlying tentorium (Figs.
1 and 2). The
latency at this site was used for the calculation of conduction
velocity (c.v.), based on a conduction distance from the ganglion
measured at 12.5 mm. For statistical analyses, neurons were classified
as C units (c.v.
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
units [conduction velocity (c.v.) 5-18 m/s] but
only 50% (5/10) of slow A-
units (1.5 <c.v.<5 m/s) and 13%
(2/15) of C units (c.v.
1.5 m/s). The mean TTX concentration that
produced complete suppression of mechanoresponsiveness was ~270-fold
higher in C units than in fast A-
units. In contrast, no significant
difference was found between C and A-
units in the concentration of
lidocaine required for complete suppression of mechanoresponsiveness,
indicating that the greater TTX resistance of mechanoresponsiveness in
C units is not attributable to differences in safety factor unrelated
to channel subtype. These data offer indirect evidence that a
TTX-resistant channel subtype is expressed in the terminal axonal
branches of many of the more slowly conducting (C and slow A-
) dural
afferents. The channel appears to be present in these fibers, but not
in the faster A-
fibers, in sufficient numbers to support the
initiation and propagation of mechanically induced impulses. Comparison
with previous data on the absence of TTX resistance in peripheral nerve
fibers suggests that the TTX-resistant sodium channel may be a
distinctive feature of the receptive rather than the conductive portion
of the sensory neuron's axonal membrane.
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
; Bossu and Feltz 1984
; Caffrey et
al. 1992
; Christian and Togo 1995
; Elliott and Elliott 1993
; Ikeda and Schofield
1987
; Ikeda et al. 1986
; Kostyuk et al.
1981
; McLean et al. 1988
; Ogata and
Tatebayashi 1992a
,b
; Rizzo et al. 1994
;
Roy and Narahashi 1992
; Roy et al. 1994
;
Stea and Nurse 1992
, 1993
; Yoshimura and
De Groat 1996
; Yoshimura et al. 1996
), which
blocks voltage-gated sodium current in most other central and
peripheral neurons (Yoshida 1994
). This sensory-neuron-specific TTX-resistant sodium current is both
pharmacologically and kinetically distinct from sodium currents that
have been described in other neural and nonneural cells (Yoshida
1994
). Recently a sensory neuron-specific sodium channel
(SNS/PN3) was cloned that is resistant to TTX and exhibits kinetics
that match those of the TTX-resistant current of small-diameter sensory
neurons (Akopian et al. 1996
; Sangameswaran et
al. 1996
). Expression of the channel is highly restricted, in
that it is present only in the small-diameter neurons of peripheral
sensory ganglia (dorsal root and trigeminal and nodose ganglia) but not
in the large diameter sensory neurons or in other peripheral or central
neurons, glia, or nonneural tissues.
) and that the TTX-resistant current is enhanced by agents such as
prostaglandin E2 that produce pain or hyperalgesia in vivo
(England et al. 1996
; Gold et al. 1996b
).
Consequently, the SNS/PN3 sodium channel has attracted great interest
as a possible pharmacological target for producing a selective
suppression of activity in nociceptive neurons. However,
electrophysiological studies have typically found that TTX resistance
is present in the cell bodies but not in the peripheral axons of
small-diameter sensory neurons (Villiere and McLachlan
1996
; Yoshida and Matsuda 1979
), an observation that was recently corroborated by immunohistochemical localization of
the SNS/PN3 channel (Novakovic et al. 1998
). This
restricted cellular distribution of the TTX-resistant sodium channel
raises some question about its potential role in nociception, since
impulse activity in the cell bodies of pseudounipolar sensory
neurons is not thought to be necessary for the propagation of sensory signals to the CNS.
and C fibers originating in the
trigeminal ganglion and is a convenient preparation for testing the
effects of chemical agents on sensory nerve endings in vivo
(Strassman et al. 1996
). Because the degree of TTX
resistance of axonal branches could potentially be affected by factors
other than channel subtype that affect impulse conduction and
generation, the selectivity of TTX for A-
versus C-fiber neurons was
compared with that of lidocaine, which blocks both TTX-sensitive and
-resistant channels (Roy and Narahashi 1992
).
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
).
1.5 m/s), slow A-
units (1.5 < c.v. < 5 m/s), and fast A-
units (c.v.
5 m/s) (Strassman et al.
1996
).
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Fig. 1.
Drawing of structures within the rat cranium, with the dura partially
cut away and the brain removed for purposes of illustration only. The
tentorial nerve is shown exiting the trigeminal ganglion at the base of
the skull and coursing caudally and dorsally up the tentorium to reach
the outer dural surface of the transverse sinus. In the experimental
preparation, the brain and dura were intact, and recording
microelectrodes were driven into the trigeminal ganglion while stimuli
were applied to the dura overlying the transverse sinus.
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Fig. 2.
Map of response latencies (in ms) evoked by single-shock stimuli at
different sites on the right transverse sinus for a dural afferent
neuron recorded in the trigeminal ganglion. The dotted line indicates
the area from which the neuron could be activated by punctate
mechanical stimulation, and the stippled area indicates the site of
lowest mechanical threshold (0.38 g). Approximately 6 mm of the right
transverse sinus is shown. Rostral is toward the top. The conduction
velocity of this neuron's main axon from the trigeminal ganglion to
the dura was calculated from the shortest dural response latency (27 ms) as 0.46 m/s.
Identification of site with lowest mechanical threshold
Neurons were then tested for responses to mechanical stimulation
of the dura by stroking the surface of the dura with a blunt probe and
indenting it with von Frey monofilaments. Only neurons for which a
mechanical receptive field could be found were included in this study.
For such neurons, the dura was probed with von Frey hairs of
progressively decreasing intensities until the site or sites with the
lowest von Frey threshold were located. The receptive field typically
consisted of a single spot (<1 mm diameter) of lowest threshold, which
was surrounded by an area of up to several millimeter radius from which
responses could be evoked only by more intense stimuli. A few neurons
exhibited two to four such spots separated by distances of 6 mm.
After the spot(s) with the lowest mechanical threshold was identified,
the distance of each of these spots from the shortest latency site
(identified with electrical stimulation as described previously) was
measured (±1 mm). This was used as a rough estimate of the length of
the axonal branch that coursed across the dura to the mechanosensitive ending and thus the length of axon that would be exposed to solutions applied topically to the dura (see Figs. 1 and 2). It should be noted
that, as explained previously, the calculation of conduction velocity
was determined from the site with the shortest response latency (which
presumably corresponds to the site at which the main axon first reaches
the dura) and thus does not reflect the conduction velocity of the
axonal branches within the dura, which was generally much lower.
Testing of suppression of mechanical responses by lidocaine and TTX
For threshold determinations, individual von Frey hairs were applied three to six times at 5-s intervals (e.g., Figs. 3 and 4). The von Frey hairs were applied in increasing order until threshold was reached, defined as the lowest force that evoked a response in >50% of the stimulus applications.
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|
Baseline measurements of the von Frey threshold at the lowest threshold
spot were made three or four times at ~10-min intervals. Threshold
measurements were then repeated in the presence of progressively increasing concentrations of lidocaine applied topically to the outer
surface of the dura. Lidocaine concentration was increased until
mechanical responsiveness was completely abolished, as defined by lack
of response to 4-g stimulation, which was the highest intensity tested.
Higher intensities were not used to avoid damage to the dura. The
lidocaine concentrations used were 1.25, 2.5, 5, 10, 20, 40, and 100 mM, in SIF. Each concentration was initially applied for 10 min, and
the threshold was then determined. If the threshold showed a change,
the drug exposure was continued, and threshold measurements were
repeated at ~3- to 5-min intervals until no further changes were
observed. (More frequent testing was not done to avoid
stimulation-induced changes in sensitivity.) In most cases the drug
effects were complete within 10 min. The next concentration in the test
series was then applied, with no washout period between different
concentrations. Recovery from lidocaine was determined by return to
baseline von Frey threshold. After recovery from the lidocaine
(typically 30-60 min), baseline measurements were again made, and the
testing process was repeated with increasing concentrations of TTX
(108, 10
7, 10
6,
10
5, and 10
4 M).
The effect of TTX on response threshold to electrical stimulation of
the dura was also determined in some neurons. Threshold was determined
with single-pulse stimuli delivered at 0.5 Hz through bipolar
electrodes with a constant-current stimulus isolation unit that could
deliver up to 10 mA at a compliance of 100 V (Winston Electronics).
Pulse width was 0.02-0.1 ms for A- neurons and 0.1-0.5 ms for C
neurons. Pulse width was chosen so that baseline threshold was ~500
uA. Baseline was determined as the mean of three to four measurements.
Threshold measurement was repeated after suppression of
mechanoresponsiveness by TTX.
Data analysis
For each neuron, two parameters of drug sensitivity were analyzed, 1) the lowest applied drug concentration that produced partial suppression of mechanoresponsiveness, defined as an increase in von Frey threshold, and 2) the lowest applied drug concentration that produced complete suppression of mechanoresponsiveness, defined as a lack of response to 4-g stimulation. For neurons with more than one mechanosensitive spot in their dural receptive field, partial suppression was considered to have occurred if any spot exhibited an increased threshold, whereas complete suppression was not considered to have occurred until all spots showed complete loss of responsiveness.
Because drugs were applied in log increments of concentration, log
values of applied concentrations were used for statistical comparisons
and correlations between TTX and lidocaine concentrations and
conduction velocity (log10 for TTX and log2 for
lidocaine and conduction velocity). For purposes of statistical
analyses, neurons that did not show suppression at the highest drug
concentrations applied were assigned a value 1 log unit higher
(103 M for TTX and 200 mM for lidocaine). Analysis of
variance (ANOVA) with the Bonferroni/Dunn posthoc correction for
multiple comparisons was used for comparisons between fast A-
, slow
A-
, and C-fiber neurons.
2 was used for comparing the
incidence of TTX resistance in the three classes of neurons. Fisher's
r-to-Z calculation was used to determine the
significance values of correlations. Statistical analyses were carried
out with StatView 4.57 (Abacus Concepts).
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RESULTS |
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General observations on suppression of mechanosensitivity by TTX and lidocaine
Extracellular unit recordings were obtained from 38 mechanosensitive dural afferent neurons in the trigeminal ganglion that were identified by their response to single shock stimulation of the
dura overlying the ipsilateral transverse sinus. These included 16 C
units (c.v. 1.5 m/s), 10 slow A-
units (1.5 m/s < c.v. < 5 m/s), and 12 fast A-
units (c.v. 5-18 m/s). All neurons in this
study exhibited mechanical receptive fields on the dura overlying or
immediately adjacent to the ipsilateral transverse sinus or the
caudalmost part of the superior sagittal sinus (e.g., Fig. 2). Baseline
response thresholds to dural stimulation ranged from 0.03 to 2.9 g
(mean ± SD, 0.59 ± 0.73 g; median, 0.38 g), with
no significant differences between C, slow A-
, and fast A-
units
(P > 0.1, ANOVA).
The effect of TTX (n = 36) and lidocaine (n = 32), applied topically to the dura, was tested on the neurons' responsiveness to mechanical stimulation of the dura. Thirty of these neurons were tested with both TTX and lidocaine. TTX (0.01-100 µM) produced a decrease in mechanoresponsiveness, as shown by an increase in response threshold, in 34 of 36 neurons. The remaining two neurons, which were both C units, showed no change in threshold at the highest TTX concentration applied (100 µM). Of the 34 neurons that showed an increased threshold, 31 showed a complete loss of responsiveness, and the remaining 3 neurons (all C units) continued to show some mechanoresponsiveness at the highest TTX concentration applied. The elevation in response threshold developed in a graded manner with increasing TTX concentration in 24 neurons (e.g., Figs. 3B, 4, and 5, A-C), whereas in the remaining 10 neurons the entire increase in threshold occurred at one increment in applied concentration (e.g., Figs. 3A and 5D).
|
Lidocaine (1.25-100 mM) produced an increase in mechanical response threshold in all neurons tested and a complete loss of mechanoresponsiveness in 27 neurons. The increase in threshold developed in a graded manner with increasing lidocaine concentration in 23 neurons (Fig. 5A), whereas in the remaining 9 neurons the entire increase in threshold occurred at one increment in applied concentration.
Relationship of TTX sensitivity to conduction velocity
Neurons with slowly conducting axons were relatively resistant to
TTX. Application of 1 µM TTX was sufficient to produce complete suppression of mechanoresponsiveness in all (11/11) fast A- units (c.v. > 5 m/s) but only 50% (5/10) of slow A-
units (1.5
c.v.
5 m/s) and 13% (2/15) of C units (c.v. < 1.5 m/s) (Figs.
6B and
7A; P < 0.001,
2). The mean concentration that produced complete
suppression was ~25-fold higher in C units than in slow A-
units
and 270-fold higher in C units than in fast A-
units (ANOVA,
P < 0.01, and P < 0.0001, respectively; mean ± SD in log units of molarity,
4.2 ± 1.2,
5.6 ± 1.0, and
6.6 ± 0.8, for C, slow A-
, and
fast A-
, respectively). Figure 7A illustrates the graded
relationship between TTX sensitivity and conduction velocity. The
concentration of TTX that produced complete suppression of
mechanoresponsiveness showed a strong inverse correlation with
conduction velocity (r =
0.77, P < 0.0001).
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|
Although mechanoresponsiveness was not completely suppressed in most C
units with application of 1 µM TTX, this concentration produced at
least partial suppression (i.e., increased response threshold) in 67%
(10/15) of C units and 80% (8/10) of slow A- units (and complete
suppression in 100% of fast A-
units, as noted previously) (Fig.
6A). The TTX concentration that produced partial suppression
of mechanoresponsiveness tended to be higher in more slowly conducting
neurons, although the correlation with conduction velocity
(r =
0.49, P < 0.01) was much weaker
than that found for the concentration that produced complete
suppression. The concentration that produced partial suppression was
significantly higher in C units than in fast A-
units (ANOVA,
P < 0.05;
6.0 ± 1.7,
6.8 ± 1.2, and
7.5 ± 0.7, respectively, for C, slow A-
, and fast A-
in
log units of molarity).
Relationship of lidocaine sensitivity to conduction velocity
Slowly conducting neurons tended to be more resistant to
lidocaine, but this relationship was much weaker than that found for
TTX (Figs. 6 and 7). The concentration of lidocaine required for
complete suppression of mechanoresponsiveness was not significantly correlated with conduction velocity, but there was a nonsignificant trend for this concentration to be higher in more slowly conducting neurons (Fig. 7B; r = 0.33, P > 0.06). There was no significant difference in this concentration
between A-
and C units (P > 0.3, ANOVA). C units as
a group showed a (nonsignificant) twofold greater resistance to
lidocaine than fast A-
neurons, with respect to the applied
concentration required for complete suppression.
The concentration of lidocaine that produced partial suppression of
mechanoresponsiveness was weakly correlated with conduction velocity
(r = 0.47, P < 0.01). There was no
significant difference in this concentration between A-
and C units
(P > 0.06, ANOVA).
Time course of TTX suppression
The relative TTX resistance of C units could in principle
result from differential access of TTX, as might occur if a diffusion barrier were present around C units but not A- units (see
DISCUSSION). In such a case, a temporary failure to observe
suppression in C units could be explained by a relative delay in access
that would disappear as exposure time was increased.
However, the time course of TTX suppression did not support this
possibility. As noted previously, TTX did produce an increase in
threshold in 87% of C units. In all cases, these increases in
threshold occurred within 10 min of exposure and appeared to have
reached an endpoint within a maximum of 15 min (Fig. 5,
A-D), just as they did in A- units. More
prolonged exposures were never observed to produce further increases in
threshold.
The absence of a progressive suppression of C units over time is
illustrated in Fig. 5B by a C unit that continued to show mechanoresponsiveness after prolonged exposure to 106 M
TTX, a concentration sufficient to produce complete suppression in
100% of fast A-
units. The neuron showed partial suppression after
just 10 min of exposure to 10
8 M TTX but then showed no
further increase in threshold after >6 h of exposure to a 100-fold
increase in concentration (10
6 M). In view of the rapid
effect of a much lower concentration (10
8 M), the lack of
further suppression by 10
6 M cannot be explained by
inadequate access or exposure time.
Figure 5, C and D, illustrates two C units that
showed only partial suppression after exposure to TTX concentrations of
10
6 M for 50 and 69 min, respectively. As in all
neurons, the increases in threshold could be observed within 10 min of
a concentration increment, and there was no tendency for progressive
threshold increases with exposures times >15 min.
Exposure length
In studies of differential sensitivities of nerve fibers to local
anesthetics, the concentration required to produce impulse blockade
depends strongly on the length of the axonal segment that is exposed to
the drug (Raymond et al. 1989). In this study the length
of exposed axon could not be controlled, but an estimate of this length
was made for each neuron by measuring the distance from the
mechanosensitive site on the dura to the site at which the main axon
appeared to reach the dura in its course from the tentorium below
(based on measurements of response latency to single shock stimuli, as
described in the METHODS). A negative correlation was found
between the length of the axonal branch that was exposed to the drug
and the applied concentration required for complete suppression of
mechanoresponsiveness for both TTX (Fig.
8A; r =
0.57, P < 0.001) and lidocaine (Fig. 8B;
r =
0.58, P < 0.001).
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To determine whether this effect of exposure length on drug
sensitivity might contribute to the differences in drug sensitivity between C- and A- fibers, the relationship between exposure length and conduction velocity was examined. There was no significant difference in exposure length between A-
and C fibers (ANOVA, P > 0.1) and no significant correlation between
exposure length and conduction velocity. However, there was a trend for
slowly conducting fibers to have shorter exposure lengths which,
although not significant (r =
0.34, P > 0.05), might still contribute to the higher TTX resistance of C
fibers. Therefore partial correlations were calculated between exposure
length, conduction velocity, and TTX sensitivity (applied concentration
of TTX required for complete suppression of mechanosensitivity) to
remove the effect of exposure length from the correlation between
conduction velocity and TTX sensitivity. The correlation between
conduction velocity and TTX sensitivity was only slightly reduced by
this calculation (r =
0.74). Stepwise regression
analysis indicated that differences associated with conduction velocity
accounted for 59% of the variance in TTX sensitivity, and differences
in exposure length accounted for an additional 11%.
Sensitivity to TTX was significantly correlated with sensitivity to lidocaine (Fig. 7C; r = 0.59, P < 0.001). Partial correlation analysis indicated that approximately one-third of this correlation was accounted for by the effect of exposure length on drug sensitivity. Overall, the statistical analyses indicate that 1) TTX and lidocaine sensitivity were influenced by a number of common factors, one of which is exposure length, and 2) sensitivity to TTX, but not to lidocaine, was also strongly dependent on conduction velocity, which was more predictive of TTX sensitivity than all other factors combined.
Effect of calcium removal
Because calcium currents could in theory generate
TTX-resistant impulses (see DISCUSSION), the effect of the
removal of calcium from the bathing solution on mechanosensitivity was
investigated in three neurons (2 C and 1 A-). Removal of calcium by
application of calcium-free SIF with 5 mM ethylene
glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic
acid (EGTA) had no detectable effect on mechanical response threshold,
although it produced an increase in ongoing discharge (in agreement
with the results of Pozo et al. 1992
in corneal
afferents). In one of these neurons, a C unit, TTX at 10
6
M was applied first in normal SIF and then for 40 min in
calcium-free/EGTA SIF, with no change in von Frey threshold.
Effect of TTX on electrical stimulation thresholds
It is theoretically possible that the differential TTX sensitivity
was entirely due to an action of TTX on mechanotransduction and that
impulse conduction was not affected by TTX in either A- or C
neurons. If this were the case, the threshold for electrical stimulation of the dura should be relatively unaffected by TTX concentrations that suppress mechanoresponsiveness. To test this possibility, response threshold to electrical stimulation of the dura
was measured before and after suppression of mechanoresponsiveness in 6 fast A-
neurons and 5 C neurons. The TTX concentrations that
produced complete suppression of mechanoresponsiveness also produced a
large elevation in electrical threshold (13.3 ± 10.8 and 8.3 ± 3.9, for A-
and C neurons, respectively, expressed as multiples
of baseline threshold), consistent with an effect on conduction. The
responses to these elevated currents occurred at shortened latencies,
indicating that they result from current spread to the parent axons in
the underlying tentorial nerve, which was not exposed to the TTX (see
DISCUSSION).
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DISCUSSION |
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Differential sensitivity to TTX
The results demonstrate a differential sensitivity to TTX
according to conduction velocity and fiber classification in
mechanosensitive dural afferent neurons. An applied TTX concentration
of 1 µM, which blocks TTX-sensitive sodium channels in dorsal root
ganglion cells, produced complete suppression of mechanoresponsiveness in all fast A- fibers (
5 m/s) but not in most C fibers. The majority of C-fibers showed a partial suppression at 1 µM, but most
C-fibers as well as many of the slower A-
fibers required higher
concentrations for complete suppression of mechanoresponsiveness. The
concentration of TTX required for complete suppression of mechanoresponsiveness was ~270-fold higher in C fibers than in fast
A-
fibers.
A similar differential sensitivity to TTX has also been found in dorsal
root ganglion, for impulses evoked by intracellular current injection.
Most of the small ganglion cells are resistant to suppression by TTX
concentrations (0.1-3 µM) that block activity in the larger cells
(Caffrey et al. 1992; Yoshida and Matsuda 1979
; Yoshimura et al. 1996
). In dorsal root
ganglion, the basis for this differential TTX sensitivity was
determined in whole cell patch clamp studies of dissociated neurons,
which showed that TTX-resistant sodium channels are present in most of
the small ganglion cells, whereas larger cells possess only
TTX-sensitive channels (Caffrey et al. 1992
;
McLean et al. 1988
; Rizzo et al. 1994
;
Roy and Narahashi 1992
; Yoshimura et al.
1996
). Some small cells possess both TTX-sensitive and
-resistant channels. The TTX-sensitive current is entirely blocked by
TTX concentrations of <0.1 µM, whereas the TTX-resistant current is
unaffected at 0.1 µM and requires concentrations of
60-100 µM
for 50% suppression (Akopian et al. 1996
;
Elliott and Elliott 1993
; Ogata and Tatebayashi 1993
; Roy and Narahashi 1992
;
Sangameswaran et al. 1996
).
Direct measurement of single-cell membrane currents has not been
accomplished in small-diameter nerve fibers, so inferences about the
presence of TTX-resistant channels in such fibers have had to rely on
recordings of impulse activity. However, in nerve fibers the
sensitivity of impulse activity to sodium channel blockers is affected
by a number of factors in addition to sodium channel subtype, such as
the locus and density of sodium channels and other channels, fiber
geometry, and cable properties (Raymond and Gissen
1987). For example, myelinated fibers might be more susceptible
to blockade as a result of the restricted distribution of sodium
channels to nodal regions of the axonal membrane, with long internodes
of inexcitable membrane. If, as a result of such factors, C fibers had
greater conduction safety than A-
fibers (Gissen et al.
1982
) or stronger mechanical transduction, they could in
principle exhibit a greater resistance to TTX without having
TTX-resistant sodium channels. We assessed the importance of these
other factors by determining the differential sensitivity of A-
and
C fibers to lidocaine, which acts on both TTX-sensitive and -resistant
channels (Roy and Narahashi 1992
). If the differential TTX sensitivity resulted from general differences in conduction safety
or impulse generation, a comparable differential sensitivity should
also be found with lidocaine. This was not the case. The difference in
lidocaine sensitivity between A-
and C fibers was <1% as large as
the difference in TTX sensitivity. From this it would appear that
factors other than channel subtype can account for only a small part of
the differential TTX sensitivity.
Two additional factors should be noted that could potentially affect
this comparison between TTX and lidocaine. First, concomitant blocking
of potassium channels, which occurs with lidocaine and not with TTX,
would partially offset the effect of sodium channel blockade. However,
the predicted effect would be relatively small (Raymond
1992) and would tend to produce a greater differential block of
A-
over C fibers, given that potassium currents appear to be larger
in C than in A-
fibers (Gorke and Pierau 1980
;
Kirchhoff et al. 1992
). Such an effect would be in the
opposite direction needed to explain our results, which showed a far
greater differential block of A-
fibers by TTX than by lidocaine.
Second, differential sensitivity to TTX could in principle result from
differential access, as might occur if a diffusion barrier to TTX were
present around C units but not A- units. Any such barrier would have
to selectively impede diffusion of TTX but not the relatively more
lipophilic lidocaine, since there was little differential sensitivity
to lidocaine. No such lipid barrier is known in any tissue, including
the dura (Andres et al. 1987
). Rather, it is A-
fibers, not C fibers, that are known to have a lipid barrier (myelin).
Furthermore, both TTX and lidocaine are thought to diffuse in the
aqueous interstitial fluid rather than in lipid, and both have access
to myelinated and unmyelinated fibers. Aside from these theoretical
considerations, the time course of the TTX effects is incompatible with
a selective failure of access of TTX to C fibers. To explain the data
by differential access, there would have to be a much longer time
course for any effect of TTX on C fibers as compared with
A-
fibers. This was not the case. TTX, even at concentrations of
1
µM, increased the threshold within 10 min of drug exposure in C
fibers as well as A-
fibers. These data are incompatible with the
idea that TTX has significantly less access to C fibers than to A-
fibers. Conceivably, C fibers might have one population of sodium
channels that is highly accessible and another population along the
same axons that is inaccessible, but such a novel hypothesis of
spatially segregated channels that differ vastly in accessibility is
generally invoked only for myelinated axons, not unmyelinated ones. In
contrast, the alternative explanation of separate channel populations
that differ in TTX affinity is consistent with the known properties of
sodium channel subtypes in small-diameter sensory neurons.
The results of this study thus offer strong indirect evidence for the
presence in dural C fibers of an ion channel that is resistant to TTX
but not to lidocaine. In addition to a TTX-resistant channel, the
majority of C-fibers also appear to possess TTX-sensitive sodium
channels, since they showed partial suppression of
mechanoresponsiveness with 1 µM TTX. However, the TTX-resistant
channels apparently are present in sufficient numbers to support the
initiation and propagation of mechanically induced impulses in most C
fibers, as well as in many slow A- fibers, but not in fast A-
fibers.
These results do not directly demonstrate the identity of the
TTX-resistant channel in the dural afferents, but the voltage-gated TTX-resistant sodium channel found in small diameter dorsal root ganglion cells seems the most likely candidate among known ion channels. It is conceivable that a C fiber-specific voltage-gated calcium channel could also contribute to the differential TTX resistance of C fibers because voltage-gated calcium channels are
resistant to TTX (Dichter and Fischbach 1977;
Heyer and Macdonald 1982
; Matsuda et al.
1978
; Ransom and Holz 1977
; Yoshida et
al. 1978
) and are suppressed by lidocaine (Oyama et al.
1988
), although not as strongly as sodium channels. However, it
is unlikely that calcium channels would be capable of supporting
impulse propagation on their own, as axonally propagating calcium
spikes have been observed only rarely, primarily in invertebrates, and
usually only in the presence of potassium channel blockers (Horn
1978
; Stockbridge and Ross 1986
; Yoshida
and Matsuda 1980
). In addition, our observations in a few
neurons that the removal of calcium did not affect TTX susceptibility
or mechanoresponsiveness, as well as similar observations on
mechanosensitivity in small-diameter corneal afferents (Pozo et
al. 1992
), make it unlikely that calcium currents have a major
role in the TTX-resistant responses we observed.
Cellular localization of TTX resistance
In the preparation used in this study, the axons of the dural
afferent neurons reach the dura through the tentorial nerve, which
courses caudally from the trigeminal ganglion along the base of the
skull and then dorsally up the tentorium to reach the dura covering the
dorsal surface of the brain (Andres et al. 1987;
unpublished observations). On reaching the dura, the axons give rise to
fine branches that travel horizontally across the dura for variable
distances of up to several millimeters to supply mechanosensitive
endings (see Figs. 1 and 2). Solutions applied topically to the outer
surface of the dura would be expected to access the dural branches but
not the main axons in the tentorial nerve below. TTX resistance in this
preparation thus requires that mechanical transduction, impulse
initiation, and impulse propagation from the mechanosensitive ending to
the point where the dural branch joins the main axon are all TTX
resistant. These findings thus demonstrate TTX resistance in the nerve
endings and the dural axonal branches supplying them but provide no
evidence on the TTX sensitivity of the main (parent) axons in the
tentorial nerve.
A number of previous electrophysiological studies found that mammalian
peripheral nerve fibers in nerve trunks are not resistant to TTX. In
recordings of the compound action potential in rabbit sciatic and vagus
nerves, it was found that the C-fiber component was slightly less
sensitive to TTX than the A- fiber component, but this difference
was no greater than that found in the sensitivity to lidocaine, and all
fibers were blocked at TTX concentrations (1-3 µM) far below that
required to suppress the TTX-resistant sodium current present in the
cell bodies of sensory neurons (Colquhoun and Ritchie
1972
; Gaumann et al. 1992
; Gissen et al.
1980
). Intracellular recording studies in preparations of
rodent dorsal root ganglion with peripheral nerve attached have found
that TTX blocked axonally conducted impulses in all neurons studied,
including C- and A-
fiber neurons whose cell bodies were TTX
resistant (Ritter and Mendell 1992
; Villiere and
McLachlan 1996
; Yoshida and Matsuda 1979
).
Conduction through the central axonal processes in the dorsal roots was
also blocked by TTX. Similarly, Noda et al. (1997)
found that TTX
blocked propagation of impulses evoked by application of bradykinin to
the distal processes of small-diameter cultured dorsal root ganglion
cells. On the other hand, recordings of the compound action potential
in peripheral nerve biopsy material from patients with peripheral
neuropathies found that the C-fiber wave was partially resistant to TTX
(Quasthoff et al. 1995
), although it is not known
whether the property of TTX resistance was affected by the nerve
pathology (Novakovic et al. 1998
). In addition, one study reported that the synaptic response of rat spinal cord neurons to
stimulation of C fibers in peripheral nerve was not blocked by
application of 0.5 µM TTX to the nerve (Jeftinija
1994
). However, the length of peripheral nerve exposed to TTX
in this study, although not reported, was clearly much shorter than
that used in the studies cited previously and may have been inadequate
for producing impulse blockade. Alternatively, there may exist a
subpopulation of TTX-resistant C fibers in peripheral nerve that were
not sampled in the single-cell recording studies. Overall, the weight
of available electrophysiological evidence indicates that the
peripheral axonal branches of most mammalian C fibers do not possess
TTX-resistant channels in sufficient numbers to support impulse
propagation. This conclusion is further supported by the recent
observation that PN3, the sensory neuron-specific TTX-resistant sodium
channel, can be localized immunohistochemically in small-diameter
dorsal root ganglion cells but cannot be detected in peripheral nerve
unless the nerve has been damaged, in which case the channel
accumulates at the site of injury (Novakovic et al.
1998
).
This study is the first to document TTX resistance in peripheral
endings of C-fiber neurons. These results, considered together with the
evidence cited previously on the TTX sensitivity of peripheral nerve
fibers, suggest that the TTX-resistant sodium channel may be
selectively distributed in the peripheral terminals and preterminal axonal branches of C-fibers, as well as in the cell body, but not in
the main axon. A selective subcellular distribution (somal vs. axonal)
has been found previously for different subtypes of the TTX-sensitive
sodium channel (Westenbroek et al. 1989). The current
results are consistent with the idea that the cell body of
small-diameter dorsal root ganglion cells may be used as a model for
the study of the electrophysiological properties of the C-fiber
peripheral nerve terminal (Gold et al. 1996a
). It is
possible that TTX-resistant channels may be a distinctive feature of
the receptive rather than the conductive portion of the axonal membrane
and that their density in the preterminal axonal branch decreases at
progressively greater distances from the terminal. This would be
consistent with the finding that neurons with longer axonal branches
within the dura tend to be more susceptible to suppression by TTX.
Interpretation of applied drug concentrations
In an in vivo preparation such as that used in this study, the
actual drug concentration at the site of action on the exposed nerve
fibers will inevitably be lower than the concentration that was
applied, as a result of factors such as connective tissue diffusion
barriers, dilution by tissue fluids, and transport in the circulation.
Such differences in absolute drug concentration do not affect the
determination of relative drug susceptibilities. For lidocaine, this
difference between the applied concentration and the effective
concentration at the site of action in our preparation may be estimated
on average at roughly one order of magnitude, based on comparison of
our mean applied blocking concentration (18 mM) with steady-state
blocking concentrations in mammalian peripheral nerve in vitro
(Huang et al. 1997; Raymond et al. 1989
). Individual nerve fibers might differ considerably in their relative accessibility to topically applied solutions, for example, as a result
of differences in the depth of the mechanosensitive nerve ending within
the dura. Such differences in accessibility might contribute to the
large interneuron variability in lidocaine sensitivity and particularly
might account for the few neurons in which lidocaine failed to produce
complete suppression of mechanosensitivity. However, such interneuron
variation in drug accessibility apparently was not strongly related to
fiber conduction velocity, because no significant relationship was
found between conduction velocity and lidocaine sensitivity.
Subdivision of A- neurons
A- neurons were subdivided into fast (>5 m/s) and slow groups
in our initial study of meningeal afferents to describe the variation
in the incidence of mechanosensitivity across the population (Strassman et al. 1996
). This subdivision was retained
because of its utility in describing the presence of TTX resistance
(i.e., TTX resistance was only found in neurons slower than 5 m/s). It should be noted that the conclusions of this study are also supported by correlation analyses based on conduction velocity without separation into neuronal classes. Other studies of primary afferents have also
treated the more slowly conducting A-
neurons as a separate group,
either explicitly or implicitly by omission. Many studies of A-
nociceptors in skin and other tissues did not examine fibers having
conduction velocities below 5 m/s (e.g., Burgess and Perl 1967
; Hoheisel et al. 1989
; Light and
Perl 1979
; Lynn and Carpenter 1982
; Meyer
et al. 1991
; Perl 1968
; Rethelyi et al.
1982
), whereas others described the slower A-
fibers as a
separate class (Hoheisel and Mense 1987
; Liang
and Terashima 1993
; Liang et al. 1995
;
Lynn et al. 1995
). One intracellular labeling study
presented light microscopic evidence that fibers in the lower end of
the A-
range were unmyelinated on both the peripheral and central
sides of their axonal bifurcation in the dorsal root ganglion
(Hoheisel and Mense 1987
). Thus this as well as several
previous studies found that in some cases slow A-
fibers can be more
similar to C fibers than to the faster A-
fibers with respect to
certain anatomic and physiological properties, including myelination. Therefore it may be warranted in some cases to regard them as a
separate, intermediate group when investigating potential differences between C and A-
fibers.
Implications for selective block of peripheral nociception
These findings support the possibility that, if a blocking agent
can be developed for the sensory neuron-specific TTX-resistant sodium
channel, it would selectively inhibit impulses in slowly conducting
sensory fibers in the periphery and thus might function as a
peripherally acting analgesic. Such an agent might not produce complete
block because TTX-sensitive sodium channels are also present in these
fibers (as discussed previously), but it would be expected to inhibit
activity evoked through all transductive mechanisms, in contrast to
agents acting at other potential C fiber-specific targets such as the
capsaicin receptor. Such an agent might thus provide for the first time
the long-sought differential blockade of small-diameter nociceptive
fibers, which is not produced by any of the existing blocking agents
such as lidocaine (Raymond and Gissen 1987).
Conclusion
The mechanosensitive endings and distal axonal branches of most
dural C fibers are resistant to TTX, whereas those of fast A- fibers
are not. The TTX resistance of C fibers relative to fast A-
fibers
is too large to be explained by a difference in safety factor, as
demonstrated by the relatively small difference in sensitivity to
lidocaine. The explanation for the differential TTX sensitivity that
appears to be most consistent with currently known properties of
sensory neurons is that TTX-resistant sodium channels are present in
the distal axonal branches of most dural C-fibers, as well as many of
the slower A-
fibers, but not fast A-
fibers in sufficient
numbers to support the initiation and conduction of mechanically
induced impulses.
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ACKNOWLEDGMENTS |
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The authors thank G. Bove for comments on the manuscript and figures and L. Gerry for the artwork in Fig. 1.
This work was supported by the National Headache Foundation and by National Institute of Neurological Disorders and Stroke Grant NS-32534.
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FOOTNOTES |
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Address for reprint requests: A. Strassman, Dept. Anesthesia, DA-717, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215.
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 24 July 1998; accepted in final form 22 October 1998.
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NOTE ADDED IN PROOF |
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Similar results were recently reported for corneal afferents (Brock et al. J. Physiol. (Lond.) 512: 211-217, 1998).
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REFERENCES |
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