1Department of Cell and Animal Biology, Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 91904, Israel; and 2Physiologisches Institut, Christian-Albrechts Universitat, 24098 Kiel, Germany
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ABSTRACT |
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Liu, Chang-Ning, Martin Michaelis, Ron Amir, and Marshall Devor. Spinal Nerve Injury Enhances Subthreshold Membrane Potential Oscillations in DRG Neurons: Relation to Neuropathic Pain. J. Neurophysiol. 84: 205-215, 2000. Primary sensory neurons with myelinated axons were examined in vitro in excised whole lumbar dorsal root ganglia (DRGs) taken from adult rats up to 9 days after tight ligation and transection of the L5 spinal nerve (Chung model of neuropathic pain). Properties of subthreshold membrane potential oscillations, and of repetitive spike discharge, were examined. About 5% of the DRG neurons sampled in control DRGs exhibited high-frequency, subthreshold sinusoidal oscillations in their membrane potential at rest (Vr), and an additional 4.4% developed such oscillations on depolarization. Virtually all had noninflected action potentials (A0 neurons). Amplitude and frequency of subthreshold oscillations were voltage sensitive. A0 neurons with oscillations at Vr appear to constitute a population distinct from A0 neurons that oscillate only on depolarization. Axotomy triggered a significant increase in the proportion of neurons exhibiting subthreshold oscillations both at Vr and on depolarization. This change occurred within a narrow time window 16-24 h postoperative. Axotomy also shifted the membrane potential at which oscillation amplitude was maximal to more negative (hyperpolarized) values, and lowered oscillation frequency at any given membrane potential. Most neurons that had oscillations at Vr, or that developed them on depolarization, began to fire repetitively when further depolarized. Spikes were triggered by the depolarizing phase of oscillatory sinusoids. Neurons that did not develop subthreshold oscillations never discharged repetitively and rarely fired more than a single spike or a short burst, on step depolarization. The most prominent spike waveform parameters distinguishing neurons capable of generating subthreshold oscillations, and hence repetitive firing, was their brief postspike afterhyperpolarization (AHP) and their low single-spike threshold. Neurons that oscillated at Vr tended to have a more prolonged spike, with slower rise- and fall-time kinetics, and lower spike threshold, than cells that oscillated only on depolarization. The main effects of axotomy were to increase spike duration, slow rise- and fall-time kinetics, and reduce single-spike threshold. Tactile allodynia following spinal nerve injury is thought to result from central amplification ("central sensitization") of afferent signals entering the spinal cord from residual intact afferents. The central sensitization, in turn, is thought to be triggered and maintained in the Chung model by ectopic firing originating in the axotomized afferent neurons. Axotomy by spinal nerve injury enhances subthreshold membrane potential oscillations in DRG neurons, augments ectopic discharge, and hence precipitates neuropathic pain.
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INTRODUCTION |
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Ectopic afferent discharge is widely believed to
be a major contributor to chronic pain following peripheral nerve
injury [neuropathic pain; reviewed in Devor and Seltzer
1999)]. This motivates a deeper understanding of the neural
processes responsible for the discharge, and the specific cellular role
played by nerve injury. The most prominent sites of origin of ectopic
discharge in axotomized primary afferent neurons are the site of injury itself (e.g., the nerve end neuroma) and the dorsal root ganglion (DRG)
(Burchiel 1984
; Govrin-Lippmann and Devor
1978
; Kirk 1974
; Study and Kral
1996
; Wall and Devor 1983
; Wall and
Gutnick 1974
). Interestingly, the relative contribution of
these two sources depends on the location of the nerve injury.
Following sciatic nerve injury, at a distance from the DRG, the neuroma
is the most prominent impulse generator. However, following spinal
nerve injury, in which afferent neurons are axotomized close to their
soma, most of the ectopic barrage originates in the DRG (Liu et
al. 1999
, 2000
).
Ectopic afferent discharge originating in neuromas and DRG neurons
contributes to neuropathic pain in several ways (Devor and
Seltzer 1999). The spontaneous component of the pain is
presumed to result from spontaneous discharge of injured afferents.
Pain on movement and on deep tissue palpation, on the other hand, is probably due to the mechanosensitivity of sites of ectopic
electrogenesis. Finally, tissue tenderness ["allodynia," defined
as pain in response to normally nonpainful stimuli (Merskey
1986
)] is thought to result from spinal amplification of
afferent signals that arise in normal low-threshold sensory endings of
neurons that survived the injury. According to this hypothesis, the
spinal amplification process ("central sensitization") is triggered
and maintained by the ectopic afferent barrage (Gracely et al.
1992
; Liu et al. 2000
; Rowbotham and
Fields 1996
; Sheen and Chung 1993
; Woolf
and Thompson 1991
; Yoon et al. 1996
).
All three of these pain components are probably present in the Chung
model of neuropathic pain, an experimental preparation in which the
L5 (or L5 + L6) spinal nerves are tightly ligated and then
cut (Kim and Chung 1992). There is direct evidence in the Chung model of a role for ectopic afferent firing. For example, Chung and collaborators report that abnormal sensory symptoms are
eliminated when the ectopic activity is prevented from entering the
spinal cord by selective dorsal rhizotomy (Sheen and Chung 1993
; Yoon et al. 1996
). Likewise, we have
recently shown that there is a sudden increase in the intensity of
ectopic afferent discharge at precisely the time of onset of tactile
allodynia, 16-24 h after transection of the spinal nerve (Liu
et al. 2000
).
The repetitive discharge of DRG neurons depends on intrinsic resonance
properties of the cell membrane (Amir et al. 1999). Most
neuronal somata in the DRG are incapable of generating repetitive spike
trains on sustained depolarization. Step depolarization usually evokes
only a single spike, or a brief burst, and slow ramp depolarization
rarely evokes any discharge at all. Only a relatively small
subpopulation of cells, those with subthreshold membrane potential
oscillations, are able to fire repetitively. In the present study we
show that the time of onset of tactile allodynia in the Chung model is
marked by a sudden increase in the proportion of DRG neurons with
subthreshold oscillations. The increased prevalence of oscillatory
behavior, in turn, leads to an augmentation in repetitive discharge in
DRG neurons, both at resting membrane potential and during sustained
depolarization. These observations strengthen the link between
oscillatory behavior in DRG neurons, ectopic firing, and positive
sensory symptoms in neuropathy.
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METHODS |
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Animals and surgery
Experiments were carried out using 68 adult (230-520 g) male
rats of the Wistar-derived Sabra strain (Lutzky et al.
1984). All procedures followed national and University
regulations for the care and use of laboratory animals, and the ethical
guidelines of the International Association for the Study of Pain
(Zimmermann 1983
). Many of the animals underwent tight
ligation and transection of the L5 spinal nerve
as described by Kim and Chung (1992)
. Briefly, under
Nembutal anesthesia (50 mg/kg ip) and using aseptic precautions, the
left paraspinal muscles were separated from the spinous processes at
the L3-S2 levels. The
L5 transverse process was removed using a fine
Lempert nipper and the point of convergence of the
L4 and L5 spinal nerves was
identified. The L5 spinal nerve was then tightly
ligated with 5-0 silk and transected with iridectomy scissors just
distal to the ligature. The ligature was placed 3-5 mm proximal to the
junction with L4 nerve, 5-10 mm distal to the
L5 DRG. The incision was then closed in layers,
and antibiotics were administered prophylactically (topical
bacteriostatic powder and penicillin 50 kilounits/kg im). Six
rats underwent sham surgery that involved the identical surgical
exposure but without ligation or transection of the spinal nerve.
Following surgery rats were returned to the animal colony and
maintained postoperatively in standard transparent plastic shoebox cages bedded with wood shavings, with a 12:12 light:dark cycle, and
with food and water available ad libitum. Some of the rats were tested
behaviorally to confirm the development of tactile and thermal
allodynia (Liu et al. 2000).
Electrophysiological recording
Between 9 h and 9 days postoperatively (hpo, dpo) animals were overdosed with pentobarbital sodium (Nembutal) and killed by carotid exsanguination. DRGs L4 and L5 were rapidly excised with their dorsal roots (DRs) and spinal nerve attached, and in controls also with a variable length of sciatic nerve. L4 DRGs from operated animals were used as control tissue, along with L5 ganglia from unoperated and sham-operated rats. During dissection and immediately afterward, the tissue was immersed in ice-chilled modified Krebs solution. This solution was allowed to equilibrate to room temperature over the subsequent 1-2 h. The Krebs solution contained (in mM) 124 NaCl, 26 NaHCO3, 3 KCl, 1.3 NaH2PO4, 2 MgCl2, and 10 dextrose, bubbled with 95% O2-5% CO2 (pH 7.4; 290-300 mosmol). Ganglia were then transferred to a recording chamber and superfused with the same Krebs solution to which 2 mM CaCl2 was added (2-4 ml/min, room temperature). The connective tissue capsule was carefully removed from the upper surface of the DRG using a fine blade.
Intracellular recordings from DRG neurons were obtained using sharp
micropipettes made from filament-containing borosilicate glass tubing.
The pipettes were pulled with a Brown-Flaming puller (model P-87;
Sutter Instruments) and filled with 3 M KCl (40-60 M, DC). Neurons
were impaled by advancing the microelectrode in 5- or 10-µm steps and
applying a small capacitance buzz. Cells were examined in order, as
impaled, accepting only those exhibiting a resting membrane potential
(Vr) more negative than
45 mV, and an overshooting action potential. Action potentials (spikes) were evoked using 0.05-ms pulses (for A-neurons) delivered to the peripheral nerve through an Ag-AgCl electrode pair, or by injecting current through the amplifier bridge (Axoclamp-2A, Axon Instruments). Current-voltage (I-V) curves were generated by measuring
voltage responses to constant current pulses (80 ms, +0.2 nA to
1.2
nA in 0.2-nA steps) using pCLAMP software (v6.0.3, Axon Instruments). Input resistance (Rin) was calculated
from the linear portion of the I-V curve in the
hyperpolarizing direction. A suction electrode on the DR was used to
monitor the compound action potential evoked by nerve stimulation.
Potentials were stored digitally for off-line analysis. Typically,
about 10 neurons were examined per DRG.
The following additional parameters were measured: 1) spike amplitude, measured from the baseline membrane potential to the positive peak of the spike, 2) spike width (duration) at half-amplitude, 3) slope of the rising limb of the action potential (dV/dt), measured by analog differentiation (i.e., peak of the corresponding deflection), 4) slope of the falling limb of the action potential, 5) afterhyperpolarization (AHP) amplitude, measured from baseline, 6) AHP duration measured at 75% amplitude decay, and 7) threshold (in nA) for evoking single spikes using intracellular 1-ms depolarizing pulses of gradually increasing current strength. Repetitive firing capability was assessed by applying a slow depolarizing ramp as described below.
Neurons were categorized by axonal conduction velocity (CV) and the
shape of the intracellularly recorded spike (Amir and Devor
1997; Koerber and Mendell 1992
; Villiere
and McLachlan 1996
). CV was calculated by dividing the
propagation distance by spike latency following just suprathreshold
pulses to the spinal or sciatic nerve. Neurons were assumed to have a
myelinated axon (A-neurons) if CV >1 m/s; C-neurons had CV
1 m/s.
A-neurons were further categorized as Ainf
neurons if they had an inflection on the falling phase of the action
potential as assessed by analog differentiation, and
A0 neurons if there was no inflection.
A proportion of neurons exhibited subthreshold oscillations in their
membrane potential that sometimes gave rise to action potentials
(Amir et al. 1999). Membrane potential in actively spiking neurons was taken as the asymptotic potential between spikes.
To assess subthreshold oscillations, spike-free
4-s epochs were
band-passed at 1-10 kHz, digitized at 5 kHz, and analyzed for power
spectral density using the fast Fourier transform (FFT, CP Analysis
v5.1, DataWave Technologies). Alternatively, particularly when spiking
interrupted runs of subthreshold oscillations, peak-to-peak oscillation
amplitude was averaged from a sample of 30-40 cycles. The first cycle
in the sample was chosen at random, and then subsequent cycles were
measured every 40 ms except where spikes were present. Oscillations
were usually obvious, but when necessary we used as a formal criterion
that amplitude peaks be at least 1.5 times the amplitude of the
background noise level present during brief pauses in the oscillations,
and/or that there be a distinct peak in the FFT plot at the frequency
expected from visual inspection of the voltage trace.
All cells were first examined at Vr.
They were then depolarized in a slow ramp (about 20 mV/s) by
intracellular current injection until oscillations and/or repetitive
spiking occurred, or until 20 mV. Data are expressed as means ± SD. Differences were compared using
2 or
two-tailed t-tests with a significance criterion of
P = 0.05 except in the case of multiple comparisons
(Table 4) in which a criterion of P = 0.02 is
recommended (Sigmastat v2.0, Jandel Scientific).
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RESULTS |
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In this report we focus exclusively on A-neurons because a prior
study in vivo showed that at 9 dpo these cells alone contribute to
the ectopic barrage responsible for hindlimb allodynia in the Chung
model of neuropathic pain (Liu et al. 2000
).
Subthreshold oscillations
CONTROL GANGLIA.
Neurons from the three types of control preparations
(L5 DRG from unoperated, n = 140, and sham operated animals, n = 33, and
L4 DRG from animals with L5
cut, n = 56) behaved similarly (Table
1) and will therefore be considered as a
group. Most had a stable membrane potential at
Vr. However, a minority (12/229, 5.2%) exhibited high-frequency, subthreshold sinusoidal oscillations in their membrane potential (Fig. 1). All
of these were A0 neurons (Table
2). Oscillations were generally
sustained, but some had intermittent brief pauses of 200 ms.
Oscillation frequency formed a single major peak in FFT histograms,
what we call the "dominant oscillation frequency" (Fig.
2). The dominant frequency varied somewhat among neurons, and it was voltage sensitive (see VOLTAGE SENSITIVITY OF OSCILLATION FREQUENCY AND THE EFFECT OF AXOTOMY). At Vr the dominant oscillation
frequency was 95.5 ± 27.6 (SD) Hz with a peak-to-peak amplitude
of 1.9 ± 1.1 mV (Table 3). These values are very similar to ones reported previously in a separate sample of DRG neurons in intact juvenile rats (Amir et al.
1999
).
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AXOTOMY ENHANCES SUBTHRESHOLD OSCILLATIONS.
Spinal nerve section caused a striking increase in the proportion of
neurons with oscillations at Vr and on
depolarization. Moreover, this change occurred quite suddenly; much of
the increase took place within the narrow time window of 16-24 h
postoperative (Table 2, Fig.
3A). Before this time, during
the period 9-16 hpo, the overall incidence of cells with oscillations
was not significantly different from control (P > 0.2). However, 16-24 hpo there was nearly a doubling of the incidence
of oscillating neurons. Values remained high until at least 9 dpo.
Counting 1-9 dpo, 38 of the 126 A0 neurons
sampled (30.2%) had oscillations at rest or on depolarization
(P < 0.001 compared with control, 2).
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VOLTAGE SENSITIVITY OF OSCILLATION AMPLITUDE AND THE EFFECT OF
AXOTOMY.
As shown previously (Amir et al. 1999), oscillation
amplitude changed systematically when the cells were depolarized.
Depolarization from the membrane potential at which subthreshold
oscillations were first observed (either
Vr or positive to
Vr) always led to an increase in the
peak-to-peak oscillation amplitude. This continued until a maximum
amplitude was reached. Depolarization beyond this "optimal"
membrane potential caused the amplitude to decline until oscillations
were no longer discernable above the background noise (Figs. 2 and
4A). In all neurons that
oscillated at Vr the oscillations were
quenched by hyperpolarization.
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VOLTAGE SENSITIVITY OF OSCILLATION FREQUENCY AND THE EFFECT OF
AXOTOMY.
Like oscillation amplitude, oscillation frequency increased
systematically when the cells were depolarized (Amir et al.
1999). However, unlike amplitude, there was no optimum value.
Rather, oscillation frequency continued to increase with
depolarization, in a monotonic manner (Fig. 4C). The
dependency of frequency on membrane potential in control neurons was
0.84 ± 0.66 Hz/mV (8 cells). This value was not significantly
affected by axotomy [0.72 ± 0.36 Hz/mV (10 cells,1-9 dpo,
P > 0.2)].
Ectopic spiking and the effect of axotomy
Subthreshold membrane potential oscillations often gave rise to
repetitive spike discharge. In all cases spikes arose from the rising
(depolarizing) phase of the oscillatory sinusoid (Table 2). In neurons
with oscillations at Vr (both intact
and axotomized) oscillation amplitude was often already large enough to
trigger spontaneous spike discharge (Fig. 3B). In neurons
that generated oscillations only on depolarization, repetitive spiking
usually appeared when the membrane was depolarized by an additional
2-4 mV beyond the threshold for appearance of oscillations (Figs. 5 and 6).
In both cases depolarization had its effect by 1) increasing oscillation amplitude and 2) bringing the peaks of the
oscillatory sinusoids closer to spike threshold. Overall, 66 of the 70 neurons studied (94%) that had subthreshold oscillations also fired
repetitively at rest or on depolarization (Table 2, Fig. 3). For the
cells that were not already firing at
Vr, the threshold for repetitive firing was 34.3 ± 7.4 (n = 10) in controls and
43.8 ± 8.3 mV (n = 13) in Chung preparations
(P = 0.01). At first, firing rate increased when the
cells were depolarized beyond firing threshold. However, still deeper
depolarization led to a reduction in oscillation amplitude and
eventually to the cessation of spiking (Fig. 5).
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Repetitive firing often took the form of irregular discharge of single spikes, where individual oscillation sinusoids triggered spikes at irregular intervals. Alternatively, there were trains of spike bursts ("interrupted autorhythmicity," Fig. 1). The mean interval between spikes or spike bursts decreased as the cell was depolarized. Likewise, the probability of burst firing increased, as did the duration of bursts. The factors that control discharge pattern will be presented in detail elsewhere.
In contrast to neurons with subthreshold oscillations, neurons that did
not either oscillate at Vr or begin to
generate oscillations on depolarization never showed
repetitive spiking (410 of 410 cells observed). This includes 313 A0 neurons (176 in control DRGs, 137 in
axotomized DRGs) and 97 Ainf neurons (31 in
control DRGs, 66 in axotomized DRGs). Despite their inability to
support repetitive firing, all of the nonoscillating
A0 and Ainf neurons generated a single action potential at the beginning of a
suprathreshold depolarizing step, and in some there was a short burst
of action potentials. The association of repetitive spiking with
subthreshold oscillations was, of course, highly significant
statistically (P < 0.001, 2).
The one axotomized Ainf neuron encountered that
had oscillations fired repetitively on depolarization.
Since axotomy increased the proportion of neurons with subthreshold
oscillations, it also increased the overall ectopic impulse barrage
(Table 2, Fig. 3B). This increase was particularly prominent at Vr, and hence relevant to the
intensity of the spontaneous ectopic barrage in vivo (Figs. 4,
A and B, and 6). There was nearly a fourfold
increase in the prevalence of spontaneous firing of A0 neurons at Vr
in axotomized compared with control ganglia (5.6 vs. 20.6% 1-9 dpo).
We have reported a similar dramatic increase in ectopic discharge in
vivo (Liu et al. 2000). This appears to be the principal
factor responsible for positive neuropathic symptoms in the Chung model
of neuropathic pain.
Biophysical characteristics of oscillating neurons and effects of axotomy
We made systematic measurements of a number of cellular parameters thought to be associated with neuronal excitability aiming to identify the ones most closely associated with oscillatory behavior. Results for A0 neurons are given in Table 4. Differences in Vr were noted above and will not be repeated here.
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OSCILLATIONS AT Vr. Considering first only cells with oscillatory capability, neurons that oscillated only when depolarized from rest had narrower spikes than those with oscillations at Vr. This was so in both control and axotomized DRGs and was associated with faster rates of rise and fall of the spike and larger spike amplitude. A small part of these differences is accounted for by the slightly more positive Vr of the neurons that oscillated at Vr. In direct measurements we found that spikes became broader on depolarization (by 16 µs/mV) and that dV/dt of the rising and falling phase of the spike decreased (by 3.4 and 1.8 V/s/mV, respectively). However, this factor accounts for only about 20% of the observed difference in spike waveform (Table 4). Moreover, as noted above, oscillations emerged in the neurons requiring depolarization at potentials far positive to the Vr of the cells that oscillated at Vr, and Rin did not differ between the two cell groups. Finally, threshold for evoking action potentials, both single spikes and sustained firing, was higher in the cells that oscillated only on depolarization. These data suggest that the neurons with oscillations at Vr constitute a distinct population, different from those that oscillate only on depolarization (see DISCUSSION).
NEURONS WITH VERSUS WITHOUT OSCILLATIONS.
For this comparison we pooled data from the two groups of oscillating
neurons (those with oscillations at Vr
and those requiring depolarization), and from control and axotomized
preparations, after ascertaining that the key differences were present
in each group independently (Table 4). Single spike threshold was much higher in the nonoscillating neurons despite the fact that the spike
rise time was faster and there was no consistent difference in
Rin. These cells did not fire
repetitively at any membrane potential, of course. Probably the most
interesting difference between neurons with and without oscillations,
however, was in the hyperpolarization that followed each spike (AHP).
This was significantly smaller in amplitude, and more
prolonged, in nonoscillating neurons (Table 4; Fig.
7). Moreover, some of the nonoscillating neurons (36/259, 14%) had a second, slow AHP phase that lasted for
many tens of milliseconds (Amir and Devor 1997). None of
the oscillating neurons had a slow AHP of this sort (P = 0.006,
2).
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INTACT VERSUS AXOTOMIZED NEURONS.
Axotomy had similar effects on all three groups of neurons, those
oscillating at Vr, those with
oscillations only on depolarization, and those without oscillations.
These were generally consistent with many prior reports (Kim et
al. 1998; Stebbing et al. 1999
; Titmus
and Faber 1990
). Specifically, axotomy caused a broadening of
the action potential, due to slowing of both the rising and the falling
limbs of the spike. There was no consistent change in spike height or
in Rin. The amplitude of the AHP was
significantly reduced, but AHP duration was unchanged. Most
importantly, there was a significant reduction in both the single spike
thereshold (Table 4) and the threshold for evoking sustained firing
(Fig. 6).
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DISCUSSION |
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Afferent discharge arising ectopically in DRG neuronal somata is
believed to contribute significantly to spontaneous dysaesthesias, pain
on movement, and tissue tenderness in patients with neuropathy (Devor and Seltzer 1999). During the first few days
after nerve injury in the Chung model of neuropathic pain virtually all
of the ectopic activity that originates in the DRG occurs in neurons with myelinated axons (A-neurons) (Liu et al. 2000
). In
the present study we recorded intracellularly from A-neurons in excised
DRGs to evaluate the process whereby axotomy gives rise to this
ectopia. The most striking observation was that repetitive firing in
DRG neurons does not result from the classical (Hodgkin-Huxley)
repetitive firing process in which a sustained (generator)
depolarization repeatedly draws the membrane potential to spike
threshold (Jack et al. 1985
). Rather, it is due to
subthreshold oscillations in the resting membrane potential
(Amir et al. 1999
). Spiking occurs when individual
oscillation sinusoids reach threshold. Nearly all neurons with
subthreshold oscillations fired repetitively either at
Vr or when depolarized; none of the
cells without subthreshold oscillations fired repetitively at any
membrane potential. Spinal nerve injury enhances the subthreshold
oscillations in DRG neurons triggering intensified ectopic discharge
and hence neuropathic paresthesias and pain.
Categories of A0 neurons
The relation between repetitive firing in DRG neurons and
subthreshold membrane potential oscillations was first reported by
Amir et al. (1999), although related resonance behavior
has been noted previously in primary afferent neurons in the trigeminal ganglion and in the mesencephalic nucleus of the trigeminal brain stem
(Pedroarena et al. 1999
; Pelkey and Marshall
1998
; Puil and Spigelman 1988
). Consistent with
the observations of Amir et al. (1999)
, we found that in
DRG A-neurons, subthreshold oscillations occur almost exclusively in
(noninflected) A0 neurons. Moreover, these appear
to be a specific A0 subpopulation, statistically distinguishable from A0 neurons without
oscillatory capability on the basis of definable biophysical
characteristics. Specifically, A0 neurons
incapable of generating oscillations have a smaller and more prolonged
AHP than those with oscillations. In Ainf
neurons, which virtually never show subthreshold oscillations, the AHP is much longer than in A0 neurons (Amir
and Devor 1997
; Liu et al. 2000
). We conclude
that at least one requirement for oscillations is rapid recovery from
the postspike hyperpolarization.
A priori, the somata of DRG neurons, particularly
A0 neurons, are poorly adapted for sustained
firing (Eng et al. 1988; Everill et al.
1998
; Kocsis et al. 1982
; Stansfeld et
al. 1986
). They are relatively large cells, with a large
membrane area (and capacitance) and have a very prominent delayed
(outward) rectification. The outwardly rectifying conductance opposes
inward (generator) currents and decreases
Rin. Both effects cause the trajectory
of depolarization after the first spike to be sufficiently shallow that
membrane accommodation tends to prevent a second and third spike from
being triggered. For this reason step depolarization rarely triggers more than one or two action potentials (at the beginning of the pulse)
unless the delayed rectifier is blocked pharmacologically.
Oscillatory behavior can be viewed as a means of enabling repetitive
firing despite the strong outward rectification. Specifically, the
rapid rise time of the depolarizing limb on the oscillatory sinusoid,
which scales with oscillation frequency, has the effect of overcoming
membrane accommodation. In this way oscillations simulate rapid-fire
(tetanic) step depolarizations. Depolarization brings oscillation peaks
closer to threshold, hence favoring repetitive discharge. However, an
even more important effect of depolarization is to increase oscillation
amplitude and the slope (dV/dt) of the
depolarizing phase of the oscillatory sinusoids. When cells become too
deeply depolarized, however, oscillation amplitude declines, and the
Na+ channels responsible for the rising limb of
the action potential become progressively inactivated. As a result,
spike generation begins to fail, despite the continued presence of
oscillations. Possible reasons why evolution provided some DRG neuronal
somata with this special repetitive firing capability are discussed
elsewhere (Devor 1999).
Among the A0 neurons with oscillatory capability
in intact DRGs, about one-half showed subthreshold oscillations at
Vr, while in the remainders it
was necessary to apply tonic depolarization. Since the resting membrane
potential of neurons with oscillations at
Vr was itself relatively depolarized
(Table 4), we initially presumed that these two groups form a continuum
of oscillatory A0 neurons. However, we now
believe that they constitute distinct functional subtypes on the basis
of two observations. First, in neurons requiring depolarization,
subthreshold oscillations became detectable at a mean of 36.5 ± 9.0 mV, a value almost non-overlapping with the resting potential of
cells with oscillations at Vr
(
56.8 ± 5.9 mV, P < 0.001). Second, neurons
requiring depolarization had much narrower action potentials than those
with oscillations at Vr, although AHP
characteristics were no different (Table 4). These differences cannot
be explained on the basis of resting membrane potential alone.
Axotomy greatly increased the population of neurons with oscillations, shifting cells from the nonoscillating to the two oscillating categories. Interestingly, early in this process (1-2 dpo), essentially all of the newly oscillating neurons oscillated at Vr (80% of all oscillating neurons in this time window oscillated at Vr). Only later (2-9 dpo) did the ratio between the two categories return to its control value of about 50% (Fig. 3A). Moreover, the cells newly oscillating at Vr had spike waveform characteristics very similar to the overall value for cells with oscillations at Vr [0.91 ± 0.43 ms (n = 17 cells, 1-2 dpo) vs. 0.95 ± 0.53 ms (n = 34), P > 0.2, Table 4], while the cells that oscillated only on depolarization 1-2 dpo were very similar to the overall population of cells with oscillations only on depolarization [0.52 ± 0.06 ms (n = 6 cells, 1-2 dpo) vs. 0.57 ± 0.24 ms (n = 25), P > 0.2, Table 4]. This means that the first change in cellular membrane characteristics induced by axotomy causes an increase in spike duration. This observation strengthens the link between spike width and oscillatory phenotype. By the same token, it suggests that individual A0 neurons can shift between the three categories (i.e., nonoscillating and 2 categories of oscillating neurons).
The existence of distinct A0 neuronal subtypes is
of considerable practical significance since electrical properties of
the DRG cell soma tend to correlate with those of the peripheral
sensory ending (Harper 1991; Reeh and Wadell
1990
). Therefore resonance properties of the soma may reflect
the type of afferent signal carried by the neuron in question. For
example, A0 neurons with oscillations at
Vr may correspond to slowly or
nonadapting afferent types such as muscle spindle afferents, while
A0 neurons that oscillate only on depolarization
may correspond to low-threshold mechanoreceptors, which have more rapid
adaptation. The quality of abnormal sensations resulting from ectopic
firing, in health and disease, depends on the functional class of the
active neurons. It is not known yet whether oscillatory behavior also
underlies ectopic repetitive firing in neuroma endbulbs or normal
sensory endings (but see Kapoor et al. 1997
). However,
if so, this difference could account for the observation that
spontaneous activity in DRG neurons and neuroma endings originates
largely in muscle afferents, while most injured cutaneous afferents
require a depolarizing stimulus to evoke ectopic firing (Johnson
and Munson 1991
; Michaelis et al. 2000
;
Proske et al. 1995
; Tal et al. 1999
).
Effects of axotomy
The prevalence of subthreshold oscillations observed here in
intact (control) DRGs was similar to that reported by Amir et al. (1999), although the present study includes a much larger sample of A-neurons. Combining data from the two studies, the proportion of A0 neurons with oscillations and
spiking at Vr was 4-5%. This
corresponds well with the prevalence of spontaneous discharge
originating in the DRG of intact rats in vivo (Wall and Devor
1983
). Likewise, the proportion of A0
neurons with oscillations and spiking on depolarization was nearly
identical to values reported by Amir et al. (1999)
.
Interestingly, the effects of axotomy were also similar despite the
fact that Amir et al. (1999)
cut the sciatic nerve
(rather than the spinal nerve), severing axons much farther from the
cell soma. The most prominent differences were the timing of the most
intense ectopic barrage (later in the case of sciatic nerve injury),
and its intensity (less at the peak in the case of sciatic nerve
injury). Detailed comparisons of the effects of distal versus proximal
axotomy are given in Liu et al. (2000)
.
Axotomy increased the incidence of oscillations and firing at
Vr. This effect, however, was not
caused primarily by shifting Vr in the
direction of depolarization. Rather, it was mostly a result of the
shift in the voltage dependence of oscillation amplitude toward
Vr (Fig. 4). An additional effect of
axotomy was to lower the frequency of oscillations at any given
membrane potential. Amir et al. (1999) showed that the
depolarizing limb of the oscillatory sinusoid is dependent on
Na+ conductance. The specific resonance
characteristics of different DRG neurons, including the heterogeneity
in the voltage dependence of oscillation amplitude and frequency, are
presumably related, at least in part, to the heterogeneity in the
Na+ channel subtypes expressed in different
classes of DRG neurons. Axotomy is known to differentially alter the
expression of certain Na+ channel subtypes,
although for other subtypes the nature of the change, if any, has not
yet been determined (Waxman et al. 1999
).
Unfortunately, the currently known changes in Na+
channel subtypes do not account in any obvious way for the observed
alterations in spike waveform and oscillatory phenotype. For example,
it is known that the kinetically fast TTX-sensitive type III
Na+ channels are up-regulated following axotomy
(Cummins and Waxman 1997; Rizzo et al.
1995
; Waxman et al. 1994
), and that the slower TTX-resistant PN3/SNS and NaN/SNS2 Na+ channels
are downregulated (Cummins and Waxman 1997
;
Dib-Hajj et al. 1998
; Novakovic et al.
1998
; Waxman et al. 1999
). This might have lead
one to predict that an early effect of axotomy would be to decrease
spike width, and increase rates of spike rise and fall
(dV/dt). In fact, the opposite was observed.
Shortly after axotomy (1-2 dpo), newly oscillating neurons showed an
increase in spike width, which only later (2-9 dpo) narrowed. Type III Na+ channels are normally expressed at very low
levels in adult DRG neurons (Waxman et al. 1994
).
Perhaps the early increase in spike width reflects reduced
K+ conductance (Everill and Kocsis
1999
), with the subsequent narrowing due to up-regulated type
III Na+ channels. Alternatively, an early change
in some other TTX-sensitive Na+ channel subtype
may be involved.
It is likely that the marked reduction in oscillation frequency induced
by axotomy is more closely related to changes in
K+ conductance than in changes in
Na+ conductance (unpublished
observations). Axotomy is known to downregulate the expression of at
least some K+ channel transcripts, and to reduce
whole cell K+ conductance, including both
IK and
IA, by about 50% (Everill and
Kocsis 1999; Ishikawa et al. 1999
). This change
also probably contributes to the overall facilitation of resonance
behavior and firing in axotomized neurons (Devor et al.
1994
; Amir et al., unpublished observations). Finally, in
addition to voltage-sensitive Na+ and
K+ channels, axotomy alters the expression of a
variety of other channel and receptor subtypes in DRG neurons (e.g.,
Baccei and Kocsis 2000
; Hokfelt et al.
1997
; Kelly et al. 1986
). The observed changes
in oscillatory phenotype no doubt result from the integrated effects of
the entire spectrum of axotomy-induced changes in membrane electrical properties.
Relation to neuropathic pain
In most cases A0 neurons are the cell bodies
of low-threshold mechanoreceptors (e.g., Koerber and Mendell
1992). Ectopic firing in these neurons is therefore expected to
be felt as touch, pressure, stretch (including proprioception), or
vibration. However, it has been well documented in recent years that in
the presence of central sensitization, afferent input along
low-threshold afferents can give rise to a sensation of pain (A
pain) (Campbell et al. 1988
; Cook et al.
1987
; Koltzenburg et al. 1994
; Torebjork
et al. 1992
). Indeed, A
pain is probably the most important
component of tenderness (tactile allodynia) in everyday minor injuries
(within the zone of primary as well as secondary hyperalgesia).
Therefore spontaneous discharge originating in DRG
A0 neurons and ectopic activity evoked by
depolarization (e.g., secondary to mechanical stimulation of the DRG,
crossed- afterdischarge or sympathetic efferent activity) may well
contribute to frank pain in addition to neuropathic paresthesias and
dysaesthesias (Devor and Seltzer 1999
).
The question remains as to the origin of central sensitization in
neuropathy. At least two options present themselves. First, in the
event of nerve injury, central sensitization may be triggered and
maintained by C-nociceptor activity originating within the DRG, the
nerve injury site, or residual intact afferents (Devor and
Seltzer 1999). Second, there is accumulating evidence that following axotomy, the structural and neurochemical phenotype of
A0 neurons may change in such a way that they,
too, can trigger and maintain central sensitization (Liu et al.
2000
; Ma and Woolf 1996
; Noguchi et al.
1995
; Woolf 1992
). Thus the enhancement of cellular resonance followed by nerve injury, and the augmented ectopic
discharge that it triggers, may well be the most fundamental of the
cellular changes that underly neuropathic pain.
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ACKNOWLEDGMENTS |
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This work was supported by grants from the United States-Israel Binational Science Foundation, the German-Israel Foundation for Research and Development, and the Hebrew University Center for Research on Pain. C.-N. Liu was supported by a Golda Meir postdoctoral fellowship. M. Michaelis received a Heisenberg-Fellowship from the Deutsche Forschungsgemeinschaft.
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FOOTNOTES |
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Address for reprint requests: M. Devor (E-mail: marshlu{at}vms.huji.ac.il).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 18 November 1999; accepted in final form 22 March 2000.
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REFERENCES |
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