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 Previous Article
The Journal of Neuroscience, February 1, 2002, 22(3):1187-1198
Burst Discharge in Primary Sensory Neurons: Triggered by
Subthreshold Oscillations, Maintained by Depolarizing
Afterpotentials
Ron
Amir1,
Martin
Michaelis2, and
Marshall
Devor1
1 Department of Cell and Animal Biology, Institute of
Life Sciences, Hebrew University of Jerusalem, Jerusalem 91904, Israel,
and 2 Physiologisches Institut, Christian-Albrechts
Universitat, 24098 Kiel, Germany
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ABSTRACT |
Afferent discharge generated ectopically in the cell soma of dorsal
root ganglion (DRG) neurons may play a role in normal sensation, and it
contributes to paraesthesias and pain after nerve trauma. This activity
is critically dependent on subthreshold membrane potential
oscillations; oscillatory sinusoids that reach threshold trigger
low-frequency trains of intermittent spikes. Ectopic firing may also
enter a high-frequency bursting mode, however, particularly in the
event of neuropathy. Bursting greatly amplifies the overall ectopic
barrage. In the present report we show that subthreshold oscillations
and burst discharge occur in vivo, as they do in
vitro. We then show that although the first spike in each burst
is triggered by an oscillatory sinusoid, firing within bursts is
maintained by brief regenerative post-spike depolarizing afterpotentials (DAPs). Numerical simulations were used to identify the
cellular process underlying rebound DAPs, and hence the mechanism of
the spike bursts. Finally, we show that slow ramp and hold (tonic)
depolarizations of the sort that occur in DRG neurons during
physiologically relevant events are capable of triggering sustained
ectopic bursting, but only in cells with subthreshold oscillatory
behavior. Oscillations and DAPs are an essential substrate of ectopic
burst discharge. Therefore, any consideration of the ways in which
cellular regulation of ion channel synthesis and trafficking implement
normal sensation and, when disrupted, bring about neuropathic pain must
take into account the effects of this regulation on oscillations and bursting.
Key words:
depolarizing afterpotential; dorsal root ganglion; ectopic firing; neuropathic pain; pain; paresthesia; subthreshold
oscillations
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INTRODUCTION |
Sensory signals normally originate
at axonal transducer endings in skin, muscle, and other peripheral
tissues. The somata of sensory neurons in segmental dorsal root ganglia
(DRGs) also generate afferent activity, but this is sparse in intact
animals and its sensory consequences are likely to be minor (Wall and Devor, 1983 ; Devor, 1999). After nerve injury, however, discharge originating ectopically within DRGs is greatly augmented and can be a
major contributor to neuropathic dysesthesias and chronic pain (Kirk,
1974; Wall and Devor, 1983; Burchiel, 1984; Nordin et al., 1984;
Kuslich et al., 1991; Kajander et al., 1992; Sheen and Chung, 1993; Na
et al., 1995; Blenk et al., 1997; Devor and Seltzer, 1999; C.-N. Liu et
al., 2000a,b; Lyu et al., 2000; Zhang et al., 2000; Boucher and
McMahon, 2001) (but see Li et al., 2000). The underlying process is
therefore of considerable practical interest.
The process is also of theoretical interest. DRGs are located within a
bony foramen, protected from external stimuli. Moreover, even when
artificially depolarized, few DRG neurons are capable of firing more
than a brief spike burst. The slow, tonic depolarizations normally
caused by physiological stimuli rarely evoke any spikes at all
(Villiere and McLachlan, 1996; Amir et al., 1999; C.-N. Liu et al.,
2000a). How, then, does ectopic afferent discharge occur? We showed
recently in vitro that DRG neurons with repetitive firing
capability have a unique property. Rather than their discharge being
triggered by the classical rhythmogenic process whereby a sustained
depolarizing current repeatedly draws the membrane potential toward
spike threshold, firing in these cells is caused by sinusoidal
oscillations in the membrane potential. Spikes arise when peaks of
normally subthreshold sinusoids reach threshold (Amir et al., 1999;
C.-N. Liu et al., 2000a). We now confirm that this also applies
in vivo.
In vivo and in vitro, oscillation sinusoids
normally reach threshold only intermittently, yielding slow, irregular
firing (Wall and Devor, 1983). Axotomy enhances firing by increasing the number of oscillating neurons and shifting the membrane potential at which oscillations occur closer to rest
(Vr) (Amir et al., 1999; C.-N.
Liu et al., 2000a). Additional amplification results from replacement
of the usual slow intermittent firing pattern with high-frequency
bursts. In principle, bursting could simply result from a higher
proportion of oscillation sinusoids reaching threshold as proposed
recently by Wu et al. (2001) on the basis of recordings from the
mesencephalic nucleus of the trigeminal nerve (MesV), but this is not
the case. Rather, we show that bursting in DRG neurons reflects
recurrent spike triggering by post-spike depolarizing afterpotentials
(DAPs). The first spike in each burst is triggered by an oscillation
sinusoid; the burst itself is maintained by DAPs.
In addition to augmenting spontaneous ectopic discharge, oscillations
and DAP-triggered bursts render somata of sensory neurons responsive to
physiologically relevant stimuli that were previously inert. We
illustrate this by demonstrating sustained burst discharge in response
to artificially generated slow ramp and hold depolarizations. Finally,
we document sustained ectopic bursting in response to natural stimuli
to which DRGs are subject in situ despite their privileged
anatomical location.
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MATERIALS AND METHODS |
Animals and surgery
Experiments were performed using male and female rats of the
Wistar-derived Sabra strain, both mature animals (>300 gm) and animals
2-5 weeks of age (20-145 gm). All protocols were in accordance with
national and University regulations for the humane care and use of
laboratory animals and followed the ethical guidelines of the
International Association for the Study of Pain (Zimmermann, 1983).
Animals used for in vivo recording, and most of those used
for in vitro recording, were intact (nonoperated). However,
for the in vitro experiment on adrenergic stimulation (see
Results), we used eight rats that had undergone unilateral neurectomy
2-10 d before electrophysiological evaluation. Briefly, under
pentobarbital anesthesia (Nembutal, 50 mg/kg, i.p.), the sciatic nerve
was exposed in the lower part of the popliteal fossa, tightly ligated
with 5-0 silk, and cut just distal to the ligature (six rats).
Alternatively, the L4 or L5 spinal nerve was exposed 5-10 mm distal to
the ganglion, tightly ligated with 5-0 silk, and cut just distal to the
ligature (two rats). In both cases, ~5 mm of the distal nerve stump
was excised. Surgical wounds were closed in layers, and the animals were treated with a topical bacteriostatic powder and penicillin (50,000 U/kg, i.m.). Recovery was uneventful. All animals were maintained under standard colony conditions, two to three per cage, in
clear plastic shoebox cages bedded with pine shavings. Food and water
was available ad libitum. The light/dark cycle was 12 hr.
Electrophysiological preparations
In vivo preparation. Intact rats
(n = 10) were anesthetized with Nembutal (50 mg/kg,
i.p., followed by ~20
mg · kg 1 · hr1
as needed). A tracheotomy was performed, one carotid artery was cannulated for monitoring arterial pressure and for administration of
fluids, and the animal was mounted prone in a spinal frame with legs in
extension. Electrocardiogram and heart rate were monitored, and
rectal temperature was maintained at 37.5°C using a
feedback-controlled radiant heater. Dorsal roots (DRs) L3-6 and either
the L4 or L5 DRG were exposed in a lower lumbar laminectomy and covered
with warmed paraffin oil (34°C) in a pool formed of muscle and skin
edges. To reduce movement artifacts, in most experiments the animal was
paralyzed with gallamine triiodoethylate (Flaxedil, Specia, ~10
mg/kg) or pancuronium bromide (~2.0 mg/kg) and artificially respirated. End tidal PCO2 in the trachea was
recorded using a Beckman LB-2 CO2 monitor.
In vitro preparation. Rats were overdosed with Nembutal
(~80 mg/kg, i.p.) and killed by carotid exsanguination. DRGs L4 or L5
were excised with the DR, spinal nerve, and a variable length of the
sciatic nerve still attached. After ~1 hr recovery in a modified
Krebs' solution containing (in mM): NaCl 124, NaHCO3 26, KCl 3, NaH2PO4 1.3, MgCl 2, dextrose 10, and saturated with 95% O2 and 5%
CO2 (pH 7.4, 290-300 mOsm, room temperature),
the ganglia were mounted in a recording chamber and superfused with the
Krebs' solution (1-2 ml/min, room temperature or 37°C) to which 2 mM CaCl2 was added. In some
experiments, as noted, adrenaline (Teva) was added to the superfusion
medium at a concentration of 10 µM, 100 µM,
or 1 mM for periods of 1-2 min. We recognize that the 1 mM concentration is probably well above physiological levels.
Recording. Sharp glass microelectrodes were used for
intracellular recording and stimulation (20-40 M filled with 3 M
KCl). Stimuli delivered through the recording microelectrode were
either brief pulses (1 msec), prolonged steps (80 msec, 100 msec, or >2 sec), or slow ramp and hold stimuli (see below). Signals were bandpass filtered at DC-10 kHz and recorded digitally on
magnetic videotape (Neurodata DR-484) for off-line analysis. We
categorized DRG neurons as A- or C-neurons by axon conduction velocity
(CV) and the waveform of the intracellularly recorded spike as
described elsewhere (Koerber and Mendell, 1992; Amir and Devor, 1996).
A-neurons were further categorized: Ainf neurons,
which have an inflection on the falling phase of the action potential
as assessed by analog differentiation, and relatively low CV (Amir and
Devor, 1996), include many A afferents and probably most of the
myelinated nociceptors. A0 neurons, neurons
without an inflection and with faster CV, are predominantly A
afferents, most of which are low-threshold mechanoreceptors (Koerber
and Mendell, 1992; Amir and Devor, 1996). Some neurons showed a
post-spike DAP. DAP amplitude was measured from the pre-spike baseline
to the DAP peak. DAP latency was measured from the DAP peak to the peak
of the preceding spike.
To test for oscillations, each cell was examined at
Vr (resting membrane potential) and
then the membrane potential was depolarized in a slow ramp (~20
mV/sec) and hold (>2 sec) until oscillations occurred or until more
than or equal to  20 mV. Potentials obtained were further bandpass
filtered at 1 Hz-1 kHz and digitized at 5 kHz (pClamp 6.0.3, Axon
Instruments). Frequency components were quantified as power spectral
density using a Fast Fourier Transform (FFT) routine (CP Analysis 5.1, Datawave). This analysis was applied to 1-4 sec epochs that did not
contain spikes. Oscillations were usually obvious, but when necessary
we used as a formal criterion that amplitude peaks be at least 1.5×
the amplitude of the background noise level present during brief pauses
in the oscillations or that there be a distinct peak in the FFT plot at
the frequency expected from visual inspection of the voltage trace,
or both.
Cross-excitation. Most DRG neurons show transient
depolarization during tetanic stimulation of neurons that share the
same DRG (Utzschneider et al., 1992; Amir and Devor, 1996). Here, two alternative stimulation protocols were used to induce this "DRG cross-excitation." In the first, pulses were delivered through an
Ag/AgCl electrode pair placed across the sciatic nerve. By adjusting
stimulation intensity and polarity, a substantial proportion of the
myelinated axons in the nerve could be activated without stimulating
the axon of the impaled neuron itself. The proportion of neighboring
neurons activated was assessed from the proportion of the maximal
compound action potential (CAP) evoked by the stimulus as monitored
through a recording suction electrode placed on the DR. In the second
protocol, pulses were delivered to the DR while the CAP was monitored
from the sciatic nerve. Stimulus pulses were monophasic 0.1- to
0.2-msec square-waves of  7mA, delivered in 10 sec tetani at 50 or 100 Hz. The stimuli were not sufficient to activate unmyelinated axons, as
judged from both the latency of CAP components and the failure to
record evoked spikes in intracellular recordings from C-neurons without
an additional substantial increase in stimulation strength.
Computational model and statistics
Burst firing was simulated using a modified Hodgkin-Huxley
(H-H) compartmental model. The simulations modeled a 50-µm-diameter isopotential cell using NEURON software [version 4.2.1; (Hines, 1989),
www.neuron.yale.edu]. Specific membrane capacitance was 1 µF/cm2, specific longitudinal resistance
was 110 -cm, and temperature was 20°C. Membrane electrical
properties, including the various kinetic terms, were based on values
from the reconstructed action potential of the squid giant axon
(Hodgkin and Huxley, 1952). However, some values were modified to more
closely resemble mammalian DRG neurons as follows. (1) The maximal
sodium conductance
(gNa+max) was
decreased to 50.0 mS/cm2 (Caffrey et al.,
1992). (2) The leak conductance was increased to 0.7 mS/cm2 (Scroggs et al., 1994). (3) The
reversal potential of the leak was set at 77.5 mV to approximate the
K+ battery. (4) The maximal
voltage-sensitive potassium conductance (gK+max) was
decreased to 2.6 mS/cm2. Prolonged
depolarizing steps (usually 600 msec) were given under current-clamp
conditions. We used the Crank-Nicholson second-order accuracy method
for integration (dt = 0.01 msec). For accuracy of
calculations we followed the rule of thumb that the number of segments
(computational compartments) should be more than or equal to section
length/0.05  (Luscher et al., 1994; Segev and Burke, 1998). Our cell
model was thus a single computational compartment.
Statistical comparisons are based on two-tailed t,
 2, and Fisher exact probabilities tests
using a significance criterion of p = 0.05. Values are
given ± the SD unless noted. We focused on A-neurons because
these have been shown to be the main component of neuropathic discharge
originating in the DRG (C.-N. Liu et al., 2000b; X. Liu et al. 2000;
Boucher and McMahon, 2001).
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RESULTS |
Subthreshold oscillations and repetitive firing recorded
in vivo
We have reported previously that in vitro, 5.5% of
A-neurons (17 of 308; A0 + Ainf) from intact DRGs exhibit high-frequency subthreshold oscillations at Vr
(~100 Hz), with an additional 9.4% (29 of 308) beginning to
oscillate on depolarization. Oscillation sinusoids typically remained
phase-locked with a pure sine wave for only a few tens of cycles,
before drifting by up to several Hertz. Defining a stable mean
frequency requires sampling over epochs of several seconds (Amir et
al., 1999; C.-N. Liu et al., 2000a). Oscillation amplitude was voltage
sensitive, with a peak of 3-6 mV at membrane potentials of 55 to
30 mV. Most cells with oscillations fired long trains when
depolarized by 2-3 mV from the potential at which they first showed
oscillations. In contrast, cells without oscillations never fired
repetitively, no matter how strongly they were depolarized.
We now document similar oscillatory behavior in vivo.
Intracellular recordings were made from 16 neurons (14 A0, 2 Ainf) in DRGs from 10 intact adult rats. Two of these neurons (13%; one A0, one Ainf) exhibited
oscillations of 50-55 Hz and repetitive firing at
Vr (Fig.
1). The Ainf neuron
fired singlet spikes at irregular intervals averaging 0.4 Hz, and the
A0 neuron fired long bursts, typically lasting
several seconds, with a fixed interspike interval (ISI) of 15 msec (67 Hz). Bursts were interrupted by brief pauses of 50-200 msec. The
singlet action potentials and the spike bursts were always triggered by
the rising (depolarizing) limb of oscillation sinusoids (Fig.
1B). As in vitro, hyperpolarization from
Vr decreased overall firing frequency
by reducing oscillation amplitude, and hence reducing burst duration
and lengthening the periods of spike-free oscillations between bursts
(Fig. 1). Firing frequency within bursts also decreased (ISI = 15 msec at 43 mV, 20 msec at 50 mV).
Vr of the cell with burst firing at
rest was 43.0 mV. Vr of the other
neurons was 46.6 ± 5.7 mV.
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Figure 1.
Subthreshold oscillations trigger sustained spike
discharge in vivo. A, Recording setup
showing dorsal (DR) and ventral (VR)
roots and the sciatic nerve (SCN) innervating the
hindpaw. The intracellular micropipette is used for recording and
stimulation (R&Sic). Spontaneous burst discharge at
Vr is shown together with a dot
display of the corresponding interspike intervals.
B, Oscillation amplitude and the prevalence of evoked
spikes decrease with a negative shift in the holding potential. Two
epochs are shown at 50 mV, one in which spikes were present, and one
without spikes. The neuron is an A0 neuron; the sciatic
nerve was cut acutely.
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An additional four A0 neurons had spike discharge
originating at peripheral receptor endings (probably muscle
proprioceptors; firing frequency = 0.25, 5, 18, and 22 Hz). This
activity was easily distinguished from the activity of somatic origin
because no oscillations were present, and neither depolarization nor
hyperpolarization affected firing frequency. The remainder of this
report is based on recordings in vitro.
Burst discharge and depolarizing afterpotentials
Singlet versus burst discharge pattern
From previous teased fiber recordings in vivo we know
that ~5% of DRG A-neurons in intact rats discharge spontaneously
(Wall and Devor, 1983; Burchiel, 1984; Michaelis et al., 2000). Most fire slow irregular trains of singlet spikes (75%; 0.5-5 Hz), whereas
others fire in a bursty (19%) or tonic (6%) pattern (ISI = 20-100 msec) (Wall and Devor, 1983). In vitro recordings
(DRGs from intact rats) in the present study yielded the same overall level of activity (5%; 12 of 236 units fired spontaneously at Vr). However, firing patterns were
somewhat different in vitro: 42% of the cells had slow
irregular discharge (5 of 12; 0.1-2.5 Hz; four
A0 and one Ainf), 58%
fired in on-off bursts (7 of 12; all A0); and
none fired tonically.
In cells with slow irregular firing, individual spikes were triggered
by individual oscillation sinusoids that reached threshold (Figs. 1,
2). Mean firing rate mostly reflected the
fraction of sinusoids that were suprathreshold, a value that depended
on membrane potential and the distribution of sinusoid amplitudes. When
cells were depolarized, sinusoid amplitude increased, and peaks came closer to threshold (Amir et al., 1999), increasing the rate of firing.
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Figure 2.
Slow irregular firing and burst discharge recorded
from DRGs of intact (unoperated) rats in vitro.
A, Irregular discharge triggered by subthreshold
oscillations at Vr (A0 neuron;
Vr = 47 mV). B,
C, Spikes evoked in the same cell by an intracellular
stimulus pulse and by axonal stimulation on the sciatic nerve
(asterisk). Three superimposed traces are shown in each
panel. This cell did not generate a DAP sufficient to trigger a second
spike; subsequent spikes in the train are generated by subthreshold
oscillations. D, Spontaneous burst firing in another
cell (at Vr = 45 mV). Each burst is
triggered by an oscillatory sinusoid that reached threshold.
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In cells with burst firing, the first spike in each burst was likewise
triggered by an oscillatory sinusoid (Figs. 1, 2D). However, subsequent spikes in the burst were not generated by this
mechanism because the ISI within bursts did not match the period of the
subthreshold oscillations seen just before the burst (Fig.
3). It was invariably shorter; hence,
instantaneous firing frequency early in each burst was always higher
than the oscillation frequency.
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Figure 3.
The interspike interval within bursts matches the
DAP latency, not the oscillation period. The two top
panels show spike bursts from two different A0
neurons (both at Vr = 45 mV). In each
panel, two bursts are superimposed. The shorter of the two bursts ends
in a subthreshold DAP (asterisk). The two top
panels also show a run of subthreshold oscillations taken from
just before the burst, showing that the oscillation period is greater
than the ISI. The graph below plots corresponding data
for 21 cells.
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Burst mechanism
We identified post-spike DAPs as the mechanism of burst discharge.
In approximately one-third of cells checked, the action potential
evoked by intracellular or axonal stimulation at
Vr was followed by a small DAP of
3.1 ± 2.4 mV (Fig. 4,
arrows). A burst was triggered whenever an oscillatory
sinusoid triggered a spike in a neuron that generated DAPs of
sufficient amplitude. That is, if DAP amplitude was large enough to
trigger a second spike, the DAP after the second spike triggered a
third spike, the third a fourth, and so on for the duration of the
burst. Within bursts the first ISI, i.e., the ISI between the first and
second spikes in the train, exactly matched the latency of the DAP
(6.4 ± 1.0 vs 6.5 ± 1.0 msec; n = 7;
Vr, p > 0.2;
r = 0.96; p < 0.005); it did not match
the period of oscillations immediately preceding the burst (Fig. 3)
(6.4 ± 1.0 vs 8.3 ± 1.0 msec; p < 0.05;
r = 0.6; p > 0.2). Bursts always ended
with a DAP that was just below spike threshold. From this we infer that
what terminated bursts was a decline in DAP amplitude during the course
of the burst (see below). Burst termination restored oscillations,
setting the stage for triggering of the next burst. In all cells that did not fire bursts, the post-spike DAP was either small or nonexistent (Fig. 2A-C).
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Figure 4.
Afterdischarge bursts evoked by stimulus pulses
are caused by DAPs. A, A brief (1 msec) depolarizing
pulse was delivered intracellularly. In one trace, a single spike was
evoked, followed by a post-spike DAP (arrow). In a
second (superimposed) trace, the DAP was suprathreshold and triggered a
second spike (Vr = 60 mV).
B, Same cell but examined at several holding potentials.
A brief (1 msec) hyperpolarizing pulse, which was subthreshold at
Vr, triggered a single anodal-break
spike at 55 mV and a spike burst followed by a DAP and damped
oscillations at 38 mV. Stimulus pulses are offset to aid visibility.
The DAP (arrow) increased in amplitude with
depolarization. C, Similar behavior after single-pulse
(0.1 msec) axonal stimulation on the sciatic nerve
(asterisk). D, Single-pulse axonal
stimulation in this neuron produced a discharge burst (two sweeps
superimposed). As during spontaneous bursts, spikes were synchronous
with the DAP (arrow) but not with subthreshold
oscillations (the epoch of oscillations shown was taken from just
before the burst). All cells were A0; spikes in
B-D are truncated.
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The presence and amplitude of DAPs, and hence the likelihood of burst
firing, correlated with other properties of the cell (Table
1). These parameters were also sensitive
to membrane potential. DAPs occurred preferentially in
A0 neurons, in cells with a brief (<20 msec)
rather than a more prolonged post-spike afterhyperpolarization (AHP),
and in cells with subthreshold oscillations. DAP amplitude increased
systematically with depolarization (0.37 ± 0.26 mV/mV; n = 24) to a peak at 35.8 ± 5.9 mV, and then it
declined (Figs. 4, 5). As a consequence,
slow ramp depolarization revealed DAPs in cells in which they were not
initially visible. Thus, although at
Vr 31% of cells had DAPs, 96% had
them on depolarization (8 of 26 vs 25 of 26;
2, p < 0.001). Not
surprisingly, cells with a DAP at Vr
had a more depolarized Vr than cells
without (52.8 ± 7.7 mV vs 58.8 ± 6.3 mV;
p < 0.005). Correspondingly, many cells that fired
singlet spikes at Vr became bursters
on depolarization, with burst duration increasing with increasing
depolarization (Fig. 1). Depolarization also decreased DAP latency
(0.21 ± 0.17 msec/mV; n = 17), causing firing
frequency within bursts to increase.
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Figure 5.
DAP amplitude varies with membrane potential.
Symbols represent data from 11 individual A0
neurons.
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Wu et al. (2001) argued recently that burst firing in MesV neurons
arises directly from runs of oscillatory sinusoids that cross the spike
threshold. We note, however, that in their results, as in ours, the
period between spikes in a train is consistently shorter than the
oscillation period (their Fig. 2, our Fig. 3). Rather, as we have
shown, an ISI within bursts matches the DAP latency, and bursts are
maintained by DAPs rather than by oscillations. This distinction is
significant; oscillations and DAPs are not the same thing. By
definition, a DAP is a brief depolarizing rebound potential that
follows a spike. Subthreshold oscillations are ongoing trains of
sinusoidal variation in the membrane potential, often completely
dissociated from spike activity. On the other hand, these two waveforms
appear to be reflections of the same underlying neuronal resonance
characteristic [see below, and Amir et al. (2002)], and they
sometimes appear in close association with one another (Table 1). For
example, spikes are sometimes followed by a damped train of post-spike
DAPs that appear to transition seamlessly into ongoing subthreshold
oscillations (Figs. 1, 4B, D).
Cessation of bursts
What causes bursts to terminate? During the course of a burst,
particularly a high-frequency burst, spike amplitude usually decreased
and ISI increased, reducing instantaneous firing frequency (Fig.
6A). The presence of
spikes precluded measurement of DAP amplitude during bursts. However,
each burst terminated in a DAP that was slightly below spike threshold.
The failure of this final DAP to trigger a subsequent spike cut short
the regenerative process that maintained the burst. Among the factors
responsible for the apparent attenuation of DAP amplitude during
bursts, and hence for shifting the peak of the DAP away from spike
threshold, is a hyperpolarizing shift that developed during the course
of the burst (Fig. 6). We have shown previously that this shift is
caused primarily by a Ca2+-activated
K+ conductance engaged during the burst
(Amir and Devor, 1997).
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Figure 6.
Mechanism of repetitive burst firing in DRG
neurons. A, This neuron was quiescent at
Vr, but subthreshold oscillations and
burst discharge developed as a depolarizing ramp and hold current
brought the cell toward 43 mV. Traces marked
1-5 show at a higher sweep speed the
parts of the main record that are marked with arrows.
The first oscillation sinusoid to reach threshold triggered a prolonged
spike burst (trace 2). During the course of the burst,
ISI increased (spike rate accommodation). This can be seen by comparing
ISI in traces 2 and 3, and also from the
ISI dot display and the fast traces shown
above the main record. As the burst progressed, membrane potential
shifted several millivolts in the hyperpolarizing direction, leading to
termination of the burst in a subthreshold DAP (trace
3). As the membrane subsequently drifted back toward the
holding potential (43 mV), the amplitude of oscillations increased,
and ultimately a second burst was triggered (traces 4
and 5). B, A similar process occurs when
the initial depolarizing shift is caused by cross-depolarization. This
cell did not fire when held at 45 mV. However, tetanic stimulation of
the axons of neighboring neurons evoked cross-depolarization and
repetitive burst firing (dashed line; 10 sec, 100 Hz
pulses to the sciatic nerve at an intensity subthreshold for the axon
of the impaled neuron but suprathreshold for the axons of
neighbors).
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After the cessation of each burst, the now hyperpolarized membrane
revealed smaller oscillations than those that occurred immediately
before the burst, or no oscillations at all. With time, membrane
potential drifted back in the depolarizing direction, and as this
occurred, oscillations reappeared and progressively grew in amplitude.
The first oscillation sinusoid to reach spike threshold triggered a
second DAP-driven burst, completing the burst cycle (Fig.
6A).
Afterdischarge bursts
An additional consequence of DAPs is "afterdischarge."
In vivo, 2-5% of DRG neurons fire a burst of action
potentials in response to single axonal stimulus pulses (intact and
axotomized rats) (Devor and Wall, 1990; Delio et al., 1992). Such
afterdischarge bursts were recorded here in vitro in a
similar proportion of neurons [intact rats, 3% (8 of 236) neurons at
Vr; all A0]
(Fig. 4). The bursts consisted of trains of 2 to >20 spikes after
single brief axonal or intracellular stimulus pulses.
In vivo, afterdischarge is most common in neurons that
already have ongoing burst discharge, and hence those that probably have subthreshold oscillations and DAPs (Devor and Wall, 1990). In such
cells application of a stimulus pulse during a silent period triggers a
burst, resetting the endogenous burst cycle and entraining it to the
applied stimulus (Lisney and Devor, 1987). Afterdischarge in
vitro was also associated with repetitive firing capability. Most
neurons that showed afterdischarge at
Vr also fired spontaneously (5 of 8).
In comparison, only 7 of the remaining 228 neurons, cells that did not
show afterdischarge at Vr, fired spontaneously (p < 0.001). An additional 5 of
the 228 neurons fired afterdischarge bursts when depolarized (by 6-26
mV from Vr; all
A0 neurons), and 3 of them fired repetitively.
All five developed subthreshold oscillations on depolarization.
All cells that generated afterdischarge bursts had DAPs. The stimulus
pulse triggered the first spike in each burst; subsequent spikes were
triggered by DAPs. As with spontaneous bursts (Fig. 3), the first ISI
in afterdischarge bursts matched the latency of the DAP (both 7.2 ± 1.7 msec; n = 9; r = 1.0) (Fig.
4D) more closely than they matched the period of
oscillations (8.4 ± 1.2 msec; r = 0.67).
Computational model of the DAP
Both oscillatory behavior and associated spike bursts persist in
the presence of wide-spectrum blockers of
Ca2+ conductances but are eliminated by
Na+ channel blockers (TTX, lidocaine) or
reduced [Na+]o
(Amir et al., 1999; Pedroarena et al., 1999; Wu et al., 2001). The DAPs
responsible for bursting are therefore distinct from the prolonged
Ca2+-dependent afterdepolarizations that
sometimes occur in DRG neurons (see Discussion) (White et al.,
1989). To gain insight into the membrane dynamics responsible for DAPs
and bursting, we ran computational simulations of mammalian DRG neurons
as described in Materials and Methods.
Just suprathreshold depolarizing steps triggered a single action
potential followed by a DAP. The latency and amplitude of the DAP were
like those observed in vitro (compare Fig.
7, A and B,
with Fig. 4A), and as in vitro,
DAP amplitude was voltage sensitive. Increasing the amplitude of the
depolarizing step increased the amplitude of the DAP, eventually
triggering brief spike bursts. A further increase led to tonic
discharge that persisted for as long as the stimulus was maintained (at
least 2 sec) (Matzner and Devor, 1992).
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Figure 7.
Post-spike DAP simulated numerically using the
H-H formalism. Cells held at Vr (77.5 mV)
were brought to near 55 mV at t = 0 using a 1 sec
depolarizing current step. At t = 500 msec, a
suprathreshold depolarizing pulse was given (0.2 msec, 10 nA) to
trigger a spike. A, Two superimposed simulations using
slightly different current steps. In one (1.030 nA), the post-spike DAP
was subthreshold; in the second (1.033 nA), the DAP reached threshold
and evoked a second spike. B, Enlargement of the
trace in A with the subthreshold DAP.
Note the close resemblance to DAPs recorded from real DRG neurons (Fig.
4A). C, Calculated transmembrane
leak, Na+, and K+ currents
underlying the voltage trace in B.
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Plots of individual (calculated) transmembrane currents (Fig.
7C) reveal the mechanism of the DAP in this model. After the spike, outward K+ and leak current induced
a brief AHP during which Na+ current fell
below its pre-spike value. This reprimed
Na+ channels, making more of them
available for opening. At the end of the AHP, when the membrane moved
back in the depolarizing direction toward the holding potential, a
small inward Na+ current was generated
yielding the DAP. In cases where the DAP was relatively large, this
process repeated itself, yielding damped oscillations (Matzner and
Devor, 1992). Indeed, the H-H simulation can yield ongoing
oscillations with frequency, amplitude, and voltage sensitivity similar
to those recorded in vitro (Amir et al. 2002). The resonance
characteristics underlying the DAP and subthreshold oscillations
therefore appear to be related.
Repetitive firing capability: response to step depolarizations
The presence of subthreshold oscillations and DAPs critically
determines whether a DRG neuron is capable of sustained firing in
response to tonic artificial or physiological depolarizing stimuli.
All neurons tested fired a single spike at the beginning of a
depolarizing step (n = 78; 48 A0,
30 Ainf; current at threshold 0.8 ± 0.3 nA
generated a depolarization of 5.6 ± 2.0 mV), and about half of
these fired a burst (i.e., more than or equal to two spikes) when
current strength was increased (40 of 78; threshold 1.7 ± 1.1 nA;
11.9 ± 9.3 mV). The remainders never fired more than one spike,
even using currents up to 10 nA, that generated depolarizations of
30-50 mV. Bursts occurred only in cells that generated a DAP.
A0 neurons were more likely to fire a burst than Ainf neurons (33 of 48 vs 7 of 30;
p < 0.001).
Burst duration and frequency were voltage sensitive. At threshold,
bursts consisted of two or three spikes with a mean ISI of 7.2 ± 2.2 msec (equivalent to an instantaneous firing frequency of 138 ± 40 Hz). With increasing current strength, burst duration increased,
persisting in some cells for the duration of the stimulus pulse (80 or
100 msec). However, using prolonged stimuli (>2 sec), bursts rarely
lasted more than ~200 msec (~40 spikes) (Fig. 9A). Instantaneous firing frequency (reciprocal of the first ISI) increased with stimulation current along a continuous negatively accelerating frequency-current trajectory (Jack et al., 1983; Matzner and Devor, 1992). Bursts always ended in a subthreshold DAP, followed by damped
oscillations in some cells (Fig. 8).
Spike rate accommodation, i.e., the gradual increase in ISI during the
course of a burst, occurred sometimes, especially during prolonged
bursts. Although bursts occurred in cells without oscillations, longer
burst durations were associated with oscillatory behavior. Thus, among
cells with bursts of >15 spikes, 6 of 9 had oscillations. This
compares with only 4 of 23 cells with bursts of fewer than 15 spikes
(p < 0.005; all 32 neurons were
A0).
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Figure 8.
DAPs maintain spike bursts during depolarizing
steps. The top panel shows three superimposed burst
responses that contained two, three, and four spikes, respectively (80 msec, 1.4 nA depolarizing steps). Asterisks mark the
subthreshold DAP that terminated the burst in each case. The
bottom panel is similar (data from a second neuron)
except that current steps of 1.0, 1.4, and 1.9 nA were used. Burst
duration increased with stimulus strength, persisting throughout the
step in the case of the 1.9 nA stimulus. Again, bursts ended in a DAP.
Both cells are A0; all spikes are truncated.
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The ability of cells to fire in a sustained manner, for >200 msec, was
critically dependent on the presence of subthreshold oscillations. For
example, in the cell illustrated in Figure
9B, an initial burst was
followed by a series of shorter bursts that persisted indefinitely.
Four such cells were encountered. In each case the recurrent bursts
were a product of the oscillation/DAP process described above.
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Figure 9.
Persistent firing during prolonged depolarizing
steps depends on the presence of subthreshold oscillations and
post-spike DAPs. A, Step depolarization (3 nA) triggered
a single spike burst. Numbered traces show that
oscillations present immediately after the burst damped out rapidly
(traces 2-8). B, Under similar
conditions, neurons with sustained subthreshold oscillations
(traces 2-6) generate repeated spike bursts.
Both neurons are A0; all spikes are truncated.
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Physiologically relevant depolarizing stimuli
Under physiological and pathophysiological conditions in
vivo, various processes are capable of depolarizing DRG somata
despite their protected location (see Discussion). However, because all of these yield slow ramp and hold-like generator currents, without fast
transients, it is likely that only oscillating cells respond with
ectopic repetitive firing. We tested this prediction.
Ramp and hold depolarization
In response to ramp and hold depolarization, most neurons did not
fire even a single spike. This includes cells that did fire an initial
burst on step depolarization (Fig.
10A). Spiking was initiated, however, in a subset of 14 of the 61 cells tested (23%; all
A0). Three cells fired a single burst during the
ramp and then fell silent. The remaining 11 fired repetitively during
both ramp and hold phases, with either irregularly spaced single spikes (n = 1) or repeated bursts (n = 10)
(Fig. 6A). Firing threshold was 9.4 ± 6.6 mV
positive to Vr (range 3-28 mV;
n = 14). In all 14 cells, repetitive firing was a
product of subthreshold oscillations and DAPs.
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Figure 10.
Burst firing during step versus ramp and hold
depolarization. A, This neuron consistently fired a
brief discharge burst in response to depolarizing steps
(left). No spikes were ever evoked by the equivalent
depolarization delivered as a slow ramp and hold
(right). B, Likewise, neurons that fired
in response to depolarizing steps (left) failed to
respond to slow physiological depolarizations, evoked in this case by
tetanic activation of neighboring neurons (cross-depolarization,
right). The exception is neurons with subthreshold
oscillations (Fig. 6B). Both neurons were
A0.
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DRG cross-depolarization
In vivo, ectopic discharge originating in DRG neurons
is facilitated by stimulation of the axons of adjacent neurons that share the same ganglion or by stimulation of the skin innervated by the
adjacent neurons (Devor and Wall, 1990). In vitro as well, most DRG neurons (~90%) are transiently depolarized by spike
activity in neighbors (Utzschneider et al., 1992; Amir and Devor, 1996, 2000). However, in the previous in vitro studies of this
"cross-depolarization" phenomenon, we failed to observe repetitive
firing. In retrospect, this failure may have been attributable to our
lack of appreciation at the time of the essential role of subthreshold
oscillations, and hence the use of inadequate stimulation protocols.
For example, we typically used 10 sec tetani of 50-100 Hz, which
produced peak cross-depolarizations of ~4-5 mV (Utzschneider et al.,
1992; Amir and Devor, 1996). This is considerably less than the
9.4 ± 6.6 mV threshold for repetitive firing using ramp-like
stimuli (above), and in any event would have been ineffective unless
oscillating cells had been chosen specifically for study.
To test this explanation we selected eight A0
neurons that showed subthreshold oscillations at
Vr (n = 1) or on
depolarization (n = 7; 14.9 ± 16.5 mV positive to
Vr). Each cell was depolarized to its
oscillatory threshold. Tetanic stimulation was then applied to the
axons of neighboring neurons to evoke cross-depolarization (which
averaged 3.7 ± 2.1 mV). In 5 of the 8 cells tested (63%), repetitive burst firing was evoked (Fig. 6B). One of
the five cells was further depolarized until it began to fire, and in
this case cross-depolarization accelerated the firing (Fig.
11). In each case, firing consisted of
spike bursts triggered by subthreshold oscillations and maintained by
DAPs (as in Fig. 6A). For comparison, 13 neurons (11 A0, 2 Ainf) were similarly
depolarized (by 15.4 ± 7.7 mV), but without the appearance of
subthreshold oscillations. None of these showed spike discharge
(2; p < 0.005),
although the cross-depolarization evoked was comparable in amplitude
(4.4 ± 3.3 mV vs 3.7 ± 2.1 mV; p > 0.2).
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Figure 11.
Cross-excitation within the DRG. Held at 45 mV,
this A0 neuron (Vr = 60
mV) fired occasional spike bursts. Cross-depolarization evoked by
tetanic stimulation of the axons of neighboring neurons (dashed
line) enhanced the amplitude of subthreshold oscillations and
increased the prevalence of bursts. Firing rate during bursts was not
affected (ISI dot display is shown above
each burst). Sample traces 1-4 (bottom)
are taken from the times indicated. Short vertical lines
are stimulus shock artifacts from the nerve stimulation.
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The tetani used in this exercise were intense (10 sec trains at 50-100
Hz). However, the activity evoked is not outside of the range that can
be evoked by strong natural stimuli or that may arise spontaneously as
sites of ectopic activity in injured nerves. Moreover, as noted, we
have shown previously that cross-excitation among DRG neurons can be
generated using considerably weaker tetani, including physiological
stimuli such as brushing the skin (Devor and Wall, 1990; Utzschneider
et al., 1992).
Adrenaline
Activation of sympathetic post-ganglionic efferents and direct
application of  -adrenergic agonists have been reported to depolarize
DRG neurons and evoke discharge, particularly in nerve-injured animals
(Burchiel, 1984; Devor et al., 1994; Petersen et al., 1996). Do
subthreshold oscillations play an essential role? We tested 24 DRG
A0 neurons, 5 from intact adult rats and 19 from rats that had undergone previous nerve injury. Cutting of sciatic (16 cells) or spinal nerves (3 cells) had similar effects, so results were
combined. In most cases, we confirmed that the cell under study had
indeed been axotomized by observing spikes in response to electrical
stimulus pulses delivered to the cut nerve end.
None of the neurons from intact rats oscillated or fired at
Vr, either before or after adrenaline
application. However, they did show a small depolarization within a few
seconds of the start of adrenaline flow (10 µM;
3.0 ± 1.8 mV; n = 5). Depolarization did not
evoke oscillations or sustained firing. In axotomized DRGs, 2 of the 19 neurons tested had oscillations at Vr
in drug-free medium, and an additional 12 oscillated when depolarized.
All 14 of the oscillating cells fired repetitively when depolarized still further, whereas none of the 5 without oscillations did so.
Adrenaline induced depolarization from
Vr in 15 of the 19 neurons (79%;
3.6 ± 3.0 mV; n = 15). These included cells that generated oscillations in drug-free medium (10 of 14) and those that
did not (5 of 5). Of the remaining four cells, two were hyperpolarized and in two there was no change in membrane potential.
Adrenaline affected oscillatory behavior and firing in 5 of the 19 axotomized neurons (26%; 4 sciatic, 1 spinal nerve injured). Remarkably, however, its effects were not mediated by membrane depolarization. In four of the five neurons adrenaline evoked oscillations and repetitive firing despite minimal or no depolarization (3.0, 1.0, 0 mV) (Fig.
12A), or even slight
hyperpolarization in one case (4.0 mV). In the fifth neuron, activity
was suppressed by adrenaline. Before administration the cell oscillated
and fired repetitively at Vr.
Adrenaline induced depolarization (100 µM, 3 mV; 1 mM, 4 mV) but a decrease in ongoing
firing frequency (Fig. 12B). In vivo too,
adrenaline sometimes suppresses spike discharge in DRG neurons (38%)
(Burchiel, 1984; Devor et al., 1994). Adrenaline can apparently affect
membrane resonance and firing properties through a mechanism
independent of depolarization.
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Figure 12.
Repetitive firing in response to adrenergic
stimulation is mediated by subthreshold oscillations. A,
Bath-applied adrenaline (10 µM, between
arrows) evoked an intense and prolonged (>1 min)
discharge burst in this cell. Note that the adrenaline facilitated
subthreshold oscillations and triggered the burst, without causing
membrane depolarization. The burst was maintained by DAPs.
B, In this cell, adrenaline (1 mM, between
arrows) induced depolarization but a decrease in firing
frequency. Firing was in singlets or doublets rather than bursts and
was driven by subthreshold oscillations. Both neurons are
A0, 3 d (A) and 10 d
(B) after sciatic nerve section.
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Unusual oscillatory modes
Slow oscillations
We encountered 3 neurons of 93 checked for this property that
developed "slow" oscillations on depolarization from
Vr (3%; all A0;
in vitro; oscillation thresholds = 38, 43, 48
mV). In addition to oscillation frequency being low (~25 vs ~100 Hz in the cells described above) (Fig.
13), the frequency appeared to be
unaffected by depolarization. Oscillation amplitude, on the other hand,
was voltage sensitive as in typically oscillating neurons. Slow
oscillations did not trigger spikes. DAPs were absent in these cells at
Vr but emerged on depolarization.
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Figure 13.
Some DRG neurons generate low-frequency
subthreshold membrane potential oscillations. A,
"Typical" high-frequency oscillations. B,
Low-frequency (24 Hz) oscillations. In both types of neurons,
oscillation amplitude is voltage sensitive.
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Nonsinusoidal voltage fluctations
Irregular low-frequency membrane potential fluctuations were seen
at Vr in 3 of the 93 neurons (two
A0, one Ainf; 3%).
However, most of the others, both A0 and
Ainf, also developed fluctuations when they were
strongly depolarized (>80%) (Fig.
14A). This includes neurons that had sinusoidal oscillations at more negative membrane potentials. Nonsinusoidal fluctuations were easily distinguished from
subthreshold oscillations: they did not show an amplitude peak in their
Fourier spectrum, and their power was concentrated at lower frequencies
(<25 Hz) (Fig. 14B,C). With
depolarization, the amplitude and hence the power density of the
fluctuations increased, with the overall effect of increasing membrane
instability.
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Figure 14.
Nonsinusoidal fluctuations in the membrane
potential increase in amplitude with depolarization. This is
illustrated in original traces in A and in the
corresponding FFT profile in B. C, Unlike
neurons with typical high-frequency sinusoidal oscillations
(filled circles) [data from Amir et al.
(1999)], cells with nonsinusoidal fluctuations
(triangles) did not show an amplitude peak in their FFT
spectrum. In both A0 cells shown, FFT analysis was
performed at the potential at which the oscillation/fluctuation
amplitude was maximal (35 mV, circles; 13 mV,
triangles). FFT plots in B were
individually normalized relative to the maximal power level observed in
the particular cell. In C, normalization was relative to
the cell indicated with filled circles.
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Voltage fluctuations seen at Vr did
not trigger spontaneous firing, and they rarely occasioned spikes on
depolarization. There are two likely reasons. First,
fluctuations tended to occur at membrane potentials sufficiently
positive that most of the Na+ channels
required for spike electrogenesis would have been inactivated. Second,
the slope of the transient depolarization (dV/dt)
at the leading edge of fluctuations was shallower than in the case of oscillations. Steep dV/dt aids in overcoming the
prominent membrane accommodation characteristic of DRG A-neurons. This
is a key attribute of high-frequency sinusoidal oscillations that
permits them to facilitate repetitive firing (C.-N. Liu et al., 2000a).
We note, however, that voltage fluctuations do trigger spikes in
C-neurons (Amir et al., 1999). Nonsinusoidal fluctuations have been
reported previously as a substrate for spiking in small-diameter DRG
neurons in dissociated cultures (Mathers and Barker, 1984; Study
and Kral, 1996).
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DISCUSSION |
Ectopic discharge generated in afferent A0
neurons in the DRG is a major contributor to chronic dysesthesias and
pain in neuropathy, and it may also play a small role in normal
sensation (see introductory remarks). We have shown in
vitro, and now also in vivo, that subthreshold membrane
potential oscillations are a critical variable linking depolarization
to ectopic firing. Only cells with oscillations are capable of firing
in a sustained manner, spontaneously or in response to the sorts of
tonic physiological stimuli that exacerbate neuropathic sensory
symptoms in the clinical setting.
Oscillations alone sustain firing at low intensity. The full fury of
ectopia is unleashed only when oscillations trigger afferent burst
discharges (C.-N. Liu et al., 2000b). Our data indicate that bursting
is a regenerative consequence of post-spike DAPs. This is so for
spontaneous bursts, bursts generated at the onset of a depolarizing
step, for afterdischarge bursts after brief stimulus pulses, and for
bursting during tonic depolarization. Oscillations and DAPs act
synergistically. The similarity of ectopic firing patterns in DRG
somata and in neuromas and other sites of nerve trauma suggests that
oscillations and DAPs may also underlie ectopic firing at these
locations (Devor and Seltzer, 1999). Any consideration of the ways in
which ion channel regulation and trafficking implement normal sensation
and, when disrupted, bring about neuropathic pain (Devor and Seltzer,
1999; Waxman et al., 1999) must take into account the effects of this
regulation on oscillations and burst discharge.
Depolarizing afterpotentials
Afterdepolarizations have been reported previously in afferent
somata. Studying enzymatically dissociated rat DRG neurons with patch
electrodes, White and Lovinger and colleagues (Lovinger and White,
1989; White et al., 1989) described a subpopulation of neurons with an
afterdepolarization 70-200 msec in duration, and tens of millivolts in
amplitude, generated by a transient (T-type)
Ca2+ conductance. Short spike bursts,
limited by the duration of the afterdepolarization, rode on this
potential. A similar prolonged afterdepolarization was reported by
Kocsis and coworkers in afferent axons bathed in
4-aminopyridine. This potential, which is not Ca2+ dependent, was attributed to a slow
Na+ conductance (Honmou et al., 1994).
The brief, short-latency, rebound DAP that we have described here is
clearly different. Although it appears frequently in published records,
the only reports we are aware of that make mention of it are by Puil
and coworkers in rat trigeminal ganglion slices (Puil et al., 1989) and
by Pedroarena et al. (1999) in primary sensory neurons of MesV. In both
studies, as in ours, fast rebound DAPs were associated with membrane
resonance and subthreshold oscillations and were not blocked by
Ca2+ channel antagonists or low
[Ca2+]o. Rather,
they appear to be caused entirely by inward
Na+ and outward
K+ (or leak) currents, and as we show
here, they are accurately simulated by our modified H-H model.
The DAP as a signal amplifier
Oscillations per se drive ectopic discharge at low frequency
(usually <5 Hz) and with an irregular ISI. This is the most common spontaneous firing pattern at Vr in
vitro and hence in DRGs in vivo (Wall and Devor, 1983;
Devor and Wall, 1990). The normally low level of electrogenesis in DRGs
is amplified many-fold when irregular singlet spikes trigger
high-frequency DAP-driven spike bursts (Amir et al., 1999; C.-N. Liu et
al., 2000b). Within bursts, instantaneous firing rates often surpass
100 Hz. Bursting has at least two distinct amplifying effects: it
augments the overall ectopic afferent barrage, and it powerfully
reinforces marginally effective synaptic links (Lisman, 1997).
DAP-sustained bursts usually damp out after the first few tens of
spikes. However, when damping is weak and the cell is depolarized to a
level at which its DAP is prominent, spike-triggered bursts may be
prolonged. Indeed, at least in vivo, DRG neurons sometimes continue firing for extended periods of time after a brief trigger pulse (Devor and Wall 1990; C.-N. Liu et al., 2000b; X. Liu, 2000). Entry into this self-sustained DAP-triggered firing mode may underlie common and previously unexplained sensory peculiarities in patients with peripheral neuropathies, such as touch-triggered pain paroxysms (in trigeminal neuralgia) and "hyperpathia" (Noordenbos, 1959; Rappaport and Devor, 1994).
Patients with hyperpathia often report sensations evoked by brief
tactile stimuli that long outlast the stimulus itself. Likewise, response to repeated tapping on the skin may summate, sometimes building up to an intensely painful crescendo (Noordenbos, 1959). Such
abnormalities, traditionally explained as CNS processes, are
readily explained by endogenous rhythmogenesis in injured primary
afferents, triggered by afferent spikes and sustained by DAPs. Although
we by no means question the possible role of the CNS in hyperpathia,
our results emphasize the contribution of PNS processes and
specifically point to resonance mechanisms as promising targets for
analgesic drug development.
DAPs and spontaneous repetitive firing in DRG neurons
In vitro, sustained firing at
Vr and on tonic depolarization (evoked
firing) occurs only in neurons that generate subthreshold oscillations.
Our recordings in vivo establish that repetitive spike
discharge in DRGs, in the whole animal, also derive from subthreshold
oscillations. Moreover, they confirm that oscillations are not an
artifact of tissue excision or other aspects of the in vitro preparation.
The correspondence of this novel electrogenic process in
vivo and in vitro rationalizes a large body of
observations on ectopic firing made using teased fiber methodologies in
whole animals (for review, see Devor and Seltzer, 1999). Qualitative
and quantitative similarities include the magnitude of the ectopic
barrage (percentage of neurons involved), potentiation of activity
after axotomy, patterns of spike discharge, types of neurons that are
active (primarily A at early postoperative times), afterdischarge
after single pulse stimuli, DRG cross-excitation, response to
sympathetic stimulation, etc., but some parameters differ. These
include the higher instantaneous frequency during bursts in
vitro, the relatively high incidence in vivo of slow
irregular firing versus bursting, and the rarity of tonic, as opposed
to burst firing, in vitro. We suspect that these differences
all derive from a single source: less prominent and more delayed DAPs
in vivo. Small DAPs are less likely to trigger spike bursts
and hence favor the slow irregular firing pattern most prevalent
in vivo. Likewise, increased DAP latency reduces
instantaneous firing frequency during bursts. Lower firing frequency,
in turn, is expected to reduce the hyperpolarizing shift responsible
for burst termination. This may explain why in vivo, ectopic
firing sometimes persists indefinitely (ongoing, spontaneous
discharge). Note that once initiated, tonic firing is maintained by the
H-H process (i.e., by DAPs) and does not require subthreshold
oscillations. Quantitative differences in DAP parameters in
vitro versus in vivo could be caused by such factors as
constituents of the extracellular medium, temperature, and effects of
cell impalement that might alter Vr
compared with values in vivo. DAPs and bursting are enhanced
at relatively depolarized potentials.
Evoked repetitive firing in DRG neurons
Although the ability to generate subthreshold oscillations is a
necessary condition for responsiveness to tonic stimuli in DRG
A-neurons, it is not a sufficient condition. Except for cells that fire
at Vr, there must also be a source of
sustained depolarization, equivalent to the generator potential at
sensory endings. Depolarization has two effects: it increases the
amplitude of oscillatory sinusoids, and it brings them closer to the
firing threshold of the neuron (Amir et al., 1999). Many conditions
in vivo are capable of inducing generator depolarizations in
the DRG cell soma despite the protection of the bony intervertebral
foramen. These include ischemia, hypoxia, systemic -adrenergic
agonists (e.g., serum adrenaline), sympathetic efferent discharge,
elevated serum [K+], inflammatory
mediators, spike activity in neighboring DRG neurons evoked by natural
cutaneous stimuli or ectopia, and mechanical strain from relative
movement of adjacent vertebrae during activity (Devor and Seltzer,
1999). Nonetheless, in healthy individuals, the DRG is only a minor
source of afferent input. This is presumably because of the small
number of intact neurons that have subthreshold oscillations at
Vr and the small amplitude of
generator depolarizations produced in DRGs by physiological stimuli.
In the event of neuropathy, however, these relations can change
radically. Axotomy greatly increases the population of neurons with
oscillations at Vr, it shifts
oscillation and firing threshold toward
Vr, and it enhances DAPs (Amir et al.,
1999; C.-N. Liu et al., 2000a,b). The net effect is to augment
spontaneous discharge and render neurons responsive to physiological
stimuli that would otherwise have had no effect. There may also be an
increase in the amplitude of generator depolarizations. For example,
foramenal stenosis, disk herniation, or iatrogenically produced
adhesions may increase the transmission of activity-generated strain
forces from the vertebrae to the DRG and of traction forces transmitted by nerves or spinal roots, e.g., during straight leg lifting (Nordin et
al., 1984; Kuslich et al., 1991; Nordin and Balagué, 1996; Nowicki et al., 1996; Zhang et al., 2000). Nerve injury is also known
to trigger sprouting of postganglionic sympathetic endings within the
DRG, a condition that may augment the local release of adrenergic
agonists (McLachlan et al., 1993; Shinder et al., 1999). Finally,
inflammation, either systemic or in local spinal tissues, may enhance
ectopic firing originating in the DRG by increasing intraganglionic
concentrations of an array of inflammatory mediators and by lowering
tissue pH (Levine and Reichling, 1999). In this context, it is
interesting to note that the DRG is uniquely devoid of a blood-nerve
barrier (Allen and Kiernan, 1994; Devor, 1999). In light of its
intrinsic oscillatory and burst-initiating properties, the DRG emerges
as a novel and thus far unexploited target for pain control.
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FOOTNOTES |
Received April 18, 2001; revised Nov. 26, 2001; accepted Oct. 23, 2001.
This work was supported by grants from the United States-Israel
Binational Science Foundation and the German-Israel Foundation for Research and Development. M.M. received a Heisenberg fellowship from the Deutsche Forschungsgemeinschaft.
Correspondence should be addressed to Prof. Marshall Devor, Department
of Cell and Animal Biology, Institute of Life Sciences, Hebrew
University of Jerusalem, Jerusalem 91904, Israel. E-mail: marshlu{at}vms.huji.ac.il.
M. Michaelis's present address: Aventis Pharma Deutschland,
Industriepark Hoechst, H821 65926 Frankfurt/Main, Germany.
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TOP
ABSTRACT
INTRODUCTION
