GABAA and 5-HT3 Receptors Are Involved in Dorsal Root Reflexes:
Possible Role in Periaqueductal Gray Descending Inhibition
YUAN BO PENG,1 JING WU,3 WILLIAM D. WILLIS,3 AND DANIEL R. KENSHALO2
1
Pain and Neurosensory Mechanisms Branch, National Institute of Dental and Craniofacial Research and 2Center for
Scientific Review, National Institutes of Health, Bethesda, Maryland 20892; and 3Department of Anatomy and
Neurosciences, University of Texas Medical Branch, Galveston, Texas 77555-1069
Received 24 October 2000; accepted in final form 20 March 2001
INTRODUCTION
Somatosensory information is generally considered to originate in the peripheral terminals of primary afferent neurons
and is then transmitted to the spinal cord or brain. The primary
afferents make synapses with second-order neurons that relay
the information to higher centers. However, under certain
circumstances, activity in primary afferent neurons can be
generated within the spinal cord and can travel antidromically
toward the periphery along the sensory fibers, a phenomenon
Address reprint requests to W. D. Willis (E-mail: [email protected]).
www.jn.org
called the dorsal root reflex (DRR). DRRs were first described
by Gotch and Horsley (1891), following which there was a
detailed study by Barron and Matthews (1935a,b, 1938a– c).
The DRR is triggered by suprathreshold depolarization of the
central terminals of primary afferent fibers, or primary afferent
depolarization (PAD) (Barron and Matthews 1935; Eccles et
al. 1961; Schmidt 1971; Toennies 1938). The negative dorsal
root potential (DRP) reflects the primary afferent depolarization and is considered to underlie one form of presynaptic
inhibition (Eccles et al. 1962, 1963a; Lloyd 1952; Lloyd and
McIntyre 1949; reviewed by Rudomin and Schmidt 1999).
PAD can be detected by intracellular recordings from primary
afferent fibers (Eccles and Krnjevic 1959; Eccles et al. 1963a;
Koketsu 1956), by extracellular field potentials (Eccles et al.
1963a), and by measurement of enhanced excitability of the
primary afferent fibers (Eccles et al. 1963a; Madrid et al. 1979;
Rudomin et al. 1980; Wall 1958; Willis et al. 1976). PAD has
been detected not only in large myelinated afferents but also in
fine afferent fibers, including both A␦ and C fibers (Calvillo et
al. 1982; Carstens et al. 1979; Fitzgerald and Woolf 1981;
Hentall and Fields 1979; Whitehorn and Burgess 1973). While
DRRs have usually been recorded from large myelinated fibers
(Brooks and Koizumi 1956; Eccles et al. 1961; Toennies
1938), there is evidence that DRRs can also be conducted in
A␦ fibers and in C fibers (Jänig and Zimmermann 1971; Lin et
al. 2000; Toennies 1938). DRRs in fine afferents may contribute to neurogenic inflammation by releasing substance P and
calcitonin gene-related peptide from sensory nerve endings and
causing plasma extravasation and vasodilation (Lin et al. 1999;
Rees et al. 1995; Sluka et al. 1993; reviewed by Willis 1999).
It has been suggested that PAD results from the action of
GABA released from inhibitory interneurons and acting on
GABAA receptors located on the synaptic terminals of primary
afferent fibers because PAD can be blocked by administration
of GABAA-receptor antagonists, such as bicuculline or picrotoxin (Curtis and Lodge 1982; Curtis et al. 1971, 1982; Duchen
1986; Eccles et al. 1963b; Levy 1974, 1977; Levy and Anderson 1972; Levy et al. 1971; Sivilotti and Nistri 1991). Behavioral and DRR studies using a rat inflammation model also
demonstrated the involvement of GABAA receptors (Rees et
al. 1995; Sluka et al. 1993, 1994). GABAA antagonists only
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49
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Peng, Yuan Bo, Jing Wu, William D. Willis, and Daniel R. Kenshalo. GABAA and 5-HT3 receptors are involved in dorsal root
reflexes: possible role in periaqueductal gray descending inhibition. J
Neurophysiol 86: 49 –58, 2001. The dorsal root reflex (DRR) is a
measure of the central excitability of presynaptic inhibitory circuits in
the spinal cord. Activation of the periaqueductal gray (PAG), a center
for descending inhibition of spinal cord nociceptive transmission,
induces release of variety of neurotransmitters in the spinal cord,
including GABA and serotonin (5-HT). GABA has been shown to be
involved in generation of DRRs. In this study, pharmacological agents
that influence DRRs and their possible mechanisms were investigated.
DRRs were recorded in anesthetized rats from filaments teased from
the cut central stump of the left L4 or L5 dorsal root, using a
monopolar recording electrode. Stimulating electrodes were placed
either on the left sciatic nerve or transcutaneously in the left foot.
Animals were paralyzed and maintained by artificial ventilation.
Drugs were applied topically to the spinal cord. A total of 64 units
were recorded in 34 Sprague-Dawley rats. Peripheral receptive fields
were found for nine of these units. In these units, DRRs were evoked
by brush, pressure, and pinch stimuli. Nine units were tested for an
effect of electrical stimulation in the periaqueductal gray on the
DRRs. In eight cases, DRR responses were enhanced following PAG
stimulation. The background activity was 4.2 ⫾ 1.9 spikes/s (mean ⫾
SE; range: 0 –97.7; n ⫽ 57). The responses to agents applied to the
spinal cord were (in spikes/s): artificial cerebrospinal fluid, 7.1 ⫾ 3.6
(range: 0 – 86.9; n ⫽ 25); 0.1 mM GABA, 16.8 ⫾ 8.7 (range: 0 –191.0;
n ⫽ 22); 1.0 mM GABA, 116.0 ⫾ 26.5 (range: 0.05–1001.2; n ⫽ 50);
and 1.0 mM phenylbiguanide (PBG), 68.1 ⫾ 25.3 (range: 0 –1,073.0;
n ⫽ 49). Bicuculline (0.5 mM, n ⫽ 27) and ondansetron (1.0 mM, n ⫽
10) blocked the GABA and PBG effects, respectively (P ⬍ 0.05).
Significant cross blockade was also observed. It is concluded that
GABAA receptors are likely to play a key role in the generation of
DRRs, but that 5-HT3 receptors may also contribute. DRRs can be
modulated by supraspinal mechanisms through descending systems.
50
Y. B. PENG, J. WU, W. D. WILLIS, AND D. R. KENSHALO
Data acquisition
EXTRACELLULAR SINGLE-FIBER RECORDINGS FROM THE CENTRAL
STUMP OF THE DORSAL ROOT. For electrophysiological recordings,
a silver wire hook electrode was used to record extracellular singleunit discharges in filaments of the L4 or L5 dorsal roots. A small strand
of the dorsal root was teased centrally from the main trunk and was
further separated into a filament containing a single active fiber (Fig.
1). The dorsal root filament was wrapped around the recording electrode. In some cases, DRRs could be evoked by mechanical stimulation of a receptive field located in the skin. In those units, DRRs were
recorded in response to graded intensities of mechanical stimuli
delivered to the receptive field. Background activity and responses to
brush, pressure, and pinch stimuli were recorded for 10 s each with a
20-s inter-stimulus interval. The rationale for examining receptive
fields was to show that activation of the primary afferent fibers in the
receptive field could evoke antidromic action potentials in different
primary fibers, which implies an interaction between primary afferents.
The SPIKE2 computer software program and CED 1401Plus, a
multi-channel data-acquisition system by Cambridge Electronic Design, were used to record the timing and amplitude of action potentials
and to analyze data on- or off-line. The advantage of this program is
that it can differentiate between action potentials of different dorsal
root units by amplitude and assign a colored and numbered template
to each spike. Each channel can be duplicated to demonstrate various
plotting modes simultaneously. The most commonly used features of
the SPIKE2 software in this experiment were wavemarks (the raw
spike data) and frequency histograms.
ELECTRICAL STIMULATION. To evoke DRRs from the periphery, a
tripolar electrode was used to stimulate the sciatic nerve. We used a
tripolar electrode to minimize the stimulus artifact and to avoid current
spread. The cathode was in the middle of the array, and two anodes, one
on each side of the cathode, were separated from the cathode by 1 mm.
The stimulus intensity was set at 1–2.5 times the threshold at various
frequencies (0.1–50 Hz) and durations (0.1–1.0 ms).
PAG is well known to be a source of descending inhibition of spinal
cord nociceptive neurons (Basbaum and Fields 1979; Fardin et al.
1984; Liebeskind et al. 1973; Liebman et al. 1970; Reynolds 1969;
Richardson and Akil 1977). Stimulating in the PAG can cause a
METHODS
Animal preparation
Adult Sprague-Dawley rats were initially anesthetized with pentobarbital sodium (50 mg/kg ip). A catheter was placed in the jugular
vein for continuous administration of anesthetic and a tracheotomy
allowed insertion of a tracheal cannula for artificial ventilation. In
some animals, craniotomy was performed to allow placement of a
PAG-stimulating electrode. The left sciatic nerve was also exposed for
stimulation in some experiments. A 4-cm-long laminectomy was
performed over the lumbosacral enlargement to expose the spinal
cord. The rat was held in a stereotaxic frame to prevent any movement
during recording. The skin over the laminectomy was elevated to form
a pool that was filled with light mineral oil. Continuous anesthesia was
accomplished by giving a mixture of 50 mg sodium pentobarbital in
9 ml 0.9% NaCl at a rate of 1.0 ml/h. Paralysis of the musculature was
achieved by intravenous injection of 0.5 ml (1 mg/ml) pancuronium
bromide every 2 h. The end tidal CO2 was maintained at around 30
mmHg. The animal’s body temperature was maintained at 37°C by a
feedback-controlled electric heating blanket.
All procedures used in this study were approved by the Animal
Care and Use Committees of National Institute of Dental and Craniofacial Research and the University of Texas Medical Branch and
followed the guidelines for the treatment of animals of the International Association for the Study of Pain (Zimmermann 1983).
FIG. 1. Illustration of method for dorsal root reflex (DRR) recordings. A
single strand of teased central stump of the dorsal root was placed over a
recording electrode from which single fiber activity was recorded, as indicated
by A, inset, where the top panel was the rate histogram of the spike wavemark
seen in the bottom panel. Pharmacological agents were applied to the dorsal
surface of the spinal cord. In a separate protocol, a stimulating electrode was
placed around the sciatic nerve and evoked DRR activity was recorded from
the central stump of the dorsal root filament, as indicated by B, inset, in which
2 different units were recorded at different latencies. Œ, the stimulus artifact.
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reduce but do not abolish the DRP (Wall 1994). Following
intra-arterial administration of either GABAA or 5-HT2 receptor antagonists, the DRP was reduced to 20% of the control
level (Thompson and Wall 1996), whereas 5-HT1A and 5-HT3
receptor antagonists had no significant effect. Radioligand
binding studies have demonstrated the presence of binding
sites for 5-HT1, 5-HT2 and 5-HT3 receptor ligands in the dorsal
half of the spinal cord (Glaum and Anderson 1988; Leysen et
al. 1982; Monroe and Smith 1983; Pazos et al. 1985). A high
density of 5-HT3 receptors is found in the superficial dorsal
horn at all levels of the spinal cord (Hamon et al. 1989). They
are mainly restricted to lamina I (Hamon et al. 1989; Kidd et al.
1993). These may reflect the presence of 5-HT3 receptors on
primary afferent fibers because it is known that 5-HT3 receptors are present on dorsal root ganglion cells, including those
belonging to C fibers (Todorovic and Anderson 1990a,b).
5-HT3 receptors are directly linked to the opening of nonselective monovalent cation channels (Derkach et al. 1989;
Yaksh 1985). Opening of the channels by 5-HT3 permits the
passage of Na⫹ and K⫹ and thus depolarizes the cell (Wallis
and Elliott 1991).
GABAA and 5-HT3 receptors have been shown to be involved in descending inhibition of spinal cord nociceptive
neurons following stimulation in the periaqueductal gray
(PAG) (Peng et al. 1996c; cf. Proudfit et al. 1980). In a
preliminary study, GABAA and 5-HT3 receptors were found to
be involved in the initiation of the DRRs when agonists of
those receptors are applied to the dorsal surface of the spinal
cord (Peng et al. 1996d). The hypothesis of this study is that
PAG activation leads to PAD and the generation of DRRs
through release of GABA and serotonin, which activate
GABAA and 5-HT3 receptors on the central terminals of primary afferent fibers. The aim of this study is to understand the
mechanisms underlying the generation of DRRs by PAG stimulation.
DORSAL ROOT REFLEXES IN RATS
51
reduction of dorsal horn neuronal activity through the release of
serotonin (5-HT), norepinephrine, GABA, glycine, and opioids (Peng
et al. 1996a-c; Yaksh 1979), and both GABA and 5-HT produce PAD
(Eccles et al. 1963b; Proudfit et al. 1980). To determine whether or not
direct electrical stimulation of the PAG will elicit DRRs, a monopolar
or a bipolar electrode was used to stimulate the PAG (7 mm posterior
to bregma, 0.2 mm lateral, and 4.5–5 mm deep from the surface of the
cerebral cortex; stimulus parameters: train of stimulus from 1 to 333
Hz at duration of 0.1–1 ms). A test of correct position of the PAG
stimulating electrode was carried out by extracellular recording of
single dorsal horn neurons; these were inhibited by PAG stimulation
(Peng et al. 1996a– c).
MANIPULATIONS. Pharmacological agents
were dissolved in artificial cerebrospinal fluid (ACSF, pH ⫽ 7.2). The
ACSF contained (in mM) 151.1 Na⫹, 2.6 K⫹, 0.9 Mg2⫹, 1.3 Ca2⫹,
2⫺
122.7 Cl⫺, 21.0 HCO⫺
3 , and 2.5 HPO4 .
Since it is known that GABA and 5-HT are released by PAG
stimulation (Cui et al. 1999), it is important to determine whether
GABA and 5-HT3 agonists will generate DRRs following direct
application to the dorsal surface of the spinal cord and whether
receptor-selective antagonists of GABAA and 5-HT3 receptors will
block the generation of DRRs elicited by the agonists. Pharmacological agents used in this study were: GABA, 1.0 mM (Sigma); 1-phenylbiguanide (PBG, 5-HT3 agonist), 1.0 mM (RBI); bicuculline, 0.5
mM (Sigma, GABAA antagonist); and ondansetron, 1.0 mM (5-HT3
antagonist), which was kindly provided by Glaxo Group Research.
Each drug was released through a needle by dropping the drugcontaining solution over the spinal cord around the dorsal root entry
zone. Controls were done by releasing ACSF. The duration of drug
action varies from experiment to experiment. Residual drugs were
removed, along with cerebrospinal fluid, by gentle suction. The time
between successive applications of drugs was based on the observation of the recovery of spontaneous activity to the control level.
PHARMACOLOGICAL
The stored digital record of unit activity was retrieved and analyzed
off-line. For single-fiber DRR recordings, responses to mechanical stimuli applied to the receptive field for 10 s were calculated by subtracting
the preceding 10 s of background activity to yield a net increase in
discharge rate. For responses to pharmacological applications, agonist or
antagonist-induced activities were calculated in a fixed time interval of
10 s including the peak if the effect lasted longer than 10 s. If the effect
was less than 10 s, it was averaged from the start to the end of the drug
effect. The effect of PAG stimulation on DRRs was evaluated by calculating the ratio of the spike rate during PAG stimulation to the spike rate
when PAG stimulation was turned off and reporting this as a percentage.
Statistical significance was tested using one-way ANOVA followed by
Dunn’s test. A change was judged significant if P ⬍ 0.05. All values are
presented as means ⫾ SE.
RESULTS
A total of 64 units were recorded in 34 Sprague-Dawley rats.
Receptive fields were identified for nine units from nine animals (Fig. 2). Three of these units responded only to brush, one
unit only to pressure and pinch, and five units to brush, pressure, and pinch. For these nine units with receptive fields, the
average background activity and responses to brush, pressure
and pinch were 0.19 ⫾ 0.1 (range: 0 – 0.8 spikes/s), 12.0 ⫾ 3.6
(range: 0 –32.7 spikes/s), 17.6 ⫾ 6.2 (range: 0 –50.8 spikes/s),
and 20.5 ⫾ 7.2 (range: 0 –57.5 spikes/s), respectively. In one
unit, high-intensity mechanical stimulation by squeezing the
nose, ears, front and hind paws elicited DRRs (Fig. 3).
Nine units from nine animals were tested for the effects of
FIG. 2. Peripheral receptive fields were found for 9 units. DRRs were evoked
by brush, pressure, and pinch stimuli. Top: the rate histogram (bars), spike
wavemarks (the raw action potentials shown as vertical lines), and the receptive
field of the DRR are shown. The wavemark is seen on a fast time base in D. B: the
measurements of DRRs recorded from 9 individual units for baseline activity and
responses to brush, pressure, and pinch of the receptive field. C: the responses of
these 9 units. Activity is indicated as mean spike/s (⫾SE).
PAG stimulation on DRRs. The average antidromic spontaneous firing rate in dorsal root axons was 3.4 ⫾ 2.1 (range:
0.25–19.5 spikes/s) when PAG stimulation was off, and the
average antidromic discharge frequency during PAG stimulation was 20.4 ⫾ 7.7 (range: 1.3– 64.7 spikes/s), a statistically
significant increase during PAG stimulation (paired t-test, P ⬍
0.05; Fig. 4).
In 20 units, the minimum latency of DRRs evoked in the
dorsal root filament was measured following stimulation of the
ipsilateral sciatic nerve. There was typically a random shift of
the latency of the responses to a series of sciatic nerve stimulations, as indicated by the two vertical lines in Fig. 5. The
response latency in this circuit depends on three components:
the latency of the primary afferent discharges from the stimulating electrode to the spinal cord, the central synaptic delay,
and latency from the central terminals to the recording electrode.
DRRs evoked by electrical stimulation in the periphery were
frequency dependent (Fig. 6). Sometimes there was a wide
range of stimulus intensities that could evoke DRRs. In this
representative unit, ipsilateral sciatic nerve stimulation at 1.0-
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Data analysis
52
Y. B. PENG, J. WU, W. D. WILLIS, AND D. R. KENSHALO
Hz, 0.1-ms duration, and 2.0-V intensity elicited a doublet
DRR from a single unit. When the stimulation frequency was
increased to 10 Hz, there was only one spike with a slightly
delayed latency. However, when the stimulation frequency was
increased to 20 Hz, the evoked spike was abolished. It reappeared immediately after the frequency was reset to 1.0 Hz.
When droplets containing GABA or 5-HT3 agonists and
antagonists were applied to the dorsal surface of the spinal
cord, antidromic discharges were recorded from primary afferent fibers. Typical antidromic dorsal root discharges evoked by
release of GABA or PBG are shown in Fig. 7, A and B. The
average spontaneous background activity was 4.2 ⫾ 1.9
(range: 0 –97.7; n ⫽ 57), and the response to ACSF was 7.1 ⫾
3.6 (range: 0 – 86.9; n ⫽ 25; Fig. 7C). There was a very large
response to GABA application (Fig. 7C), which was dose
dependent. The average responses to 0.1 and 1.0 mM GABA
were 16.8 ⫾ 8.7 (range: 0 –191.0; n ⫽ 22) and 116.0 ⫾ 26.5
(range: 0.1–1001.2; n ⫽ 49), respectively. The average response to 1.0 mM PBG, which is a 5-HT3–receptor specific
agonist, was 68.1 ⫾ 25.3 (range: 0 –1073.0; n ⫽ 49; Fig. 7C).
The increases in the antidromic dorsal root activity evoked by
1.0 mM GABA or 1.0 mM PBG were significant as compared
with the background activity (1-way ANOVA, followed by
Dunn’s test, ***P ⬍ 0.001; *P ⬍ 0.05). No significant changes
were detected in the ACSF and 0.1 mM GABA groups.
When the GABAA receptor antagonist, bicuculline, was
used at a concentration of 1.0 mM, in 27 units, the GABAinduced antidromic dorsal root activity was significantly attenuated from the control level (agonist only) of 166.3 ⫾ 43.5
(range: 0.4 –1,001.2; n ⫽ 27) to 16.9 ⫾ 4.8 (agonist and
antagonist; range: 0 –72.3; n ⫽ 27, P ⬍ 0.05), and there was
recovery (agonist only) to 81.3 ⫾ 29.5 (range: 0.3–312.4; n ⫽
15; Figs. 8A and 9). Similarly in 10 units, when the 5-HT3
receptor antagonist, ondansetron, was used at a concentration
of 1.0 mM, the PBG-induced antidromic dorsal root activity
was attenuated significantly from a control level (agonist only)
FIG. 5. DRRs evoked by stimulating the ipsilateral sciatic nerve. The
minimum afferent conduction velocity was 30.1 ⫾ 6.3 m/s (range: 3.7–118.3
m/s; n ⫽ 20). The DRRs showed a marked tendency toward latency shifting.
Top: the spike evoked by single stimulation at the sciatic nerve. Bottom: the
spikes evoked by repeated stimulation at 1.0 Hz in the same unit. Note the
variation of the latencies over about 2 ms.
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FIG. 3. In 1 unit, high-intensity mechanical stimulation of the ears and front
and hindpaws all elicited DRR responses, suggesting an involvement of
descending systems. The 7 panels show the responses evoked by pinching at
different places as indicated. Top: a rate histogram (spikes/s). Bottom: the
wavemarks (vertical lines in mV).
FIG. 4. Nine units were tested for an effect of electrical stimulation of the
periaqueductal gray (PAG) on the DRRs. In 8 cases, there were enhanced
responses following PAG stimulation. One example is shown in A, in which
the short bars above the figure indicate the period of PAG stimulation (1-s
trains at 333 Hz, 800-␮A intensity, 0.2 ms duration separated by 2 s). B: the
sizes of the DRR discharges of 9 units are shown with (PAG-on) and without
(PAG-off) PAG stimulation. The activity was generally lower when PAG
stimulation was off than when it was on. C: a summary of the results and shows
the mean firing rates in the absence and presence of PAG stimulation.
DORSAL ROOT REFLEXES IN RATS
53
of 210.4 ⫾ 103.5 (range: 9.8 –1,073.0; n ⫽ 10) to 21.4 ⫾ 11.0
(agonist and antagonist; range: 0.1–96.3; n ⫽ 10, P ⬍ 0.05),
and there was only a slight recovery (agonist only) to 34.2 ⫾
13.8 (range: 3.2–119.6; n ⫽ 8; Figs. 8C and 9). The effect of
bicuculline on PBG-induced antidromic dorsal root activity for
control (agonist only), drug (agonist and antagonist), and after
washout (agonist only) was 146.6 ⫾ 51.1 (range: 0.2–1,073.0;
n ⫽ 24), 31.4 ⫾ 12.3 (range: 0 –269.5; n ⫽ 24, P ⬍ 0.05), and
21.9 ⫾ 5.3 (range: 0.1–74.5; n ⫽ 19), respectively. The effect
of ondansetron on GABA-induced antidromic dorsal root activity for control, drug, and after washout was 82.9 ⫾ 31.5
(range: 5.4 –318.7; n ⫽ 9), 16.6 ⫾ 7.2 (range: 0 –58.2; n ⫽ 9,
P ⬍ 0.05), and 13.3 ⫾ 6.4 (range: 0.2– 47.9; n ⫽ 7), respectively. There were significant cross actions between the agonists and antagonists of these two receptors, i.e., bicuculline
reduced PBG-induced antidromic dorsal root activity, and ondansetron markedly reduced GABA-induced antidromic dorsal
root activity (Fig. 9). There was no obvious recovery.
DISCUSSION
DRRs evoked by peripheral stimulation
In previous work, DRRs were evoked in the medial articular
nerve in arthritic rats (Rees et al. 1994) as well as in cats and
monkeys (Sluka et al. 1995) when the tissue around the joint or
even on the lower limb was stimulated mechanically by firm
pressure or repeated brisk taps. The same stimuli failed to
evoke any activity in anesthetized animals that were not arthritic or on the contralateral side in acutely arthritic animals
(Rees et al. 1994). DRRs have also been recorded from dorsal
root filaments in response to cutaneous stimulation in rats
following intradermal injection of capsaicin (Lin et al. 1999,
2000). Some DRR activity was seen prior to capsaicin injection, but the DRR responses were increased during the acute
inflammation. In this study, we were also able to record DRRs
from dorsal root filaments in noninflamed rats following stimulation of the skin or of the sciatic nerve. We could record
responses following mechanical stimuli that were applied ipsilaterally (Fig. 2) or contralaterally (Fig. 3), although not from
every single dorsal root fiber examined. Thus our results are
consistent with cross talk between two sides of the body. This
could occur at the spinal level or at a supraspinal level. There
are other reports that DRRs can be induced by natural stimulation, including light touch, pressure, vibration, and muscle
stretch (Millar 1979). Toennies (1938) described DRRs evoked
in the saphenous nerve following stimulation of the saphenous
nerve itself, adjacent dorsal roots, or other sensory nerves in
the ipsilateral or contralateral limb. There was a 4-ms delay of
the reflex, suggesting a polysynaptic pathway. Spread of the
DRR both along and across the cord has been noted in several
studies (Bagust et al. 1989, 1993; McCouch and Austin 1958;
Toennies 1938). In the isolated hamster spinal cord, this spread
extends over at least 16 segments from lower lumbar to upper
thoracic roots, in both rostrocaudal and caudorostral directions
from the stimulated dorsal root, and to the contralateral side of
the cord (Bagust et al. 1989).
DRRs evoked by PAG stimulation
It has been reported that spinalization increases the antidromic discharge rate in sensory axons to a level about four
times higher than the control rate; this change could last for
2 h, suggesting a tonic inhibition by supraspinal structures (Lin
and Fu 1998). We did not observe a similar phenomenon in this
study because we did not transect the spinal cord. On the
contrary, we found that stimulation of the PAG, the origin of
one of the major descending inhibitory systems, could evoke or
enhance DRRs.
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FIG. 6. The electrically evoked DRRs were frequency dependent. In this representative unit, ipsilateral sciatic nerve stimulation
at 1.0-Hz, 0.1-ms duration, and 2.0-V intensity elicited a doublet discharge. When the stimulation frequency was increased to 10
Hz, there was only 1 spike with a delayed latency (the smaller spikes that appeared occasionally should be ignored). However, when
the stimulation frequency was increased to 20 Hz, the evoked spike was abolished, and it reappeared immediately after the
frequency was reset to 1.0 Hz.
54
Y. B. PENG, J. WU, W. D. WILLIS, AND D. R. KENSHALO
Latency shift and frequency dependency of peripherally
evoked DRRs
The ipsilateral sciatic nerve was stimulated to determine the
minimum conduction velocity of the afferents that evoked the
DRRs, although the way we measured this might not be accurate because we were measuring the total latency of the reflex,
and we assumed that the incoming and outgoing volleys propagated at the same conduction velocity. We found that the
threshold was difficult to determine. Sometimes there was a
wide range of stimulus intensities that could evoke DRRs.
Usually the very first stimulus required a lower intensity than
the subsequent ones. We also found that the latency of the
DRR in a specific dorsal root fiber shifted from stimulus to
stimulus (Fig. 5). Lin and Fu (1998) observed a similar phenomenon. They recorded DRRs at L5, and the threshold was
determined by recording the afferent volley in the L6 dorsal
root when the sciatic nerve was stimulated. When the stimulus
intensity was changed between 2 and 40 times threshold, the
latency of the afferent volley at L6 did not change, but the
latency between the incoming volley and DRR at L5 changed
GABAA and 5-HT3 receptors are involved in DRRs
In this study, we have focused on two major neurotransmitter receptors, GABAA and 5-HT3. Pharmacological agents
were applied by directly dropping them on the dorsal surface of
the spinal cord near the dorsal root entry zone. We usually
applied ACSF as a control first before other pharmacological
agents were applied.
In one experiment, three separate units were recorded simultaneously when the sciatic nerve was stimulated at 1 Hz and 80
V. When 0.3 ml of 0.5 mM bicuculline was administered
intravenously, the DRRs evoked in these three units were
almost eliminated (a total of 12 evoked spikes after a series of
60 stimuli), as compared with control (58/63) and recovery
(60/60). Although the effect of bicuculline was dramatic, we
were not confident about the site of drug action. Instead of
systemic administration of agents, later in our experiments, we
chose to use local applications of the drugs. In agreement with
other studies (Curtis and Lodge 1982; Curtis et al. 1971, 1982;
Duchen 1986; Eccles et al. 1963b; Levy 1974, 1977; Levy and
Anderson 1972; Levy et al. 1971; Sivilotti and Nistri 1991;
Sluka et al. 1993, 1994), we found that GABA induced a large
DRR response (Fig. 8A). This response was significantly, but
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FIG. 7. Representative antidromic dorsal root activity evoked by topical
application of GABA (A) or PBG (B) over the spinal cord. A summary is
shown in C. A statistically significant increase of spontaneous antidromic
dorsal root activity occurred when 1.0 mM GABA or 1.0 mM PBG was
applied.
from 7.5 ms at 5 times threshold to 3 ms at 20 times threshold.
However, they did not indicate whether there was a smear shift
or a jump of the latency.
In addition, we found that the DRRs evoked by stimulation
of the sciatic nerve were frequency dependent. In the fiber
illustrated in Fig. 6, stimulation at 1.0 Hz evoked a doublet
DRR; at 10 Hz, only one spike was evoked with a slightly
longer latency; at 20 Hz, DRRs were totally abolished. Previous studies reported similar findings. For example, Eccles and
Willis (1962) found that DRRs in group Ia afferent fibers can
release enough neurotransmitter to excite motoneurons in kittens. When a number of different peripheral nerves were stimulated at low (0.3 Hz) and high (10 Hz) frequencies, DRRs
elicited at low frequencies evoked large excitatory postsynaptic
potentials (EPSPs) that sometimes caused the motoneuron to
discharge, but the EPSPs were subthreshold when elicited at a
high frequency (Eccles and Willis 1962). This phenomenon
suggests a dynamic change of the excitability of the spike
generation sites near the central terminals of primary afferent
fibers and is consistent with the involvement of polysynaptic
circuitry in DRR generation. In a hemisected spinal cord in
vitro preparation, it was found that stimulation of a lumbar
dorsal root evokes a reflex in up to four adjacent spinal segments in both rostral and caudal directions, and a period of
depressed activity was demonstrated following both evoked
and spontaneous discharges, which could be as long as 600 ms
(Bagust et al. 1985). Since the fascicles from which recordings
were made were disconnected from the periphery, DRRs
evoked by stimulating the sciatic nerve were speculated to be
generated either by collaterals of afferent fibers interacting
with the fiber from which recordings are made through an
interneuronal pathway in the spinal cord or by activating a
descending system from brain stem structures, such as the
PAG. Both shifting of latency and frequency dependence of
DRRs would suggest synaptic delays in the interneuronal circuitry within the dorsal horn or even the involvement of
ascending and descending systems.
DORSAL ROOT REFLEXES IN RATS
55
not completely, blocked by the GABAA antagonist, bicuculline, applied locally to the surface of the spinal cord prior to
GABA administration (Fig. 9). An explanation of the failure of
bicuculline to eliminate the GABA responses completely
would be that too low a dose reached the GABAA receptors.
Various 5-HT receptors have been demonstrated on dorsal
root ganglion cells (Todorovic and Anderson 1990a,b) and in
the dorsal half of the spinal cord (Glaum and Anderson 1988;
FIG. 9. Summary of the effects of bicuculline or ondansetron on GABAand PBG-induced antidromic dorsal root activity. Control, agonist only; drug,
agonist plus antagonist; recovery, agonist only.
Leysen et al. 1982; Monroe and Smith 1983; Pazos et al. 1985).
Thompson and Wall (1996) found that the DRP was reduced to
20% of control level by a 5-HT2 receptor antagonist, whereas
5-HT1A and 5-HT3 receptor antagonists had no significant
effect. The dose of granisetron (a 5-HT3 antagonist) used was
0.1 mg/kg with arterial injection of 0.1 ml, which seems to be
a low dose as compared with the dose of ondansetron that we
used. Furthermore, it is unclear where the site of action was
since the drug was delivered systemically. The actual concentration at the spinal cord level would be much lower than the
injected concentration. However, a high density of 5-HT3
receptors is found in the superficial dorsal horn at all levels of
the spinal cord (Hamon et al. 1989; Kidd et al. 1993). It has
been demonstrated that 5-HT3 receptors produce depolarization by opening nonselective monovalent cation channels
(Derkach et al. 1989; Wallis and Elliott 1991; Yaksh 1985). At
least some of the 5-HT3 receptors in the dorsal horn are likely
to be on primary afferent terminals (Todorovic and Anderson
1990a,b).
Midbrain PAG-induced descending antinociception (Hayes
et al. 1979; Oliveras et al. 1974; Reynolds 1969) has been
shown to be mediated in part by descending serotonergic
pathways (Carstens et al. 1981; Yezierski et al. 1982). We have
implicated 5-HT3 and 5-HT1A receptors (Lin et al. 1996; Peng
et al. 1996c), as well as ␣2-adrenoreceptors (Peng et al. 1996b),
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FIG. 8. Representative experiment showing the effects of bicuculline or ondansetron on GABA- or PBG-induced antidromic
dorsal root activity. A: effect of bicuculline on GABA-induced antidromic dorsal root activity. B: effect of bicuculline on
PBG-induced antidromic dorsal root activity. C: effect of ondansetron on PBG-induced antidromic dorsal root activity. D: effect
of ondansetron on GABA-induced antidromic dorsal root activity. Control, agonist only; drug, agonist plus antagonist; recovery
(the term indicates the process of recovery, whether or not recovery occurs), agonist only.
56
Y. B. PENG, J. WU, W. D. WILLIS, AND D. R. KENSHALO
might be attributed to tachyphylaxis from repeated agonist
applications.
Conclusion
FIG. 10. Illustration of the dorsal root circuitry that may underlie the
generation of DRRs by GABA and serotonin.
and GABAA and glycine receptors in PAG inhibition of dorsal
horn neurons (Lin et al. 1994; Peng et al. 1996a). The inhibition of dorsal horn neurons could be the result of both pre- and
postsynaptic inhibition. In addition to GABAA receptors,
5-HT3 receptor activation is a possible contributor to this
mechanism. A schematic drawing of the possible dorsal horn
circuitry is shown in Fig. 10. The results of our study showed
that, similar to the application of GABA, application of the
5-HT3 receptor agonist, PBG, induced large DRR responses.
These were partially but significantly blocked by ondansetron,
a 5-HT3 antagonist, applied to the surface of the spinal cord.
Since there are both GABAA and 5-HT3 receptors on the
central terminals of the primary afferent fibers, application of
the GABAA receptor antagonist, bicuculline, significantly reduced GABA-induced antidromic dorsal root activity but not
the tonic activity from direct activation of 5-HT3 receptors on
the central terminals of the primary afferents. The reason why
bicuculline significantly reduced PBG-induced antidromic dorsal root activity (Fig. 9) was because even though GABA may
have been released by activation of 5-HT3 receptors on the
GABAergic interneurons (Fig. 10), its effect on the central
terminals of the afferents would have been reduced due to the
presence of bicuculline. On the other hand, when 5-HT3 antagonist, ondansetron was applied, it would block 5-HT3 receptors on the central terminals to prevent the generation of
DRRs. Furthermore, we speculate that ondansetron also had a
secondary effect by blocking 5-HT3 receptors on GABAergic
interneurons to reduce the release of GABA, which had acted
on GABAA receptors on the primary terminals to generate
DRRs (Fig. 10). This would explain how ondansetron could
reduce the antidromic dorsal root activity, but externally applied GABA could still have some activity on the GABAA
receptors on the primary afferent terminals. The fact that there
was significant cross blockade of GABAA activity by a 5-HT3
receptor antagonist and vice versa suggests these two systems
play synergistic roles in the generation of DRRs. While there
was partial recovery of GABA-induced antidromic dorsal root
activities following bicuculline, the absence of recovery following antagonist application in the other groups of animals
The authors thank G. Gonzales for assistance with the illustrations.
This work was supported by National Institute of Neurological Disorders
and Stroke Grant NS-09743 and by the National Institute of Dental and
Craniofacial Research Division of Intramural Research.
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