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 the Excitability of Rat Dorsal Root Ganglion Neurons. J. Neurophysiol. 85: 630-643, 2001. The spontaneous, ectopic activity in sensory nerves that is induced by peripheral nerve injury is thought to contribute to the generation of "neuropathic" pain in humans. To examine the cellular mechanisms that underlie this activity, neurons in rat L4-L5 dorsal root ganglion (DRG) were first grouped as "large," "medium," or "small" on the basis of their size (input capacitance) and action potential (AP) shape. A fourth group of cells that exhibited a pronounced afterdepolarization (ADP) were defined as AD-cells. Whole cell recording was used to compare the properties of control neurons with those dissociated from rats in which the sciatic nerve had been sectioned ("axotomy" group) and with neurons from rats that exhibited self-mutilatory behavior in response to sciatic nerve section ("autotomy" group). Increases in excitability in all types of DRG neuron were seen within 2-7 wk of axotomy. Resting membrane potential (RMP) and the amplitude and duration of the afterhyperpolarization (AHP) that followed the AP were unaffected. Effects of axotomy were greatest in the small, putative nociceptive cells and least in the large cells. Moderate changes were seen in the medium and AD-cells. Compared to control neurons, axotomized neurons exhibited a higher frequency of evoked AP discharge in response to 500-ms depolarizing current injections; i.e., "gain" was increased and accommodation was decreased. The minimum current required to discharge an AP (rheobase) was reduced. There were significant increases in spike width in small cells and significant increases in spike height in small, medium, and AD-cells. The electrophysiological changes promoted by axotomy were intensified in animals that exhibited autotomy; spike height, and spike width were significantly greater than control for all cell types. Under our experimental conditions, spontaneous activity was never encountered in neurons dissociated from animals that exhibited autotomy. Thus changes in the electrical properties of cell bodies alone may not entirely account for injury-induced spontaneous activity in sensory nerves. The onset of autotomy coincided with alterations in the excitability of large, putative nonnociceptive, neurons. Thus large cells from the autotomy group were much more excitable than those from the axotomy group, whereas small cells from the autotomy group were only slightly more excitable. This is consistent with the hypothesis that the onset of autotomy is associated with changes in the properties of myelinated fibers. Changes in Ca2+ and K+ channel conductances that contribute to axotomy- and autotomy-induced changes in excitability are addressed in the accompanying paper.
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INTRODUCTION |
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Peripheral nerve damage can invoke
"neuropathic" pain in humans (Kauppila 1998;
Millan 1999
). This type of chronic pain is thought to be
initiated by persistent spontaneous activity in primary afferent fibers
(Nordin et al. 1984
; Nystrom and Hagbarth 1981
). Experiments using various animal models suggest that
ectopic activity originates both from the neuroma that develops at the site of nerve injury and from sensory neuron cell bodies in dorsal root
ganglia (DRG) (Babbedge et al. 1996
; Kajander and
Bennett 1992
; Millan 1999
; Study and Kral
1996
; Wall and Devor 1983
). Several laboratories
have therefore examined how nerve injury affects the
electrophysiological properties of individual DRG neurons (Czeh
et al. 1977
; Gallego et al. 1987
; Gurtu
and Smith 1988
; Kim et al. 1998
; Stebbing
et al. 1999
; Study and Kral 1996
; Zhang
et al. 1997a
). The most consistent finding is that nerve injury
reduces the amount of depolarizing current that is required to
discharge an action potential (AP) (Gallego et al. 1987
;
Gurtu and Smith 1988
; Kim et al. 1998
;
Stebbing et al. 1999
; Study and Kral
1996
; Zhang et al. 1997a
). Other observations
are less consistent. For example, some authors report clear-cut
increases in spike width (Gallego et al. 1987
;
Kim et al. 1998
; Stebbing et al. 1999
), whereas others report either small changes (Gurtu and Smith
1988
) or no effect (Oyelese and Kocsis 1996
;
Oyelese et al. 1997
; Zhang et al. 1997a
).
This inconsistency likely reflects differences in the response of
different types of sensory neuron to axotomy (Abdulla and Smith
1997, 1999
; Gurtu and Smith 1988
;
Kajander and Bennett 1992
; Kim et al.
1998
; Oyelese and Kocsis 1996
). Variability may
also devolve from the use of sharp microelectrodes to study DRG in vivo
or in vitro in some experiments and the use of patch electrodes to
study dissociated cells in others (see Stebbing et al.
1999
). Species differences may also be an issue. While some
groups have used Wistar rats (Everill and Kocsis 1999
;
Waxman et al. 1994
) or Sprague-Dawley rats
(Kajander and Bennett 1992
; Xie et al. 1995
; Zhang et al. 1997a
), Gurtu and
Smith (1988)
used hamsters, and Gallego et al.
(1987)
used cats. Even the choice of the strain of rat may lead
to inconsistency because the behavioral response of Sprague-Dawley rats
to nerve injury is more intense than that of Wistar rats (Carr
et al. 1992
). Moreover, several different types of nerve injury
model have been employed. For example, Wall and Devor
(1983)
, Gurtu and Smith (1988)
, Kim et
al. (1998)
and Stebbing et al. (1999)
used a
straightforward nerve-section axotomy model, whereas others capped or
ligated the cut nerve to encourage neuroma formation (Babbedge
et al. 1996
; Cummins and Waxman 1997
; Oyelese et al. 1997
; Wall and Gutnick
1974
; Zhang et al. 1997a
). Study and Kral
(1996)
used a chronic nerve constriction or
"loose-ligature" model developed by Bennett and Xie
(1988)
. Because the spontaneous activity induced by nerve
injury peaks at 13 days and returns to a lower level after 30 days
(Govrin-Lippmann and Devor 1978
), differences in the
time between the initial nerve perturbation and the examination of DRG
cells in different studies presents yet another source of
inconsistency. There is also the possibility that the distance between
the site of injury and the DRG may effect the intensity of changes seen
in cell bodies (Kim et al. 1998
). Last, differences in
the ages of animals used in different studies may have been a factor.
Peripheral axotomy promotes much more cell death in the DRG of young
animals than it does in older animals (Himes and Tessler
1989
).
Most recent studies have used patch-clamp or molecular biological
techniques to examine sciatic nerve injury-induced changes in
Na+, Ca2+, and
K+ channel properties and/or expression
(Baccei and Kocsis 2000; Black et al.
1997
; Cummins and Waxman 1997
; Everill
and Kocsis 1999
; Ishikawa et al. 1999
;
Rizzo et al. 1995
; Sleeper et al. 2000
;
Waxman et al. 1994
). Because of the differences in
response of different types of DRG neuron to axotomy (Abdulla
and Smith 1997
, 1999
; Gurtu and Smith
1988
; Kajander and Bennett 1992
; Kim et
al. 1998
; Oyelese and Kocsis 1996
), changes in
AP configuration must be correlated with changes in ionic currents in
the same subgroup of neurons. Up until now, few attempts have been made to do this. Oyelese and Kocsis (1996)
studied identified
cutaneous afferent neurons and showed that sciatic nerve damage reduced the proportion of neurons exhibiting an inflection on their
repolarizing phase of their APs. This was correlated with a decrease in
the expression of slowly inactivating tetrodotoxin-insensitive
Na+ current in the same identified neuronal
subset (Oyelese et al. 1997
). In general, however,
differences in the sensory neuron classification criteria used in
different laboratories (Gallego et al. 1987
;
Gurtu and Smith 1988
) have made it difficult to
correlate the older current-clamp data with contemporary voltage-clamp
findings. Gallego et al. (1987)
studied neurons with
conduction velocities >2 m/s, which exhibited a "hump" on the
repolarizing phase of their APs. Gurtu and Smith (1988)
divided DRG neurons into those with long APs and those with short APs,
whereas Study and Kral (1996)
classified DRG cells into
"small," "medium," and "large" on the basis of cell body
diameter. Some of the more recent voltage-clamp experiments have
studied identified unmyelinated "C"-type neurons (Cummins
and Waxman 1997
; Zhang et al. 1997a
) or
identified cutaneous afferent neurons (Baccei and Kocsis
2000
; Everill and Kocsis 1999
). As yet, however,
no study of the effects of peripheral nerve injury has attempted to
correlate current-clamp and voltage-clamp data for the whole DRG population.
We have therefore divided rat L4 and
L5 DRG neurons into four groups; small, medium,
large, and AD-cells and have studied the effect of sciatic nerve
section on the "classical" electrophysiological properties of each
group. We have used the same cell identification procedure and
recording technique in the accompanying paper where we examine changes
in K+ channel currents,
Ca2+ channel currents, and
Ca2+-sensitive K+
conductances (Abdulla and Smith 2001). This approach has
also allowed us to apply the same experimental manipulation to similar populations of animals in the two studies and to thereby secure clearer
correlation between current-clamp and voltage-clamp data (Abdulla and Smith 2001
). Moreover, we are better able
to document the differences in magnitude of the effects of axotomy
among different groups of DRG neurons.
A second aspect of our work is to study the relationship between
nerve injury-induced changes in DRG cells and a self-mutilatory behavior that has been termed "autotomy" (Coderre et al.
1986; Wall et al.1979
). This
phenomenon involves biting or gnawing the digits of a denervated limb.
Although the appearance of autotomy is regarded, by some, as an
appropriate animal model for human neuropathic pain, the exact
relationship between the two phenomena continues to be the subject of
vigorous debate (Coderre et al. 1986
; Kauppila
1998
; Rodin and Kruger 1984
). As already
mentioned, spontaneous activity in damaged sensory nerves originates
from both the site of the axon injury and from the cell bodies in the DRG. Wall et al. (1979)
suggested that this aberrant
activity invokes both autotomy in animals and neuropathic pain in
humans. Surprisingly, however, application of local anesthetic to the neuroma that forms at the site of an amputation fails to prevent spontaneous "phantom limb pain" in humans (Nystrom and
Hagbarth 1981
). This suggests that autotomy is not always
directly related to ongoing sensory activity that originates at
the site of nerve injury and infers that it may be causally
related to aberrant activity originating centrally and/or within DRG
(Kauppila 1998
). If this is so, the initiation of
autotomy should correlate with the onset of alterations in the
properties of DRG neuron cell bodies. To test this hypothesis, we have
also investigated the possible relationship between axotomy-induced
changes in DRG cells and the appearance of autotomy.
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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) and were reviewed and approved by the University of Alberta animal welfare committee. This committee is responsible for maintaining standards set forth by the Canadian Council for Animal Care.
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. Operated animals were inspected twice daily for foot
mutilation and the degree of autotomy was recorded. Autotomy was scored
according to the scale devised by Wall et al. (1979). A
score of 1 was given for the removal of one or more nails. The score
was increased by 1 for injury to each distal digit, and by another 1 for injury to each proximal digit. Although the maximum score permitted
under IASP guidelines is 11, all of our animals were killed before they
attained a score of 8. No abnormal behavior or premature deaths
occurred in the 7-wk study period. No unoperated limbs were mutilated
by any of the rats. For electrophysiological analysis, rats were killed by decapitation, and L4 and
L5 DRG were removed. These ganglia were selected
because 98% of sensory fibers in the sciatic nerve have cell bodies in
the L4 and L5 DRG
(Swett et al. 1991
). It should be noted, however, that
the axons of as many as 30% of the neurons in the
L4 and L5 DRG originate
from afferent nerves other than the sciatic (Himes and
Tessler 1989
). Although we therefore cannot guarantee
that all cells studied in the axotomy experiments actually had severed
axons, statistically significant differences were found between neurons
from control ganglia and axotomized ganglia. If axons of some
L4 and L5 neurons were not
cut, this would tend to minimize the differences between the control
and axotomy groups. This would produce, if anything, an underestimate of the effect of axotomy. Once they were removed from the rat, ganglia
were minced 2-3 times with iridectomy scissors before being incubated
for 40 min at 35°C in 5 ml of Dulbecco's modified Eagle's medium
containing 0.55 mg/ml trypsin type III (Sigma), 1.1 mg/ml collagenase
type 1A (Sigma), and 0.11 mg/ml DNase type III (Sigma) in plastic
culture bottles in a heated water bath. The culture bottles, which were
agitated at about 2 Hz throughout the dissociation procedure, were
removed from the water bath once every 10 min and subject to vigorous
shaking by hand. Soybean trypsin inhibitor type IIs (10 mg, Sigma) was
added on termination of incubation. Cells were plated into 3.5-mm
plastic Petri dishes ("NUNCLON SURFACE," Canadian Life
Technologies, Burlington, ON, Canada) and used for electrophysiological
recording within 2-10 h of plating. Whole cell recordings (at room
temperature, 22°C) were made using an AXOCLAMP 2A amplifier in
bridge-balance current-clamp mode as described previously
(Abdulla and Smith 1997
). The total volume of fluid in
the recording dishes was about 1 ml. The Petri dishes were superfused
with external solutions at a flow rate of 2 ml/min.
Data were acquired using PCLAMP 5.5 (Axon Instruments, Foster City,
CA), and final data records were produced using ORIGIN 5.0 (Microcal,
Northampton, MA). The ORIGIN/PCLAMP package (Microcal) was used to
transfer data between the two programs. 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 after the
amplifier was switched to discontinuous single-electrode voltage-clamp
mode (for details, see Abdulla and Smith 1997,
2001
). For action potential (AP) recording, 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, 1 CaCl2, and 10 HEPES-KOH (pH 7.2; osmolarity 310-320 mOsm). This solution would be
expected to buffer intracellular Ca2+ at about 10 nM. APs, generated using 2-ms depolarizing current pulses, were
recorded within 1 min of establishing whole cell recording.
Current-clamp data were filtered to
3 db at 30 kHz. Rheobase was
defined as the minimum amount of depolarizing current that discharged
an AP in 50% of trials. Spike width (AP duration) was measured at 50%
maximum amplitude. The full duration of the afterhyperpolarization
(AHP) that followed the AP was difficult to measure because its
approach to resting membrane potential (RMP) was so gradual. The
duration was therefore measured at 75, 50, and/or at 25%
repolarization. To quantify changes in excitability in response to a
500-ms current pulse, we measured the latency between the first two APs
(1st interspike interval) and the last two APs when the discharge rate
was assumed to have reached a "steady state" (see Fig.
4E). We have adopted the terminology of Heyer and
Llinás (1977)
and have used the term "gain" to
describe the relationship between the amplitude of injected current and the frequency of AP discharge.
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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Autotomy score
A detailed study of the rate of development of autotomy following sciatic nerve transection was done in 16 rats over a period of 7 wk. Seventy-five percent of the rats studied for this time period exhibited autotomy. Although there was considerable variation between different individuals in the rate of onset of autotomy, only 1 of the 16 experimental animals (6.3%) exhibited autotomy within the 1st week after axotomy, whereas the response was detectable in 7 of 16 animals (43.8%) within 2 wk. The percentage of animals exhibiting autotomy are represented by the bars in Fig. 1. The axotomy scores of these animals are represented by the overlaid linear plot. By 4 wk after axotomy, 12 rats (75%) exhibited autotomy scores of 3 or more, and the mean autotomy score at the 7th week was 6.1 ± 0.9 (means ± SE; Fig. 1).
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Classification of DRG neurons
Control DRG neurons were initially divided into three groups on the basis of their AP waveform: the first group had an AP duration of <3 ms with no deflection in the falling phase, the second group had an AP duration of 3-5 ms with a deflection or shoulder on the falling phase, and the third group had an AP duration of more than 5 ms (Fig. 2A). These differences in AP duration and shape were reflected as differences in cell size as estimated from Cin. Thus the first group, in which Cin was always >90 pF, were defined as large neurons, the second group, in which Cin was 70-90 pF, were defined as medium neurons, and the third group, which had the widest spikes and had Cin < 70 pF, were defined as small neurons.
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The APs recorded in 16.7% of all control neurons exhibited an
afterdepolarization (ADP) rather than an afterhyperpolarization (AHP;
Fig. 2A). AD-neurons of this type have been described by White et al. (1989) and Cardenas et al.
(1995)
. Although the Cin of
most of these cells places them in the medium neuron category, some
such cells clearly belonged in the small or large neuron categories.
This, and the fact that the AP characteristics of AD-neurons were
radically different from small, medium, or large neurons suggested that
they should be placed in a fourth, separate category. Because the
presence of an ADP often caused AD-neurons to generate bursts of two or
more spikes in response to a brief (2 ms) depolarizing current command
(Fig. 2A), we were interested to see whether such cells
became more excitable after axotomy.
Characteristics of control DRG neurons
Figure 2A illustrates representative recordings of APs elicited in small, medium, large and AD-neurons by a 2-ms pulse of depolarizing current. Note long duration (broad spike width) of small neuron AP and large ADP in the AD-neuron that promotes the discharge of three APs despite the brevity of the stimulus. The electrophysiological characteristics of 259 control large, medium, small, and AD-neurons are summarized in Table 1.
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Although strict size (Cin) and AP shape and duration criteria were applied to the classification of DRG neurons, there was considerable variability in the amplitude, shape, and duration of the AHP and its rate of onset in control large neurons. Although 15.6% of large neurons had a multiphasic AHP, neurons with "intermediate" AHPs (duration at 50% repolarization <50 ms and amplitude <15 mV) were most frequently encountered. Such cells comprised 50.3% of the total, control, large neuron population. By contrast the AHP of small and medium neurons exhibited much less variability. The mean AHP duration of large neurons was also shorter than that of small neurons (P < 0.005 at 75 and 50% repolarization and <0.05 at 25% repolarization). There were no significant differences between the AHP duration of medium neurons and that of large or small neurons (Table 1).
Apart from their spike width, all other electrophysiological properties of control DRG neurons were quite similar. Thus there were no significant differences in the spike amplitudes, rheobase or RMP of large, medium, small, and AD-neurons (Table 1).
Because the ADP of AD-cells is the consequence of activation of T-type
Ca2+ current (White et al. 1989)
and this current is partly inactivated at the typical RMP of DRG
neurons (
55 to
60 mV; Table 1) (see Fox et al.
1987
), we injected current into some neurons to see whether
they would exhibit an ADP when their membrane potential was
hyperpolarized to
90 mV. By this procedure, we were never able to
induce an ADP in a neuron that did not exhibit an ADP when studied at
its normal resting potential.
Effects of axotomy
The morphological and functional effects of axotomy on DRG neurons
start as early as 3 days and continue for more than 15 wk
(Titmus and Faber 1990). In the present study, we
investigated effects that occur within a period ranging from 2 to 7 wk
postaxotomy in age-matched control rats. Initially, we attempted to
make a detailed time course study of the electrophysiological changes induced by axotomy; there was, however, little significant difference between groups sampled at successive weekly intervals over the 2- to
7-wk postaxotomy period. We did note, however, that DRG neurons from
rats exhibiting autotomy were more affected than those from axotomized
rats that did not exhibit autotomy. The division of data into three
groups was therefore made without reference to the postoperative time
according to the following terminology: 1) data from control
animals, 2) data from axotomized animals that did
not exhibit autotomy ("axotomized" group), and 3) data from axotomized animals that exhibited autotomy
("autotomy" group).
Because Cin is a primary criterion for cell identification, it was important to establish that cell size was not altered by axotomy. Figure 3, A-C, shows distribution histograms for Cin for control, axotomized, and autotomy group cells. There are clear cut distributions for small, medium, and large cells, and the peaks do not appear to shift after axotomy or in the autotomy group.
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Effects of axotomy and the appearance of autotomy on spike width (AP duration)
In previous studies of hamster DRG neurons using sharp
micoelectrodes (Gurtu and Smith 1988), it was noted that
AP duration tended to increase after axotomy, but this increase fell
short of attaining statistical significance. In rat neurons, spike
width also tended to increase, but the difference was only significant for small cells and not for large, medium, or AD-cells (Table 1). It is
possible that changes that occurred in these groups were masked by the
pooling of data for statistical analysis. When the data are analyzed in
a different way, an upward trend in all spike widths can be
seen. This is illustrated in Fig. 3, D-F, which shows the
distribution of spike widths in control neurons, axotomized neurons,
and neurons from animals exhibiting autotomy. Figure 3D
shows that the peak spike width for the whole population of control
neurons falls within the 1- to 2-ms range, and this represents 55% of
all cells studied. Figure 3E shows that after axotomy, the
peak of spike width between 1 and 2 ms represents only 40% of the
population. There are also rather few control neurons with spike widths
>6 ms (about 4% of the total neuronal population, Fig.
3D). After axotomy however, about 17% of the cells exhibit
spike widths >6 ms (Fig. 3E).
The trend toward broad spike widths is greater in the autotomy group (Fig. 3F). Here, only 8% of the total population have spike widths in the 1- to 2-ms range, and some neurons have spike widths of 15-16 ms. Moreover, the AP duration of small, medium, large, and AD-cells from animals that exhibited autotomy were significantly greater than those of both the axotomy and control groups (Table 1).
The typical recordings of APs from the four types of control neurons in Fig. 2A are compared with those from axotomized neurons in Fig. 2B and with those from neurons in animals that exhibited autotomy in Fig. 2C. Note the very broad APs recorded from neurons in the autotomy group and that the increase in spike width devolved from a slowing of AP repolarization rather than from an increase in time to peak.
Effects of axotomy and autotomy on spike height (AP amplitude)
The AP amplitudes of small, medium, and AD-neurons were significantly increased by axotomy (Table 1). Axotomy did not, however, change the AP amplitude of large neurons. By contrast, the AP amplitude of all four cell types in the autotomy group were significantly increased compared with controls. Additionally, the AP amplitude of large neurons were significantly larger in the autotomy group compared with the axotomy group (Table 1).
Effect of axotomy and autotomy on AHP amplitude and duration
Although axotomy attenuates the amplitude and
duration of the AHP in both frog sympathetic B-neurons
(Gordon et al. 1987) and in cat slow motoneuron
(Kuno et al. 1974
), it produces little or no change in
the AHP of hamster DRG neurons (Gurtu and Smith 1988
).
Similarly the duration and amplitude of the AHP in rat DRG neurons were
not significantly altered in either the axotomy or the autotomy groups
(Table 1). In view of the complex shape of the AHP in some large cells,
we measured AHP duration in three ways; at 25, 50, or 75%
repolarization. Despite this, no significant differences were seen for
any measure of AP duration in large, small, or medium cells.
Effect of axotomy and autotomy on AD-cells
AD-cells comprised 16.7% of the control neuronal population, 14.3% of the axotomy population, and 9% of the autotomy population. It is unlikely therefore that cells that exhibited an AHP prior to axotomy developed an afterdepolarization once the sciatic nerve was cut. AD-neurons from control animals occasionally exhibited more than one AP in response to a brief pulse of depolarizing current (Fig. 2). There was no increased tendency for AD-neurons in the axotomy or autotomy groups to behave this way. Thus control AD-neurons cells fired an average of 1.55 ± 0.10 APs in response to brief depolarizing stimuli (n = 44), axotomized neurons fired 1.61 ± 0.10 APs (n = 44), and neurons from rats that developed autotomy fired 1.50 ± 0.15 APs (n = 12). Examination of the data using ANOVA showed that there are no significant differences between the three groups: F(2) = 0.20, n.s.
Spontaneously active cells
A very small proportion of control dissociated neurons (0.4%) and slightly more (2.1%) axotomized cells exhibited spontaneous activity. This spontaneous discharge of APs was not seen in any cells derived from animals that exhibited autotomy. Overall, spontaneously active cells comprised 0.8% of all neurons studied.
Effect of axotomy and autotomy on excitability
Because DRG are thought to be a source of ectopic activity in
damaged sensory nerves (Kajender and Bennett 1992;
Study and Kral 1996
; Wall and Devor 1983
;
Zhang et al. 1997a
), we examined axotomy-induced changes
in excitability. Three different types of discharge patterns were seen
in response to a 500-ms depolarizing current command. Some control
cells exhibited a phasic response in that they discharged a single AP
and then accommodated and remained silent throughout the remainder of
the depolarizing current command. This occurred in 55.7% of control
large cells, 55.6% of control medium cells, 46.2% of control small
cells, and 53.6% of control AD-cells. Other cells were slightly more
excitable and exhibited two APs before accommodating. This occurred in
14.4% of large cells, 13.9% of medium cells, 19.2% of small cells,
and 17.9% of AD-cells. The third group of cells exhibited more than two APs and continued to discharge throughout the application of
applied current. The rate of discharge of APs slowed and reached a
steady-state frequency after 4 or 5 spikes. This type of sustained discharge occurred in 29.9% of large cells, 30.6% of medium cells, 34.6% of small cells, and 28.6% of AD-cells. These numbers are summarized in Fig. 4, A-D.
Figure 4, E-G, shows examples of sustained discharge in
large neurons from the control (Fig. 4E), axotomy (Fig.
4F), and autotomy groups (Fig. 4G). The graphs in
Fig. 4, A-D, also show that after axotomy, more large,
medium, small, or AD-cells tended to exhibit sustained discharge (>2
spikes), whereas fewer cells fired only one or two spikes. Moreover,
this trend was exaggerated in cells from the autotomy group, in this situation over 70% of small, medium, large, and AD-cells exhibited sustained discharge, whereas only 10.7% of small cells (Fig.
4A), 8.7% of medium cells (Fig. 4B), 16.7% of
large cells, and 11.1% of AD-cells exhibited a single AP and
accommodation. Axotomy thus produced an overall increase in
excitability, and this effect was exaggerated in cells from the
autotomy group.
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The effects of axotomy and autotomy on repetitive discharge was analyzed more quantitatively by constructing frequency-current (f-I) curves for the first and steady-state interspike intervals for small, medium, large, and AD-neurons (see Fig. 4E). Cells that discharged only one AP in response to sustained current have an infinite initial and steady-state interspike interval. They were therefore assigned initial and steady-state discharge frequencies of 0 Hz. In cells that exhibited 2 APs, the interval between them yielded the initial discharge frequency. Since these cells stopped firing after 2 spikes, the steady-state interval was infinite, and a steady-state frequency of 0 Hz was assumed. Results for the first interspike interval are illustrated in Fig. 5, A-D, and those for the steady-state interspike interval in Fig. 5, E-H. The slopes of f-I curves for both intervals were steeper for the axotomy group and steeper still for the autotomy group; i.e., the gain of cells from the autotomy group was greater than that of axotomized cells, which was, in turn, greater than that of control cells. An idea of the level of significance of increases in excitability can be obtained by examining the extent of error bar overlap in Fig. 5. Differences are more apparent for the steady-state intervals (Fig. 5, E-H), especially with larger currents. Figure 5, A and E, shows that axotomy produces a large increase in the excitability of small cells. Modest differences are seen between control and axotomized medium, large, and AD-neurons (Fig. 5, B-D, and F-H). This pattern is not preserved, however, when axotomized cells are compared with those from the autotomy group. Small and medium cells from the autotomy group have only slightly higher gain than those from the axotomy group (Fig. 5, A and E, and B and F), whereas the large neurons and AD-cells from animals that exhibit autotomy have a much higher gain than those from the axotomy group (Fig. 5, C and G, and D and H). This important observation implies that the appearance of autotomy is associated with a change in the properties of the larger, nonnociceptive cells rather than with a change in the small cells that are presumably predominantly nociceptive. To underline this point, some data from Fig. 5E for small cells and from Fig. 5G for large cells have been replotted in Fig. 6. The presence of autotomy is associated with a significant alteration in the steady-state discharge frequency of large cells but not in that of small cells.
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|
Effects of axotomy and autotomy on rheobase
The amount of current required to evoke AP generation in all neuronal types was significantly decreased in the axotomy group and was further decreased in the autotomy group. Thus after axotomy, rheobase was lowered by 47% in small cells, by 45% in medium and AD-cells, and by 40% in large cells. In the autotomy group, rheobase was lowered by an additional 31% in small cells, an additional 33% in large and medium cells, and an additional 40% in AD-cells (Table 1). Decreases in rheobase seen in the axotomy and autotomy groups are also illustrated in the typical recordings shown in Fig. 7. The top records are a series of 10 voltage responses induced by the 10-incrementing current commands shown in the bottom records. In the control large cell (Fig. 7A), the first seven current commands produce only passive responses (shaded in gray), and only the three most intense currents elicit APs. In the axotomized large cell (Fig. 7B), only the first four current commands fail to generate APs, whereas in the neuron from the autotomy group (Fig. 7C) all current pulses stimulate the generation of one or even two APs.
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Effects of axotomy and autotomy on time-dependent rectification
Many DRG neurons, especially the larger cells, exhibit a
time-dependent change in voltage or "sag" in response to
hyperpolarizing current (Czeh et al. 1977; Gurtu
and Smith 1988
; Villière and McLachlan
1996
). In the present experiments, we observed this phenomenon
in 49/49 control large cells, 33/37 control medium cells, 14/23 control
small cells, and 27/30 control AD-cells. A typical record from a large
control neuron is shown in Fig. 8A. The initial transient peak
voltage response (VPeak) settles to a
less negative steady-state level (VSS)
within 300 ms. This rectification results from the presence of a
hyperpolarization-activated, inwardly rectifying, time-dependent,
nonselective cation current (H-current,
IH) (Abdulla and Smith
2001
; Jafri and Weinreich 1998
; Mayer and Westbrook 1983
; Scroggs et al.
1994
). The graph in Fig. 8B shows the relationship
between peak (VPeak) and steady-state (VSS) voltage responses and
hyperpolarizing current commands for control large neurons
(n = 49). The time-dependent rectification is
pronounced as the peak voltage response to
0.5 nA is over
100 mV,
whereas the steady-state voltage response is only
60 mV.
|
Previous studies in the cat (Czeh et al. 1977) showed
that time-dependent rectification in DRG cells was attenuated after peripheral nerve axotomy. Our results show that this also happens in
rats. A typical recording from an axotomized large cell is illustrated
in Fig. 8C. Note the attenuation of rectification in these
records compared with the records from a control neuron shown in Fig.
8A. Not all large axotomized cells exhibited the rectification (23/28), and the response was observed less frequently in
medium (16/21), small (10/24), and AD-cells (18/23). Figure 8D illustrates the attenuation of rectification after
axotomy in all large cells studied. There is much more difference
between VPeak and
VSS in the control large neurons
(n = 23; Fig. 8B) than in axotomized large
neurons (Fig. 8D).
No further attenuation of time-dependent rectification was seen in cells from the autotomy group. The sample data record from a large cell in an animal that exhibited autotomy (Fig. 8E) is similar to that seen after axotomy (Fig. 8C). The rectification response was still present in 20/25 large cells, 14/20 medium cells, 10/26 small cells, and 8/11 AD-cells. The amplitude of the rectification is illustrated for 20 large cells from the autotomy group illustrated in Fig. 8F is similar to that seen in axotomized cells (Fig. 8D).
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DISCUSSION |
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Classification of DRG neurons
Both Cin and AP shape were used to distinguish small, medium, and large cells in the control DRG neuron population. Because very few cells exhibited Cin between 61 and 70 pF, the use of 70 pF as a cutoff clearly distinguishes the small and medium cell populations (Fig. 3A). The use of 90 pF as a point of distinction between medium and large cells therefore seems more arbitrary as there is no obvious gap in the size distribution at this point. It was noted, however, that all cells with a clear inflection on their falling phase exhibited Cin <90 pF. The combination of inflection and Cin >70 pF and <90 pF was therefore used to define medium cells and to distinguish them from large cells in the control DRG neuron preparation. After axotomy, however, spike shape changes (Fig. 2) so that Cin is the only available criterion for cell identification. Nevertheless, the size distinction between small and medium cells is preserved both after axotomy (Fig. 3B) and in the autotomy group (Fig. 3C). In both cases, few cells are distributed in the 61- to 70-pF range. This implies that cell size is unaffected by axotomy. It would therefore seem reasonable to use Cin as the criterion for distinguishing small, medium, and large cells in the axotomy and autotomy groups.
The relationship between cell body size, axonal conduction velocity,
functionality, and AP configuration of different types of DRG neuron is
a topic of ongoing debate (Harper and Lawson 1985;
Ritter and Mendell 1992
; Rose et al.
1986
; Villière and McLachlan 1996
).
Sensory neurons are usually divided into three groups: rapidly
conducting A
/
cells that transmit nonnociceptive sensory
information; A
-cells that are responsible for the relatively rapid
transfer of nociceptive stimuli that might be elicited by a pin prick;
and slowly conducting C-cells that transmit longer lasting,
"burning" pain that may be associated with inflammation (Bessou and Perl 1969
). While it is generally agreed
that A
/
cells have the shortest spike width and largest soma
diameter (Gallego and Eyzaguirre 1978
; Gurtu and
Smith 1988
; Kim et al. 1998
; Koerber et
al. 1988
; Rose et al. 1986
) and that nociceptive C-cells have the greatest spike width and smallest soma diameter (Gallego and Eyzaguirre 1978
), the distinction is not
absolute. This is because the soma diameter of some A
-cells fall
into the A
/
or C-cell range (Harper and Lawson
1985
; Villière and McLachlan 1996
). It is
therefore not appropriate to equate the medium cell population used in
our study with A
-cells identified in vitro or in vivo. It is likely,
however, that nociceptive neurons predominate in the small cell group,
whereas nonnociceptive neurons reside within the large cell group.
Characteristics of dissociated DRG neurons
One advantage of whole cell recording for comparing the
electrophysiological properties of different groups of neurons is that
APs can be recorded with high fidelity and there are no problems with
"penetration" injury that can be promoted by using sharp microelectrodes (Ikeda et al. 1986). However, APs
recorded in DRG with sharp microelectrodes are generally of shorter
duration than those recorded with patch electrodes. While our
measurements of spike width in control DRG neurons (1.37 ± 0.37 ms for large cells and 6.21 ± 0.24 ms for small cells; Table 1)
are similar to those seen in other studies done with whole cell
recording (Caffrey et al. 1992
;
Cardenas et al. 1995
; Ikeda et al.
1986
), they are considerably shorter than values reported by
Villière and McLachlan (1996)
. Using
microelectrodes, these authors observed spike widths as little as 0.5 ms in A
/
(large) cells, and spike widths in small cells (C-cells)
were always <4 ms. The experiments of Villière and
McLachlan (1996)
were done at 35°C, whereas ours were done at
room temperature. Since cooling from 37 to 20°C can double AP
duration in DRG neurons (Amir and Devor 1996
),
differences between our data and those of Villière and
McLachlan (1996)
may be explained, at least in part, in terms
of differences in temperature.
Our finding that about 30% of all types of control neurons
show sustained discharge in response to depolarizing current (Fig. 4,
A-D) also differs from results obtained with
microelectrodes. For example, Villière and McLachlan
(1996) found sustained discharge in only 4 of 56 (7%) of
A
/
neurons studied with microelectrodes at 35°C, whereas we
found it in 29.9% of large control neurons studied at 20°C (Fig.
4C). Voltage-insensitive, low-conductance gK,Cas that underlie slow AHPs
(SKCa channels) limit repetitive discharge in a
subpopulation of vagal afferent neurons (Cohen et al.
1994
; Cordoba-Rodriguez et al. 1999
;
Fowler et al. 1985
; Moore et al. 1998
),
but such channels are not seen under our experimental conditions. We
saw no effect of the SKCa blocker, apamin, on
repetitive discharge or AP shape of DRG cells studied with patch
pipettes at room temperature (M. J. Stebbing and P. A. Smith,
unpublished observations). Loss of SKCa function
during recording with patch electrodes has been reported in other
neuronal types (Zhang et al. 1994
; but see also
Gold et al. 1996
). It is therefore tempting to speculate
that the absence of functional SKCa channels in
our studies accounts for the observed propensity for sustained
discharge (Fig. 4). This does not, however, explain the lack of
accommodation seen in 29.9% of control large cells (Fig.
4C) as these cells have relatively short AHPs (Table 1) and
do not express SKCa channels (Gold et al.
1996
). The presence of other types of channels presumably
underlies spike-frequency accommodation in large cells. If these
channels were damaged during enzymatic dissociation procedures, this
may contribute to both increased excitability and to increased spike
width in dissociated DRG neurons.
Changes produced by axotomy
Axotomy tended to increase excitability, spike height, and spike width in all neuronal types, whereas RMP or AHP were unaffected (Table 1). Increases in excitability included increases in gain and decreases in accommodation and rheobase (Figs. 4 and 5). For all AP parameters that were altered, changes were most pronounced in small cells and least pronounced in the large cells. The altered properties of medium and AD-cells laid between those of large and small cells. Thus axotomy promoted a marked increase in the excitability of small cells, less pronounced increases in medium and AD-cells, and small increases in the excitability of large cells. Similarly for changes in spike height. The effects of axotomy were highly significant in the small and AD-cells (P < 0.001), significant at the P < 0.05 level in medium cells yet insignificant in large cells. Axotomy also significantly increased the spike width of small cells, and although its effect on large, medium, and AD-cells did not attain statistical significance (Table 1), there is a clear trend toward wider spikes in the frequency histograms shown in Fig. 3, D-F. Axotomy decreased rheobase in all cell types by the same level of significance (Table 1).
The trend toward increased excitability, reduced rheobase, increased
spike height, and increased spike width has been reported to a greater
or lesser extent by others who have studied the response of DRG cells
to axotomy or other injury. For brevity, our findings have been
compared in tabular form with those already published (Table
2). There are two apparent discrepancies
that should be discussed. First, whereas several groups, including
ourselves, saw increases in spike width in axotomized neurons,
Oyelese and Kocsis (1996) showed that sciatic nerve
injury reduced the proportion of cutaneous afferent neurons exhibiting
a deflection on their repolarizing phase. The overall spike width in
this group of neurons did not, however, appear to change, and there was
a significant increase in the spike width of identified muscle
afferents. Thus the more subtle changes seen by Oyelese and
Kocsis (1996)
may have been obscured by our cell identification
procedure. Second, our study is the only one to report clear cut
increases in spike height, an effect that was especially clear in small
cells. One explanation for this is that microelectrode penetration
injury often results in attenuation of spike height, and this effect is
especially apparent in small cells. This type of attenuation would not
have occurred in our experiments that were done with patch electrodes
that allow high quality recording of APs from small cells. Indeed, an
increase in spike height is a characteristic consequence of axotomy in
a variety of neuronal types (Titmus and Faber 1990
).
Thus the observed increase in spike height in DRG cells is not without
precedent and may reflect the marked increase in
Na+ current seen in these cells after axotomy
(Rizzo et al. 1995
; Zhang et al. 1997a
;
but see also Cummins and Waxman 1997
).
|
Nevertheless, the fact that there are differences between our current-clamp data and those obtained in other studies (Table 2) reemphasizes the need to compare the same groups of cells in voltage- and current-clamp studies.
Changes seen with autotomy
The changes in electrophysiological properties promoted by axotomy
were always expressed more clearly in cells from the animals that
exhibited autotomy (Table 1). Although the differences between axotomized small cells and those from the autotomy group are modest, this is not the case for large cells. There are obvious and significant differences between the properties of axotomized large cells and large
cells from autotomy group. This distinction applies to spike height,
rheobase (Table 1) excitability (Figs. 5 and 6), and to the changes in
certain voltage-sensitive conductances described in the accompanying
paper (Abdulla and Smith 2001). The development of
autotomy therefore seems to be associated with a change in properties
of nonnociceptive cells; i.e., autotomy is initiated without
a change in the properties of small, putative nociceptive cells. This
finding parallels the observation that destruction of peripheral
C-fibers with capsaicin fails to prevent the autotomy induced by a
prior nerve injury (Nagy et al. 1986
). It is also consistent with the clinical observation that large myelinated afferents seem to signal the mechanical hyperalgesia associated with
nerve injury (Campbell et al. 1988
). If alterations in
the properties of myelinated neurons correlate both with the onset of
pain and with the onset of autotomy, this would support the contention
that autotomy is a useful experimental model for human neuropathic pain
(Coderre et al. 1986
).
Increased excitability but lack of spontaneous activity in axotomized neurons
Because only 0.8% of the whole population of control, axotomized,
or autotomy group neurons exhibited spontaneous activity, changes in
the electrophysiological properties of DRG cell bodies may not entirely
account for the ectopic activity that originates in injured DRG in vivo
(Wall and Devor 1983). It should be noted, however, that
the dissociated neurons used in this study lack an axon hillock region
that is characterized by low-threshold Na+
channels. These channels could perhaps drive spontaneous activity in
axotomized DRG in vivo. Other mechanisms may also contribute. These
include the actions of norepinephrine released from perivascular sympathetic fibers that sprout into DRG neurons after injury
(Devor et al. 1994
; McLachlan et al.
1993
). Although these fibers seem to abut primarily the larger
neurons, both small and large neurons start to express excitatory
2-adrenoceptors (Abdulla and Smith 1997
; Birder and Perl 1999
; Petersen et
al. 1996
; Xie et al. 1995
; Zhang et al.
1997b
). These receptors may be important in generating ectopic
activity because stimulation of sympathetic nerves invokes spontaneous
activity in axotomized sensory nerves (Devor et al. 1994
; McLachlan et al. 1993
; Xie et al.
1995
). Other potential excitatory mediators include substance
P, which is released from the cell bodies of DRG neurons (Huang
and Neher 1996
) and exerts powerful excitatory actions within
sensory ganglia (Dray and Pinnock 1982
; Inoue et
al. 1995
). Neuropeptide Y (NPY)-containing axons are also
abundant in axotomized DRG (Wakisaka et al. 1991
,
1992
), and the excitatory actions of this peptide on DRG
neurons are increased after sciatic nerve section (Abdulla and
Smith 1999
). Actions of any or all of these substances may
contribute to the chemical cross-excitation that has been reported to
occur in axotomized DRG in vivo (Amir and Devor 1996
).
Our findings with sciatic nerve section axotomy differ from those of
Study and Kral (1996), who showed that about 20% of
neurons dissociated from animals that have received chronic
constriction injury to their sensory neurons displayed spontaneous
activity ex vivo. This may simply reflect methodological differences
between our studies and theirs.
Possible mechanisms for the effects of axotomy
Although the present current-clamp experiments say little about
the specific mechanisms underlying the increased excitability of
axotomized neurons, certain possibilities can be ruled out. For
example, axotomized cells are not chronically depolarized (see
Kim et al. 1998), and AD-cells that tend to generate
repetitive action potentials under control conditions do not display
this tendency to a greater extent after axotomy (Fig. 2). The maximum discharge rate elicited in AD cells in the autotomy group (Fig. 5,
D and H) is no greater than that seen in small
(Fig. 5, A and E), medium (Fig. 5, B
and F), or large cells (Fig. 5, C and
G). Also, time-dependant rectification (Czeh et al.
1977
; Villière and McLachlan 1996
), which
could contribute to increased excitability at certain membrane
potentials, is attenuated rather than enhanced after axotomy (Fig. 8).
Axotomy-induced lowering of rheobase (Fig. 7) goes along with increased
excitability. The lack of effect of axotomy on AHP amplitude and
duration was somewhat unexpected because it occurs in at least two
other neuron types (Gordon et al. 1987
; Kuno et
al. 1974
), and increased neuronal excitability is often a
consequence of AHP attenuation. On the other hand, no decreases in AHP
amplitude or duration were seen in our previous study of the effects of
axotomy on hamster DRG neurons (Gurtu and Smith 1988
).
We show, however, in the accompanying paper (Abdulla and Smith
2001
) that axotomy attenuates Ca2+
channel current (ICa), as well as Ca
2+-sensitive K+
conductances (gK,Ca) that depend on
ICa. Because some types of gK,Ca are voltage sensitive
(Pennefather et al. 1985
), it is possible that changes
in this conductance can alter repetitive discharge without affecting an
AHP recorded at a relatively negative membrane potential. This is
likely to be the case in rat DRG cells as attenuating influx of
Ca2+ through N-type Ca2+
channels with Cd2+ (Abdulla and Smith
1997
) or
-conotoxin GVIA or attenuating "maxi" (voltage-sensitive) gK,Ca with
charybdotoxin (Abdulla and Smith 1999
) or iberiotoxin
(Scholz et al. 1998
) increases their excitability.
The voltage-clamp studies described in the accompanying paper
(Abdulla and Smith 2001) address axotomy-induced changes
in various ionic channels in DRG neurons and provide additional
information as to how changes in Ca2+ and
K+ currents might contribute to the observed
changes in excitability of axotomized neurons.
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ACKNOWLEDGMENTS |
---|
We thank Drs. W. F. Colmers and Martin Stebbing for comments on an early version of the manuscript.
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 |
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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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