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Groups II and III Metabotropic Glutamate Receptors
Differentially Modulate Brief and Prolonged Nociception
in Primate STT Cells
Volker Neugebauer, Ping-Sun Chen and William D. Willis
J Neurophysiol 84:2998-3009, 2000. ;
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Groups II and III Metabotropic Glutamate Receptors Differentially
Modulate Brief and Prolonged Nociception in Primate STT Cells
VOLKER NEUGEBAUER, PING-SUN CHEN, AND WILLIAM D. WILLIS
Department of Anatomy and Neurosciences and Marine Biomedical Institute, The University of Texas Medical Branch,
Galveston, Texas 77555-1069
Received 21 July 2000; accepted in final form 11 September 2000
Address for reprint requests: W. D. Willis, Jr., Dept. of Anatomy and
Neurosciences and Marine Biomedical Institute, The University of Texas
Medical Branch, 301 University Blvd., Galveston, TX 77555-1069 (E-mail:
[email protected]).
2998
tion because they modulate responses of sensitized STT cells but have
no effect under control conditions.
INTRODUCTION
G-protein-coupled metabotropic glutamate receptors (mGluRs)
play important roles in various forms of neuroplasticity and are
becoming novel therapeutic targets for certain neurological and
psychiatric disorders associated with neuroplastic changes (Bordi
and Ugolini 1999; Conn and Pin 1997; Knöpfel et al. 1995;
Nicoletti et al. 1996). The heterogeneous mGluR family consists
of eight cloned subtypes, which are classified into three groups.
Group I receptors (mGluRs 1 and 5) couple to phospholipase C
and regulate neuronal excitability and synaptic transmission.
Group II (mGluRs 2 and 3) and group III (mGluRs 4, 6, 7, and 8)
receptors inhibit adenylyl cyclase and reduce neuronal excitability
and synaptic transmission (Bushell et al. 1999; Conn and Pin
1997; Gereau and Conn 1995; Knöpfel et al. 1995; Macek et al.
1996; Miller 1998; Neugebauer et al. 1997, 2000; Schoepp et al.
1999; Schoppa and Westbrook 1997; Schrader and Tasker 1997).
An emerging field of research implicates mGluRs in nociception and hyperalgesia. Whereas the first reports on the
involvement of mGluRs in spinal nociceptive processing relied
on a relatively nonselective mGluR antagonist (Neugebauer et
al. 1994; Young et al. 1994), recent electrophysiological and
behavioral studies used more selective agents to demonstrate a
role of group I mGluRs and, in particular, the mGluR1 subtype
in prolonged nociception in the spinal cord (Budai and Larson
1997; Fisher and Coderre 1996; Fundytus et al. 1998; Neugebauer et al. 1999; Young et al. 1997, 1998).
The role of group II and group III mGluRs in the modulation
of spinal nociceptive processes is not clear yet. Activation of
these receptors has been shown to inhibit neuronal excitability
and synaptic transmission in many brain areas (see references
in Conn and Pin 1997; Knöpfel et al. 1995; Miller 1998;
Neugebauer et al. 1997; Pin and Duvoisin 1995; Schoepp et al.
1999) as well as in spinal cord in vitro preparations (Cao et al.
1997; Dong and Feldman 1999; Jane et al. 1996; King and Liu
1996). With regard to nociception, an electrophysiological
study of wide-dynamic-range (WDR) spinal dorsal horn neurons in vivo described inhibitory effects of a group II mGluR
agonist on C-fiber-evoked discharges in rats with carrageenan
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Neugebauer, Volker, Ping-Sun Chen, and William D. Willis.
Groups II and III metabotropic glutamate receptors differentially
modulate brief and prolonged nociception in primate STT cells. J
Neurophysiol 84: 2998 –3009, 2000. The heterogeneous family of
G-protein-coupled metabotropic glutamate receptors (mGluRs) provides excitatory and inhibitory controls of synaptic transmission and
neuronal excitability in the nervous system. Eight mGluR subtypes
have been cloned and are classified in three subgroups. Group I
mGluRs can stimulate phosphoinositide hydrolysis and activate protein kinase C whereas group II (mGluR2 and 3) and group III
(mGluR4, 6, 7, and 8) mGluRs share the ability to inhibit cAMP
formation. The present study examined the roles of groups II and III
mGluRs in the processing of brief nociceptive information and capsaicin-induced central sensitization of primate spinothalamic tract
(STT) cells in vivo. In 11 anesthetized male monkeys (Macaca
fascicularis), extracellular recordings were made from 21 STT cells in
the lumbar dorsal horn. Responses to brief (15 s) cutaneous stimuli of
innocuous (brush), marginally and distinctly noxious (press and pinch,
respectively) intensity were recorded before, during, and after the
infusion of group II and group III mGluR agonists into the dorsal horn
by microdialysis. Different concentrations were applied for at least 20
min each (at 5 ␮l/min) to obtain cumulative concentration-response
relationships. Values in this paper refer to the drug concentrations in
the microdialysis fibers; actual concentrations in the tissue are about
three orders of magnitude lower. The agonists were also applied at
10 –25 min after intradermal capsaicin injection. The group II agonists
(2S,1⬘S,2⬘S)-2-(carboxycyclopropyl)glycine (LCCG1, 1 ␮M-10 mM,
n ⫽ 6) and (⫺)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY379268; 1 ␮M-10 mM, n ⫽ 6) had no significant effects on
the responses to brief cutaneous mechanical stimuli (brush, press,
pinch) or on ongoing background activity. In contrast, the group III
agonist L(⫹)-2-amino-4-phosphonobutyric acid (LAP4, 0.1 ␮M-10
mM, n ⫽ 6) inhibited the responses to cutaneous mechanical stimuli
in a concentration-dependent manner, having a stronger effect on
brush responses than on responses to press and pinch. LAP4 did not
change background discharges significantly. Intradermal injections of
capsaicin increased ongoing background activity and sensitized the
STT cells to cutaneous mechanical stimuli (ongoing activity ⬎
brush ⬎ press ⬎ pinch). When given as posttreatment, the group II
agonists LCCG1 (100 ␮M, n ⫽ 5) and LY379268 (100 ␮M, n ⫽ 6)
and the group III agonist LAP4 (100 ␮M, n ⫽ 6) reversed the
capsaicin-induced sensitization. After washout of the agonists, the
central sensitization resumed. Our data suggest that, while activation
of both group II and group III mGluRs can reverse capsaicin-induced
central sensitization, it is the actions of group II mGluRs in particular
that undergo significant functional changes during central sensitiza-
GROUPS II AND III
MGLURS
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METHODS
Animal preparation and anesthesia
Adult male monkeys (Macaca fascicularis, n ⫽ 11, 2.1–2.9 kg)
were initially tranquilized with ketamine (10 mg/kg im). Anesthesia
was induced with a mixture of halothane, nitrous oxide, and oxygen
followed by ␣-chloralose (60 –90 mg/kg iv) and maintained by continuous intravenous infusion of pentobarbital sodium (5 mg 䡠 kg⫺1 䡠
h⫺1 ). After a tracheotomy, animals were paralyzed with pancuronium
(0.4 – 0.5 mg/h iv) and ventilated artificially to maintain the end-tidal
CO2 between 3.5 and 4.5%. A bilateral pneumothorax minimized
movements caused by respiration. Arterial oxygen saturation was
monitored with a rectal oxymeter probe and kept between 96 and
100%. Throughout the experiment, the level of anesthesia was monitored by frequently examining pupillary size and reflex responses and
by continuously recording CO2 levels and electrocardiogram (ECG).
Core body temperature was kept at ⬃37°C using a thermostatically
controlled heating blanket.
A laminectomy exposed the lumbar enlargement. A pool was
formed with the skin flaps overlying the cord and filled with mineral
oil, which was kept at 37°C using a heating device. The dura mater
was opened and reflected to expose the cord. A craniotomy was
performed for stereotaxic placement of a monopolar stimulating electrode into the ventroposterolateral (VPL) nucleus of the thalamus. The
stereotaxic coordinates were: A: 8 mm; L: 8 mm; 16 –18 mm from the
cortical surface. To ensure correct placement, the thalamic electrode
was initially used to record the potentials evoked by electrical stimulation of the contralateral dorsal columns and responses to cutaneous
stimulation of the contralateral hind limb.
Microdialysis
For drug application, three microdialysis fibers (Spectrum Scientific, 18-kDa cutoff) were positioned in the lumbar enlargement in
areas most responsive to stimulation of the lower hindlimb. The fibers
were made from Cuprophan tubing (150 ␮m ID; wall thickness, 9
␮m). The tubing was coated with a thin layer of silicone rubber
(3140RTN, Dow Corning) except for a 1-mm-wide gap that was
positioned in the gray matter of the ipsilateral spinal dorsal horn. Each
fiber was pulled through the spinal cord just below the dorsal root
entry zone using a stainless steel pin cemented in the fiber lumen. The
fibers were placed in different segments of the lumbar enlargement
(L5-L7), about 10 mm apart. Using polyethylene tubing, the microdialysis fibers were connected to a syringe seated in a Harvard infusion
pump and were continuously perfused with artificial cerebrospinal
fluid (ACSF), containing (in mM) 125.0 NaCl, 2.6 KCl, 2.5 NaH2PO4,
1.3 CaCl2, 0.9 MgCl2, 21.0 NaHCO3, and 3.5 glucose, at a rate of 5
␮l/min. The ACSF was oxygenated and equilibrated to pH 7.4 with a
mixture of 95% O2-5% CO2. In the beginning of each experiment,
ACSF was pumped through the fibers for at least 2 h to allow the
neurochemical milieu to reach equilibrium.
Administration of drugs
After stable control responses of a neuron were recorded during
administration of ACSF into the dorsal horn for 0.5–1.5 h, the following drugs (concentrations given in parentheses) were administered
by microdialysis at 5 ␮l/min for 20 –30 min through the fiber closest
to the recorded STT neuron (for mGluR subgroup selectivity of the
compounds, see Cartmell et al. 1999, 2000; Conn and Pin 1997; Monn
et al. 1999; Schoepp et al. 1999): (2S,1⬘S,2⬘S)-2-(carboxycyclopropyl)glycine (LCCG1, a traditional group II mGluR agonist; 1 ␮M to
10 mM); (⫺)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate
(LY379268, a novel potent and selective group II mGluR agonist; 1
␮M to 10 mM); L(⫹)-2-amino-4-phosphonobutyric acid (LAP4, a
potent and selective key group III mGluR agonist; 0.1 ␮M to 10 mM).
LCCG1 and LAP4 were purchased from Tocris. LY379268 was a
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inflammation, whereas mixed (excitatory or inhibitory) effects
were observed in control rats without inflammation (Stanfa and
Dickenson 1998). A behavioral study found that the nociceptive responses in the second phase of the formalin test were
potentiated by a group II agonist but slightly reduced by a
group III agonist (Fisher and Coderre 1996).
The present electrophysiological study of primate spinothalamic tract (STT) cells is the first to address the role of group
II and group III mGluRs in brief nonnociceptive and nociceptive transmission and in prolonged nociception evoked by
intradermal capsaicin. Intradermal injection of capsaicin in
humans results in primary mechanical and thermal hyperalgesia at the injection site as well as in secondary mechanical
hyperalgesia (increased pain from noxious stimuli) and mechanical allodynia (pain evoked by innocuous stimuli) in an
area surrounding the zone of primary hyperalgesia (LaMotte et
al. 1991; Simone et al. 1991). Primate STT neurons develop
enhanced responses to cutaneous mechanical stimuli and increased background activity after intradermal capsaicin injection (Dougherty and Willis 1992; Simone et al. 1991). Central
nervous system changes, termed central sensitization, are believed to underlie capsaicin-induced secondary hyperalgesia
and allodynia since afferent nerve fibers supplying the area
outside of primary hyperalgesia zone do not sensitize (Baumann et al. 1992; LaMotte et al. 1992).
Capsaicin-induced central sensitization of STT cells involves the activation of N-methyl-D-aspartate (NMDA) and
non-NMDA glutamate receptors, group I metabotropic glutamate receptors of the mGluR1 subtype, and neurokinin 1 and 2
receptors (Dougherty et al. 1992, 1994; Neugebauer et al.
1999; Rees et al. 1998). Intracellular signal transduction systems such as the protein kinase C (PKC), cAMP-dependent
protein kinase A (PKA), and the nitric-oxide-activated cGMPdependent protein kinase G (PKG) pathways also play important roles in the sensitization of STT neurons after intradermal
capsaicin (Lin et al. 1996b, 1997, 1999; Sluka 1997a; Sluka
and Willis 1998; Sluka et al. 1997; Wu et al. 1998).
Activation of the G-protein-coupled group II and group III
mGluRs has been shown to inhibit cAMP formation in expression systems, brain slices, and neuronal cultures (Conn and Pin
1997; Pin and Duvoisin 1995; Schoepp et al. 1999). It is not
clear, however, whether group II and/or group III mGluRs
actually couple to inhibition of neurotransmitter-induced
cAMP increases in native systems. If so, agonists at these
receptors may be useful in downregulating the enhanced responses of nociceptive neurons in those instances of central
sensitization that involve the cAMP-PKA pathway. The functional role of group II and group III mGluRs in spinal nociceptive processing and central sensitization was addressed in
the present study. In anesthetized monkeys, extracellular recordings were made from STT cells. The responses to brief
cutaneous stimuli were tested before, during, and after the
application of selective group II and group III mGluR agonists
into the dorsal horn by microdialysis. Cumulative concentration-responses curves were measured. The agonists were also
applied 10 –25 min after intradermal injections of capsaicin to
analyze the potential usefulness of agents targeting groups II
and III mGluRs in the treatment of prolonged pain associated
with central sensitization. Preliminary results have been reported in abstract form (Chen et al. 1998).
AND CENTRAL SENSITIZATION
3000
V. NEUGEBAUER, P.-S. CHEN, AND W. D. WILLIS
generous gift from Eli Lilly. Stock solutions (100 mM) were prepared
that were diluted in ACSF to the indicated final concentrations on the
day of the experiment. Numbers given throughout this paper refer to
the drug concentrations within the microdialysis fiber. The amount of
drug diffusing across the dialysis membrane was estimated in vitro.
These experiments have shown that in a chamber containing 100 ␮l
ACSF the concentration of these drugs is about 1–3% of that being
perfused at a rate of 5 ␮l/min for 30 min. It is estimated that due to
diffusion in the tissue the concentration reached at the recorded
neuron is another order of magnitude lower, i.e., about 1/1,000 of the
drug concentration in the microdialysis fiber (see Benveniste and
Huttemeier 1990; Neugebauer et al. 1999; Sluka et al. 1997; Sorkin et
al. 1988).
Recording of STT cells
Experimental protocol
Once an STT cell was identified and isolated, ongoing background
activity and responses to graded mechanical stimuli were recorded,
Data analysis
Recorded activity was analyzed off-line from peristimulus rate
histograms using Spike2 software. Background activity was subtracted from the evoked responses. The effects of drugs and capsaicin
on the responses to cutaneous mechanical stimuli were similar for the
selected test sites (see Figs. 1, 2, and 4). Therefore the responses
evoked from the different stimulation sites were averaged for each of
the mechanical stimuli (brush, press, pinch). All averaged values are
given as the means ⫾ SE. Cumulative concentration-response relations were constructed by averaging the mean frequency of the responses for each drug concentration; the averages were then expressed
as percentage of predrug control values (set to 100%). EC50s and the
respective 95% confidence intervals were calculated from sigmoid
curves fitted to the cumulative concentration-response data using the
FIG . 1. (2S,1⬘S,2⬘S)-2-(carboxycyclopropyl)
glycine (LCCG1), a group II metabotropic glutamate receptor (mGluR) agonist, has no significant
effects on the ongoing activity and responses of a
spinothalamic tract (STT) neuron to brief cutaneous
mechanical stimuli. Extracellular recordings from 1
STT neuron of the wide-dynamic-range (WDR)
type in the dorsal horn (1,606 ␮M) of the lumbar
enlargement. The STT cell could be activated antidromically by electrical stimulation (T ⫽ 500 ␮A)
in the ventroposterolateral (VPL) nucleus of the
thalamus (not shown). Bin width of the histograms
in A–C is 0.1 s. A: responses to brief (15 s) graded
mechanical stimuli (brush, press, pinch) applied to 3
test points within the cutaneous receptive field (see
inset) in the presence of normal artificial cerebrospinal fluid (ACSF) administered by microdialysis
into the dorsal horn. B and C: administration of
LCCG1 (1 and 10 mM, respectively, dissolved in
ACSF) by microdialysis did not alter the responses
to mechanical stimuli.
Downloaded from http://jn.physiology.org/ at Penn State Univ on February 23, 2013
STT neurons were recorded extracellularly in the lumbar enlargement of the spinal cord using carbon filament electrodes (resistance: 4
M⍀). Cells were recorded within 0.5–1 mm of a microdialysis fiber to
ensure that the drugs administered by microdialysis would reach the
neuron in a short period of time at a sufficient concentration. Single
STT neurons were isolated following antidromic activation from the
contralateral VPL nucleus by square-wave current pulses (1 Hz, 1
mA, 200 ␮s) as described previously (Neugebauer et al. 1999).
Criteria for antidromic activation were as follows (see Trevino et al.
1973): constant latency of the evoked spike; ability to follow highfrequency (333–500 Hz) stimulation; and collision of orthodromic
spikes with antidromic spikes. The extracellularly recorded signals
were amplified and displayed on analog and digital storage oscilloscopes. Signals were also fed into a window discriminator whose
output was processed by an interface (CED 1401) connected to a
Pentium PC. Spike2 software (CED) was used to create peristimulus
rate histograms on-line and to store and analyze digital records of
single-unit activity off-line. Throughout the experiment spike size and
configuration were continuously monitored on the digital oscilloscope
and with the use of Spike2 software to confirm that the same neuron
was recorded and that the relationship of the recording electrode to the
neuron remained constant.
and the cutaneous receptive field in the hindlimb was mapped. Cells
were characterized by their responses to the following stimuli applied
to the most responsive sites in the receptive field: brush (brushing the
skin with a soft-hair artist’s brush in a stereotyped manner), press
(firm pressure using a large arterial clip to apply 1,005 g/8 mm2,
which is marginally painful when applied to the skin in humans), and
pinch (using a small arterial clip to apply 2,660 g/4 mm2, which is
clearly painful without causing overt damage to the skin). All cells
included in this study were WDR STT cells, which responded consistently to innocuous stimuli but were more strongly activated by
noxious stimuli.
The stimuli were delivered at three to five test points chosen to span
the receptive field. Each stimulus was applied for 15 s followed by a
15-s pause before the next test site was stimulated. To minimize a
potential “human factor” bias, the experimenter who applied the
mechanical stimuli did not observe the oscilloscope or the computer
monitor and was unaware of the response magnitude. The entire
sequence of mechanical stimuli (brush, press, and pinch) was repeated
three times before drug application and before capsaicin injection,
respectively. The stimuli were also applied during and after the
application of different drug concentrations and after capsaicin injection. Capsaicin (0.1 ml, diluted in Tween 80 and saline at 3%) was
injected intradermally between two test sites at the most responsive
portion of the receptive field. Care was taken that the test stimuli were
applied to areas away from the capsaicin injection site, i.e., in the zone
of secondary hyperalgesia.
GROUPS II AND III
MGLURS
following formula for nonlinear regression (Prism 3.0, GraphPad
Software, San Diego, CA): y ⫽ [A ⫹ (B – A)]/[1 ⫹ (10C/10X)D],
where A ⫽ bottom plateau, B ⫽ top plateau, C ⫽ log (EC50), D ⫽
slope coefficient. Significance of the effects of LAP4 on ongoing and
evoked activity was determined using a repeated-measures ANOVA
to analyze the nonlinear concentration-response relationships. An F
test (Prism 3.0, GraphPad Software) was used to analyze the linear
regression fitted to the concentration-response data for LCCG1 and
LY379268 since no successful curve fits were achieved using nonlinear regression analysis. A repeated-measures ANOVA, followed by
Newman-Keuls multiple comparison test where appropriate (Prism
3.0, GraphPad Software), was performed on raw data to test for
statistical significance of drug effects on capsaicin-induced changes in
the responses to the mechanical stimuli and ongoing background
activity. Statistical significance was accepted at the level P ⬍ 0.05.
RESULTS
Recordings were made from 21 STT cells of the WDR type
in the lumbar enlargements (L5-L7) of 11 monkeys. These
neurons were recorded at depths of 840 –1,606 ␮m from the
dorsal surface of the spinal cord. Based on the correlation
between recording depth and laminar position established for
STT cells in this laboratory (see Lin et al. 1999), 15 neurons
were in superficial dorsal horn (840 –1,193 ␮m) and 6 neurons
were in the deep dorsal horn (1,388 –1,606 ␮m). STT cells
were identified by antidromic activation from the VPL nucleus
of the thalamus as described in METHODS. The mean threshold
for antidromic activation was 449 ⫾ 44 ␮A (150 –740 ␮A); the
mean latency was 6.1 ⫾ 0.5 ms (4.0 –10.0 ms). All 21 STT
cells had cutaneous receptive fields on the ipsilateral foot,
including the toes in 15 cases. The receptive fields of 11
neurons also included parts of the lower leg; 7 of these neurons
had cutaneous receptive fields around the knee and on the
thigh. Ongoing activity ranged from 1.6 to 33.6 Hz (mean ⫽
9.9 ⫾ 2.5 Hz). Each cell was recorded within 0.5–1 mm of a
microdialysis fiber to ensure that the drugs administered by
microdialysis reached the neuron in a short period of time at a
sufficient concentration. Recordings were conducted no sooner
than 3 h following insertion of the microdialysis fiber, which is
sufficient time for the stabilization of extracellular neurotransmitter levels (Sorkin and McAdoo 1993; Sorkin et al. 1988).
Activation of group III mGluRs, but not group II mGluRs,
inhibits the responses of STT cells to brief innocuous and
noxious cutaneous mechanical stimuli
GROUP II MGLURS. The modulation of ongoing background
activity and of the responses to brush, press, and pinch by
group II mGluRs was studied using the agonists LCCG1 (6
STT cells) and LY379268 (6 STT cells). Many previous studies have relied on LCCG1 as a highly potent key group II
mGluR agonist with EC50 values at mGluR2 and mGluR3 in
the upper nanomolar range, although higher concentrations of
LCCG1 have now been shown to affect other mGluR subtypes
as well (see Conn and Pin 1997; Schoepp et al. 1999). Therefore in the present study we also tested the novel selective and
highly potent group II mGluR agonist LY379268, which demonstrates EC50 values in the low nanomolar range at mGluR2
and mGluR3 (Cartmell et al. 1999, 2000; Monn et al. 1999).
Application of LCCG1 into the dorsal horn by microdialysis
3001
had no significant effects on the ongoing activity and responses
of STT cells to innocuous and noxious cutaneous stimuli.
Figure 1 shows a typical example. The STT neuron was recorded extracellularly in the deep dorsal horn (1,606 ␮M) of
the lumbar enlargement. Ongoing activity and responses to
graded mechanical stimuli (brush, press, pinch; see METHODS)
applied at selected test points of the neuron’s cutaneous receptive field (see Fig. 1, inset) were recorded in the presence of
normal ACSF administered by microdialysis into the dorsal
horn (Fig. 1A). Administration of LCCG1 by microdialysis,
even at the high concentrations of 1 and 10 mM, did not change
ongoing activity or the responses to mechanical stimuli (Fig. 1,
B and C).
Similarly, the novel group II mGluR agonist LY379368 had
no effect on the ongoing background activity and responses of
STT neurons to mechanical stimulation of the skin. A typical
example is shown in Fig. 2. This STT neuron was recorded in
the superficial dorsal horn (988 ␮M) of the lumbar enlargement. Ongoing activity and responses to graded mechanical
stimulation (brush, press, pinch; see METHODS) of the skin at the
indicated test points (see Fig. 2, inset) were recorded in the
presence of normal ACSF administered by microdialysis into
the dorsal horn (Fig. 2A). Administration of relatively high
concentrations of LY379368 (1 and 10 mM) by microdialysis
did not change ongoing activity and evoked responses of the
STT neuron (Fig. 2, B and C).
Cumulative concentration-response relationships show that
neither LCCG1 (up to 10 mM; n ⫽ 6; see Fig. 3A) nor
LY379268 (up to 10 mM; n ⫽ 6; see Fig. 3B) altered significantly ongoing activity (⫻) or the responses to mechanical
cutaneous stimuli (brush, press, and pinch represented by different symbols) of STT neurons recorded under control conditions, i.e., in the absence of capsaicin-evoked central sensitization (see Figs. 6 and 7). Linear regression analysis of the
concentration-response curves using an F test and the “runs”
test, which determines whether the data differ significantly
from a straight line (Prism 3.0, GraphPad Software; see METHODS), showed that the slope was not significantly different from
zero and the data were not significantly nonlinear (P ⬎ 0.05 in
each case). These tests indicate that linear regression analysis
was appropriate and there was no significant change of ongoing
or evoked activity associated with the different agonist concentrations.
MGLURS.
In contrast, the selective group III mGluR
agonist LAP4 (see Conn and Pin 1997; Schoepp et al. 1999)
inhibited the responses to graded mechanical stimuli in a
dose-dependent manner without a significant effect on the
ongoing discharges. Figure 4 shows extracellularly recorded
spikes of one STT neuron in the lumbar spinal dorsal horn
(depth 1,030 ␮M). Ongoing activity and the responses to
graded mechanical stimulation (brush, press, pinch; see METHODS) of selected test points in the neuron’s cutaneous receptive
field (see Fig. 4, inset) were recorded in the presence of normal
ACSF administered by microdialysis into the dorsal horn (Fig.
4A). Administration of LAP4 (100 ␮M) by microdialysis inhibited the responses to brush more than to pinch and press but
had no clear effect on ongoing activity (Fig. 4B). The effects of
LAP4 were reversible after washout with normal ACSF by
microdialysis (Fig. 4C).
Figure 5 shows the cumulative concentration-response rela-
GROUP III
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Sample of STT neurons
AND CENTRAL SENSITIZATION
3002
V. NEUGEBAUER, P.-S. CHEN, AND W. D. WILLIS
tionships for LAP4 measured in recordings from 6 STT neurons in the lumbar dorsal horn. LAP4 inhibited the responses to
brief mechanical cutaneous stimuli (brush, press, pinch; represented by different symbols), but not ongoing background
activity (⫻), in a concentration-dependent manner. Cumulative
concentration-response relations were obtained from six WDR
STT cells recorded in the lumbar dorsal horn. Cumulative
concentration-response relations were constructed by averaging the mean frequency of the responses for each concentration
of LAP4; the averages were then expressed as percentage of
predrug control values (set to 100%); the EC50s were calculated using the formula given in METHODS. LAP4 was more
FIG. 3. LCCG1 (A, n ⫽ 6) and LY379268 (B, n ⫽ 6), agonists at group II
mGluRs, do not significantly change the ongoing activity (⫻) and responses of
WDR-type STT cells to brief cutaneous mechanical stimuli (brush, press, and
pinch; see different symbols). Cumulative concentration-response relationships were obtained by averaging the mean frequency of the responses for each
concentration of LCCG1 and LY379268, respectively; the averages were then
expressed as percentage of predrug control values (set to 100%). Linear
regression analysis of the concentration-response curves using an F test and the
“runs” test (see text) showed that the slope was not significantly different from
0 and the data were not significantly nonlinear (P ⬎ 0.05 in A and B). Values
refer to the drug concentrations in the microdialysis fiber; concentration at the
recorded neurons is an estimated 3 orders of magnitude lower (see METHODS).
Symbols and error bars represent means ⫾ SE.
potent in inhibiting the brush responses (EC50, 1.0 ␮M; 95%
confidence interval, 0.6 –1.9 ␮M) than the responses to press
(EC50, 5.8 ␮M; 95% confidence interval, 1.0 –35.8 ␮M), and
pinch (EC50, 4.2 ␮M; 95% confidence interval, 1.7–10.6 ␮M;
see METHODS). The effects of LAP4 on the responses to brush,
press, and pinch were highly significant (P ⬍ 0.0001 in each
case, repeated-measures ANOVA) whereas ongoing background activity was not significantly changed (P ⫽ 0.7699,
repeated-measures ANOVA).
Group II and III mGluR agonists reverse capsaicin-induced
central sensitization in STT cells
The ability of group II and III mGluRs to modulate capsaicin-induced sensitization of STT cells was studied by administering the group II agonists LCCG1 (100 ␮M, 15 min; n ⫽ 5)
and LY379268 (100 ␮M, 15 min; n ⫽ 6) and the group III
agonist LAP4 (100 ␮M, 15 min; n ⫽ 6) into the spinal dorsal
horn at 10 min after intradermal capsaicin (3%) injection. Each
agonist was able to reverse the enhanced responses to brush
and press following capsaicin.
GROUP II MGLURS. Figure 6 shows the typical effects of LCCG1
(A) and LY379268 (B) on two different STT cells. In Fig. 6A,
extracellular recordings were made from an STT neuron in the
superficial dorsal horn (1,080 ␮M) of the lumbar enlargement.
Brief (15 s) graded mechanical stimuli (brush, press, pinch)
were applied to the skin of the neuron’s receptive field. Consistent with previous studies from this laboratory (Dougherty
and Willis 1992; Simone et al. 1991), intradermal injection of
capsaicin (3%) clearly enhanced the ongoing activity and the
responses to brush and press to 252, 192, and 170%, respectively, of the control values before capsaicin; the pinch-evoked
responses increased to only 112%. Administration of LCCG1
(100 ␮M; 15 min) into the dorsal horn by microdialysis reduced the enhanced responses almost to the levels before
capsaicin. The responses increased again after washout of drug
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FIG. 2. (⫺)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY379268), a novel selective and highly potent group
II mGluR agonist, does not change the ongoing activity and
responses of an STT neuron to brief mechanical stimulation of
the skin. Extracellular recordings from 1 STT neuron of the
WDR type in the dorsal horn (988 ␮M) of the lumbar enlargement. The STT cell could be activated antidromically by electrical stimulation (T ⫽ 250 ␮A) in the VPL nucleus of the
thalamus (not shown). Bin width of the histograms in A–C is
0.1 s. A: responses to brief (15 s) graded mechanical stimuli
(brush, press, pinch) applied to three test points in the cutaneous
receptive field (see inset) in the presence of normal ACSF
administered by microdialysis into the dorsal horn. B and C:
administration of LY379268 (1 and 10 mM, respectively, dissolved in ACSF) by microdialysis had no apparent effect on the
responses to mechanical stimuli.
GROUPS II AND III
MGLURS
AND CENTRAL SENSITIZATION
3003
with normal ACSF and returned to baseline at about 2 h after
capsaicin. Without any pre- or posttreatment, the capsaicininduced sensitization of STT cells typically lasts for about 2 h
(Dougherty and Willis 1992; Lin et al. 1999; Simone et al.
1991).
Figure 6B shows the effects of the novel group II mGluR
agonist LY379268 on central sensitization in another STT cell
recorded at 1,116 ␮M in the superficial dorsal horn of the
lumbar enlargement. Ongoing activity and responses to brief
(15 s) graded mechanical stimuli (brush, press, pinch) applied
FIG. 5. The group III mGluR agonist LAP4 inhibits the responses to brief
mechanical cutaneous stimuli (brush, press, pinch; represented by different
symbols), but not ongoing background activity (⫻), in a concentration-dependent manner. Cumulative concentration-response relationships were obtained
from 6 WDR STT cells recorded in the lumbar dorsal horn. Cumulative
concentration-response relations were constructed by averaging the mean
frequency of the responses for each concentration of LAP4; the averages were
then expressed as percentage of predrug control values (set to 100%); the
EC50s were calculated using the formula given in METHODS. Values refer to the
drug concentrations in the microdialysis fiber; concentration at the recorded
neurons is an estimated 3 orders of magnitude lower (see METHODS). Symbols
and error bars represent means ⫾ SE.
to the skin were recorded extracellularly before and after the
intradermal injection of capsaicin (3%). The capsaicin injection enhanced the ongoing activity and the responses to brush
and press, but not pinch, to 359, 151, 134, and 104%, respectively, of the control values before capsaicin. Administration of
LY379268 (100 ␮M; 15 min) into the dorsal horn by microdialysis clearly reduced the enhanced responses. The responses
increased again after washout of drug with normal ACSF.
Figure 7 summarizes the data for the group II mGluR agonists LCCG1 (A; n ⫽ 5) and LY379268 (B; n ⫽ 6). Intradermal capsaicin (3%) enhanced ongoing activity (A and B, P ⬍
0.001) and the responses of STT cells to brush (A, P ⬍ 0.01;
B, P ⬍ 0.001), press (A, P ⬍ 0.01; B, P ⬍ 0.001) but not pinch
(A and B, P ⬎ 0.05; repeated-measures ANOVA followed by
Newman-Keuls multiple comparison test; see METHODS). Application of LCCG1 (100 ␮M; n ⫽ 5; Fig. 7A) into the dorsal
horn by microdialysis for 15 min significantly reduced the
capsaicin-induced increase of ongoing activity (P ⬍ 0.001) and
the enhanced responses to brush (P ⬍ 0.01) and press (P ⬍
0.01; repeated-measures ANOVA followed by Newman-Keuls
multiple comparison test). Similarly, LY379268 (100 ␮M; n ⫽
6; Fig. 7B) administered into the dorsal horn by microdialysis
for 15 min significantly reduced the enhanced ongoing activity
(P ⬍ 0.001) and the responses to brush (P ⬍ 0.001) and press
(P ⬍ 0.001; repeated-measures ANOVA followed by Newman-Keuls multiple comparison test). After washout of
LCCG1, ongoing activity and responses to brush and press
increased again significantly at 60 min post capsaicin (P ⬍
0.05, compared with the values during drug administration at
25 min post capsaicin; P ⬍ 0.05– 0.001, compared with control
values before capsaicin; repeated-measures ANOVA followed
by Newman-Keuls multiple comparison test). Similarly, after
washout of LY379268 ongoing activity and brush- and pressevoked responses increased again and were significantly different from the control values before capsaicin (P ⬍ 0.05;
repeated-measures ANOVA followed by Newman-Keuls multiple comparison test). The responses to brush and press at 60
min post capsaicin were also significantly different from the
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FIG. 4. The group III mGluR agonist L(⫹)-2amino-4-phosphonobutyric acid (LAP4) inhibits the
brush responses more than the pinch and press responses. Extracellular recordings from 1 STT neuron
in the lumbar spinal dorsal horn (depth: 1,030 ␮M).
The STT cell could be activated antidromically by
electrical stimulation (T ⫽ 350 ␮A) in the VPL
nucleus of the thalamus (not shown). Brief (15 s)
graded mechanical cutaneous stimuli were applied at
the indicated points of the neuron’s receptive field
(see inset). Bin width of the histograms in A–C is
0.1 s. A: responses to brush, press, and pinch stimuli
in the presence of normal ACSF administered by
microdialysis into the dorsal horn. B: administration
of LAP4 (100 ␮M dissolved in ACSF) by microdialysis inhibited the responses to brush, press, and
press without a clear effect on ongoing activity. C:
effects of LAP4 were reversible after washout with
normal ACSF by microdialysis.
3004
V. NEUGEBAUER, P.-S. CHEN, AND W. D. WILLIS
saicin (3%) clearly enhanced ongoing background activity and
the responses to brush and press to 187, 245, and 164%,
respectively, of the control values before capsaicin. Pinch
responses increased to only 111%. Administration of LAP4
(100 ␮M; 15 min) into the dorsal horn by microdialysis at 10
min post capsaicin reversed the increase in ongoing activity
and reduced the enhanced brush and press responses to below
the control level before capsaicin. Ongoing activity and evoked
responses increased again after washout of drug with normal
ACSF and showed a decline toward baseline levels at about 2 h
after capsaicin.
Figure 9 summarizes the data for the group III mGluR
responses during drug administration at 25 min (P ⬍ 0.01 and
P ⬍ 0.05, respectively; repeated-measures ANOVA followed
by Newman-Keuls multiple comparison test). The increase of
ongoing activity and evoked responses after washout of the
agonists suggests that the inhibition of capsaicin-induced spinal sensitization in these STT cells was due to the drug effects
and not the decay of capsaicin-induced sensitization. The similarity of the inhibitory effects obtained with each agonist
suggests that the inhibition of capsaicin-induced spinal sensitization involved the group II mGluRs.
GROUP III MGLURS. The group III agonist LAP4 was also able to
reverse capsaicin-induced central sensitization in STT cells as
shown in Fig. 8. Ongoing activity and responses to graded
mechanical stimuli (brush, press, pinch) were recorded extracellularly from an STT cell in the superficial dorsal horn (840
␮M) of the lumbar enlargement. Intradermal injection of cap-
FIG. 7. Summary of the inhibition of capsaicin-induced central sensitization by the group II mGluR agonists LCCG1 (A; n ⫽ 5) and LY379268 (B; n ⫽
6). In both samples of STT cells (A and B), intradermal capsaicin (3%)
enhanced ongoing activity and responses to brush and press, but not pinch,
significantly. A: application of LCCG1 (100 ␮M; n ⫽ 5) into the dorsal horn
by microdialysis for 15 min significantly reduced the capsaicin-induced increase of ongoing activity and responses to brush and press. B: similarly,
LY379268 (100 ␮M; n ⫽ 6) administered into the dorsal horn by microdialysis
for 15 min significantly reduced the enhanced ongoing activity and responses
to brush and press. A and B: after washout of LCCG1 and LY379268,
respectively, ongoing activity and responses to brush and press increased again
at 60 min post capsaicin and were significantly different from the control
values before capsaicin. brush- and press-evoked responses at 60 min post
capsaicin were also significantly different from the responses during drug
administration at 25 min, suggesting that the inhibition of capsaicin effects was
due to the drug effects and not the decay of capsaicin-induced sensitization. In
each sample of neurons (A and B), ongoing activity and evoked responses
recorded at the indicated times after capsaicin were averaged and expressed as
percentage of the ongoing activity and evoked responses in the control period
before capsaicin, which were averaged and set to 100%. *P ⬍ 0.05, **P ⬍
0.01, ***P ⬍ 0.001, compared with control values before capsaicin; ⫹P ⬍
0.05, ⫹⫹P ⬍ 0.01, ⫹⫹⫹P ⬍ 0.001, compared with 12 min postcapsaicin; 0P ⬍
0.05, 00P ⬍ 0.01, ongoing activity and evoked responses at 25 min compared
with 60 min post capsaicin (repeated-measures ANOVA performed on raw
data followed by Newman-Keuls multiple comparison test; see METHODS).
Columns and error bars represent means ⫾ SE.
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FIG. 6. The group II mGluR agonists LCCG1 (A) and LY379268 (B)
reverse capsaicin-induced central sensitization. A: recordings of ongoing activity and responses of 1 WDR STT cell (1,080 ␮M) to brief (15 s) graded
mechanical cutaneous stimuli (brush, press, pinch). Intradermal injection of
capsaicin (3%) enhanced the responses to brush (E) and press (●) as well as
ongoing activity (⫻); only a slight increase of the pinch-evoked responses (䡲)
was observed. Administration of LCCG1 (100 ␮M; 15 min) into the dorsal
horn by microdialysis reduced the enhanced responses almost to the levels
before capsaicin. Ongoing and evoked activity increased again after washout of
drug with normal ACSF and returned to baseline at ⬃ 2 h after capsaicin. B:
recordings of ongoing activity and responses of 1 WDR STT neuron (1,116
␮M) to cutaneous mechanical stimuli (brush, press, pinch). Intradermal injection of capsaicin (3%) strongly enhanced ongoing activity (⫻) and the responses to brush (E) and press (●). Administration of LY379268 (100 ␮M; 15
min) into the dorsal horn by microdialysis reversed the potentiating effects of
capsaicin. Ongoing activity and evoked responses increased again after washout of drug with normal ACSF and remained elevated until ⬃ 2 h after
capsaicin. A and B: each symbol represents the number of spikes per second.
Ongoing activity has been subtracted from the evoked response.
GROUPS II AND III
MGLURS
agonist LAP4 (n ⫽ 6). Intradermal capsaicin (3%) significantly
enhanced ongoing activity (P ⬍ 0.01) and the responses of
STT cells to brush (P ⬍ 0.01) and press (P ⬍ 0.05) but not
pinch (P ⬎ 0.05; repeated-measures ANOVA followed by
Newman-Keuls multiple comparison test; see METHODS). Administration of LAP4 (100 ␮M, 15 min; n ⫽ 6) into the dorsal
horn by microdialysis post capsaicin significantly reduced the
enhanced responses to brush (P ⬍ 0.001) and press (P ⬍ 0.01;
repeated-measures ANOVA followed by Newman-Keuls multiple comparison test). LAP4 also reduced the increased ongoing activity in three of six neurons, but this effect did not reach
the level of statistical significance in the whole sample of
neurons (P ⬎ 0.05). Although the pinch-evoked responses
were not significantly enhanced by capsaicin, LAP4 significantly inhibited the responses to pinch in four of six neurons
(P ⬍ 0.05; repeated-measures ANOVA followed by NewmanKeuls multiple comparison test), resembling its effects in the
absence of central sensitization (see Figs. 4 and 5). After
washout of LAP4, the responses to brush, press, and pinch
increased again significantly at 60 min post capsaicin (P ⬍
0.001, P ⬍ 0.01, P ⬍ 0.05, respectively, compared with 25 min
post capsaicin; repeated-measures ANOVA followed by Newman-Keuls multiple comparison test). Ongoing activity also
increased at 60 min post capsaicin but was not significantly
different from the values during drug application at 25 min post
capsaicin (P ⬎ 0.05). The rebound of capsaicin effects after
washout of LAP4 suggests that the inhibition of capsaicininduced sensitization in these STT cells was due to drug effects
and not the decay of capsaicin-induced sensitization.
DISCUSSION
This study addressed in primate STT cells the role of groups
II and III mGluRs in brief nonnociceptive and nociceptive
transmission and in central sensitization evoked by intradermal
capsaicin. The major findings are as follows. 1) Administration
of group II mGluR agonists into the spinal dorsal horn had no
3005
significant effect on ongoing background activity or responses
to brief cutaneous mechanical stimuli. 2) Intraspinal administration of a group III agonist, however, reduced the responses
to innocuous and noxious mechanical stimuli without a significant effect on background activity. 3) When given as posttreatment in capsaicin-induced central sensitization, the group
II mGluR agonists now inhibited the enhanced responses to
brush and press and background activity. 4) Posttreatment with
the group III agonist inhibited the enhanced responses to brush
and press and, in some cases, pinch. These data suggest that in
central sensitization of primate STT cells the functional role of
group II mGluRs changes substantially, whereas the effects
mediated by group III mGluRs remain largely unchanged.
The interpretation of our findings relies on the selectivity
and appropriate concentrations of the mGluR agonists we used.
The group II agonist LCCG1 has been used in many previous
studies as a highly potent agonist at mGluR2 and mGluR3
subtypes (see Conn and Pin 1997; Schoepp et al. 1999). We
tested LCCG1 so that our results can be compared with those
studies. Since higher concentrations of LCCG1 have been
shown to affect other mGluR subtypes as well (see Schoepp et
al. 1999), we also tested the novel potent and selective group
II mGluR agonist LY379268. LY379268 has a nanomolar
affinity and potency at mGluR2 and mGluR3 and shows a more
than 1,000-fold selectivity for group II mGluRs over other
mGluR subtypes (Cartmell et al. 1999, 2000; Monn et al.
FIG. 9. Summary of the inhibition of capsaicin-induced central sensitization by the group III mGluR agonist LAP4 (n ⫽ 6). Intradermal capsaicin (3%)
significantly enhanced ongoing activity and the responses of STT cells to brush
and press but not pinch. Application of LAP4 into the dorsal horn by microdialysis post capsaicin significantly reduced the enhanced responses to brush
and press. LAP4 also inhibited the responses to pinch in 4 neurons. Inhibition
of ongoing activity by LAP4 was seen in 3 of 6 neurons but did not reach the
level of significance in the whole sample of neurons. After washout of LAP4,
evoked responses increased again significantly at 60 min post capsaicin,
suggesting that the inhibition of capsaicin-induced sensitization in these STT
cells was due to drug effects and not the decay of capsaicin-induced sensitization. Ongoing activity also increased at 60 min post capsaicin but was not
significantly different from the values during drug application at 25 min post
capsaicin. Ongoing activity and evoked responses recorded at the indicated
times after capsaicin were averaged and expressed as percentage of the
ongoing activity and evoked responses in the control period before capsaicin,
which were averaged and set to 100%. *P ⬍ 0.05, **P ⬍ 0.01, compared with
control values before capsaicin; ⫹P ⬍ 0.05, ⫹⫹P ⬍ 0.01, ⫹⫹⫹P ⬍ 0.001,
compared with 12 min postcapsaicin; 0P ⬍ 0.05, 00P ⬍ 0.01, 000P ⬍ 0.001,
ongoing activity and responses at 25 min compared with 60 min post capsaicin
(repeated-measures ANOVA performed on raw data followed by NewmanKeuls multiple comparison test; see METHODS). Columns and error bars represent means ⫾ SE.
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FIG. 8. The group III mGluR agonist LAP4 inhibits capsaicin-induced
central sensitization. Recordings of ongoing activity and responses of 1 WDR
STT cell (840 ␮M) to brief (15 s) cutaneous mechanical stimuli (brush, press,
pinch). Intradermal injection of capsaicin (3%) enhanced the responses to
brush (E) and press (●) as well as ongoing activity (⫻). Effect on the response
to pinch (䡲) was less clear. Administration of LAP4 (100 ␮M; 15 min) into the
dorsal horn by microdialysis reversed the potentiating effects of capsaicin.
Ongoing activity and evoked responses increased again after washout of drug
with normal ACSF. Each symbol represents the number of spikes per second.
Ongoing activity has been subtracted from the evoked response.
AND CENTRAL SENSITIZATION
3006
V. NEUGEBAUER, P.-S. CHEN, AND W. D. WILLIS
mGluR antagonist has recently been reported to reduce response thresholds to noxious mechanical stimuli in awake
animals, possibly suggesting a tonic group II mGluR component in spinal cord during normal behavior (Dolan and Nolan
2000). In spinal cord in vitro preparations, presumed group II
mGluR antagonists potentiated synaptically evoked responses
of spinal motoneurons (Cao et al. 1997) but not of neurons in
lamina II of the dorsal horn (Chen and Sandkühler 2000) and
had no effect on dorsal root-evoked ventral root potentials
(Jane et al. 1996). Another explanation of the lack of group II
agonist effects involves the growing body of evidence that
mGluR-mediated signal transduction is tightly regulated by the
phosphorylation state of the receptor through various kinases
(Macek et al. 1999; Peavy et al. 1998; Saugstad et al. 1998;
Schaffhauser et al. 2000). In fact Schaffhauser et al. (2000)
have shown that mGluR2 specifically is turned off by PKA.
However, previous studies from our lab (Sluka 1997a; Sluka
and Willis 1997) and work done by others (Krieger et al. 2000;
Malmberg et al. 1997; Ohsawa and Kamei 1999; Wei and
Roerig 1998; Young et al. 1995) have not produced any evidence for a significant role of PKA or adenylyl cyclase in
normal spinal transmission.
In contrast, the group III agonist LAP4 clearly inhibited the
responses of nonsensitized STT cells. Group III mGluRs can
function as presynaptic autoreceptors, inhibit adenylyl cyclase,
and modulate ion channels (Conn and Pin 1997; Miller 1998;
Pin and Duvoisin 1995). Inhibition of cAMP production is
unlikely to be involved in the inhibitory effects of LAP4 on
nonsensitized STT cells because neither peripheral nor spinal
blockade of the cAMP-PKA pathway affect normal responses
and nociceptive thresholds in animals (Ahlgren and Levine
1993; Sluka and Willis 1997). Further, if cAMP was involved,
group II and III agonists should have similar effects since both
subgroups couple negatively to adenylyl cyclase. A general
reduction of neuronal excitability by activation of potassium
currents is also unlikely since LAP4 inhibited only the evoked
responses but not ongoing activity.
The stronger inhibition by LAP4 of the brush than the press
and pinch responses could involve L-type or both L- and
N-type calcium channels. L- and N-type calcium channel
blockers reduce the responses of dorsal horn neurons to innocuous and noxious mechanical stimuli with L-type blockers
having stronger effects on the nonnociceptive responses
(Neugebauer et al. 1996). LAP4 may also influence transmitter
release “downstream” of calcium entry (Bushell et al. 1999).
Differences in the laminar distribution and agonist affinity of
the group III subtypes could then explain the stronger effect of
LAP4 on the brush responses: mGluR7 has a much lower
affinity for glutamate and LAP4 than mGluR4 and is localized
on nociceptive afferent terminals in the superficial dorsal horn,
whereas the laminar distribution of mGluR4 is consistent with
a localization on nonnociceptive primary afferents and spinal
interneurons (Kinoshita et al. 1998; Li et al. 1996, 1997; Ohishi
et al. 1995a,b). No other group III subtypes have been detected
in the spinal cord (Berthele et al. 1999; Valerio et al. 1997).
In capsaicin-induced central sensitization, both group II and
III agonists inhibited the enhanced responses of STT cells. This
effect may be related to inhibition of cAMP accumulation.
Both groups of mGluRs are characterized by their negative
coupling to adenylyl cyclase (see Conn and Pin 1997; Miller
1998; Pin and Duvoisin 1995; Schoepp et al. 1999), and
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1999). For the study of group III mGluRs, we used LAP4, a
potent and selective key agonist for group III mGluRs (Conn
and Pin 1997; Pin and Duvoisin 1995; Schoepp et al. 1999).
LCCG1 and LAP4 have been shown by others to inhibit
transmitter release in the brain when administered by microdialysis with a time course and at concentrations similar to our
study (Cozzi et al. 1997; Hu et al. 1999; Maione et al. 1998).
Additional evidence suggests that our approach allowed the
pharmacological discrimination of groups II and III mGluRs.
We used micromolar to low millimolar drug concentrations in
the microdialysis fiber, which produce effective nanomolar to
low micromolar concentrations in the tissue at the recorded
neuron (see METHODS). Such low concentrations have been
shown to be selective in in vitro studies (Conn and Pin 1997;
Monn et al. 1999; Schoepp et al. 1999). We have measured the
amount of drug diffusing across the dialysis membrane in vitro
to be only 1–3% of the perfused concentration (see METHODS).
This concentration decreases by another order of magnitude
after diffusion of the drug away from the microdialysis fiber
into the dorsal horn close enough to affect the recorded STT
neuron (cf. Sluka et al. 1997; Sorkin et al. 1988). Furthermore
both group II mGluR agonists, LCCG1 and LY379268, had
very similar effects but behaved differently than the group III
agonist LAP4 in that the group II agonists had no significant
effects under normal conditions, whereas LAP4 reduced the
responses to cutaneous mechanical stimuli.
The differential modulation of the responses of nonsensitized STT neurons by group II and group III mGluRs is an
unexpected but intriguing finding. It is not clear why group II
agonists had no effect under normal conditions. Group II
mGluR2 and mGluR3 subtypes have been detected in the
dorsal horn at the messenger RNA (Berthele et al. 1999; Ohishi
et al. 1993a,b; Valerio et al. 1997) and the protein level (Jia et
al. 1999; Tang and Sim 1999). The cellular localization, however, of group II mGluRs in the preterminal area distant from
the transmitter-release site rather than on the presynaptic terminals per se (Shigemoto et al. 1997) may play a role in the
lack of group II agonist effects we observed. It is also possible
that activation of spinal group II mGluRs produces both inhibitory and excitatory effects with the result of a zero net effect.
Electrophysiological studies have found excitatory, inhibitory,
dual, and mixed effects of group II mGluRs on spinal motoneurons and unidentified dorsal and ventral horn neurons
(Bond and Lodge 1995; Bond et al. 1997; Cao et al. 1995,
1997; Dong and Feldman 1999; King and Liu 1996; Stanfa and
Dickenson 1998). Group II mGluRs can act as auto- as well as
hetero-receptors to inhibit glutamatergic and GABAergic synaptic transmission (see Conn and Pin 1997; Miller 1998; Pin
and Duvoisin 1995). STT cells receive both glutamatergic and
GABAergic inputs (Lekan and Carlton 1995) and group II
mGluRs are present on glutamatergic and GABAergic terminals in the spinal cord (Jia et al. 1999). In addition, the
combination of group II mGluR-mediated postsynaptic excitation (see Conn and Pin 1997; Miller 1998; Pin and Duvoisin
1995) and postsynaptic inhibition (Dutar et al. 1999; Holmes et
al. 1996; Keele et al. 1999; Knoflach and Kemp 1998; Sharon
et al. 1997) may contribute to a zero net effect of group II
agonists.
It is also conceivable that the group II agonists have little
effect because the available group II receptors are under some
level of activation. The intrathecal application of a group II
GROUPS II AND III
MGLURS
3007
conditions. The present study suggests that group II mGluR
agonists may be among those agents. In contrast, group III
agonists (this study) and group I antagonists (Neugebauer et al.
1999) can inhibit central sensitization of STT cells but also
affect normal neurotransmission in these neurons. The significant change of the functional role of group II mGluRs in
central sensitization suggests that mGluR2 and/or mGluR3
may be useful targets for the development of drugs to relieve
persistent pain associated with central sensitization.
We thank K. Gondesen and G. Robak for excellent technical assistance and
G. Gonzales for help with the artwork. We also thank Dr. Smriti Iyengar at Eli
Lilly and Company for the generous gift of LY379268.
This work was supported by National Institutes of Health Grants NS-09743
and MH-10322.
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blockade of the cAMP-PKA pathway can reduce capsaicininduced mechanical hyperalgesia and allodynia (Sluka 1997a;
Sluka and Willis 1997) and central sensitization (Sluka et al.
1997a). Increased cAMP accumulation can result from the
activation of group I mGluRs, especially mGluR1 (see also
Conn and Pin 1997), which plays a crucial role in the capsaicin-induced central sensitization of STT cells (Neugebauer et
al. 1999).
The dramatic change in the effects of the group II mGluR
agonists following intradermal injection of capsaicin may suggest a change in the functional role of group II mGluRs in
central sensitization. Similarly, Stanfa and Dickenson (1998)
found that the group II agonist 1S,3S-ACPD had mixed inhibitory and excitatory effects on electrically (C-fiber)-evoked
activity in dorsal horn neurons in control animals but predominantly inhibitory effects in the carrageenan model. Several
mechanisms may account for this change: inhibition of the
cAMP-PKA transduction cascade through group II mGluRs in
central sensitization but not under normal conditions; upregulation of group II mGluRs in central sensitization; inhibition of
high-voltage-activated calcium channels, e.g., P/Q-type channels, which can be inhibited by group II mGluRs (see Conn and
Pin 1997; Miller 1998) and are, at the level of the spinal cord,
involved in central sensitization but not in normal behavior or
neurotransmission (Nebe et al. 1997; Sluka 1997b); and imbalance of inhibition of excitatory and inhibitory transmission
in central sensitization, which is characterized by an increased
glutamatergic transmission combined with a decreased
GABAergic tone (Dougherty and Willis 1992; Dougherty et al.
1992; Lin et al. 1996a) so that inhibition of excitatory transmission by group II agonists would prevail over disinhibition.
It is important to note that capsaicin-induced central sensitization of STT cells resumed after the group II and group III
agonists were washed out. This finding implies some form of
tonic activation that attempts to produce central sensitization
and is only temporarily blocked by the activation of group II
and III mGluRs. Capsaicin-induced central sensitization of
STT cells involves a variety of neurotransmitter and neuromodulators (Dougherty et al. 1992, 1994; Neugebauer et al.
1999; Rees et al. 1998) and signal transduction pathways (Lin
et al. 1996b, 1997, 1999; Sluka 1997a; Sluka and Willis 1998;
Sluka et al. 1997; Wu et al. 1998). Since manipulation of each
individual component of the sensitization process has profound
effects, these elements appear to operate in series rather than in
parallel. This hypothesis is consistent with the various welldocumented interactions of ionotropic and metabotropic glutamate receptors, glutamate and neuropeptide receptors, metabotropic glutamate receptors, and second-messenger systems, and
among signal transduction pathways in the nervous system (for
recent reviews, see Conn and Pin 1997; Herrero et al. 2000;
Hobbs et al. 1999; Hollmann and Heinemann 1994; Majewski
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1998; Schoepp et al. 1999; Tanaka and Nishizuka 1994; Van
Rossum et al. 1997; Willis et al. 1996; Xia and Storm 1997).
In conclusion, both group II and III agonists inhibit capsaicin-induced central sensitization of STT cells whereas the
group III, but not group II, mGluR agonists also affect the
responses of nonsensitized neurons. For the treatment of pain
states, the ideal drug should be effective in pain-related central
sensitization but have no or minimal effects under normal
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