J Neurophysiol 89: 1588 –1602, 2003;
10.1152/jn.00855.2002.
Similar Electrophysiological Changes in Axotomized and
Neighboring Intact Dorsal Root Ganglion Neurons
CHAO MA, YOUSHENG SHU, ZHENG ZHENG, YONG CHEN, HANG YAO, KENNETH W. GREENQUIST,
FLETCHER A. WHITE, AND ROBERT H. LAMOTTE
Department of Anesthesiology, Yale University School of Medicine, New Haven, Connecticut 06510
Submitted 19 July 2002; accepted in final form 2 November 2002
In animal models of neuropathic pain involving peripheral
nerve injury, the measure of pain is typically a reflex withdrawal to cutaneous stimulation. Despite the fact that it is the
intact neurons that transmit cutaneous information to the CNS,
electrophysiological studies of primary sensory neurons have
focused more on the neurons that are axotomized. Among
these axotomized neurons are those with hyperexcitable cell
bodies (somata) that can become a source of ectopic discharges
(Devor and Seltzer 1999; Kajander et al. 1992; Kirk 1974;
Wall and Devor 1983).
The spinal-nerve ligation (SNL) model provides a means of
separating the somata of neurons with axotomized and intact
axons (Kim and Chung 1992). Of the axotomized somata, only
those with thickly myelinated axons, and not those with unmyelinated axons, became hyperexcitable (Liu et al. 2000,
2002). In addition, there is evidence that the electrophysiological properties of neighboring, intact L4 DRG neurons can also
change after an L5 SNL. For example, abnormal spontaneous
activity has been observed in L4 DRG neurons with myelinated
axons (Boucher et al. 2000) as well as those with unmyelinated
axons (Ali et al. 1999; Wu et al. 2001). Possible changes in the
membrane properties of the somata of intact neurons that are in
proximity to axotomized neurons have received relatively little
attention and are the subject of the present investigation.
Injury to dorsal roots, when compared with peripheral nerve
injury, appears to have less of an effect on the properties of
DRG neurons (Black et al. 1999; Sleeper et al. 2000). Subsequently, hyperalgesia after a complete transection of the dorsal
root is more likely due to central changes triggered by a loss of
afferent input rather than to enhanced excitability of the
transected DRG neurons that have lost their central connection
while retaining their peripheral trophic support (Eschenfelder
et al. 2000; Li et al. 2000; Wu et al. 2001). Partial rhizotomy
(PR) may be an exception, as injured and intact axons are
adjacent to each other, analogous to the situation for degenerating and intact sciatic axons after an L5 spinal nerve transection. Thus it would be useful to compare the membrane properties of the somata of axotomized and intact neurons in the
same DRG after PR.
None of the above studies have investigated the receptive
field properties of the somata of neighboring intact neurons
after a nerve injury, nor has there been a comparison of the
effects of peripheral versus central axotomy on the electrophysiological properties of intact DRG somata. With these
objectives in mind, the present experiments used two models of
neuropathic pain. For one model, SNL, the somata of axotomized and intact neurons were in different ganglia (L5 and L4,
respectively), whereas in the other model, PR, the somata of
axotomized and intact neurons were in the same ganglion.
Extracellular recordings of dorsal root fibers and intracellular
recordings of DRG somata were made from axotomized and
intact neurons. A novel preparation was developed that al-
Address for reprint requests: R. H. LaMotte, Dept. of Anesthesiology, Yale
University School of Medicine, New Haven, CT 06510 (E-mail: robert.
[email protected]).
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.
INTRODUCTION
1588
0022-3077/03 $5.00 Copyright © 2003 The American Physiological Society
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Ma, Chao, Yousheng Shu, Zheng Zheng, Yong Chen, Hang Yao,
Kenneth W. Greenquist, Fletcher A. White, and Robert H. LaMotte. Similar electrophysiological changes in axotomized and
neighboring intact dorsal root ganglion neurons. J Neurophysiol 89:
1588 –1602, 2003; 10.1152/jn.00855.2002. We investigated electrophysiological changes in chronically axotomized and neighboring
intact dorsal root ganglion (DRG) neurons in rats after either a
peripheral axotomy consisting of an L5 spinal nerve ligation (SNL) or
a central axotomy produced by an L5 partial rhizotomy (PR). SNL
produced lasting hyperalgesia to punctate indentation and tactile allodynia to innocuous stroking of the foot ipsilateral to the injury. PR
produced ipsilateral hyperalgesia without allodynia with recovery by
day 10. Intracellular recordings were obtained in vivo from the cell
bodies (somata) of axotomized and intact DRG neurons, some with
functionally identified peripheral receptive fields. PR produced only
minor electrophysiological changes in both axotomized and intact
somata in L5 DRG. In contrast, extensive changes were observed after
SNL in large- and medium-sized, but not small-sized, somata of intact
(L4) as well as axotomized (L5) DRG neurons. These changes included (in relation to sham values) higher input resistance, lower
current and voltage thresholds, and action potentials with longer
durations and slower rising and falling rates. The incidence of spontaneous activity, recorded extracellularly from dorsal root fibers in
vitro, was significantly higher (in relation to sham) after SNL but not
after PR, and occurred in myelinated but not unmyelinated fibers from
both L4 (9.1%) and L5 (16.7%) DRGs. We hypothesize that the
changes in the electrophysiological properties of axotomized and
intact DRG neurons after SNL are produced by a mechanism associated with Wallerian degeneration and that the hyperexcitability of
intact neurons may contribute to SNL-induced hyperalgesia and
allodynia.
SIMILAR CHANGES IN AXOTOMIZED AND INTACT DRG NEURONS
lowed the visualization of DRG somata in vivo, thereby providing the opportunity of determining the receptive field properties of neurons while maintaining unusually stable conditions
for intracellular recording. Some preliminary results of the
present study have been published in abstract form (Ma et al.
2001; Zheng et al. 2000).
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dye was washed out by artificial cerebrospinal fluid (ACSF), and a
small piece of gelfoam (sterile sponge, Upjohn) was put on the surface
of dorsal roots to prevent compression and adhesion from the surface
tissues. The incision was closed in layers. Eleven rats received a sham
surgery (PR-sham) consisting of the same surgical exposure but
without either the transection of the dorsal root or the loading of dye.
Behavioral tests
METHODS
Surgical procedures
Seventy-one adult female Sprague-Dawley rats weighing 150 –250
g were used. Groups of three or four animals were housed together in
a climate-controlled room under a 12-h light/dark cycle. The use and
handling of animals were in accordance with guidelines provided by
the National Institutes of Health and the International Association for
the Study of Pain and received approval from the Institutional Animal
Care and Use Committee of the Yale University School of Medicine.
tion and transection of the right L5 spinal nerve using a modification
of the surgical procedure described by Kim and Chung (1992).
Briefly, under pentobarbital sodium (Nembutal) anesthesia (50 mg/kg
ip) and using aseptic precautions, the L5 transverse process was
removed and the L5 and L4 spinal nerves identified. The L5 spinal
nerve was separated and tightly ligated with 5-0 silk sutures and
transected just distal to the ligature. The ligature was located approximately 3– 4 mm proximal to the junction with L4 nerve and 5– 6 mm
distal to the L5 DRG. The incision was closed in layers, and antibiotics were administered prophylactically. Another group of eight rats
underwent sham surgery (SNL-sham) that involved the identical surgical exposure without a ligation or transection of the spinal nerve.
MEASUREMENT OF THE INCIDENCE OF WITHDRAWAL TO INNOCUOUS STROKING. A wisp of cotton, pulled up but still attached to
a cotton swab, was stroked mediolaterally across the middle of the
plantar surface of the hindpaw at a velocity of approximately 10 mm/s
(Fig. 4C). A total of six strokes were given to each foot, alternating
between feet for each stroke with an interstimulus interval of 10 –15
s. Because this type of stimulus never elicited a withdrawal in normal,
control rats, it was considered to be a valid test for the presence of
tactile allodynia. The number of the six strokes that elicited a withdrawal was expressed as a percentage that was taken as an index of
tactile allodynia for each foot.
MEASUREMENT OF THE THRESHOLD FORCE ELICITING WITHDRAWAL TO PUNCTATE INDENTATION. A series of Von Frey–type
monofilaments each delivering a different bending force in ascending
order (5, 10, 20, 40, 60, 80, 100, and 120 mN) but having the same tip
diameter of 0.1 mm were delivered to designated loci on the skin
(LaMotte et al. 1998; Zhang et al. 1999). Each filament was applied
for 1 s, alternately to each foot at intervals of 10 –15 s, to each of 10
sites distributed across the plantar surface of the rat hindpaw (Fig.
4A). A Hill equation was fitted (Origin Version 6.0, Microcal Software) to the function relating the percentage of indentations eliciting
a withdrawal to the force of indentation. From this equation, the
threshold force was obtained, defined as the force corresponding to a
50% withdrawal. Cutaneous hyperalgesia was defined as a postoperative decrease in threshold of 20 mN from the mean of the three
preoperative thresholds.
CENTRAL AXOTOMY: L5 DORSAL ROOT PR (FIG. 1B). Fifteen rats
received a transection of the caudal half of L5 dorsal root on the right
side. Anesthesia and aseptic procedures were as described. The spinous process was exposed at the level of L1 to L3, and a laminectomy
was made at L2. The dura mater was opened, and the L5 dorsal root
was identified at the point where its 4 –5 rootlets entered the spinal
cord. The caudal 2–3 rootlets were then separated and transected
approximately 3 mm distal to the point of entry into the spinal cord.
The distal cut ends of the rootlets were immersed for 10 min in a
fluorescent dye, Oregon-Green dextran (Molecular Probes, 2–3 ␮l,
10% in double-distilled water with 20% DMSO). The fluorescent dye
was selectively absorbed by the cut dorsal rootlets and could be
retrogradely transported to the somata within 5 days. The rest of the
Intracellular electrophysiological recording in vivo
FIG. 1. Two methods of axotomizing L5 sensory neurons. A: peripheral
axotomy. The spinal nerve was ligated and transected [spinal nerve ligation
(SNL)] approximately 8 mm distal to the L5 dorsal root ganglion (DRG) and
electrophysiological recordings made from the axotomized (L5) and intact
(L4) neurons 3–7 days after surgery. B: central axotomy. The caudal half of the
L5 dorsal root was cut approximately 3 mm from its entry to the spinal cord
[partial rhizotomy (PR)] and the distal, cut end loaded with Oregon green dye.
The circled area was further enlarged in the right lower corner of each panel.
Electrophysiological recordings were obtained from the axotomized (dyelabeled) and intact L5 neurons 3–7 days after surgery.
Three to 7 days after an L5 SNL (n ⫽ 9 rats), SNL-sham (n ⫽ 8),
PR (n ⫽ 9), or PR-sham (n ⫽ 7) operation, animals were anesthetized
with pentobarbital (Nembutal, initial dose of 50 mg/kg ip followed by
20 mg/kg per hour as needed). The L4 and L5 spinal nerves and the
sciatic nerve were exposed and separated from adjacent tissues down
to the level of the knee joint. All the other branches of sciatic nerve
above the knee joint were cut. A laminectomy was performed at the
levels of L2–L6. The L4 and L5 dorsal root ganglia (DRGs) and their
corresponding dorsal roots were identified and exposed. The L4 –L5
dorsal roots were transected just prior to their entry to the spinal cord.
Oxygenated ACSF was dripped periodically onto the surface of the
ganglia during the surgical procedure. The ACSF contained (in mM)
130 NaCl, 24 NaHCO3, 3.5 KCl, 1.25 NaH2PO4, 1.2 MgCl2, 1.2
CaCl2, and 180 dextrose, bubbled with 95% O2-5% CO2 and having
a pH of 7.4 and an osmolarity of 290 –310 mosM. The L4 –L5 dorsal
roots, ganglia, and spinal nerves were isolated from the surrounding
tissues and transferred to a chamber filled with oxygenated ACSF.
The L5 ganglion alone was used for the partial rhizotomy experiments. The sciatic nerve, which still connected to the lower leg, was
brought out of the chamber through a petroleum jelly wall and was
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PERIPHERAL AXOTOMY: L5 SPINAL NERVE LIGATION AND TRANSECTION (SNL) (FIG. 1A). Nine rats received a unilateral, tight liga-
Behavioral tests were made on each of 3 consecutive days before
and 1, 4, and 7 days after one of the following operations: SNL,
SNL-sham, PR, or PR-sham surgery. Six PR and six PR-sham rats
were additionally tested 10 and 14 days after operation. Tests were
carried out without knowledge of the type of surgery and electrophysiological results. Before testing, the rat was placed in a clear plastic
cage with a metal mesh floor. After about 15 min of accommodation,
the following tests were performed.
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MA ET AL.
FIG. 2. Method of obtaining intracellular electrophysiological recordings and receptive field properties from visualized
DRG somata in the anesthetized rat. The dorsal roots, ganglia,
spinal nerve, and the upper portion of the sciatic nerve were
dissected free and placed in a recording chamber that was
perfused with temperature controlled artificial cerebrospinal
fluid (ASCF). The chamber and electrode-positioning microdrives were mounted on 1 platform and the rat lay on a 2nd
one, mechanically isolated from the 1st. The 2nd platform and
a clamp that pinned the knee allowed the lower limb to be
manipulated during the search for receptive fields without
transmitting unwanted mechanical stimuli to the electrodes or
recording chamber.
CRITERIA FOR
ACTIVITY. For
CLASSIFYING
A
NEURON
AS
cut end of dorsal roots or by injecting current through the amplifier
bridge to the somata. The latency of APs electrically evoked from the
suction electrode was divided into the distance between the cut end of
the dorsal root and the center of the DRG to obtain the conduction
velocity (CVdr, m/s). For animals having received a PR, a neuron was
identified as having a cut dorsal root axon by the presence of Oregon
Green fluorescence (Fig. 3, B and E) and/or APs elicited by electrical
stimulation of the formerly axotomized dorsal rootlets.
A current-voltage (I-V) function was obtained from the voltage responses to
DETERMINATION OF THE CURRENT-VOLTAGE FUNCTION.
SPONTANEOUS
each neuron isolated for study, a continuous recording was obtained for 3 min without the delivery of any external
stimulus. If during this period, some pattern of spontaneous discharge
(regular, bursting, or irregular) was observed, the neuron was classified as spontaneously active (SA) only if the activity could not be
attributable to normal, ongoing activation of peripheral receptors. For
example, the tonic activity of a muscle spindle afferent was easily
manipulated by changing the length of the muscle. Any “injury
discharge” that appeared on occasion immediately after electrode
insertion and lasted for ⬍30 s but never came back within 3 min was
ignored.
DETERMINATION OF CONDUCTION VELOCITY. Action potentials
(APs) were evoked either by delivering current pulses (0.1–5 mA,
0.05– 0.5 ms duration) through the suction electrodes attached to the
J Neurophysiol • VOL
FIG. 3. Visualization of DRG somata during intracellular recording. DRG
somata were viewed with a CCD camera combined with infrared DIC (A, C,
and E) or fluorescence (B, D, and F) microscopy. A: view of entire DRG under
low magnification (40⫻). B: same view under fluorescence reveals many
somata filled with Oregon green that was retrogradely transported from cut
dorsal rootlets. C: medium-sized soma (arrow) viewed during intracellular
recording under 400⫻ magnification. D: same view under fluorescence reveals
that the cell was filled with Oregon green and therefore had a transected
dorsal-root axon. E: electrode inserted into soma of a high-threshold, cutaneous mechanoreceptive neuron innervating the dorsum of the foot. Note the
functioning blood vessels as indicated by the presence of red blood corpuscles
moving through capillaries below and to the right. F: same cell viewed under
fluorescence after the 1st electrode had been withdrawn and a 2nd one now
inserted and used with iontophoresis to fill the cell with tetramethyl rodamine.
Scale bar in A and B: 20 ␮m (40⫻), C–F: 200 ␮m (400⫻).
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kept moist by periodic applications of ACSF through a strip of gauze.
The skin incisions on the back and thigh were closed.
Under the dissecting microscope, the sheath covering the surface of
the DRG (perineurium and epineurium) was carefully removed using
fine forceps and scissors. A metal frame with nylon mesh was used to
gently hold the ganglia in the center of the recording chamber. The
chamber was then transferred and mounted to a fixed platform under
a light microscope (Olympus, model BX50WI; Fig. 2). The rat lay on
a second platform, mechanically isolated from the first, to which a
clamp pinning the knee was attached. This platform and clamp allowed the lower limb to be manipulated during the search for receptive fields without transmitting unwanted mechanical stimuli to the
electrodes or recording chamber (Fig. 2). The ganglia were continuously perfused at a rate of 3– 4 ml/min with oxygenated ACSF. The
temperature of the ACSF in the chamber was maintained at 36 ⫾ 1°C
by a heater and controller (Warner Instrument, TC-344A).
Intracellular recordings were obtained only from the somata of
neurons on the surface of DRG, which were visualized under differential interface contrast (DIC) mode (Fig. 3). The recording sharp
electrode was filled with 1.0 M KCl (impedance: 50 – 80 M⍀) and
positioned by a Narashige (MC-35A) microdrive. Prior to electrode
insertion, the size of a soma to be studied was visually classified as
small (ⱕ30 ␮m), medium (31– 45 ␮m), or large (⬎45␮m) (Zhang et
al. 1999). Electrophysiological recordings were collected with singleelectrode continuous current clamp (AxoClamp-2B, Axon Instruments), stored digitally via a Digidata 2200 interface, and analyzed
off-line with pClamp 8 software (Axon Instruments). A neuron was
accepted for study only when it exhibited a resting membrane potential (RMP) more negative than ⫺45 mV.
SIMILAR CHANGES IN AXOTOMIZED AND INTACT DRG NEURONS
Classification of the receptive field properties
of DRG neurons
The receptive field properties of DRG neurons were classified using
hand-held stimulators according to standard criteria (Burgess and Perl
1967; Lawson et al. 1997). Muscle spindle afferents were classified by
their characteristic responses to changes in muscle length, probing of
the muscle belly, and slight taps to the tendon. Cutaneous afferents
were identified as low or high threshold (LT or HT) by their responses
to soft brush, hair movement (hair follicle fibers), or gentle pressure
versus mild pinching, mild (37°C) versus noxious (51°C) heat stimulation (5s), or noxious cold (ice-water, 0°C, 20 s). Because mechanical stimuli were used as the primary search stimuli for most of the
cases in our study, the search procedure was biased against finding
mechanical insensitive units.
Extracellular electrophysiological recording in vitro
Microfilament recordings were made from dissected dorsal root
fiber strands in 12 SNL rats and 4 SNL-sham rats 3–7 days after
surgery. The L4 and L5 DRG with the attached dorsal roots, spinal
nerves, and sciatic nerve were removed from the rat. The sciatic nerve
was cut at the mid-thigh level, and for SNL rat, the L5 spinal nerve
was cut approximately 1 mm proximal to the injury (ligated) site. The
ganglia and nerves were placed in a recording chamber and perfused
with oxygenated and warmed (36 ⫾ 1°C) ACSF (Zhang et al. 1997).
A suction electrode was placed on the distal end of peripheral nerve
to apply electrical stimulation. The dorsal roots were led into an
adjacent mineral oil-filled chamber where microfilament dissection
and extracellular fiber recording was performed. Each chamber was
separated with petroleum jelly. In each experiment, the dorsal root of
each ganglion was divided into bundles, each having an approximate
diameter of 50 ␮m (as judged from the known diameter of the
recording electrode, which was 75 ␮m). After isolating a fiber for
study, recordings were obtained for 3 min in the absence of any
electrical stimulation of the nerve or dorsal root. The criteria for SA
were an ongoing pattern of spontaneous discharge (e.g., regular,
bursting, or irregular) that persisted for ⱖ3 min. For each fiber bundle,
the spinal nerve was stimulated with a gradually increasing intensity
of current (0.1– 0.5 ms square-wave pulses, 1–2 Hz) up to 10 mA. The
number of different AP waveforms was counted, and the incidence of
SA in A- or C-fibers was defined as the percentage of dorsal root
fibers activated by electrical stimulation of the nerve that exhibited
SA. Electrophysiological recordings were collected and stored digiJ Neurophysiol • VOL
tally via an Axon interface (Digidata 2100, Axon Instruments) and
pCLAMP software (Version 6.0, Axon Instruments) for off-line analyses. Usually 20 bundles could be dissected from each dorsal root, and
an average of 5 fibers (range, 0 –20) were recruited from each bundle.
Extracellular electrophysiological recording in vivo
Three to 7 days after an SNL (n ⫽ 9) or SNL-sham (n ⫽ 3)
operation, animals were anesthetized with pentobarbital (Nembutal)
with an initial dose of 50 mg/kg ip followed by 20 mg/kg per hour as
needed. The L4 spinal nerve was exposed and gently separated from
adjacent tissues. A small pledget of cotton soaked in saline was placed
around the L4 spinal nerve at approximately 8 mm distal to the L4
DRG. A laminectomy was performed at the levels of L2–L6. Both L4
and L5 dorsal roots were exposed and covered with a pool of warmed
paraffin oil (35 ⫾ 2°C) formed by the edges of the skin sewn to a ring.
Extracellular recordings were made from teased L4 and L5 dorsal root
microfilaments. The procedure was similar to the in vitro extracellular
recording described above. The experiment began with the dorsal root
intact, after which microfilaments were cut close to entry into the
spinal cord and divided to achieve bundles of about 50 ␮m in
diameter. Recordings were obtained within 6 h from neurons with
unanesthetized, intact peripheral axons, or from dorsal roots with an
anesthetized spinal nerve (10 min after replacing the cotton pledget
around the L4 spinal nerve with one soaked with saline containing 1%
chloroprocaine), or from dorsal roots with acutely axotomized spinal
nerve fibers (10 min after transection of the anesthetized L4 spinal
nerve). The criteria for classifying a neuron as SA are the same as
those described for intracellular recording.
Immunohistochemistry for c-Jun
Animals were terminally anesthetized at 5 days after SNL (n ⫽ 2)
or SNL-sham operation (n ⫽ 2) and perfused transcardially with 4%
paraformaldehyde. L4/L5 DRGs ipsilateral and contralateral to the
injury were dissected and frozen immediately for immunohistochemistry. Cryosections (20 –30 ␮m thick) of the DRG were mounted on
Silane-coated slides (Sigma, St. Louis, MO), air-dried, and fixed for
30 min in 4% paraformaldehyde at 4°C before preincubating in a
dilution buffer (0.1 M PBS, 0.8% bovine serum albumin, 0.25%
Triton X-100, and 5% normal goat serum) for 1 h. After three rinses
in PBS, sections were incubated in rabbit anti-c-jun (1:100; Oncogene
Research Products, Cambridge, MA) antibody. Immunoreactive cells
were visualized with Cy3-conjugated and fluorescein isothiocyanate
(FITC)-conjugated anti-rabbit secondary antibodies. Entire ganglia
sweeps through every section were made using a 40⫻ objective, and
every c-Jun-labeled neuron was counted. Sections from transected L5
DRG were used as positive control.
Statistical analyses
SigmaStat software (Version 2.03, SPSS) was used to determine the
statistical significance of differences in the mean threshold forces for
foot withdrawal to punctate indentation as a function of time and
between experimental groups by means of repeated measures analyses
of variance (RMANOVA) followed by post hoc pairwise comparisons (Student-Newman-Keuls Method). Student’s t-test or one-way
ANOVAs with post hoc pairwise comparisons were used to test the
significance of differences between experimental conditions in mean
values obtained in the electrophysiological experiments. ␹2 tests were
used to assess differences between experimental groups in the incidence of SA or incidence of foot withdrawal to the cotton wisp. A
probability of 0.05 was chosen as the criterion for significance.
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hyperpolarizing and depolarizing current pulses of ⫺1.0 – 4.0 nA (100
ms duration) delivered in increments of 0.05 nA until an AP was
evoked (or reached 4 nA). The following measurements (1–3) were
obtained from the I-V function, and 4 –9 were obtained from the AP
evoked by stimulating the dorsal root or the receptive fields: 1) the
input resistance (Rin, M⍀), calculated from the slope of a steady-state
I-V curve between ⫺1.0 and 0.2 nA; 2) the threshold current (nA) of
the AP, defined as the minimal current injected that elicited an AP; 3)
the threshold voltage of the AP (mV) defined as the first point on the
rising phase of the spike at which the change in voltage exceeded 50
mV/ms; 4) the duration of the AP (APD50, ms) measured at one-half
of the peak amplitude; 5) the maximal rising rate of the AP (dV/dt r.,
mV/ms) or the maximal slope of the rising limb of the AP, measured
by analog differentiation; 6) the maximal falling rate of the AP (dV/dt
f., mV/ms) or maximal slope of the falling limb of the AP; 7) the peak
amplitude of the afterhyperpolarization (AHP amp., mV); 8) the
duration of the afterhyperpolarization (AHP50, ms), measured at
one-half of the peak amplitude; and 9) the presence or absence of an
inflection on the falling phase of the AP as determined by analog
differentiation.
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RESULTS
Effects of SNL on withdrawal responses to mechanical
stimulation of the foot
Effects of PR on withdrawal responses to mechanical
stimulation of the foot
The mean force thresholds for foot withdrawal to punctate
indentation were analyzed separately for each foot for PR and
PR-sham rats with a two-way ANOVA (group ⫻ days) with
repeated measures on days. Post hoc comparisons were made
between means obtained on each postoperative day and the
mean of the 3 consecutive preoperative days of tests.
Before surgery, there was no significant difference for the
mean foot withdrawal threshold either between PR and PRsham groups or between the ipsilateral and contralateral feet
within groups. The mean force thresholds on postoperative
days 1, 4, and 7 were each significantly lower than the mean
preoperative threshold, but remained unchanged on the contralateral foot and on either foot in the sham-operated group
(Fig. 5A). However, by the 10th and 14th postoperative day,
thresholds ipsilateral to the PR had returned to preoperative
values. Thus, unlike the SNL that reportedly maintains a lower
than normal threshold on the ipsilateral foot for upward of 10
wk (Kim and Chung 1992; Liu et al. 2000), the behavioral
effects of PR lasted little more than 1 week.
None of the rats exhibited reflex withdrawal to stroking with
the cotton wisp before surgery. Postoperatively, there was no
significant tactile allodynia after either the PR or the PR-sham
surgeries (Fig. 5B). Although a reflex withdrawal was observed
ipsilateral (4 –14%) or contralateral (0 – 4%) to the PR in 6 of
15 rats, the mean percentage withdrawal did not reach statis-
FIG. 4. The effects of spinal-nerve ligation
(SNL) on withdrawal responses to mechanical
stimulation of the foot. A: Von Frey filaments
with different bending forces, each with a tip
diameter of 0.1 mm, were delivered to 10
points on the plantar surface of the hindfoot in
the indicated numerical sequence. (L, M ⫽
lateral, medial; shaded area: foot pads). B:
mean threshold force for withdrawal to Von
Frey stimulation before (PRE: mean of 3 consecutive preoperative days) and ⱕ7 days after
SNL or sham surgery. Each data point is the
mean threshold (⫾SE) for each group of rats
tested on the foot ipsilateral (Ipsi.) or contralateral (Cont.) to SNL or sham surgery. C:
a wisp of cotton was stroked 6 times mediolaterally across the plantar surface of each
hind foot. D: mean percentage withdrawal to
the cotton wisp obtained before and ⱕ7 days
after SNL or sham surgery. Each data point is
the number of withdrawals expressed as a
percentage of the 6 strokes and averaged for
each group. *P ⬍ 0.01, SNL ipsilateral foot
compared with the preoperative means.
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The mean force thresholds for foot withdrawal to punctate
indentation were analyzed separately for each foot for SNL and
SNL-sham rats with a two-way ANOVA (group ⫻ days) with
repeated measures on days. Post hoc comparisons were made
between means obtained on each postoperative day and the
mean of the 3 consecutive preoperative days of tests. Before
surgery, there was no significant difference for the mean foot
withdrawal threshold either between SNL and sham or between
the ipsilateral and contralateral feet within each group. The
mean withdrawal thresholds obtained ipsilateral to the SNL
decreased significantly from the preoperative means on the first
postoperative day and remained significantly lower up to the
seventh postoperative day (P ⬍ 0.01; Fig. 4B). In contrast,
there were no significant changes in withdrawal thresholds
contralateral to the SNL or for either foot in the sham-operated
group.
None of the SNL or sham-operated rats exhibited reflex
withdrawal to stroking with the cotton wisp prior to surgery
(Fig. 4D). Occasionally a rat would move away from stimulus,
but this response could easily be discriminated from a reflexive
lifting of the foot. In contrast, after surgery, most of the SNL
rats, but none of the shams, exhibited significant tactile allodynia. Most SNL rats exhibited a reflex withdrawal to at least
some of the strokes on the foot ipsilateral to the injury on
postoperative days 1–7 (Fig. 4D), indicative of significant
tactile allodynia. A few SNL rats exhibited withdrawal on
occasion in response to the strokes delivered to the contralateral foot. The percentage of withdrawals on postoperative days
1, 4, and 7 on the foot ipsilateral to the SNL was significantly
greater than it was on any preoperative test and was also
significantly greater than on the foot contralateral to the SNL
(␹2 tests, P ⬍ 0.01). The percentage of withdrawals on postoperative days 4, but not 1 and 7, was significantly greater on
the foot contralateral to the SNL than it was on any preoperative test.
SIMILAR CHANGES IN AXOTOMIZED AND INTACT DRG NEURONS
1593
FIG. 5. The effects of partial rhizotomy
(PR) on withdrawal responses to mechanical
stimulation of the foot. A: mean threshold
force for withdrawal to Von Frey stimulation
before and ⱕ14 days after PR or sham surgery.
Same format as in Fig. 4B. *P ⬍ 0.01, PR
ipsilateral foot compared with the preoperative
means. B: mean percentage withdrawal to a
cotton wisp obtained before and ⱕ14 days
after PR or sham surgery. Same format as in
Fig. 4D.
conduction velocities are in accordance, respectively, with
conduction in A␤-, A␦-, and C-fibers.
Effects of SNL on the membrane properties of axotomized
and intact DRG somata recorded in vivo
whether the electrophysiological properties of DRG somata
from sham rats were similar for neurons with, as opposed to
without, peripheral receptive fields. Those without receptive
fields were assumed to have received an acute peripheral
axotomy during the surgery required to place the DRG in the
recording chamber. Student’s t-test revealed no significant
differences for any of the properties listed in Table 1 for any of
the three categories of cell sizes. Thus acute axotomy had no
demonstrable effect on the membrane properties recorded from
DRG neurons in sham-operated rats.
Three to 7 days after an L5 SNL (9 rats) or a sham operation
(8 rats), intracellular electrophysiological recordings were obtained in vivo from 490 DRG somata. At total of 182 neurons
were recorded from the spinally transected L5 DRG and 152
from the adjacent L4 DRG with an intact spinal nerve. For
sham-operated rats, 156 neurons were recorded from the L4 or
L5 DRG. There were no significant differences in mean conduction velocities of dorsal root axons (CVdr) between SNL
and sham groups for each of the three somal size categories
(Student’s t-test). For SNL-sham rats, these means ⫾ SE (m/s)
were 14.35 ⫾ 1.02 (n ⫽ 33) for large-sized, 5.91 ⫾ 0.65 (n ⫽
24) for medium-sized, and 0.41 ⫾ 0.02 (n ⫽ 44) for smallsized somata (see Table 3 for combined data of CVdr). These
TABLE
EFFECTS OF ACUTE PERIPHERAL AXOTOMY ON THE MEMBRANE
PROPERTIES OF DRG SOMATA FROM SHAM RATS. We determined
EFFECTS OF THE SNL-SHAM
PROPERTIES OF DRG SOMATA.
SURGERY
ON
THE
MEMBRANE
We next examined whether the
properties of DRG somata from sham rats were similar to
published values obtained from unoperated control rats. The
published values were obtained from small-, medium-, and
1. Electrophysiological properties of DRG neurons from axotomized and intact ganglia after SNL
Size Category
Treatment
Large
Sham
DRGL4
Medium
DRGL5
Sham
Cell size, um
58.7 ⫾ 1.7
58.9 ⫾ 1.3
57.9 ⫾ 0.9
38.1 ⫾ 0.7
n
44
44
96
40
RMP, mV
⫺60.1 ⫾ 1.0
⫺60.3 ⫾ 1.1 ⫺57.1 ⫾ 0.6
⫺56.6 ⫾ 1.3
n
44
44
96*†
40
Rin, Mohm
20.2 ⫾ 1.7
27.4 ⫾ 3.0
31.7 ⫾ 2.1
29.9 ⫾ 2.7
n
36
29*
71**
33
Curr. thre., nA
1.55 ⫾ 0.22
1.16 ⫾ 0.16
0.72 ⫾ 0.05
0.88 ⫾ 0.12
n
31
21*
77**†
31
Vol. thre., mV
⫺39.9 ⫾ 1.3
⫺45.9 ⫾ 1.6 ⫺45.9 ⫾ 0.9
⫺32.4 ⫾ 2.0
n
33
24**
55**
26
APD50, ms
0.49 ⫾ 0.06
0.67 ⫾ 0.10
0.99 ⫾ 0.06
0.96 ⫾ 0.08
n
23
23*
58**†
25
dV/dt r., mV/ms
296
⫾ 27.3
248 ⫾ 28.4
199
⫾ 10.3
202
⫾ 17.3
n
23
23
58**
26
dV/dt f., mV/ms ⫺181
⫾ 18.6 ⫺133
⫾ 11.4 ⫺88.5 ⫾ 5.6 ⫺112
⫾ 12.0
n
23
23**
58**†
26
AHP50, ms
3.23 ⫾ 0.35
4.11 ⫾ 0.68
3.82 ⫾ 0.36
4.21 ⫾ 0.72
n
23
23
50
23
AHP amp., mV
⫺9.9 ⫾ 0.6
⫺9.7 ⫾ 0.5
⫺7.0 ⫾ 0.4
⫺10.8 ⫾ 1.0
n
23
23
51**††
23
Small
DRGL4
DRGL5
Sham
37.6 ⫾ 0.7
38.4 ⫾ 0.6
25.5 ⫾ 0.6
34
47
72
⫺54.1 ⫾ 1.2 ⫺53.5 ⫾ 1.0 ⫺52.3 ⫾ 0.9
34
47
72
27.5 ⫾ 2.9
38.5 ⫾ 3.7
39.7 ⫾ 2.8
31
41†
66
0.84 ⫾ 0.09
0.52 ⫾ 0.04
0.62 ⫾ 0.05
31
44**†
58
⫺33.8 ⫾ 3.0 ⫺40.5 ⫾ 2.0 ⫺21.4 ⫾ 1.2
22
31*†
50
1.56 ⫾ 0.22
1.46 ⫾ 0.12
2.73 ⫾ 0.27
22*
27*
32
138
⫾ 12.2
145
⫾ 9.0
109
⫾ 7.5
22*
27*
32
⫺74.3 ⫾ 7.8 ⫺63.2 ⫾ 4.1 ⫺60.9 ⫾ 4.4
22*
27*
32
5.54 ⫾ 0.83
5.03 ⫾ 0.53
8.71 ⫾ 1.12
21
25
31
⫺12.1 ⫾ 1.1
⫺8.78 ⫾ 0.8 ⫺10.5 ⫾ 0.9
21
25
.31
DRGL4
DRGL5
25.3 ⫾ 0.6
74
⫺51.4 ⫾ 0.9
74
44.1 ⫾ 3.9
63
0.64 ⫾ 0.06
53
⫺23.6 ⫾ 1.8
41
2.78 ⫾ 0.18
40
96.7 ⫾ 7.3
40
⫺48.1 ⫾ 4.0
40*
7.22 ⫾ 0.55
41
⫺11.1 ⫾ 0.7
41
26.7 ⫾ 0.6
39
⫺50.3 ⫾ 1.2
39
50.2 ⫾ 4.6
36
0.62 ⫾ 0.06
37
⫺34.4 ⫾ 2.2
21**††
2.80 ⫾ 0.25
21
99.1 ⫾ 10.1
21
⫺41.1 ⫾ 2.6
21**
6.31 ⫾ 0.54
18
⫺11.9 ⫾ 0.8
18
Values are means ⫾ SE, and n ⫽ sample size; SNL: L5 spinal nerve ligation and transection; DRG L4 and L5: intact (L4) and axotomized (L5) ganglia from
SNL rats, Sham: ganglion from Sham-operated rats; Cell size: mean diameter of neuron; RMP: resting membrane potential; Curr. thre.: current threshold; Vol.
thre.: voltage threshold; APD50: action potential (AP) duration at half width, dV/dt r.: maximum rising rate of AP; dV/dt f.: maximum falling rate of AP; AHP50:
afterhyperpolarization duration at half width; AHP amp.: amplitude of afterhyperpolarization. * P ⬍ 0.05, ** P ⬍ 0.01, DRG L4 or DRG L5 vs. Sham; † P ⬍
0.05, †† P ⬍ 0.01, DRG L5 vs. DRG L4.
J Neurophysiol • VOL
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tical significance and was not significantly higher than the
postoperative values obtained on the contralateral foot, or for
the sham operates, on either foot (␹2 tests).
1594
MA ET AL.
large-sized somata in intact DRGs using intracellular electrophysiological recording in vivo or in vitro. For each property,
the mean values listed in Table 1 for each size category for
sham operates were within range of published values (Abdulla
and Smith 2001a; Liu et al. 2000; Stebbing et al. 1999; Villiere
and McLachlan 1996; Zhang et al. 1999).
EFFECTS OF SNL ON THE MEMBRANE PROPERTIES OF DRG
SOMATA OF AXOTOMIZED AND INTACT NEURONS (FIG. 6). The
FIG. 6. Examples of action potentials recorded intracellularly from largesized somata (A␤ neurons) in vivo. Left: an intact A␤ neuron in L5 DRG from
a control (sham-operated) rat. Right: a peripherally axotomized A␤ neuron in
L5 DRG from an SNL rat. S: stimulation from the cut end of dorsal root. Top:
membrane potential (Vm); bottom: the analog differentiation of Vm vs. time
(dV/dt). Note that the action potential for the SNL neuron is wider and presents
an “inflection” on the falling phase, as determined by dV/dt (pointed by arrow).
Scale bar: 10 mV, 2 ms.
J Neurophysiol • VOL
Effects of PR on the membrane properties of axotomized and
intact DRG somata recorded in vivo
Three to 7 days after PR, intracellular electrophysiological
recordings were obtained in vivo from 86 DRG neurons obtained from nine rats. The central axons had been transected by
the PR for 38 of these neurons but remained intact for the
remaining 48. Thirteen cells were without the fluorescent dye
but responded to electrical stimulation of the cut root and were
therefore classified as “axotomized” neurons. Fifty-five neurons were recorded from seven rats 3–7 days after the PR-sham
operation.
EFFECTS OF THE PR-SHAM
PROPERTIES OF DRG SOMATA.
SURGERY
ON
THE
MEMBRANE
Because the mean values of each
parameter for SNL-shams (averaged for L4 and L5) were
within range of published values, they were assumed to be
normal and therefore used to assess the effects of the PR-sham
operation. The mean values of each parameter for PR-sham
somata in each category of cell size were compared with
corresponding values averaged from L4 and L5 somata of the
SNL-sham-operated rats (Student t-test). The means for most
parameters in Table 1 were not significantly different for
PR-shams and SNL-shams and were within range of published
values obtained using intracellular recording methods from the
intact DRGs of unoperated control rats (Liu et al. 2000; Villiere and McLachlan 1996; Zhang et al. 1999). There were two
exceptions. First, the mean input resistance for medium- and
large-sized somata was significantly lower for PR- than SNLshams (in Mohm, 8.98 ⫾ 1.49, n ⫽ 16 vs. 20.24 ⫾ 1.69, n ⫽
36, for large somata and 15.77 ⫾ 2.04, n ⫽ 24 vs. 29.94 ⫾
2.70, n ⫽ 33, for medium somata). Second, the mean current
threshold of medium-sized cells for PR-shams (1.76 ⫾ 0.27
nA, n ⫽ 23) was significantly higher than that of SNL-shams
(0.88 ⫾ 0.12 nA, n ⫽ 31).
89 • MARCH 2003 •
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next question was whether the effects of SNL were confined to
axotomized neurons in L5 or whether the lesion also affected
the membrane properties of intact neurons in L4. For each size
category for sham operates, the data in Table 1 represent
averages of values obtained from neurons in L5 and L4 DRGs.
For each parameter, for a given size category, the means for
sham operates were compared with the corresponding means
obtained for L5 (DRG L5) and for L4 neurons (DRG L4) from
SNL rats using one-way ANOVAs followed by post hoc comparisons. It was found that the effects of SNL on membrane
properties were similar for L4 and L5 cells, and furthermore,
were confined largely to the large- and medium-sized somata.
In comparison with corresponding measurements obtained
from sham-operated rats, large-sized somata in both L4 and L5
of SNL rats had significantly higher input resistances, lower
current thresholds, lower voltage thresholds (i.e., closer to
resting potential), and APs of wider durations and slower
maximal falling rates. To investigate the contribution of rising
and falling phases to the AP duration, we further compared the
rise and fall time of L5/L4 neurons between SNL and shamoperates. The L5 large-sized somata exhibited APs with both a
significantly longer mean rise time (0.74 ⫾ 0.06 ms, n ⫽ 58,
P ⬍ 0.05) and longer mean fall time (1.49 ⫾ 0.11 ms, n ⫽ 58,
P ⬍ 0.01) than those of the shams (rise 0.50 ⫾ 0.06 ms, fall
0.70 ⫾ 0.10 ms, n ⫽ 23). Although the large-sized somata of
L4 DRGs in SNL rats exhibited a similar trend, their rise and
fall times (0.69 ⫾ 0.12 and 0.82 ⫾ 0.10 ms, respectively, n ⫽
23), were not significantly different from those of shams.
Seven (16%) of the L4 and 13 (14%) of the L5 large-sized
somata from SNL rats exhibited an inflection on the falling
phase of the AP (Fig. 6) in contrast to only 1 (2%) large-sized
soma from sham-operative rats. However, the difference was
not statistically significant (␹2 test).
Some effects of SNL were greater for L5 than for L4
large-sized somata. L5 cells had a significantly lower resting
potentials, lower current thresholds, and APs with wider durations, lower AHP amplitudes, and slower falling rates.
For medium-sized somata, the effects of SNL were similar
to those of large-sized cells for some parameters but negligible
for others. In comparison with values for sham rats, both L4
and L5 medium-sized somata from SNL rats exhibited APs
with significantly wider durations and slower maximal rising
and falling rates. The rise and fall times of medium-sized
somata from either L5 (rise 1.78 ⫾ 0.21 ms, fall 1.97 ⫾ 0.15
ms, n ⫽ 27) or L4 (rise 2.47 ⫾ 0.62 ms, fall 1.95 ⫾ 0.29 ms,
n ⫽ 22) DRGs of SNL rats were significantly longer than those
from sham rats (rise 1.17 ⫾ 0.10 ms, fall 0.98 ⫾ 0.13 ms, n ⫽
26). The effects of SNL were slightly greater for L5 than for L4
medium-sized somata. The L5 medium-sized somata exhibited
significantly lower current and voltage thresholds than somata
of either L4 or sham cells and significantly higher input resistances than those of L4 (but not sham).
SNL had a lesser effect on the membrane properties of
small-sized neurons than on those of medium- or large-sized
cells. Only two effects were observed. First, the maximum
falling rates of the AP in small-sized somata of L5 and L4 SNL
were significantly slower than those of sham rats. Second,
small-sized somata in L5 had significantly lower voltage
thresholds than those of L4 SNL or sham (with no difference
between values obtained from L4 SNL and from sham rats).
SIMILAR CHANGES IN AXOTOMIZED AND INTACT DRG NEURONS
TABLE
1595
2. Electrophysiological properties of DRG neurons with axotomized or intact central axons after PR
Size Category
Large
Medium
Small
Sham
Uncut
Cut
Sham
Cut
Cut
Sham
Uncut
Cut
Cell size, um
n
RMP, mV
n
Rin, Mohm
n
Curr. thre., nA
n
Vol. thre., mV
n
APD50, ms
n
dV/dt r., mV/ms
n
dV/dt f., mV/ms
n
AHP50, ms
n
AHP amp., mV
n
51.7 ⫾ 1.0
18
⫺56.4 ⫾ 2.0
18
9.0 ⫾ 1.5
16
1.85 ⫾ 0.33
16
⫺44.8 ⫾ 4.6
10
0.60 ⫾ 0.04
6
198
⫾ 20.6
7
⫺133 ⫾ 11.9
7
3.35 ⫾ 1.18
7
⫺13.1 ⫾ 2.3
7
49.6 ⫾ 0.7
17
⫺57.2 ⫾ 2.0
17
13.1 ⫾ 2.0
17
2.18 ⫾ 0.34
16
⫺54.9 ⫾ 2.1
12*
0.62 ⫾ 0.06
12
225
⫾ 19.9
12
⫺131
⫾ 9.1
12
4.71 ⫾ 0.79
12
⫺11.1 ⫾ 0.8
12
51.3 ⫾ 1.3
17
⫺58.6 ⫾ 1.6
17
20.2 ⫾ 4.0
16*
1.70 ⫾ 0.38
12
⫺56.2 ⫾ 2.0
10*
0.66 ⫾ 0.11
10
229
⫾ 30.7
10
⫺144
⫾ 22.3
10
4.81 ⫾ 1.44
10
⫺10.2 ⫾ 1.1
10
39.6 ⫾ 0.8
24
⫺55.6 ⫾ 1.7
24
15.8 ⫾ 2.0
24
1.76 ⫾ 0.27
23
⫺36.4 ⫾ 3.9
20
0.93 ⫾ 0.09
18
170
⫾ 15.8
18
⫺100
⫾ 11.0
18
6.05 ⫾ 1.44
21
⫺13.5 ⫾ 0.9
21
39.1 ⫾ 0.7
17
⫺55.5 ⫾ 2.0
17
18.1 ⫾ 3.1
16
1.63 ⫾ 0.32
14
⫺54.5 ⫾ 2.3
9**
0.79 ⫾ 0.09
10
183
⫾ 21.0
10
⫺124
⫾ 14.0
10
8.15 ⫾ 2.18
10
⫺10.0 ⫾ 1.6
9
38.9 ⫾ 1.0
17
⫺56.1 ⫾ 1.6
17
28.4 ⫾ 3.7
16**†
2.08 ⫾ 0.44
13
⫺48.3 ⫾ 2.9
14*
0.83 ⫾ 0.09
15
201
⫾ 20.1
15
⫺123
⫾ 8.4
15
4.69 ⫾ 1.15
15
⫺11.5 ⫾ 0.8
15
27.2 ⫾ 0.6
13
⫺50.9 ⫾ 2.5
13
50.9 ⫾ 8.3
13
0.84 ⫾ 0.17
13
⫺29.5 ⫾ 3.8
13
1.68 ⫾ 0.17
12
155
⫾ 16.5
12
⫺69.8 ⫾ 7.8
12
6.19 ⫾ 0.80
12
⫺15.0 ⫾ 1.6
12
25.0 ⫾ 1.2
14
⫺52.6 ⫾ 2.5
14
30.4 ⫾ 3.8
14
1.83 ⫾ 0.49
11
⫺28.7 ⫾ 6.0
10
1.84 ⫾ 0.25
12
133
⫾ 17.4
12
⫺60.1 ⫾ 9.9
12
5.28 ⫾ 1.13
10
⫺12.3 ⫾ 1.9
10
28.5 ⫾ 1.4
4
⫺52.9 ⫾ 4.4
4
22.3 ⫾ 5.2
3
0.70 ⫾ 0.15
3
⫺31.0 ⫾ 11.6
2
2.22 ⫾ 0.70
3
85.6 ⫾ 36.3
3
⫺48.8 ⫾ 16.4
3
6.02 ⫾ 2.44
3
⫺11.6 ⫾ 3.7
3
PR: L5 dorsal root partial rhizotomy; uncut: neurons with intact dorsal root fibers, cut: neurons with cut dorsal root fibers in PR rats, Sham: DRG neurons
from Sham-operative rats; Cell size: mean diameter of neuron; * P ⬍ 0.05, ** P ⬍ 0.01, cut or uncut vs. Sham; † P ⬍ 0.05, cut vs. uncut. Other parameters
are same as Table 1.
EFFECTS
OF
PR
ON
THE
MEMBRANE
PROPERTIES
OF
DRG
Medium- and large-sized DRG somata were more
affected by central axotomy than were small-sized somata just
as they were after peripheral axotomy. However, in contrast to
the effects of SNL, those of PR were relatively minor (Table
2). Both axotomized and intact PR somata of medium or large
size had significantly lower voltage thresholds than somata of
corresponding size from PR-shams. In addition, the axotomized medium- and large-sized somata (but not the intact) had
a significantly higher input resistance than corresponding somata from PR-shams. The only significant difference between
intact and axotomized neurons was a higher input resistance for
axotomized medium-sized somata. PR had no significant effects, in relation to PR-sham, on the membrane properties of
small-sized somata.
SOMATA.
TABLE
Functional properties of intracellularly recorded neurons
with peripheral receptive fields
Among the 449 neurons recorded intracellularly, peripheral
receptive fields (RFs) were found in 129 neurons, including 55
with large-sized somata, 28 with medium-sized somata, and 46
with small-sized somata. The RFs were mainly distributed in
the lower leg and foot, because most of the sciatic nerve
branches above the knee joint were transected during the
surgical preparation for in vivo recording. Because there were
no significant differences in RF properties due to the nerve
lesion (SNL or PR), the RF data were pooled in Table 3 for
SNL (L4 DRG only), SNL-sham (L4 and L5 DRG), PR, and
PR-sham (L5 DRG).
Large-sized somata had muscle spindles (n ⫽ 27, 49%),
3. Electrophysiological properties of DRG neurons with functionally identified peripheral receptive fields
Size Category
Large
Medium
Small
Submodality
MS
LTM
HTM
MS
LTM
HTM
LTM
HTM
MH
Cell size, um
n
CVdr, m/s
n
Curr. Thre., nA
n
APD50, ms
n
AP amp., mV
n
AHP50, ms
n
58.2 ⫾ 2.3
27
16.6 ⫾ 1.4
17
1.19 ⫾ 0.26
14
0.48 ⫾ 0.08
11
54.4 ⫾ 3.1
11
2.35 ⫾ 0.28
11
57.1 ⫾ 1.7
27
14.7 ⫾ 1.4
25
1.41 ⫾ 0.16
16
0.53 ⫾ 0.07
18
57.1 ⫾ 2.7
17
3.69 ⫾ 0.40
18
50.0
1
10.6
1
3.40
1
0.45
1
76.5
1
15.1
1
38.9 ⫾ 1.3
7
11.0 ⫾ 1.5
2
0.58 ⫾ 0.14
4
0.55 ⫾ 0.10
6
50.5 ⫾ 1.5
5
2.01 ⫾ 0.23
6
41.1 ⫾ 0.5
11
6.56 ⫾ 1.02
11
1.21 ⫾ 0.12
10
0.77 ⫾ 0.10
11
64.5 ⫾ 3.0
10
3.21 ⫾ 0.25
10
36.6 ⫾ 1.3
9
6.21 ⫾ 1.16
7
1.76 ⫾ 0.67
7
0.92 ⫾ 0.09
8
69.1 ⫾ 3.3
8
8.11 ⫾ 1.80
8
30.0
1
0.61
1
1.20
1
1.74
1
69.3
1
3.17
1
26.3 ⫾ 0.8
27
0.41 ⫾ 0.03
20
0.87 ⫾ 0.11
18
2.90 ⫾ 0.43
15
74.4 ⫾ 3.7
15
9.24 ⫾ 1.14
14
24.4 ⫾ 1.5
17
0.38 ⫾ 0.03
11
0.56 ⫾ 0.11
11
3.31 ⫾ 0.49
9
67.7 ⫾ 3.5
9
8.96 ⫾ 2.04
9
Values are given in means ⫾ SE, and n ⫽ sample size. Neurons were classified by peripheral receptor type, i.e., muscle spindle (MS), cutaneous low-threshold
mechanoreceptor (LTM), cutaneous high-threshold mechanosensitive nociceptor (HTM), and cutaneous mechano-heat nociceptor (MH) (3 of which also
responded to cold). One mechanoheat sensitive nociceptive medium sized soma and and one heat-only nociceptive small sized soma are not included. CVdr:
dorsal root conduction velocity, AP amp.: amplitude of action potential. Other parameters are same as Table 1.
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Treatment
1596
MA ET AL.
SNL, but not PR, increased the incidence of abnormal SA
Abnormal SA was defined as discharges that persisted for
more than 3 min, typically having a bursting, tonic, or irregular
pattern. The incidence of SA was electrophysiologically recorded three different ways: 1) in vivo, intracellularly from
DRG somata; 2) in vitro, extracellularly from dorsal-root fibers; and 3) in vivo, extracellularly from dorsal root fibers. The
intracellular recordings were confined to somata on the surface
of the DRG, whereas the extracellular recordings presumably
sampled the SA of neurons with somata distributed throughout
the DRG. Consequently the extracellular recordings were
thought to yield an incidence of SA that was more representative of SA in the DRG population. The patterns of SA
(regular, bursting, or irregular) were similar for intracellular
and extracellular recordings and some examples are shown in
Fig. 7, E–G and H–J, respectively.
IN VIVO INTRACELLULAR RECORDING OF SA AFTER SNL. None of
the 84 large- and medium-sized somata of “A-neurons” from
sham-operated rats exhibited SA (Fig. 7A)—neither was the
SA recorded in any of the 72 small-sized somata of “Cneurons” from sham rats (Fig. 7B). In contrast, after SNL, 11
of 143 A-neurons and 1 of 39 C-neurons from axotomized L5
DRG exhibited SA. Most of the cells with SA were recorded
from axotomized L5 somata with only 1 of 78 A-neurons and
0 of 74 C-neurons sampled from intact somata in the L4 DRG
J Neurophysiol • VOL
(Fig. 7, A and B). The incidence of SA of L5 A-neurons was
significantly higher after SNL than after the SNL-sham operation (P ⬍ 0.05, ␹2 test). However, we never found a SA
neuron that had a RF. For neurons with RFs, any ongoing
activity, for example, the tonic activity generated by muscle
spindles, appeared normal and could always be manipulated by
applying adequate stimuli to the RF. Because we could only
access the RFs on the foot and lower leg, it is conceivable that
some of the “SA” neurons may have had RFs on the upper leg,
hip, or the back.
IN VITRO EXTRACELLULAR RECORDING OF SA AFTER SNL. In
each experiment, the dorsal root was divided into bundles each
having an approximate diameter of 50 ␮m, and the incidence
of SA A-fibers (or C-fibers) was defined as the percentage of
electrically activated dorsal root fibers that exhibited SA (see
METHODS). Extracellular recordings from dorsal root fibers of
axotomized L5 neurons revealed that 166 of 992 A-fibers
(16.7%) and 1 of 226 C-fibers (0.4%) exhibited SA (Fig. 7, C
and D). Similarly, SA was also recorded from A-fibers from
the formerly intact L4 DRG. Of those fibers recorded from the
L4 dorsal root, 98 of 1,079 A-fibers (9.1%) and 0 of 205
C-fibers exhibited SA (Fig. 7, C and D). For sham-operated
rats, the results were pooled for L4 and L5 dorsal roots. It was
found that 9 of 696 A-fibers (1.2%) and 0 of 89 C-fibers
exhibited SA. The incidence of SA in A-fibers after SNL was
significantly greater for L5 than for L4 neurons and significantly higher for both populations of neurons after SNL than
after the sham operation (P ⬍ 0.01, ␹2 tests).
IN VIVO INTRACELLULAR RECORDING OF SA AFTER PR. Only 2 of
the 86 cells recorded intracellularly after PR exhibited SA.
These two cells were medium-sized with transected central
axons and were without peripheral receptive fields. None of the
55 cells from sham rats exhibited SA.
IN VITRO EXTRACELLULAR RECORDING OF SA AFTER PR. Recordings were obtained from 618 fibers from the injured portion of the L5 dorsal root and 607 fibers from the intact portion
of L5 dorsal root for which electrical stimulation of the sciatic
nerve evoked APs. Only seven A-fibers (1.13%), recorded
from the injured portion of the dorsal root, exhibited SA. Of the
315 fibers activated from sciatic nerves from two PR-sham
rats, none exhibited SA.
Thus the incidence of SA was not increased after central
axotomy (PR) in contrast to the significant increase found in
both axotomized and intact DRG neurons after chronic peripheral axotomy (SNL).
Acute peripheral axotomy may enhance the incidence of SA
in intact A-neurons after SNL
During intracellular recordings from L4 somata, only one
cell without peripheral RFs exhibited SA. The neurons without
peripheral receptive fields were presumably acutely axotomized during the surgical procedure required to place the DRG
into the recording chamber. There was also a tendency for the
alterations in various membrane properties in cells to be more
prominent in cells without receptive fields. Among the 44
large-sized somata recorded from the intact L4 DRG after
SNL, peripheral RFs were located for 13 neurons. The remaining 31 neurons were presumably acutely axotomized during the
surgical procedure required to place the DRG into the record-
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cutaneous low-threshold mechanoreceptors (LTM, 27, 49%),
and a cutaneous high-threshold mechanoreceptor with no response to heat or cold (HTM, 2%).
Medium-sized somata had cutaneous LTMs (n ⫽ 11, 39%),
HTMs (9, 32%), muscle spindles (7, 25%), and a cutaneous
high-threshold mechanoheat receptor (MH, 4%) that was unresponsive to cold.
All but 1 of the 46 small neurons with RFs had nociceptors,
including 27 (59%) HTMs with no response to heat or cold, 14
(30%) MHs with no response to cold, 3 (7%) MHCs (MHs that
responded to cold), and 1 (approximately 2%) responsive only
to heat (H). An additional C-neuron had a low-threshold mechanoreceptor (LTM). The search procedure used mainly mechanical stimuli and was therefore biased against finding mechanically insensitive neurons.
There were certain features of the shapes of the APs that
were more characteristic of nociceptive than the nonnociceptive neurons. All the C-nociceptors (including the HTMs,
MHs, MHCs, and H) exhibited APs with an inflection on the
falling phase, as did most of the A-nociceptors (82%, with the
exception of 1 large- and 1 medium-sized HTM). In contrast,
only one medium-sized LTM (approximately 2%) and none of
the muscle spindles had APs with inflections. In addition, the
AP amplitude and AHP duration (AHP50) of A- and C-nociceptive neurons were significantly larger than those with muscle spindles or LTMs. Further comparisons of the membrane
properties of A-neurons (large- and medium-sized somata)
with RFs revealed that the current thresholds, AP durations, AP
amplitudes, and AHP durations increased in the order of those
with muscle spindles, LTMs, and HTMs (Table 3). These
findings are in general agreement with those obtained with
intracellular recording from DRG somata of cat (Koerber et al.
1988), rat (Ritter and Mendell 1992), and guinea pig (Djourhi
et al. 1998).
SIMILAR CHANGES IN AXOTOMIZED AND INTACT DRG NEURONS
1597
ing chamber. Of the 44 large-sized somata from the L4 or L5
DRG after SNL-sham operation, RFs were identified for 27,
with the remaining 17 considered to be acutely axotomized (for
L4 DRG alone, there were 15 of 26 with and 11 without RFs).
No significant differences between means for any of the electrophysiological parameters were found between the neurons
with as opposed to without RFs. A possible exception was the
higher incidence for large-sized somata without a RF to exhibit
APs with an inflection on the falling phase. After SNL, 7 of 31
neurons (22.6%) without RF had an inflection on the falling
phase of their APs, whereas 0 of the 13 neurons with RFs had
the inflection. After the sham operation, only 1 of the 17
neurons (5.9%) without RFs and 0 of the 27 neurons with RFs
had an inflection. After SNL, but not after SNL-sham, largesized somata without RFs exhibited a tendency toward lower
current thresholds (L4 without RF vs. with RF: 1.14 ⫾ 0.19
nA, n ⫽ 17 vs. 1.25 ⫾ 0.19 nA, n ⫽ 4), more negative voltage
threshold (⫺47.16 ⫾ 1.90 mV, n ⫽ 17 vs. ⫺42.96 ⫾ 2.80 mV,
n ⫽ 7), and longer APD50 (0.71 ⫾ 0.13 ms, n ⫽ 16 vs. 0.58 ⫾
0.14 ms, n ⫽ 7) than those with RFs. However, these numerical
J Neurophysiol • VOL
differences did not quite reach statistically significance. Similar tendencies were not observed for medium- or small-sized
neurons with versus without RFs.
An experiment was undertaken to determine whether SA
could be observed in A-neurons (including A␤ and A␦) with
peripheral RFs and whether the incidence of SA in intact DRG
neurons after SNL was enhanced by acute peripheral axotomy.
The criteria for “SA” would be an ongoing pattern of discharge
(e.g., bursting, irregular) not characteristic for activity generated by the activation of peripheral receptors. Extracellular
recordings from teased L4-dorsal root fibers were obtained in
vivo 3–5 days after an ipsilateral L5 SNL. The spinal nerve of
L4 was gently separated from adjacent tissues so that a small
pledget of cotton soaked either in saline or in saline with 1%
chloroprocaine could be placed around the distal portion approximately 8 mm from the DRG.
The incidence of SA was recorded under each of three
experimental conditions: the distal portion of the spinal nerve
was exposed either to saline only, to the local anesthetic, or
locally anesthetized and then transected. Attempts to electri-
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FIG. 7. A–D: effects of SNL on the incidence of spontaneous activity in intact (L4) and axotomized (L5) DRG
neurons. The incidence or percentage of spontaneously
active (SA) neurons is defined as the number of SA neurons
divided by the total number sampled for each group
(⫻100). In each panel, the numbers on which the percentages are based are given for each of the 3 groups: L5
(axotomized), L4 (intact), and sham (L5 and L4 data combined). A and B: percentages obtained from intracellular
recording (in vivo) from large- or medium-sized somata (A)
and small-sized somata (B). C and D: percentages obtained
from extracellular recording (in vitro) from dorsal root
A-fibers (C) and C-fibers (D). *P ⬍ 0.05, **P ⬍ 0.01, SNL
L4 or L5 vs. Sham; ††P ⬍ 0.01, SNL L5 vs. L4 (by ␹2 test).
E–J: different patterns of spontaneous activity recorded
from DRG neurons after SNL. E–G: in vivo intracellular
recording from L5 (axotomized) large- or medium-sized
somata. H–J: in vitro extracellular recording from L4 (intact) dorsal root A-fibers; similar results were obtained
from L5 (axotomized). E and H: regular discharge; F and I:
bursting; G and J: irregular discharge. Scale bar: E–G, 10
mV, 100 ms; H–J: 200 ms.
1598
MA ET AL.
cally stimulate the proximal, unanesthetized portion of the
nerve were not successful, owing in part to difficulties in
access to the nerve and stimulus spread to the recording electrode. Consequently, the incidence of SA was defined in terms
of the number of fibers with SA per filament bundle of approximately 50 ␮m diameter. Individual fibers were distinguished
by the shapes of their APs each of which also had to have a
minimal signal to noise ratio of 3:1.
The incidence of SA obtained in the in vitro fiber recording
experiments was expressed in Fig. 8A as the mean number of
SA fibers per bundle. The incidence of SA was significantly
higher for L5 than for L4 dorsal roots after SNL and lowest
after the sham operation.
The incidence of SA recorded in vivo from L4 dorsal root
fibers of three sham-operated rats was 0.061 (5 SA fibers in 82
intact L4 or L5 dorsal root bundles; Fig. 8B). This value was
not significantly different from that recorded in vitro from
sham dorsal roots (Fig. 8A; ␹2 test). The incidence of SA
recorded in vivo from dorsal roots 3–5 days after SNL (3 rats;
Fig. 8B) was 0.89 for axotomized L5 neurons (58 SA fibers
from 65 bundles) and 0.125 for intact L4 neurons (7 SA fibers
from 56 bundles). The latter was not significantly different
from that recorded from sham L4, but significantly lower than
that obtained from L4 with in vitro recordings (Fig. 8A; ␹2 test,
P ⬍ 0.01).
The anesthetic was effective in eliminating activity originating from peripheral tissues innervated distal to the nerve block.
After applying the anesthetic for about 10 min, the incidence of
SA in the L4 dorsal root was 0.23 (24 SA fibers from 88
bundles, 3 rats)—significantly higher than that obtained from
sham L4 (0.061), but not significantly different from that
recorded in the absence of anesthetic (0.125; ␹2 test; Fig. 8B).
However, the incidence of SA was significantly higher after the
spinal nerve was anesthetized and then transected. After
transecting the nerve the incidence was 0.55 (43 fibers of 78
bundles, 3 rats)—a value significantly greater than that obJ Neurophysiol • VOL
tained when the nerve was intact with or without the anesthetic
(␹2 test, P ⬍ 0.01; Fig. 8B).
A test for the possible injury of L4 neurons after
ipsilateral L5 SNL
Five days after SNL (2 rats) or sham operations (2 rats), cell
counts were obtained from L4 and L5 DRGs ipsilateral and
contralateral to lesion. For sham-operated rats, there was no
significant difference in the number of c-Jun immunopostitive
cells (⬍1%) between ipsilateral L4 and L5 DRG. The effect of
SNL on the number of c-Jun immunopositive cells in L5 was
dramatic. Most ipsilateral L5 neurons were immunopositive for
c-Jun 5 days after L5 spinal nerve transection (Fig. 9A). However, only about 1% of neurons were c-Jun positive in L4 DRG
ipsilateral to L5 spinal nerve transection (Fig. 9B). In ipsilateral
L4 DRG, there was a strong likelihood that the few c-Jun–
positive cells presented had peripheral targets in the dorsal skin
of the back that were severed during surgery. There was no
significant difference in the number of positive cells between
the ipsilateral L4 and contralateral L4/L5 DRG of SNL rats.
FIG. 9. C-jun immunostaining of DRG neurons after SNL. A: 5 days after
SNL, most ipsilateral L5 neurons were immunopositive for c-Jun as demonstrated by nuclear label. B: only 1% of the cells were c-Jun positive (arrow) in
L4 DRG ipsilateral to the L5 SNL.
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FIG. 8. Effects of acute transection of L4 spinal nerve on the incidence of spontaneous activity in dorsal root A-fibers from L4
and L5 DRGs in SNL rats. From 3 to 7 days after L5 SNL, the spinal nerve of the adjacent L4 DRG was anesthetized and/or cut
just before electrophysiologically recording from teased dorsal root fibers. The incidence of spontaneously active (SA) dorsal root
fibers is defined as the number of electrophysiologically isolated SA dorsal fibers divided by the number of 50-␮m dorsal root
bundles (numbers provided above each column). A: incidence of SA recorded in vitro. Left to right: L4 and L5 dorsal roots from
sham-operated rats (spinal nerve acutely transected, open bar), L4 dorsal root from SNL rats (spinal nerve acutely transected, gray
bar), and L5 dorsal root from SNL rats (spinal nerve chronically transected, black bar). B: incidence of SA recorded in vivo. Left
to right: L4 dorsal root from sham-operated rats (“sham L4”), L4 dorsal root with intact L4 spinal nerve packed with saline soaked
cotton pledget (“L4 intact”), L4 dorsal root with intact L4 spinal nerve soaked with the local anesthetic, 1% chloroprocaine (L4
anes.), L4 dorsal root with acutely transected L4 spinal nerve (column L4 anes. ⫹ cut), and L5 dorsal root with SNL. *P ⬍ 0.01,
SNL L4 or L5 vs. Sham; †P ⬍ 0.01, SNL L5 vs. L4 (by ␹2 test).
SIMILAR CHANGES IN AXOTOMIZED AND INTACT DRG NEURONS
DISCUSSION
Central axotomy produced only transient hyperalgesia and
minor changes in electrophysiological properties
of DRG neurons
Peripheral axotomy produced lasting hyperalgesia and
allodynia and major changes in the excitability of DRG
neurons with myelinated axons
SNL produced significant mechanical hyperalgesia to punctate indentation of the ipsilateral foot and allodynia to innocuous tactile stroking each lasting through the seventh postoperative day of testing. A lowered withdrawal threshold to Von
Frey stimuli ipsilateral to SNL has been amply demonstrated in
previous studies and found to persist for at least 10 wk (e.g.,
Kim and Chung 1992; Liu et al. 2000).
After peripheral axotomy by SNL, somata of large and
medium size, in both chronically axotomized and the neighboring intact DRG, exhibited a higher input resistance, lower
current and voltage threshold, longer AP duration, and slower
AP rise and falling rates. There were no major changes in the
J Neurophysiol • VOL
membrane properties of C-neurons with small-sized somata.
Correspondingly, SA was recorded in vitro from the dorsal root
fibers of A- but not C-neurons of L4 as well as L5.
Our finding of the absence of c-jun labeling in L4 (in
contrast to L5) was taken as evidence that the similarity in
electrophysiological changes between the two ganglia was not
due to the possibility that the L4 axons were stressed or injured
(Decosterd et al. 2002; Jenkins and Hunt 1991; Kenney and
Kocsis 1997a,b).
Possible mechanisms of enhanced excitability of axotomized
and intact DRG neurons after SNL
The changes in excitability and the shapes of APs of A-neurons after peripheral axotomy are well described in the literature and similar to present findings (Abdulla et al. 2001a; Kim
et al. 1998; Liu et al. 2000; Stebbing et al. 1999). A novelty of
our findings is that qualitatively similar changes occurred in
intact A-neurons with somata in the L4 DRG adjacent to the
peripherally axotomized L5. Thus it seems reasonable to postulate that similar ionic mechanisms were responsible for the
changes in membrane properties observed in both axotomized
and intact neurons. In addition, the signaling mechanisms that
trigger these changes may be similar as well. Our working
hypothesis is that there is a de novo synthesis of cytokines from
either neurons or nonneuronal cells in response to axonal injury
and that these cytokines induce changes in the cell bodies of
A-type neurons.
Voltage-clamp studies of isolated currents in dissociated
DRG cells from nerve-inured animals have focused on the
altered properties of axotomized but not intact neurons. For
example, after peripheral axotomy, the axotomized somata
exhibited an up-regulation of tetrodotoxin-sensitive (TTX-S)
Na⫹ current and down-regulation of tetrodotoxin-resistant
(TTX-R) Na⫹ currents (see Black et al. 1999; Rizzo et al.
1995; Sleeper et al. 2000; Waxman et al. 1999 for review),
together with a reduction of K⫹ and Ca2⫹ currents (Abdulla
and Smith 2001b; Baccei and Kocsis 2000; Everill et al. 1999;
Hogan et al. 2000). Although some of these findings might be
argued as contributing in various ways to the changes we
observed in the shape of the AP or the enhanced excitability of
the axotomized L5 neurons, there is scant evidence in the
literature that the same changes occur for intact L4 neurons. In
addition, explanations for the cause of the changes in current
expression in an axotomized neurons based on the loss of
trophic support (e.g., NGF) from the periphery (Dib-Hajj et al.
1998; Oyelese et al. 1997; for review, Waxman et al. 2000)
cannot be used to explain similar changes in intact neurons in
a neighboring ganglion that still has this support.
Because L4 afferent fibers comingle with degenerating L5
axons in the peripheral nerve, Wallerian degeneration may be
the cause of the events leading to the enhanced excitability of
intact neurons with somata in L4 DRG adjacent to the axotomized L5 DRG. For example, nerve growth factor (NGF) or
glial cell line-derived neurotrophic factor (GDNF), cytokines
[e.g., tumor necrosis factor (TNF)␣, interleukin (IL)-1␤], or
other inflammatory mediators released by immune cells and
Schwann cells activated by L5 axonal degeneration could
diffuse to neighboring intact axons of L4 and be either retrogradely transported or locally induce signal transduction cascades in L4 DRG, thereby influencing gene expression in these
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The transection of central axons by a PR produced transient
mechanical hyperalgesia on the ipsilateral foot, without allodynia to stroking. After surgery, the withdrawal threshold of
ipsilateral foot to Von Frey hair stimulation was significantly
lowered through the seventh day of testing, but returned to the
preoperative level by day 10. A short lasting hyperalgesia was
also obtained after a complete transection of the L5 dorsal root
(Li et al. 2000; Sheth et al. 2002). In these studies, mechanically evoked withdrawal threshold returned to normal within 7
days.
The electrophysiological effects of the partial rhizotomy
were rather small. There was no increase in the incidence of
SA in either axotomized or intact neurons, and the minor
changes in membrane properties were limited to medium- or
large-sized somata. For these, the input resistance was lowered
by the sham operation itself but increased by the effects of
axotomy, suggesting a predominantly nonspecific effect of the
surgical procedure. Possibly the opening of the dura, carried
out for both sham and experimental rats, might have produced
a local inflammation on the surface of exposed dorsal roots
that, in turn, might have altered the membrane properties of
some primary afferent neurons (DeLeo and Yezierski 2001).
Our findings of transient hyperalgesia and few significant
electrophysiological changes in axotomized and intact DRG
neurons after PR are at odds with the results obtained by Zhou
and Xie (2000) and Xie et al. (1999). These investigators
ligated one-half of the dorsal root of L5 and left the suture in
place. Ipsilateral mechanical hyperalgesia persisted through 30
days of testing. In addition, abnormal SA was recorded from
both axotomized and intact dorsal root fibers. Possibly their
implanted suture may have had a chronic inflammatory and
mechanically irritating effect on both axotomized and intact
roots. However, there is no direct evidence for this at the
present time. In any event, we conclude that the transient
mechanical hyperalgesia we observed in the present experiment was not due to a change in the excitability of primary
sensory neurons but rather to changes in central processing
caused by a loss of afferent input (Eschenfelder et al. 2000; Li
et al. 2000).
1599
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MA ET AL.
Effect of acute axotomy on the excitability
of intact L4 neurons
In rats with no previous nerve injury, a transection of the L5
spinal nerve does not result in abnormal SA until 16 –20 h after
the axotomy (Liu et al. 2000). We found that after SNL, intact
L4 neurons still connected with their peripheral receptors exhibited no SA. However, starting approximately 30 min after
acute peripheral axotomy, SA could be recorded in approximately 9.1% of the A-neurons. Boucher (2000) obtained a
similar incidence of SA in 14 ⫾ 2% of the myelinated dorsal
root fibers from L4 DRG in vivo (with spinal nerve acutely
transected) after an L5 SNL.
We consider three possibilities for the rapid appearance of
SA in certain A-neurons after acute axotomy. The simplest
explanation is that, after nerve section, it may be easier to
distinguish abnormal SA from ongoing activity produced by
certain peripheral receptors such as muscle spindles. Against
this idea was the finding that the incidence of SA was not
significantly different with versus without anesthetization of
the nerve (which eliminated, for example, the competing, normal, ongoing activity from muscle spindles). Alternatively, the
ectopic generator in the soma may be suppressed (or continuJ Neurophysiol • VOL
ally reset) by APs generated from the periphery. A third
possibility, and one that we favor, is that the nerve section
either interrupts a signal transmitted along the axon from the
periphery to the DRG that normally acts to suppress the excitability of the soma or a positive signal is generated at the
proximal stump of the nerve that is also transported anterogradely and acts to further increase the excitability of the soma.
After SNL, the intact L4 somata and/or peripheral axons may
develop an enhanced sensitivity to neuroactive agents (e.g.,
TNF␣, IL-1, ATP, bradykinin, epinephrine, and protons) released from the axotomized axons, Schwann cells, blood vessels, and other tissues in the acute transected proximal nerve
stump (see review by Stoll and Muller 1999).
Evaluation of the in vivo recording preparation
Our in vivo preparation provided a relatively stable environment for the somata of primary sensory neurons by isolating
the DRG from its original site and perfusing it with oxygenated
ACSF, while maintaining at least a portion of its blood supply.
The preparation also allows the determination of the receptive
field properties of a portion of the DRG neurons, but only those
with somata on the surface. The extent to which the properties
of these surface somata are the same of somata located deeper
in the ganglion remains to be determined. The location of the
cell bodies within the DRG was not well specified in previous
intracellular recordings from intact DRG after L5 SNL. For
most of the membrane properties recorded intracellularly with
our preparation, the mean values for each size category for
axotomized and sham-operated rats were within range of published values (Abdulla and Smith 2001a; Liu et al. 2000;
Stebbing et al. 1999; Villiere and McLachlan 1996; Zhang et
al. 1999), except for some discrepancies in AP shape (duration,
rise/fall rates and percentage of inflected A-neurons), input
resistance, and AP threshold. As noted in previous studies, for
example, Amir and Devor (1996), Stebbing et al. (1999), and
Liu et al. (2000), such discrepancies might arise from differences in the type of injury, the type of electrophysiological
recording (patch- or sharp-electrode), the temperature of the
preparation, and whether recordings are carried out in vitro or
in vivo.
We appreciate the assistance of D. Donnelly, R. Friedman, and C. Lu.
This work was supported by the National Institute of Neurological Disorders
and Stroke Grants NS-14624 and NS-38317 to R. H. LaMotte and a Christopher Reeve Paralysis Foundation Grant to F. A. White.
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SNL. NaV1.8 protein and the TTX-R C-wave of the compound
AP were up-regulated in the intact C-fibers in the sciatic nerve
(Gold et al. 2001). In addition, Wu et al. (2001) found an early
onset of SA at a low rate (approximately 7 pulses/5 min) in L4
dorsal root C-fibers after L5 SNL. This SA originated distal to
the L4 DRG and was present in nociceptive fibers with cutaneous receptive fields. We did not observe any SA in intact,
small-sized somata with C-fibers and peripheral cutaneous
nociceptors. However, the observation period we allowed for
SA, prior to continued study of a neuron, was relatively short
(3 min), leaving open the possibility that a very low rate of SA
may have been overlooked. It may also be that the C-neurons
of L4, while altered in certain ways by the SNL injury, do not
express SA unless they are repetitively activated, for example, by
the electrical search stimuli used in the study by Wu et al. (2001).
The cellular events set in motion by peripheral axotomy may
increase the excitability of second order nociceptive neurons in
the dorsal horn receiving input both from nociceptive and low
threshold mechanoreceptive DRG neurons. As discussed in the
literature, such a state of central sensitization might be initiated
and maintained by abnormal processing in central as well as
peripheral neurons (for review, see Devor 1999; Ji and Woolf
2001).
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