1Department of Pharmacology and Division of Neuroscience, University of Alberta, Edmonton, Alberta T6G 2H7, Canada; and 2Department of Physical Therapy, Tennessee State University, Nashville, Tennessee 37209
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ABSTRACT |
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Abdulla, Fuad A. and
Peter A. Smith.
Axotomy- and Autotomy-Induced Changes in Ca2+and
K+ Channel Currents of Rat Dorsal Root Ganglion Neurons.
J. Neurophysiol. 85: 644-658, 2001.
Sciatic nerve section (axotomy) increases the
excitability of rat dorsal root ganglion (DRG) neurons. The changes in
Ca2+ currents, K+ currents,
Ca2+-sensitive K+ current,
and hyperpolarization-activated cation current
(IH) that may be associated with this
effect were examined by whole cell recording. Axotomy affected the same
conductances in all types of DRG neuron. In general, the largest
changes were seen in "small" cells and the smallest changes were
seen in "large" cells. High-voltage-activated
Ca2+-channel current
(HVA-IBa) was reduced by axotomy.
Although currents recorded in axotomized neurons exhibited increased
inactivation, this did not account for all of the reduction
in HVA-IBa. Activation kinetics were
unchanged, and experiments with nifedipine and/or -conotoxin GVIA
showed that there was no change in the percentage contribution of
L-type, N-type, or "other" HVA-IBa
to the total current after axotomy. T-type (low-voltage-activated)
IBa was not affected by axotomy.
Ca2+-sensitive K+
conductance (gK,Ca) appeared to be
reduced, but when voltage protocols were adjusted to elicit similar
amounts of Ca2+ influx into control and
axotomized cells, IK,Ca(s) were
unchanged. After axotomy, Cd2+-insensitive,
steady-state K+ channel current, which primarily
comprised delayed rectifier K+ current
(IK), was reduced by about 60% in
small, medium, and large cells. These data suggest that axotomy-induced
increases in excitability are associated with decreases in
IK and/or decreases in
gK,Ca that are secondary to decreased
Ca2+-influx. Because
IH was reduced by axotomy, changes in
this current do not contribute to increased excitability. The amplitude
and inactivation of IBa in all cell
types was changed more profoundly in animals that exhibited
self-mutilatory behavior (autotomy). The onset of this behavior
corresponded with significant reduction in
IBa of large neurons. This finding
supports the hypothesis that autotomy, that may be related to human
neuropathic pain, is associated with changes in the properties of large
myelinated sensory neurons.
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INTRODUCTION |
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The spontaneous activity that is
induced in sensory nerves following peripheral nerve injury may be
related to the development of "neuropathic" pain in humans
(Kauppila 1998; Millan 1999
). Because
some of this activity arises from the dorsal root ganglia (DRG)
(Babbedge et al. 1996
; Wall and Devor
1983
), there is considerable interest in understanding how
nerve injury might influence the electrical properties of sensory
neuron cell bodies. In rats, damage to the sciatic nerve increases the
excitability of L4 and L5
DRG neurons. This is seen as an increased incidence of spontaneously active cells and lowering of threshold (Study and Kral
1996
) as well as a reduction in rheobase (Kim et al.
1998
). We have also shown that sciatic nerve section (axotomy)
increases the number of action potentials (APs) fired in response to
sustained depolarizing current (Abdulla and Smith 2001
);
i.e., it decreases accommodation and increases "gain." For those
experiments, DRG neurons were divided into four categories on the basis
of size and/or AP shape (Abdulla and Smith 2001
). Most
nociceptors were assumed to be contained within the "small" cell
population (Bessou and Perl 1969
; Gallego and
Eyzaguirre 1978
), and "large" cells were presumed to be
primarily nonnociceptive. We also defined "medium" cells that were
intermediate in size and spike width between small and large cells.
"AD"-cells were defined as those in which the AP was followed by an
afterdepolarization (ADP) (White et al. 1989
) rather
than by an afterhyperpolarization (AHP). In general, sciatic nerve
section affected small cells more than medium or AD-cells, and these
were affected more than large cells. In addition to decreasing
accommodation and increasing gain, axotomy decreased rheobase and
produced significant increases in spike height (AP amplitude) in small,
medium, and AD-cells. It also produced a significant increase in spike
width (AP duration) in small cells (Abdulla and Smith
2001
).
Decreased accommodation of AP discharge and increased gain of DRG
neurons is also seen following suppression of
Ca2+ channel currents with neuropeptide Y (NPY),
-conotoxin GVIA (
-CNTX GVIA), Cd2+,
or norepinephrine (Abdulla and Smith 1997
,
1999
). Similar effects are seen following suppression of
Ca2+-sensitive K+
conductances (gK,Ca) with blockers of
BKCa channels such as iberiotoxin (Scholz
et al. 1998
) or charybdotoxin (Abdulla and Smith
1999
). Other work has shown that blockade of slowly
inactivating K+ conductances with 4-aminopyridine
or dendrotoxin increases the excitability of visceral afferent neurons
in rat nodose ganglion (Stansfeld et al. 1986
). Since
these pharmacologically induced increases in excitability resemble
those invoked by chronic sciatic nerve section (Abdulla and
Smith 2001
), we examined the effects of axotomy on the
properties of Ca2+ currents,
Ca2+-sensitive K+ currents,
and various K+ currents in rat DRG neurons. We
have also examined the effect of axotomy on the
hyperpolarization-activated cation conductance (IH) (Mayer and Westbrook
1983
). To relate the results to injury-induced changes in
excitability and AP shape of various types of DRG neuron, we have
classified cells as small, medium, AD-, or large cells according to
criteria established in our current-clamp studies (Abdulla and
Smith 2001
).
The effects induced in DRG neurons by axotomy were intensified in
animals that exhibit a self-mutilatory behavior known as autotomy
(Coderre et al. 1986; Wall et al. 1979
).
Further increases in excitability were seen, and there was an
especially clear difference between the properties of large cells from
axotomized animals that did not exhibit autotomy and from those that
did (Abdulla and Smith 2001
). In fact, the onset of
autotomy seemed to correlate more with a shift of the properties of
large cells than with a shift in the properties of small, putative
nociceptive cells. A second aspect of the present work was to examine
which changes in ionic conductance accompanied this change in
properties of large neurons.
A preliminary report of part of this work has appeared (Abdulla
and Smith 1995).
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METHODS |
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All experimental procedures were in concordance with the
recommendations of the International Association for the Study of Pain
(IASP). Protocols were reviewed and approved by the University of
Alberta Animal Welfare Committee that maintains standards set forth by
the Canadian Council for Animal Care. As in the accompanying paper
(Abdulla and Smith 2001), adult male Sprague-Dawley rats weighing 120-170 g before surgery were housed 2 per cage with free
access to food and water under an alternating 12-h light and dark cycle
at 23°C. The rats were allowed to adapt to their home cages for 1 wk
before use. Rats were anesthetized with pentobarbital sodium (50-55
mg/kg ip), and the sciatic nerve was sectioned proximal to its
bifurcation into the tibial and the peroneal divisions. A 5- to 10-mm
segment of nerve was removed to prevent regeneration. Some of the
control animals underwent nerve exploration alone. Two to 7 wk later,
control rats or operated rats were killed by decapitation and neurons
enzymatically dissociated from L4 and L5 DRG. In a similar fashion to our previous
current-clamp study (Abdulla and Smith 2001
), data were
pooled from axotomized animals 2-7 wk postoperatively. Axotomized
animals were divided into two groups: those that exhibited
"autotomy" (Coderre et al. 1986
; Wall et al.
1979
) and those that did not. Autotomy was scored according to
the scale devised by Wall et al. (1979)
. Although the
maximum score permitted under IASP guidelines is 11, all of our animals
were killed before they attained a score of 8. Whole cell recordings
(at 22°C) were made using a single-electrode voltage-clamp amplifier
(AXOCLAMP 2A) as described previously (Abdulla and Smith 1997
). With low-resistance patch electrodes (2-5 M
), it was
possible to use high switching frequencies >30 kHz with clamp gains as high as 30 mV/nA. The fidelity of the clamp was confirmed by examining voltage recordings. Recordings from cells where the voltage trace was
slow to rise or distorted were discarded. Data were acquired using
PCLAMP 5.5 (Axon Instruments, Foster City, CA) and analyzed using
PCLAMP 6, 7, or 8. Final data records were produced using ORIGIN 5.0 (Microcal, Northampton, MA). Input capacitance
(Cin) was calculated from the membrane
time constant and input resistance or by integration of the
capacitative transient generated by a 10-mV voltage jump (for details
see Abdulla and Smith 1997
). In some experiments, APs
were recorded in bridge-balance current-clamp mode before switching to
single-electrode voltage clamp. APs were generated using a 2-ms
depolarizing current pulse. Spike width (AP duration) was measured at
50% maximum amplitude. For recording APs, instantaneous
current-voltage (I-V) relationships or
IH, external solution contained (in
mM) 150 NaCl, 5 KCl, 2.5 CaCl2, 1 MgCl2, 10 HEPES-NaOH (pH 7.4), and 10 D-glucose (osmolarity 330-340 mOsm). Internal (pipette)
solution contained (in mM) 130 KGluconate, 2 Mg-ATP, 0.3 Na-GTP, 11 EGTA, 10 HEPES-KOH (pH 7.2), and 1 CaCl2
(osmolarity 310-320 mOsm). Ca2+ channel currents
(ICa) were measured using
Ba2+ as a charge carrier
(IBa). For these experiments, external
"Ba2+" solution contained (in mM) 160 TEA-Cl,
10 HEPES, 2 BaCl2, 10 D-glucose, and
200 nM TTX, adjusted to pH 7.4 with TEA-OH, and internal solution
contained (in mM) 120 CsCl, 5 Mg-ATP, 0.4 Na2-GTP, 10 EGTA, and 20 HEPES-CsOH (pH 7.2). For
recording K+ currents, external solution
contained (in mM) 145 N-methyl-D-glucamine (NMG)Cl (pH 7.4), 10 KCl, 2.5 CaCl2, 10 HEPES,
1.0 MgCl2, and 10 D-glucose, and
internal solution contained (in mM) 100 K gluconate, 40 NMG-Cl (pH
7.2), 2 Mg-ATP, 0.3 Na2GTP, 11 EGTA, 10 HEPES,
and 1.0 CaCl2. [Predicted
K+ equilibrium potential
(EK) =
58 mV (at 20°C).]
The total volume of fluid in the recording dishes was about 1 ml. They
were superfused with external solutions at a flow rate of 2 ml/min.
Drugs were applied by superfusion. Nifedpine solutions were
administered under subdued lighting conditions from light-proof reservoirs. The drug was dissolved in DMSO (dimethyl sulfoxide) or
polyethylene glycol to make 10-mM stock solutions. These were diluted 1 in 5,000 in external solution for application to the DRG neurons.
-CNTX GVIA was added directly to the bath pipette near the neuron
under study after bath flow was halted, achieving a final concentration
of approximately 1 µM.
Clear-cut differences in the Cin
provided a criterion for classification of DRG cells (see Fig. 3 in
Abdulla and Smith 2001). Cin was always >90 pF for large
neurons, 70-90 pF for medium neurons, and always <70 pF for small
neurons. In experiments where AP solutions were used, classification of
control neurons could be verified by examining AP shape; large neurons
had AP duration <3 ms with no deflection in the falling phase of AP,
medium neurons had AP duration 3-5 ms with a deflection on the falling
phase, and small neurons had AP duration more than 5 ms. AD-neurons
were identified by the presence of an ADP in current-clamp recordings
or by the presence of a predominant T-type Ca2+
channel current (ICa,T or
IBa,T) (Scroggs and Fox
1992
; White et al. 1989
) following a voltage
step from
90 to
40 mV when studied in Ba2+
external solution. The AD-cell population likely includes the "type 2 cutaneous afferent neurons" recently defined by Baccei and
Kocsis (2000)
. The identification of AD-cells was precluded in
the solutions used to study K+ currents. Since
their size fell mainly within the medium cell range (see Scroggs
and Fox 1992
), it is presumed that AD-cells make up some of the
medium cell category investigated under these conditions. All data are
presented as means ± SE. In graphs where no error bars are
visible, the error bars are smaller than the symbols used to designate
the data points.
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RESULTS |
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Calcium channel currents
Ca2+ channel currents
(IBa) were evoked by an incremental
series of 50-ms depolarizing voltage commands
(Vc) from holding potentials (Vh) of 90,
60, or
40 mV. Figure
1, A-D, illustrates typical recordings of currents evoked at
40 and at
10 mV or
15 mV in small, medium, large, and AD-cells from
Vh =
90 mV. AD-cells are
distinguished from medium cells by the presence of a
low-voltage-activated (LVA) current (T-current,
ICa,T, or
IBa,T) with small depolarizing commands to
40 mV (Fig. 1D) (Fox et al.
1987
; Scroggs and Fox 1992
; White et al.
1989
). No current is evoked in small, medium, or large cells at
this potential (Fig. 1, A-C).
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Families of I-V plots averaged from 20 to 68 neurons of each
type are shown in Fig. 2,
A-D. The three lines in each graph represent
IBa evoked from
Vh = 40,
60, or
90 mV. Small
cells (Fig. 2A) have the smallest currents, medium-sized
cells, and AD-cells have intermediate-sized currents (Fig. 2,
B and D), and large cells have the largest
currents (Fig. 2C). The presence of
IBa,T shows up as shoulder on the
I-V plot at voltages between
60 and
30 mV. This is
marked with an asterisk in Fig. 2D (see also Fox et
al. 1987
).
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Effects of axotomy on IBa amplitude
Axotomy reduced HVA-IBa in all
types of DRG neuron. Table 1 presents a
statistical comparison of HVA IBa peak
amplitudes (recorded at 10 mV from
Vh =
90 mV) in control cells and
axotomized cells. The reduction in IBa
produced by axotomy was significant in small and medium neurons but not
in large and AD-neurons (Table 1). These effects are clearly seen by
comparing the I-V plots from control small and medium
neurons in Fig. 2, A and B, with those from small
and medium axotomized neurons in Fig. 2, E and F.
By contrast, there is little difference between the I-V
plots for control large and AD-cells (Fig. 2, C and
D) and those from axotomized large and AD-cells (Fig. 2,
G and H).
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LVA-IBa
(IBa,T) in AD-cells was unchanged by
axotomy. The current, recorded at 50 mV to minimize contamination by
HVA IBa in control AD-cells was
2.04 ± 0.18 nA (mean ± SE; n = 25) and in
axotomized cells was 1.9 ± 0.19 (n = 24;
P > 0.6). The absence of an effect on
IBa,T is further illustrated by the
persistence of the inward current shoulder on the I-V plot
for axotomized AD-cells between
60 and
30 mV (asterisk in Fig.
2H).
Representative data records from axotomized neurons are shown in
Fig. 1, E-H. HVA IBa (at
10 or
15 mV) in the axotomized cells is consistently smaller than
that in control cells (Fig. 1, A-D). There is, however,
little difference in the amplitude of
IBa,T recorded at
40 mV from the
typical control (Fig. 1D) and from the axotomized AD-cell
(Fig. 1H).
IBa amplitude in cells from animals that exhibited autotomy
The effects of axotomy on
HVA-IBa in all cell types were
intensified in animals that exhibited autotomy. This is clear from the
numerical values that are summarized in Table 1 and from the
I-V plots shown in Fig. 2, I-L. The profound
attenuation of HVA-IBa in cells from
the autotomy group (Fig. 1, I-L) compared with
axotomized neurons (Fig. 1, E-H) and to those
from the control group (Fig. 1, A-D) is also
clear from the typical records shown in Fig. 1. There was, however, no
difference between the amplitude of
LVA-IBa,T, recorded at 50 mV, in
axotomized AD-cells (1.9 ± 0.19 nA; n = 24) and
in those from animals that exhibited autotomy (1.83 ± 0.18 nA;
n = 24; P > 0.7). There was no
difference between this value and the amplitude of current seen in
control cells (2.04 ± 0.18 nA; n = 25, P > 0.4). The persistence of a robust IBa,T in AD-cells from the autotomy
group is illustrated in the data records of Fig. 1L. There
is also an obvious difference between the averaged I-V plot
evoked from
90 mV and that evoked from
60 mV for this type of cell
(marked with an asterisk in Fig. 2L). This reflects the
relatively large contribution that
IBa,T makes to the total current under
these conditions.
Voltage dependence of gBa activation
The voltage dependence of gBa
activation was examined to gain further insights into the effects of
axotomy and autotomy on Ca2+ channel properties.
A series of 50-ms voltage commands was applied from a
Vh of 90 mV and tail currents
recorded at
40 mV. Activation curves were constructed from normalized
tail current amplitudes. These were measured 0.5 ms after the
termination of the command voltage pulse. After axotomy, the activation
curves of small and medium cells were shifted by about +3 mV (data not
shown). In the autotomy group, the activation curves for large and
AD-cells were shifted by +3 to +5 mV relative to control. It is
difficult to conclude, however, that these modest shifts reflect bona
fide alterations in voltage dependence of
HVA-gBa. This is because the observed
changes in tail current amplitude may reflect differences in
inactivation during a 50-ms pulse rather than a change in voltage dependence of activation.
Effects of axotomy and autotomy on inactivation of gBa
Baccei and Kocsis (2000) recently demonstrated that
sciatic nerve axotomy increased the inactivation of
gBa in cutaneous afferent neurons of
rat DRG. An effect of axotomy and autotomy on
gBa inactivation also is apparent in
the data records from small, medium, large, and AD-cells shown in Fig.
1. In the records chosen for illustration, gBa inactivation at
10 or
15 mV in
control cells (Fig. 1, A-D) is less than that in axotomized
cells (Fig. 1, E-H), which in turn is less than that in
cells from the autotomy group (Fig. 1, I-L). Table
2 shows numerical values for percentage
inactivation of IBa in response to
50-ms pulses to
10 mV for small, medium, large, and AD-cells. These
percentages were calculated from the ratio of peak to end-of-pulse
current recorded at
10 mV. While axotomy is associated with a
relatively modest increase in inactivation in large cells (from 4.1 to
8.0%), a much greater effect is seen in small cells (from 7.1 to
34.7%). By contrast, the appearance of autotomy, in axotomized
animals, is associated with a further large change in inactivation in
large cells (from 8.0 to 32.3%) but with only a modest change in
inactivation in small cells (from 34.7 to 52.0%). This is an important
point, as it implies that the transition of axotomized animals to the
autotomy state coincides with changes in the properties of large rather
than small neurons.
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The consequences of these increases in inactivation are seen in the
families of I-V plots obtained from different values of Vh (Fig. 2). It is clear that changing
Vh has much greater effects on cells
from the axotomy or autotomy groups than on control cells. For example,
changing Vh from 90 to
60 mV has
minimal effect on maximum IBa in all
types of control cell (Fig. 2, A-D) but has pronounced
effects on IBa amplitude in neurons
from animals that exhibited autotomy (Fig. 2, I-L). Thus
for control small cells (Fig. 2A), shifting
Vh from
90 to
60 mV changes
maximum IBa (at
10 mV) by about 14%
(from 7.96 ± 0.86 nA to 6.88 ± 0.73 nA, P > 0.3). The same manipulation on small cells in the axotomy group
changes the current by about 33% (from 4.98 ± 0.51 to 3.38 ± 0.37 nA; Fig. 2E, P < 0.003).
The effects of changing Vh on peak
IBa in typical control and axotomized
small cells are further illustrated in Fig.
3A. Figure 3, A1
and A2, shows recordings of currents evoked at 10 mV from three different values of Vh. Figure
3A1 shows that altering Vh from
90 to
60 mV in a control cell has no effect on the current evoked at
10 mV; the two currents exactly superimpose. In the axotomized small cell, however (Fig. 3A2), peak
IBa is attenuated by about 30% by
shifting Vh from
90 to
60 mV.
Also, further reduction of Vh from
90 to
40 mV in the axotomized cell attenuates the current by about
85% (Fig. 3A2), whereas in the control cell (Fig.
3A1), only a 35% attenuation is seen.
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Effects of Vh on peak
HVA-IBa from small, medium, and large
control, axotomized, and autotomy group neurons are summarized in Fig.
3, B-D. AD-neurons, that exhibit
IBa,T, were excluded from this
analysis. Two points emerge. First, currents evoked from
Vh = 90 mV in all cell types under
all conditions are close to maximal. This implies that the attenuation
of the amplitude of IBa seen in
axotomy and/or autotomy (Figs. 1 and 2) cannot be accounted for by
increased inactivation alone. In other words, axotomy and autotomy
reduce IBa evoked from voltages where
inactivation is largely removed. Second,
IBa, and hence
Ca2+ influx evoked in axotomized small cells from
a Vh of
90 mV is about the same as
the Ca2+ influx invoked in control small cells
from Vh =
44 to
51 mV. The dashed
lines in Fig. 3B show that about 5 nA of inward current is
seen under both conditions. Similarly, Ca2+
influx invoked in axotomized medium cells from a
Vh of
80 mV is about the same as
that seen in control medium cells from a Vh about of
45 mV (about 6 nA in
both cases, dashed lines in Fig. 3C). This point is
revisited below during the discussion of
Ca2+-dependent K+ currents.
For large cells, the same amount of inward current is seen in both
control and axotomized cells from all values of Vh (Fig. 3D). Much less
current is seen in cells from the autotomy group.
A more traditional illustration of the effects of inactivation are shown in Fig. 3, E-G. Here, peak current amplitudes have been normalized. For small neurons, there is a large difference in the voltage dependence of inactivation between the control and axotomy group and less of a difference between the axotomy and autotomy groups (Fig. 3E). Little difference is seen for medium neurons (Fig. 3F). For large neurons (Fig. 3G), the voltage dependence of inactivation is not much altered by axotomy but is changed significantly in the autotomy group. These data are again consistent with the idea that the transition of axotomized animals to the autotomy state coincides with changes in the properties of large neurons.
Pharmacology of IBa in control, axotomized, and autotomy group neurons
The classical L-, N-, and T-subtypes of
ICa were originally defined in DRG
cells by Fox et al. (1987). It was subsequently noted
that not all types of ICa fit into
these categories (Scroggs and Fox 1992
), and the
presence of P- and Q-type ICa in DRG
neurons was demonstrated by Rusin and Moises (1995)
.
Because N-type Ca2+ channel current
(ICa,N or
IBa,N) inactivates more rapidly than L-type current (ICa,L or
IBa,L) (Fox et al. 1987
), we
asked whether the increased inactivation seen in axotomized neurons
might result from altered expression of channel subtypes. For
simplicity, HVA-IBa was divided into
IBa,N,
IBa,L, or "other"
(IBa,other) on the basis of the
effects of 1 µM
-CNTX GVIA or 1 µM nifedipine.
A typical experiment on a small control cell is illustrated in
Fig. 4. The control current of 1.8 nA was
reduced to 0.95 nA by nifedipine and reduced further to 0.4 nA by
-CNTX GVIA. This procedure was used to calculate the percentage of
IBa,N,
IBa,L, and
IBa,other, in each neuron studied. The
results are shown in Table 3. As has been
observed by others (Cardenas et al. 1995
; Scroggs
and Fox 1992
), IBa,L is more
prevalent in small cells than in other cell types. The prevalence of
IBa,other in AD-cells reflects the
presence of IBa,T (Scroggs and
Fox 1992
; White et al. 1989
). Despite the
changes in the absolute amplitude and inactivation characteristics of
IBa seen after axotomy (Figs. 1-3),
there seems to be no obvious change in relative proportions of channel
types seen after axotomy or in neurons from animals that exhibited
autotomy. Data were obtained from six to nine cells of each type under
each experimental condition.
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K+ channel currents
McFarlane and Cooper (1991) described three types
of K+ current in sensory neurons: fast and slow
A-currents (IAf and
IAs) and a noninactivating delayed
rectifier (IK). More recently,
Gold et al. (1996a)
further subdivided the currents into
six different types; they recognized
IAf and
IAs as well as a high-threshold A-current (IAht). Sustained current
was divided into three components: IKi,
IKlt, and
IKn. The differential distribution of
K+ channel types in different classes of DRG
neurons is now well-established (Everill and Kocsis
1999
; Everill et al. 1998
; Gold et al.
1996a
), and this must contribute to the diversity in AP shape
seen in these cells (Abdulla and Smith 2001
; Kim
et al. 1998
; Koerber et al. 1988
; Rose et
al. 1986
; Stebbing et al. 1999
;
Villière and McLachlan 1996
). The studies of
McFarlane and Cooper (1991)
, Gold et al.
(1996a)
, Everill et al. (1998)
, and
Everill and Kocsis (1999)
were done in solutions
containing Co2+ or Cd2+ to
block Ca2+ channel currents as well as
Ca2+-dependent conductances such as
Ca2+-dependent K+
conductances (gK,Ca). Both
voltage-sensitive large conductance (BKCa) and
low conductance gK,Cas
(gAHP) have been described in DRG
neurons (Akins and McCleskey 1993
; Fowler et al.
1985
; Gold et al. 1996b
; Gurtu and Smith
1988
; Scholz et al. 1998
). Since suppression
of gK,Ca either directly
(Scholz et al. 1998
) or indirectly, via inhibition of
gCa (Abdulla and Smith
1997
, 1999
; Akins and McCleskey
1993
), decreases spike frequency accommodation in DRG neurons,
we studied effects of axotomy on both
Ca2+-insensitive and
Ca2+-sensitive K+ currents.
The latter experiments are particularly important in the light of the
observed attenuation of ICa
(IBa) in axotomized neurons (see Figs.
1-3) (see also Baccei and Kocsis 2000
).
Outward currents were studied in a solution designed to isolate
K+ currents in the absence of
Na+ currents while preserving
Ca2+ currents. Because this solution contained
NMG rather than Na+, it was not feasible to
distinguish AD-cells by observing an ADP under current-clamp conditions
for this series of experiments. Most AD-cells were presumably included
within the medium cell population (Abdulla and Smith
2001; Scroggs and Fox 1992
). At least in the
presence of Cd2+, all outward currents recorded
in the voltage range we employed are carried by
K+ (Everill et al. 1998
).
We initially divided K+ channel currents
into noninactivating currents and those that displayed some degree of
inactivation during a 50-ms voltage command. Both types of current were
seen in small, medium, and large cells. Inactivating currents were seen
in 33/47 (70%) small cells, 18/30 (60%) medium cells, or 34/62 (55%)
large cells studied from Vh of 90 or
80 mV (Table 4).
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Typical recordings from a small cell in which outward current
inactivated are shown in Fig.
5A. Inactivation of the total outward current probably does not reflect inactivation of the delayed
rectifier K+ current
(IK) (McFarlane and Cooper
1991) but may rather reflect the presence of
IAf,
IAs, and
IAht (Gold et al.
1996a
; IAs may correspond to
the ID defined by Everill et
al. 1998
). Alternatively, Ca2+-sensitive
K+ currents may decline in amplitude with time as
a consequence of the inactivation the Ca2+
conductances responsible for their activation. This mechanism appears
to be in effect in the neuron illustrated in Fig. 5A as the
apparent inactivation is removed when the current is recorded in the
presence of 500 µM Cd2+ (Fig. 5A2).
This blocks Ca2+ influx and removes the
contribution of gK,Ca to the total
conductance. The remaining current, which does not exhibit appreciable
inactivation during the 50-ms voltage command, presumably reflects
IK. Figure 5A3 illustrates
the subtraction of the Cd2+-resistant current
shown in Fig. 5A2 from the total current shown in Fig.
5A1. This yields the Cd2+-sensitive
portion of the current that presumably reflects
IK,Ca.
|
Figure 5B shows recordings from another small neuron in which no inactivation of outward current was seen.
Inactivation often appeared to abate in the presence of
Cd2+. For example, from
Vh = 80, Cd2+
removed the apparent inactivation in 15/16 small control cells, 4/5
medium control cells, and 8/13 large cells (Table 4). This implies that
gK,Ca contributes to the outward
conductances in all neuron types and that it is especially important in
small and medium cells. The inactivation that persisted in the presence of Cd2+ was usually quite slow and presumably
reflects the presence of IAs/ID
(Everill et al. 1998
; Gold et al. 1996a
;
McFarlane and Cooper 1991
). Slowly inactivating
conductances were more characteristic of large cells than of small
cells. Table 4 shows that 5 of 13 large cells exhibited
Cd2+-resistant inactivation, whereas this was
seen in only 1 of 16 small cells.
Effects of axotomy on K+ currents
In view of the complexity and variability of
K+ currents in DRG neurons (see Akins and
McCleskey 1993; Gold et al. 1996a
), we performed
a fairly simple analysis of the effects of axotomy. We initially
concentrated on its effects on total, steady-state outward currents
evoked from a Vh of
80 mV. The
effects of axotomy were examined by constructing I-V plots
from control and axotomized small, medium and large cells
(n = 11-44; Fig. 6).
Steady-state current amplitude was recorded at the end of a 50-ms
voltage command. Because the large amplitudes of the currents may have
compromised the voltage control attained by the clamp, measured
voltages were plotted against measured currents. Figure 6 shows that
axotomy promotes the greatest attenuation of total steady-state outward current in small cells (62.4% at approximately +65 mV, Fig.
6A), less attenuation in medium cells (41.5% at
approximately +65 mV, Fig. 6B), and the least attenuation in
large cells (22.3% at approximately +65 mV, Fig. 6C).
|
Effects of axotomy on Cd2+-insensitive, steady-state K+ current (mainly IK)
The steady-state I-V plots shown in Fig. 6,
D-F, illustrate the attenuation of
Cd2+-insensitive outward current by axotomy.
These plots were obtained from the steady-state currents that were
evoked by a series of voltage commands from
Vh = 80 mV in the presence of 500 µM or 1 mM Cd2+. Axotomy produces a similar
effect in all three cell types; at approximately +65 mV, current is
attenuated by 56.4% in small cells, 64.8% in medium cells, and 60.0%
in large cells. This is atypical, as axotomy characteristically exerts
its strongest effects on ionic currents in small cells and its weakest
effects on currents in large cells (see Figs. 1-3 and 6) (see also
Abdulla and Smith 2001
). Although
Cd2+-insensitive inactivation was seen in 1/16
small cells, 1/5 medium cells, and 5/13 large cells (Table 4), the
steady-state current flowing at the end of a 50-ms command in the
presence of Cd2+ should be dominated by
IK because
IAs (at +40 mV) is about 80% inactivated at this time (McFarlane and Cooper 1991
). It
is therefore suggested that the data presented in Fig. 6,
D-F, corresponds closely to the effect of axotomy on
IK, the delayed rectifier.
Effects of axotomy on gK,Ca/IK,Ca
To examine the effects of axotomy on IK,Ca, steady-state I-V plots obtained in the presence of Cd2+ (Fig. 6, D-F) were subtracted from the total I-V plots (Fig. 6, A-C) to yield the plots for the Cd2+-sensitive component of the current (Fig. 6, G-I). As expected from its effects on ICa, axotomy attenuated steady-state IK,Ca. The effect was stronger in small and medium cells (53.1 and 55.3% attenuation, respectively, at approximately +65 mV; Fig. 6, G and H) and weak in the large cells (13.0% attenuation at approximately +65 mV; Fig. 6I).
Measurements of the ratios of Cd2+-sensitive to Cd2+-insensitive current from the graphs in Fig. 6 also show that at approximately +65 mV, IK,Ca comprises 46.6% of total outward current in small control cells, 50.8% in medium control cells, and 37.6% in large control cells.
Axotomy-induced attenuation of IK,Ca
could reflect the attenuation of ICa
described above (Figs. 1-3) with or without an additional effects on
intracellular Ca2+ buffering and/or on
gK,Ca channels themselves. To test
whether attenuation of ICa could
completely account for
IK,Ca attenuation, we compared
currents evoked in control and axotomized cells under conditions where
Ca2+ influx was unchanged. This was done by using
different values of Vh. The
experiments illustrated in Fig. 3B show that
IBa evoked from
Vh of about 50 mV (actually
44 to
51 mV) in control small cells is similar in amplitude to
IBa evoked from
90 mV in axotomized cells (about 5 nA in both cases at
10 mV). Figure
7A shows that the steady-state
IK,Ca recorded from
Vh =
90 mV in axotomized cells is of
similar amplitude to that recorded from
Vh =
50 mV in control small cells.
We also showed in Fig. 3C, that for axotomized medium cells,
a Vh of
80 mV permitted about 6 nA
of IBa to be evoked
10 mV. A similar
current amplitude was seen using a Vh of about
46 mV in control medium cells. Figure 7B shows
the effect of activating gK,Ca from
close to these two values of Vh in
axotomized and control medium cells.
IK,Ca recorded from
80 mV in
axotomized medium cells was statistically indistinguishable from that
seen from a Vh of
50 mV in control
medium cells. These results show that when changes in
Ca2+ channel properties are accounted for,
axotomized and control cells express similar amounts of
gK,Ca. This implies that
axotomy-induced changes in IK,Ca
reflect ICa attenuation alone and that
gK,Ca channels are not directly
affected by nerve injury.
|
The properties of K+ channel currents in animals that exhibited autotomy were not examined.
Effect of axotomy on H-current (IH)
DRG neurons exhibit a voltage-dependent rectification that
produces a sag in voltage responses to hyperpolarizing current pulses
(Abdulla and Smith 2001; Czeh et al.
1977
; Gallego et al. 1987
; Gurtu and
Smith 1988
). This slow sag in the voltage trajectory reflects
activation of the hyperpolarization-activated cation current,
IH (Mayer and Westbrook
1983
; Scroggs et al. 1994
). Because sciatic
nerve section attenuates the sag ("rectification") seen in current
clamp (Abdulla and Smith 2001
; Czeh et al.
1977
; Gallego et al. 1987
), we examined the
effect of axotomy on IH. The current was elicited by a series of 1,200-ms hyperpolarizing voltage commands (incremental
10-mV steps to
130 mV) from a
Vh of
50 mV. The inward current
consisted of an initial instantaneous response (IInst, see below), followed by a slow
inward relaxation that became larger and that developed more rapidly at
more negative voltages. A typical recording of
IH in a control large neuron is shown
in Fig. 8A1.
IH relaxations were seen in 49/49
large cells, 33/37 medium cells, 27/30 AD-cells, and 14/24 small cells; a distribution that parallels that of the voltage-dependent sag seen
under current clamp (Abdulla and Smith 2001
). Moreover,
IH amplitude was greatest in large
cells and least in small cells. Absolute values are listed in Table
5. The table also shows that IH was significantly reduced compared
with control in all four cell types after axotomy, and further
attenuation was seen in small and large cells from animals that
exhibited autotomy.
|
|
Figure 8A2 shows typical recordings of the attenuated IH relaxations seen in axotomized large cells. Figure 8B shows the percentages of large, AD-, medium, and small cells in the control, axotomy, and autotomy groups that display IH. The graph illustrates the two general trends; the larger the cell, the more likely it is to display IH, and axotomy reduces the frequency of occurrence of IH. Figure 8C shows IH versus VC plots for the four different cell types in the three different situations. The reduction in current amplitude after axotomy is clearly apparent.
Effects on other K+ currents
Figure 8D illustrates the effect of axotomy and the
development of autotomy on the instantaneous current
(IInst) evoked in small, medium,
large, and AD-cells in response to a series of Vc from
Vh = 50 mV. These data were obtained
from the same experiments that were used to study
IH and that are illustrated Fig. 8,
A or B. Axotomy reduced input conductance for all
cell types for the
120- to
50-mV range. Further decreases occurred
in the autotomy group. Since these effects are seen at relatively
negative potentials, this implies that axotomy decreases leak
conductance. While the instantaneous I-V relationships for
control small and large cells are close to linear over the
120- to
50-mV range, there is a progressive increase in slope with increasing
hyperpolarization in the I-V relationships of medium and
AD-cells (Fig. 8D). This presumably reflects the
pseudo-instantaneous, inwardly rectifying K+
current (IIR) that is seen more
frequently in medium cells than in small or large DRG cells
(Scroggs et al. 1994
). Because the extent of the
contribution of IIR to
IInst is not easily determined using
our experimental conditions, it cannot be stated with certainty whether
IIR is altered by axotomy.
M-current (IM) is a small,
voltage-sensitive, noninactivating K+ current
that is activated at potentials positive to 70 mV in autonomic
neurons (Adams et al. 1982
). Cells that express
M-conductance (gM) exhibit a slow
inward relaxation of membrane current (due to
gM deactivation) when the membrane
potential is stepped to
70 mV from a holding potential of
30 mV.
Because IM may appear in amphibian DRG
cells maintained in tissue culture (Tokimasa and Akasu
1990
) and it is enhanced in bullfrog sympathetic
ganglion B-neurons following axotomy (Jassar et al.
1994
), we used voltage steps to
70 mV from a
Vh of
30 mV to see whether
IM relaxations occurred in control rat
DRG neurons or whether the current was present in the axotomy or
autotomy groups. No evidence for gM was found in any of the cell types under any of the conditions used in
the present study.
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DISCUSSION |
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The effects of axotomy on K+ and
Ca2+ channel properties in identified cutaneous
afferent DRG neurons have recently been described by Everill and
Kocsis (1999) and by Baccei and Kocsis (2000)
. In the present work, we seek to extend the findings of these two studies to the whole DRG population, to incorporate effects on Ca2+-sensitive K+ channels
and other conductances activated by membrane hyperpolarization and to
relate changes in ion channel properties to the development of
autotomy. Using this approach, we find qualitatively similar changes in specific ionic conductances in small, medium, large, and
AD-cells; axotomy affects the same types of K+
and Ca2+ channels in all types of DRG neuron. The
observation that the electrical properties of small cells are more
perturbed by axotomy than large cells (Abdulla and Smith
2001
) therefore reflects quantitative differences in
the effects of axotomy on K+ and
Ca2+ channels in different cell types and from
the differential distribution of these channel types in small, medium,
AD-, and large neurons (Cardenas et al. 1995
;
Scholz et al. 1998
; Scroggs and Fox 1992
; Scroggs et al. 1994
).
The main findings were as follows: 1) HVA-IBa, and hence HVA-ICa, was reduced by axotomy, whereas LVA-IBa (ICa,T) was unaffected; 2) although HVA-IBa displayed increased inactivation in axotomized cells, this did not completely account for the observed in decrease in current; 3) the activation kinetics of gBa were unchanged and the relative contribution of IBa,N, IBa,L, and IBa,other to the total current was unchanged after axotomy; 4) the apparent reduction in gK,Ca seen after axotomy was fully attributable to changes in HVA-ICa; 5) IK and IH were reduced by axotomy; 6) changes in HVA-ICa, and hence gK,Ca were largest in small cells, less in medium and AD-cells, and least in large cells, whereas a comparable decreases in IK and IH were seen across all cell types; 7) ICa,L and gK,Ca were preferentially expressed in small DRG cells, whereas IH and inactivating K+ conductances (IA and/or ID) were seen most frequently in large cells; 8) in general, the changes in ionic currents seen in animals exhibiting autotomy were more intense than those seen in axotomized animals; and 9) the onset of autotomy was associated with a substantial change in the properties of large neurons rather than with a change in the properties of small, putative nociceptive neurons.
Several of these observations are in good agreement with those seen by
others. For control cells, the preferential expression of
ICa,L in small cells was previously
noted by Scroggs and Fox (1992), and the preferential
expression of IH in large cells was previously described by Scroggs et al. (1994)
. Also,
Scholz et al. (1998)
have shown that high conductance
gK,Ca (BKCa
channel current) is seen only in small cells that exhibited broad APs. McFarlane and Cooper (1991)
described the predominance
of inactivating K+ currents in large cells.
Axotomy-induced increases in the inactivation of HVA
ICa have recently been described in
identified cutaneous afferent neurons of rat DRG by Baccei and
Kocsis (2000)
. Increased inactivation of
gBa may therefore be a characteristic
consequence of axotomy as it is also seen in frog sympathetic neurons
(Jassar et al. 1993
) and in rat facial motoneurons
(Umemiya et al. 1993
). Everill and Kocsis
(1999)
have also shown that IK
is reduced by axotomy in identified cutaneous afferent DRG neurons.
Changes in Ca2+ channel currents
IBa activated from
Vh = 90 mV is close to maximal in
all cell types, but the maxima are smaller in axotomized cells (Fig. 3,
B-D). In other words, currents from a
Vh of
90 mV, where most of the
inactivation is removed, are still attenuated by axotomy. This implies
that the axotomy-induced increase in inactivation of
gBa cannot account for all
of the observed reduction in current. A similar conclusion was reached
by Baccei and Kocsis (2000)
in their recent study of
effects of axotomy on identified cutaneous afferent neurons.
Although there appear to be small changes in the voltage dependence of
gBa activation after axotomy, this
likely reflects increased inactivation because
gBa was measured from tail currents that followed 50-ms depolarizing voltage commands. Currents invoked by
this pulse length display noticeable inactivation (see Fig. 1 and Table
2). This would reduce tail current amplitude and yield an underestimate
of the amount of gBa that would be
available at a given voltage. Thus when inactivation is increased after axotomy, more attenuation of tail currents would occur and
gBa, at a given voltage, would appear
smaller than control. If it is accepted that axotomy does
not alter the voltage dependence of activation, this rules
out the possibility that axotomy alters opening probability of
Ca2+ channels. It is therefore suggested that
axotomy reduces the expression of functional Ca2+
channels and that those that are expressed exhibit increased inactivation. This again confirms the findings of Baccei and
Kocsis (2000) in cutaneous afferent neurons.
Because IBa,N inactivates more rapidly
than IBa,L (Fox et al.
1987), we considered the possibility that axotomy alters the relative contribution of IBa,N to the
total current. Although this hypothesis seemed to be excluded by
experiments with nifedipine and
-CNTX GVIA that showed that the
ratio of IBa,L to
IBa,N to IBa,other was preserved after axotomy
(see Table 3), slightly different results were reported in the recent
study by Baccei and Kocsis (2000)
. These authors
reported that IBa,N may be selectively attenuated in identified cutaneous afferent neurons, which are included
within the large and medium neurons of this study. The reasons for
these differences are not yet clear.
We were also particularly interested in knowing how
ICa,T might be altered by axotomy as
this current is responsible for the ADP that is the defining feature of
AD-cells (White et al. 1989). Moreover, the ADP can
promote discharge of multiple APs in response to a brief stimulus and
is therefore a potential source of repetitive discharge in DRG neurons
(Abdulla and Smith 2001
). In confirmation of the
findings of Baccei and Kocsis (2000)
, however,
IBa,T of AD-cells was unaffected by
axotomy (see Figs. 1 and 2). This lack of effect goes along with the
observation that AD-cells showed no greater tendency to discharge
multiple spikes in axotomized animals (Abdulla and Smith
2001
).
Changes in K+ channel currents
We have used 500 µM to 1 mM Cd2+ to
eliminate Ca2+-dependent components of
K+ conductances. One potential problem with this
approach is that divalent cations can interfere with gating and
inactivation of Ca2+-insensitive
K+ currents (Mayer and Sugiyama
1988). While these authors demonstrated effects of
Mn2+, Cd2+, and other
divalent cations on A-currents (IA;
presumably IAf and
IAs) in cultured rat DRG neurons,
IK was unaffected by
Mn2+ (up to 10 mM). Because
Cd2+ is about 3 times as effective as
Mn2+ in altering K+
currents (Mayer and Sugiyama 1988
),
IK should be unaffected by up to 3.3 mM Cd2+. Since we used only 0.5-1 mM
Cd2+, the Cd2+-resistant
current recorded in our experiments likely reflects relatively pure
IK. The effects of
Cd2+ on IA are
complex; it affects both the activation curve and the inactivation
curve for the underlying conductance
(gA) (Mayer and Sugiyama
1988
). Although we used low concentrations of
Cd2+ (500 µM to 1 mM), small
alterations in the properties of IA
could complicate the assumption that the amplitude of
IK,Ca may be deduced from the
difference between total outward current and
Cd2+-resistant current. Complex interactions of
Cd2+ with IA may
therefore have affected the detailed time course of some of the
currents recorded. Despite this, effects of Cd2+
are unlikely to have affected our interpretation of I-V data as these were obtained from steady-state currents flowing at the end of
a 50-ms pulse. At this time, the slowest components of IA are largely inactivated
(McFarlane and Cooper 1991
).
Unlike changes in HVA-IBa that are
small in large cells and large in small cells, the extent of
axotomy-induced reduction of IK was
independent of cell type. About a 60% reduction was seen in small,
medium, and large cells. Everill and Kocsis (1999) also
reported large decreases in IA but
little change in slowly inactivating K+ current
(ID) following axotomy. We have
insufficient data to confirm this observation in large and medium cells
that likely include the population of cutaneous afferent neurons
identified by these authors. Although
Cd2+-insensitive inactivation occurred in only 1 of 16 control small cells, it was present in 8 of 15 axotomized small
cells (Table 4). This raises the possibility that
IA and/or
ID increase in small cells but not in
large cells after axotomy (Everill and Kocsis 1999
).
This possibility remains to be tested.
Because gK,Ca links changes in
Ca2+ flux to changes in spike frequency
accommodation (Abdulla and Smith 1997,
1999
; Scholz et al. 1998
), we were
interested in knowing how axotomy-induced changes in
Ca2+ currents might affect
gK,Ca. This conductance was reduced by axotomy in small and medium cells (Fig. 6, G and
H) but not in large cells (Fig. 6I). We conclude,
however, that the gK,Ca channels themselves are unaffected and that the observed reduction in
IK,Ca is a straightforward consequence
of the decrease in Ca2+ influx produced
by axotomy. This argument is supported by the data shown in Fig. 7,
where Ca2+ influx in control and axotomized cells
was "balanced" by using different values of
Vh. Figure 3B shows that
small axotomized cells held at
90 mV allow the same amount of
Ca2+ influx as control cells held at about
50
mV. Using these two different values of
Vh, similar amounts of
IK,Ca are seen in control and
axotomized small cells (Fig. 7A). A similar "balancing"
of Ca2+ fluxes in medium cells produced the same
amount of IK,Ca before and after
axotomy (Fig. 7B).
Although some types of DRG neuron exhibit a long AHP when studied with
microelectrodes (Fowler et al. 1985; Gold et al.
1996b
; Gurtu and Smith 1988
) and these may
reflect the presence of low-conductance, voltage-insensitive
gK,Ca channels
(IAHP channels) (Pennefather et
al. 1985
), such channels are not readily recorded in
dissociated cells with patch electrodes (Jassar et al.
1994
; Zhang et al. 1994
). We therefore conclude
that most of the effects observed under our experimental conditions
devolved from high-conductance, voltage-sensitive
(BKCa) channels. This idea is supported by
observation that iberiotoxin (Scholz et al. 1998
) and/or
charybdotoxin (Abdulla and Smith 1999
) increase the
number of APs evoked by a depolarizing current in dissociated DRG
neurons studied with patch electrodes, whereas apamin has little or no
effect (M. P. Stebbing and P. A. Smith, unpublished observations).
Changes in other currents
The observation that IH is
reduced by axotomy matches our findings with current clamp, where we
observed a parallel pattern of axotomy-induced changes in the "sag"
in the voltage responses to hyperpolarizing currents (Abdulla
and Smith 2001). Because axotomy-induced reductions in
IH and attenuation of sag would result
in impairment of a depolarizing response in unclamped neurons, this
change is unlikely to be relevant to axotomy-induced increases in
excitability. In fact, the voltage range for
IH activation (Mayer and
Westbrook 1983
) is probably too negative for it to profoundly
influence the excitability of DRG neuron cell bodies.
Relationship between current-clamp and voltage-clamp data
The same criteria that were used to define small, medium, AD-, and
large cells in this study were used to define the cell types in a
previous current-clamp study (Abdulla and Smith 2001). This approach was adopted to establish the direct relationship between
changes in ionic currents and changes in excitability and AP
characteristics in each cell type and to compare the magnitude of
changes across the whole DRG population. The changes in
HVA-gCa, IK, and
gK,Ca invoked by axotomy as well as
the decrease in leak conductance (Fig. 8D) are in a
direction that would be expected to increase excitability. Decreases in
ICa, which are manifest as decreases
in gK,Ca would tend to reduce spike
frequency adaptation as does decreased
IK. In fact, decreased
IK alone (Fig. 6F) may be
responsible for increased excitability of large cells as
gK,Ca in these neurons is little
altered by axotomy (Fig. 6I).
Although we have too little data to say much about effects on
Cd2+-insensitive slowly inactivating currents
(ID or
IA,S) in large or medium cells, data
from another laboratory suggests that these currents are unchanged
(Everill and Kocsis 1999).
While this paper has concentrated on changes in
Ca2+, K+, and
Ca2+-sensitive K+ currents,
axotomy-induced changes in voltage-dependent Na+
conductances (gNa) must also play an
important role in increased excitability (Zhang et al.
1997). They are also likely to be involved in axotomy-induced
increases in spike width and height and in decreases in rheobase
(Abdulla and Smith 2001
). Although our preliminary data
suggest that Na+ currents are increased by
axotomy (unpublished observations), the issue of effects of axotomy is
complex because several different types of Na+
channels are expressed in DRG neurons (Caffrey et al.
1992
; Elliot and Elliot 1993
; Ikeda et
al. 1986
; Rush et al. 1998
), and these may be
differentially affected by axotomy. For example, there are now several
reports that axotomy impairs TTX-resistant, slowly inactivating
INa (Cummins and Waxman
1997
; Rizzo et al. 1995
) as well as
TTX-resistant, persistent INa
(Sleeper et al. 2000
). By contrast, TTX-sensitive
current was either increased (Rizzo et al. 1995
) or more
readily "reprimed" (i.e., it recovered more rapidly from
inactivation) (Cummins and Waxman 1997
). There is also
evidence that new types of Na+ channels appear
after axotomy (Waxman et al. 1994
). The exact relationship between changes in different types of
gNa and axotomy-induced changes in
spike shape, rheobase and excitability therefore remain to be elucidated.
Relationship to pain mechanisms
The axotomy-induced reduction in amplitude of
HVA-IBa and the increased inactivation
was greatest in small cells, less in medium and AD-cells, and least in
large cells (Figs. 1-3, Tables 1 and 2). This is reflected by a
similar pattern of decreases in gK,Ca
and in the excitability changes in unclamped neurons (Abdulla
and Smith 2001). By contrast,
IK and
IH are attenuated by similar amounts
in all cell types. One possible explanation for this difference is that
the changes in IBa reflect loss of retrograde availability of nerve growth factor (NGF), whereas decreases
in IK and
IH reflect some other consequence of
sciatic nerve section. This argument is supported by the fact that TrkA receptors are preferentially expressed on nociceptive DRG cells (Lewin 1996
) and that
IBa and the inactivation of
IBa are controlled by retrograde
availability of target-derived NGF in other peripheral neurons
(Lei et al. 1997
).
As was previously seen with excitability changes (Abdulla and
Smith 2001), the transition of axotomized animals to the
autotomy state coincides with large changes in
IBa inactivation in large cells (Fig.
3D, Table 2) but not in small and medium cells (Fig. 3,
B and C, Table 2). This goes along with the idea
that the induction of autotomy is more a function of alterations in the properties of neurons with myelinated axons than a function of altered
properties of nociceptors (Coderre et al. 1986
;
Kajander and Bennett 1992
; Nagy et al.
1986
). According to some authors, alterations in the properties
of myelinated fibers may also contribute to the generation of human
neuropathic pain (Campbell et al. 1988
; Kauppila
1998
; Nystrom and Hagbarth 1981
; Woolf et
al. 1992
).
Since cell body ICa is attenuated by
axotomy, this raises the possibility that
ICa may also be altered at primary
afferent terminals in the spinal cord. Since
gK,Ca channels also reside in nerve
terminals (Sun et al. 1999), it is possible that axotomy increases the excitability of primary afferent terminals. Terminals might even become a source of spontaneous activity. Alternatively, decreased Ca2+ influx may reduce neurotransmitter
release from primary afferent fibers. These interesting possibilities
remain to be investigated.
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ACKNOWLEDGMENTS |
---|
We thank P. Stemkowski for help with data analysis.
This work was supported by the Medical Research Council of Canada. F. A. Abdulla received postdoctoral fellowships from the Rick Hansen Man-in-Motion/Alberta Paraplegic Foundation and from the Alberta Heritage Foundation for Medical Research. The work was also supported by start-up funds provided by the Office of the Dean, School of Allied Health Professions and the Office of the Academic Vice President, Tennessee State University.
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FOOTNOTES |
---|
Address for reprint requests: P. A. Smith, Dept. of Pharmacology, University of Alberta, 9.75 Medical Sciences Bldg., Edmonton, Alberta T6G 2H7, Canada (E-mail: Peter.A.Smith{at}UAlberta.ca).
Received 23 May 2000; accepted in final form 18 October 2000.
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REFERENCES |
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