Spinal Nerve Injury Enhances Subthreshold Membrane Potential Oscillations in DRG Neurons: Relation to Neuropathic Pain

Chang-Ning Liu,1 Martin Michaelis,2 Ron Amir,1 and Marshall Devor1

 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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 MOmega , 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 chi 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).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Table 1. Prevalence of subthreshold oscillations and repetitive firing, at Vr and on depolarization, in the three types of control ganglia (data from A0 and Ainf neurons combined)



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Fig. 1. Subthreshold membrane potential oscillations trigger action potentials in dorsal root ganglion (DRG) neurons. Traces illustrate oscillations without spiking (A), with intermittent spiking (B), and with intermittent spike bursts (C). Recordings in all 3 cells were made at Vr. DRG, Vr, and time after spinal nerve injury were as follows: A: DRGL5, -53 mV, 33 hpo; B: DRGL5, -61 mV, 7 dpo; C: nonaxotomized DRGL4, -62 mV, 5 days after cutting the ipsilateral L5 spinal nerve. Spikes are truncated. Calibration: 100 ms/10 mV.


                              
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Table 2. Proportion of A-neurons with subthreshold oscillations and with repetitive spike discharge in intact DRGs and at various times following spinal nerve transection



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Fig. 2. The amplitude and frequency of subthreshold oscillations is voltage sensitive. A: sample epochs of subthreshold membrane potential oscillations at 4 levels of depolarization from rest (epochs at depolarized potentials were chosen to avoid action potentials). Normalized oscillation power values for this cell in various frequency bands, derived from fast Fourier transform (FFT) analysis, are shown in B. Data are from an A0 neuron recorded in the DRGL5 23 h after L5 spinal nerve section. Vr = -50 mV. Calibration: 200 ms/5 mV.


                              
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Table 3. Dominant frequency and peak-to-peak amplitude of subthreshold oscillations in neurons from control DRGs, and DRGs 9 h to 9 days after spinal nerve injury (Chung model)

The resting membrane potential (Vr) of the 12 A0 neurons that had oscillations at Vr was -56.8 ± 5.9 mV, a value depolarized by 6-7 mV compared with the remainder of the neurons in which there were no oscillations at Vr (-63.1 ± 6.8 mV for all A-neurons, -63.4 ± 6.7 mV considering only A0 neurons, both P < 0.001). This suggests that the oscillatory behavior may have been due to the depolarized state of the membrane. However, when we injected current intracellularly so as to generate a slow ramp depolarization, subthreshold oscillations appeared in only an additional 10 neurons (all A0) that did not already have them at Vr (Table 2). Moreover, in these 10 neurons oscillations were not seen until the membrane potential reached -36.5 ± 9.0 mV, far positive to the -56.8 ± 5.9 value of cells with oscillations at Vr (P < 0.001). We conclude that oscillatory behavior is not a general characteristic of DRG neurons. Rather, it can be demonstrated in only a select subpopulation of A0 neurons, even on deep depolarization.

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, chi 2).



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Fig. 3. Spinal nerve injury triggered a sharp increase in the oscillatory behavior of axotomized DRG neurons (A), and in the resulting ectopic impulse barrage (B). Bars indicate observations made at Vr () and when the cells were depolarized to at least -20 mV (). Number of cells studied at each time interval are indicated in parentheses.

As in the control ganglia, Vr of the cells with oscillations was depolarized by about 7 mV compared with the cells without oscillations (P < 0.001). And like the controls, depolarization with intracellular current injection recruited oscillations in only a small fraction of the cells that did not already have them at Vr (Table 2, Fig. 3A). Finally, as in the controls, virtually all cells with oscillations at Vr or on depolarization were of the A0 type. Only one Ainf neuron was encountered that showed subthreshold oscillations (on depolarization, in a preparation 1 dpo).

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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Fig. 4. The amplitude and frequency of subthreshold oscillations are voltage sensitive, and the dependence on voltage is altered by axotomy. A: data from 2 control neurons () and 4 axotomized neurons (1-3 dpo, open circle ). Each neuron shows an "optimal" membrane potential at which oscillation amplitude is maximal. B: the distribution of "optimal" membrane potentials is shifted toward more negative (hyperpolarized) values by axotomy. The axotomized neurons (Chung) were from preparations 9hpo-9dpo. C: the dominant oscillation frequency (obtained from FFT analysis) increases linearly as neurons are depolarized. Data for 2 axotomized neurons are shown [3 dpo, Vr = -50 mV (), 8 dpo, Vr = -52 mV ()]. D: the dominant oscillation frequency, i.e., the frequency seen at the potential at which oscillation amplitude was maximal, decreased following axotomy (9 hpo-9 dpo).

Spinal nerve injury had a marked effect on the relation of oscillation amplitude to membrane potential. First, it shifted the function to more negative (hyperpolarized) potentials. Following axotomy, fewer cells required deep depolarization before generating oscillations (Fig. 4, A and B), and the mean threshold and optimal potentials for generating oscillations shifted in the negative direction (P < 0.01, Table 3). Second, axotomy caused a net increase in oscillation amplitude (Table 3). For example, oscillation amplitude at the optimal membrane potential averaged 1.9 ± 1.0 mV (n = 21) in control neurons versus 2.9 ± 1.2 mV (n = 34) in axotomized neurons (P = 0.002).

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)].

Figure 4D plots the dominant oscillation frequency at the membrane potential at which oscillation amplitude was maximal. Axotomy caused a clear shift to lower frequencies (106.0 ± 29.3 Hz in 21 control neurons vs. 74.7 ± 26.7 in 32 axotomized neurons, P < 0.001, Table 3). Part of this change is expected from the relatively hyperpolarized optimal membrane potentials of axotomized neurons (Fig. 4B, Table 3). However, the difference averaging over all cells (about 13 Hz/mV, Table 3) was >10 times larger than expected from the above-noted dependency of frequency on membrane potential (about 0.8 Hz/mV). We conclude that axotomy lowers oscillation frequency through a process independent of membrane potential. Interestingly, in our previous study in which axotomy was carried out distally, on the sciatic nerve, we did not see a significant decline in oscillation frequency (Amir et al. 1999).

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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Fig. 5. Relation of membrane potential to repetitive firing. Depolarization in this A0 neuron led to an increase in oscillation amplitude, and the initiation of burst firing (at -60, -58, and -55 mV). Still deeper depolarization led to a decrease in oscillation amplitude (also see Fig. 2) and the cessation of firing. Spike height is truncated. Calibration: 100 ms/5 mV.



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Fig. 6. Following axotomy (9 hpo-9 dpo, ) a larger proportion of active DRG neurons fire at relatively negative membrane potentials. Among DRG neurons that supported repetitive firing, some fired at Vr (negative to -45 mV) while others began to fire only when depolarized. Bars indicate the most negative membrane potential range for each neuron in which repetitive firing was observed. The bin marked -65 to -70 mV contains neurons that fired at Vr (which was positive to -65 mV), but continued to fire when the membrane was hyperpolarized to this range. Further hyperpolarization quenched their firing.

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, chi 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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Table 4. Properties of DRG A0 neurons that had subthreshold oscillations at Vr or on depolarization, and neurons without oscillatory capability, in intact animals and in the Chung model of neuropathic pain (spinal nerve section 9 hpo to 9 dpo)

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, chi 2).



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Fig. 7. Oscillatory capability in A0 neurons is associated with large amplitude, short-duration postspike afterhyperpolarization (AHP). A and B: voltage traces at Vr and on depolarization in neurons with and without subthreshold oscillations. C: superimposed spike traces of these 2 neurons illustrate characteristic differences in AHP parameters (population data are given in Table 4).

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).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 (Abeta pain) (Campbell et al. 1988; Cook et al. 1987; Koltzenburg et al. 1994; Torebjork et al. 1992). Indeed, Abeta 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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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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ABSTRACT
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