Department of Anatomy and Neurosciences and Marine Biomedical Institute, The University of Texas Medical Branch, Galveston, Texas 77555-1069
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ABSTRACT |
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Neugebauer, Volker, Ping-Sun Chen, and William D. Willis. Role of Metabotropic Glutamate Receptor Subtype mGluR1 in Brief Nociception and Central Sensitization of Primate STT Cells. J. Neurophysiol. 82: 272-282, 1999. G-protein coupled metabotropic glutamate receptors (mGluRs) are important modulators of synaptic transmission in the mammalian CNS and have been implicated in various forms of neuroplasticity and nervous system disorders. Increasing evidence also suggests an involvement of mGluRs in nociception and pain behavior although the contribution of individual mGluR subtypes is not yet clear. Subtypes mGluR1 and mGluR5 are classified as group I mGluRs and share the ability to stimulate phosphoinositide hydrolysis and activate protein kinase C. The present study examined the role of group I mGluRs in nociceptive processing and capsaicin-induced central sensitization of primate spinothalamic tract (STT) cells in vivo. In 10 anesthetized male monkeys (Macaca fascicularis) extracellular recordings were made from 20 STT cells in the lumbar dorsal horn. Responses to brief (15 s) cutaneous stimuli of innocuous (BRUSH) and barely and substantially noxious (PRESS and PINCH, respectively) intensity were recorded before, during, and after the infusion of group I mGluR agonists and antagonists into the dorsal horn by microdialysis. Cumulative concentration-response relationships were obtained by applying different concentrations for at least 20 min each (at 5 µl/min). The actual concentrations reached in the tissue are 2-3 orders of magnitude lower than those in the microdialysis fibers (values in this paper refer to the latter). The group I antagonists were also applied at 10-25 min after capsaicin injection. S-DHPG, a group I agonist at both mGluR1 and mGluR5, potentiated the responses to innocuous and noxious stimuli (BRUSH > PRESS > PINCH) at low concentrations (10-100 µM; n = 5) but had inhibitory effects at higher concentrations (1-10 mM; n = 5). The mGluR5 agonist CHPG (1 µM-100 mM; n = 5) did not potentiate but inhibited all responses (10-100 mM; n = 5). AIDA (1 µM-100 mM), a mGluR1-selective antagonist, dose-dependently depressed the responses to PINCH and PRESS but not to BRUSH (n = 6). The group I (mGluR1 > mGluR5) antagonist CPCCOEt (1 µM-100 mM) had similar effects (n = 6). Intradermal injections of capsaicin sensitized the STT cells to cutaneous mechanical stimuli. The enhancement of the responses by capsaicin resembled the potentiation by the group I mGluR agonist S-DHPG (BRUSH > PRESS > PINCH). CPCCOEt (1 mM) reversed the capsaicin-induced sensitization when given as posttreatment (n = 5). After washout of CPCCOEt, the sensitization resumed. Similarly, AIDA (1 mM; n = 7) reversed the capsaicin-induced sensitization and also blocked the potentiation by S-DHPG (n = 5). These data suggest that the mGluR1 subtype is activated endogenously during brief high-intensity cutaneous stimuli (PRESS, PINCH) and is critically involved in capsaicin-induced central sensitization.
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INTRODUCTION |
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G-protein coupled metabotropic glutamate receptors (mGluRs) are
involved in various forms of neuroplasticity and nervous system disorders (Conn and Pin 1997; Knöpfel et
al. 1995
). Eight mGluR subtypes have been cloned to date and
are classified into groups I (mGluRs 1 and 5), II (mGluRs 2 and 3), and
III (mGluRs 4, 6, 7, and 8) based on their sequence homology, agonist
pharmacology, and coupling to intracellular effector systems
(Conn and Pin 1997
). These receptors couple to a variety
of second messenger systems and ion channels to modulate synaptic
transmission and neuronal excitability (Conn and Pin
1997
; Pin and Duvoisin 1995
; Saugstad et
al. 1995
).
An emerging field of research implicates mGluRs in nociception and
hyperalgesia. In the spinal cord, iontophoretic application of a
broad-spectrum mGluR antagonist reduced discharges of rat dorsal horn
neurons sensitized by a knee joint inflammation (Neugebauer et
al. 1994) or by repeated cutaneous administrations of mustard oil (Young et al. 1994
, 1995
). Despite an increasing
interest in the role of mGluRs in different pain models, the
contribution of individual mGluR subgroups and subtypes to spinal
nociceptive processes is not entirely clear yet. Pharmacological data
from subsequent electrophysiological and behavioral studies in rats are
consistent with the involvement of group I mGluRs in the spinal processing of sustained nociceptive input evoked by intraplantar formalin (Fisher and Coderre 1996a
) or carrageenan
(Young et al. 1997
) and by repeated cutaneous
applications of mustard oil (Young et al. 1997
). On the
other hand, a recent study in spinalized rats implies that mGluRs were
not involved in the facilitation of withdrawal reflexes induced by
intraarticular mustard oil (Silva et al. 1997
).
The use of alternative strategies suggests a rather distinct and
heterogeneous role of mGluRs in spinal nociception. Intrathecally applied anti-rat mGluR1 and mGluR5 antibodies reduced cold
hypersensitivity in a model of neuropathic pain but had no significant
effect on heat responses in the plantar test nor on pain behavior in
the formalin test (Fundytus et al. 1998). Intrathecal
application of an mGluR1 antisense oligodeoxynucleotide reagent
increased the tail-flick latency and reduced the responses of dorsal
horn neurons to repeated cutaneous mustard oil applications
(Young et al. 1998
). It is likely therefore that
individual mGluR subtypes play distinct roles in different aspects of nociception.
The present electrophysiological study on primate spinothalamic tract
(STT) cells utilized subtype selective pharmacological agents, which
have became available only recently, to address the role of group I
mGluR subtypes in brief nonnociceptive and nociceptive transmission and
in prolonged nociception evoked by intradermal capsaicin. Intradermal
injection of capsaicin in humans results in primary hyperalgesia to
heat and mechanical stimuli applied near the injection site, and 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
). STT neurons recorded
in anesthetized monkeys develop enhanced responses to cutaneous
mechanical stimuli and increased background activity after intradermal
capsaicin injection (Dougherty and Willis 1992
;
Simone et al. 1991
). CNS changes are believed to underlie capsaicin-induced secondary hyperalgesia and allodynia since
afferent nerve fibers supplying the area outside of primary hyperalgesia zone have not been shown to sensitize (Baumann et al. 1992
; LaMotte et al. 1992
).
The capsaicin-induced sensitization of STT cells can be blocked by
antagonists of N-methyl-D-aspartate (NMDA) and
non-NMDA glutamate receptors and neurokinin 1 receptors
(Dougherty et al. 1992, 1994
; Rees et al.
1998
). Consistent with this is an increased intraspinal release
of glutamate, aspartate (Sluka and Willis 1998
;
Sorkin and McAdoo 1993
) and substance P
(Gamse et al. 1979
) following capsaicin administration.
Second messenger systems also play an important role in
capsaicin-induced spinal sensitization. Recent studies from this
laboratory suggest that GTP-binding proteins (G-proteins) and protein
kinase C (PKC), cAMP-protein kinase A (PKA), and the nitric oxide
activated cGMP-protein kinase G (PKG) pathways are involved in the
sensitization of STT neurons after intradermal capsaicin (Lin et
al. 1996
, 1997
, 1999
; Sluka and Willis 1998
;
Sluka et al. 1997a
,b
; Wu et al. 1998
).
The present study tested the hypothesis that group I mGluRs, particularly the mGluR1 subtype, are involved in nociceptive processes in the spinal cord of primates. 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 I mGluR agonists and antagonists into the dorsal horn by microdialysis. Cumulative concentration-responses curves were measured. The antagonists were also applied 10-25 min after intradermal injections of capsaicin to analyze the role of group I mGluR subtypes in spinal sensitization and the potential usefulness of these agents in the treatment of persistent pain states associated with spinal sensitization.
Preliminary results have been reported in abstract form
(Neugebauer et al. 1998).
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METHODS |
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Animal preparation and anesthesia
Adult male monkeys (Macaca fascicularis,
n = 10, 2.2-2.9 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
sodium pentobarbital (5 mg · kg
1 · 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. The arterial
oxygen saturation, monitored with a rectal oxymeter probe, was kept
between 96 and 100%. The electrocardiogram (ECG) was also monitored.
The level of anesthesia was frequently checked during the experiment by examining pupillary size and reflexes and monitoring
CO2 level and ECG. Core body temperature was kept
at ~37°C using a thermostatically controlled heating blanket.
A laminectomy exposed the lumbar enlargement. The skin flaps overlying the cord were tied back to form a pool. The dura was opened and reflected to expose the cord. The pool was filled with mineral oil, which was kept at 37°C using a heating device. 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 hindlimb.
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 µm inner diameter; wall thickness 9 µm). 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 µl/min. The ACSF was oxygenated and equilibrated to pH 7.4 with a mixture of 95% O2-5% CO2. In the beginning of the experiments, ACSF was pumped through the fiber for at least 2 h to wash out substances released during fiber insertion.
Administration of drugs
The following drugs (concentrations given in parentheses) were
administered by microdialysis at 5 µl/min for 20-30 min through the
fiber closest to the recorded STT neurons (for mGluR subtype selectivity of the compounds see Casabona et al. 1997;
Conn and Pin 1997
; Doherty et al. 1997
;
Palmer et al. 1997
): 1-aminoindan-1,5-dicarboxylic acid
(AIDA, mGluR1 antagonist; 1 µM -100 mM);
7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester
(CPCCOEt, mGluR1 and mGluR5 antagonist; 1 µM -100 mM);
2-chloro-5-hydroxyphenyl-glycine (CHPG, mGluR5 agonist; 1 µM -100
mM); (S)-3,5-dihydroxyphenylglycine (S-DHPG, mGluR1 and mGluR5 agonist;
100 nM -10 mM). The antagonists were also applied from 10 to 25 min
after capsaicin injection (see below). All drugs were dissolved in ACSF
at the indicated concentrations. 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 µl ACSF the concentration of these drugs is ~1-4% of that being
perfused at a rate of 5 µl/min for 30 min (see also Sluka et
al. 1997b
; Sorkin et al. 1988
).
Recording of STT cells
STT neurons were recorded extracellularly in the lumbar
enlargement of the spinal cord using a carbon filament electrode
(resistance: 4 M). Cells were recorded within 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 (2 Hz, 1 mA, 200 µs). Criteria for antidromic
activation were as follows (see Trevino et al. 1973
)
1) Constant latency of the evoked spike. 2)
Ability to follow high-frequency (333-500 Hz) stimulation.
3) Collision of orthodromic spikes with antidromic spikes.
The extracellularly recorded signals were amplified and displayed on
analogue 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. Peristimulus rate histograms were
constructed on-line with Spike-2 software. Digital records of
single-unit activity were also stored for off-line analysis. Throughout
the experiment spike size and configuration was continuously monitored
on the digital oscilloscope and with the use of Spike-2 software to
confirm that the same neuron was recorded and that the relationship of
the recording electrode to the neuron remained constant.
Experimental protocol
Once an STT cell was identified and isolated, the background activity and responses to graded mechanical stimuli were recorded and the receptive field on the skin of 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 1005 g/8 mm2, which is near threshold for pain when applied to human skin), and PINCH (using a small arterial clip to apply 2660 g/4 mm2, which is clearly painful without causing overt damage to the skin). All cells included in this study were wide dynamic range (WDR) STT cells which responded consistently to innocuous stimuli but were more strongly activated by noxious stimuli.
The stimuli were delivered at 3-5 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 every 5-10 min for up to 2 h following 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.
Data analysis
Recorded activity was analyzed off-line from peristimulus time histograms using Spike 2 software. Background activity was subtracted from the evoked responses. The effects of drugs and capsaicin on the responses to cutaneous mechanical stimuli were similar across the receptive field. Therefore the responses evoked from the 3-5 stimulation sites were averaged for each of the mechanical stimuli to analyze drug effects before and after capsaicin. A repeated measures analysis of variance (ANOVA), followed by Newman-Keuls multiple comparison test where appropriate, was used to test for statistical significance of drug effects on capsaicin-induced changes in the responses to the mechanical stimuli. The paired two-tailed t-test was used to compare the effects of an agonist alone and in the presence of an antagonist. Statistical significance was accepted at the level P < 0.05. All averaged values are given as 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 following formula for nonlinear regression (Prism 2.01, GraphPad Software): y = A + (B -A)/[1 + (10C/10X)D], where A = bottom plateau, B = top plateau, C = log(EC50), D = slope coefficient.
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RESULTS |
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Sample of STT neurons
Recordings were made from 20 STT cells of the WDR type in the
lumbar enlargement (L5-L7)
of 10 monkeys. These neurons were recorded at depths of 828-1,606 µm
from the dorsal surface of the spinal cord. According to the
correlation between recording depth and laminar position established in
previous studies from this laboratory (see Lin et al.
1999), 12 neurons were superficial STT cells (828-1,198 µm),
presumably within lamina I, and 8 neurons were deep STT cells
(1,350-1,606 µm), which are found mostly within laminae IV-VI. STT
cells were identified by antidromic stimulation in the VPL nucleus of
the thalamus as described in METHODS. The mean threshold
for antidromic activation was 497 ± 47 µA (150-900 µA); the
mean latency was 6.7 ± 0.4 ms (3.8-10.5 ms). All 20 STT cells
had cutaneous receptive fields on the ipsilateral foot including the
toes in 10 cases. The receptive fields of 11 neurons also included the
lower leg; 6 of these neurons had cutaneous receptive fields in the
knee area. Ongoing activity ranged from 4.8 to 45.9 Hz (mean = 15.9 ± 2.3 Hz). Each cell was recorded within 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.
Activation of group I mGluR1, but not mGluR5, subtype potentiates the responses of STT cells to brief cutaneous mechanical stimuli
The modulation of the responses to BRUSH, PRESS, and PINCH by the
group I mGluR agonist S-DHPG was tested in 5 STT cells. S-DHPG is a
potent agonist at both the mGluR1 and mGluR5 subtypes with
EC50s in the low micromolar range as determined
on cell lines that express the respective subtype (Conn and Pin
1997).
Application of S-DHPG at low concentrations preferentially enhanced the responses to innocuous versus noxious cutaneous stimuli. Figure 1 shows a typical example. The STT neuron was recorded extracellularly in the deep dorsal horn (1,388 µM) of the lumbar enlargement. The STT cell could be activated antidromically by electrical stimulation in the VPL nucleus of the thalamus (see bottom left for high-frequency activation and collision test). Responses to graded mechanical stimulation (BRUSH, PRESS, and PINCH; see METHODS) of the neuron's cutaneous receptive field (see bottom right) were recorded in the presence of normal ACSF administered by microdialysis into the dorsal horn (Fig. 1A). Administration of S-DHPG (100 µM) by microdialysis potentiated the responses to graded mechanical stimuli (BRUSH > PRESS > PINCH, Fig. 1B). The effects of S-DHPG were partially reversible after wash-out with normal ACSF by microdialysis (Fig. 1C).
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Cumulative concentration-response relationships (Fig. 2A) show that S-DHPG had a concentration-dependent biphasic effect: low concentrations (<100 µM) enhanced whereas higher concentrations (1-10 mM) inhibited the responses of STT cells to cutaneous mechanical stimuli (n = 6). Whereas the maximum potentiations of the BRUSH, PRESS, and PINCH responses by S-DHPG were different, the respective EC50s were similar; 6.5 µM (95% confidence interval, 1.6-26.7), 3.7 µM (0.3-49.6), and 3.4 µM (0.1-20.9, see METHODS for details).
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In contrast, the selective group I mGluR5 agonist CHPG (Doherty
et al. 1997) had no potentiating effects but inhibited the responses to graded mechanical stimuli at higher concentrations (1-100
mM). Figure 2B shows the cumulative concentration-response relations measured in 5 STT neurons. The EC50s
for inhibition of BRUSH, PRESS, and PINCH responses by CHPG were not
significantly different; 2.5 mM (95% confidence interval, 0.1-58.7),
3.9 mM (0.6-28.0), and 3.4 mM (0.1-93.6, see METHODS for details).
Effects of group I mGluR antagonists on the responses of STT cells to brief cutaneous stimuli
Two group I mGluR antagonists of different subtype selectivity
were applied into the spinal dorsal horn by microdialysis to examine
the possible endogenous activation of these mGluRs by brief cutaneous
mechanical stimuli: AIDA, a competitive mGluR1 subtype selective
antagonist, and CPCCOEt, a noncompetitive group I mGluR antagonist with
a higher potency at mGluR1 than mGluR5 (Casabona et al.
1997; Conn and Pin 1997
).
AIDA had differential effects on the responses to graded mechanical stimuli. Figure 3 shows extracellularly recorded spikes of one STT neuron in the lumbar spinal dorsal horn (depth 1,134 µM), which could be activated antidromically by electrical stimulation in the VPL nucleus of the thalamus (see bottom left for high-frequency activation and collision test). The neuron's responses to brief graded mechanical stimulation (BRUSH, PRESS, and PINCH; see METHODS) of the cutaneous receptive field (see bottom right) were recorded in the presence of normal ACSF administered by microdialysis into the dorsal horn (Fig. 3A). AIDA (1 mM) inhibited the responses to PINCH more than to PRESS but had no effect on the responses to the innocuous BRUSH stimulus (Fig. 3B). The effects of AIDA were reversible after wash-out with normal ACSF by microdialysis (Fig. 3C).
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Cumulative concentration-response relationships were determined for AIDA (mGluR1 antagonist, n = 6, Fig. 4A) and CPCCOEt (antagonist at mGluR1 and mGluR5, n = 6, Fig. 4B). Each antagonist inhibited the responses of STT cells to high-intensity mechanical stimuli (PINCH > PRESS), but not the innocuous BRUSH-evoked responses, in a concentration-dependent manner. No differences were found between the antagonists with regard to their maximum effects, suggesting that block of the mGluR1 subtype is sufficient to produce the observed inhibition. A slight but nonsignificant difference in potency of the antagonists was noted. AIDA inhibited PINCH and PRESS responses with EC50s of 670 µM (95% confidence interval, 376 µM -1.29 mM) and 625 µM (51.5 µM -7.57 mM), respectively (see METHODS for details). The EC50s for CPCCOEt-mediated inhibition of PINCH and PRESS responses were 301 µM (95% confidence interval, 99.5-914 µM) and 462 µM (32.4 µM -6.59 mM, see METHODS).
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Effects of group I mGluR antagonists on capsaicin-induced spinal sensitization
The involvement of group I mGluRs in central sensitization was studied by the spinal administration of the mGluR1 antagonist AIDA (1 mM, n = 7) and the group I mGluR1,5 antagonist CPCCOEt (1 mM, n = 5) into the dorsal horn at 10-15 min after intradermal capsaicin (3%) injection. Each antagonist was able to reverse the enhanced responses to BRUSH and PRESS following capsaicin.
Figure 5 shows a typical example.
Extracellular recordings were made from an STT neuron in the lumbar
spinal dorsal horn (1,132 µM). Brief (15 s) graded mechanical stimuli
(BRUSH, PRESS, and PINCH) were applied to the skin of the neuron's
receptive field. In agreement with previous studies from this
laboratory (Dougherty and Willis 1992; Simone et
al. 1991
), intradermal injection of capsaicin (3%) clearly
enhanced the responses to BRUSH and PRESS; only a slight increase of
the PINCH-evoked responses was observed. Administration of AIDA (1 mM)
into the dorsal horn by microdialysis reduced the enhanced responses to
the control level before capsaicin. The responses increased again after
washout of drug with normal ACSF and returned to baseline at ~ 2 h after capsaicin.
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Figure 6A summarizes the data for AIDA. Intradermal capsaicin (3%) potentiated the responses of STT cells to BRUSH (P < 0.001), PRESS (P < 0.01), and PINCH (P < 0.05, repeated measures ANOVA followed by Newman-Keuls multiple comparison test). AIDA (1 mM, n = 7) significantly reduced the enhanced responses to BRUSH (P < 0.001), PRESS (P < 0.001), and PINCH (P < 0.01, repeated measures ANOVA followed by Newman-Keuls multiple comparison test) following capsaicin. After wash-out of the antagonist, the responses increased again significantly at 60 min post capsaicin, suggesting that the inhibition by AIDA was because of receptor block rather than the normal decay of capsaicin-induced sensitization.
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A similar result was obtained with CPCCOEt (Fig. 6B). In this sample of neurons, intradermal capsaicin (3%) injection significantly potentiated the BRUSH (P < 0.001) and PRESS (P < 0.01) but not the PINCH responses (P > 0.05, repeated measures ANOVA followed by Newman-Keuls multiple comparison test, see METHODS for details). CPCCOEt (1 mM; n = 5) administered into the dorsal horn by microdialysis 10-25 min after capsaicin injection, reduced the responses to BRUSH (P < 0.001), PRESS (P < 0.01), and PINCH (P < 0.05, repeated measures ANOVA followed by Newman-Keuls multiple comparison test). The inhibitory effects of CPCCOEt were reversible. The similarity of the inhibitory effects obtained with each antagonist suggest that the inhibition of capsaicin-induced spinal sensitization is mediated through the mGluR1 subtype.
The mGluR1 antagonist AIDA blocks the effects of the group I mGluR agonist S-DHPG but not of the mGluR5 agonist CHPG
The antagonist AIDA was also coapplied with the agonists S-DHPG and CHPG to ensure the subtype selectivity of AIDA and increase the evidence that it is in fact the mGluR1 subtype that mediates the potentiation by the group I agonist S-DHPG and the block of capsaicin-induced central sensitization by AIDA. Figure 7A shows a typical example. The BRUSH, PRESS, and PINCH responses of one STT neuron in the lumbar enlargement (depth 925 µm) were recorded before and during the spinal administrations of the following drugs (20 min each): S-DHPG, S-DHPG together with AIDA, CHPG, and CHPG together with AIDA. The mGluR1 antagonist AIDA (1 mM) blocked the potentiating effects of the group I agonist S-DHPG (10 µM). However, AIDA (10 mM) had no effect on the inhibition of the responses by the mGluR5 agonist CHPG (10 mM).
|
Figure 7B summarizes the data obtained with AIDA and S-DHPG. The potentiation of the BRUSH, PRESS, and PINCH responses by S-DHPG (10 µM, n = 5) was blocked during coapplication of the mGluR1 antagonist AIDA (1 mM, n = 5), suggesting that the effects of S-DHPG were due mainly to the activation of the mGluR1 subtype.
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DISCUSSION |
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The present study is the first to address pharmacologically the role of mGluR subtypes in brief and prolonged nociceptive processing or in primate STT cells. The main findings are as follows. 1) The mGluR1,5 agonist S-DHPG, but not the mGluR5 agonist CHPG, enhanced the responses of STT cells to brief cutaneous mechanical stimuli (BRUSH > PRESS > PINCH), suggesting that this potentiation was because of activation of mGluR1 but not mGluR5. 2) The group I mGluR antagonists AIDA (mGluR1) and CPCCOEt (mGluR1,5) inhibited the responses of STT cells to noxious PINCH and PRESS but not to innocuous BRUSH which suggests that in the spinal cord of primates the mGluR1 subtype may be activated endogenously during brief noxious but not during innocuous stimulation. 3) Intradermal capsaicin enhanced the responses of STT cells to brief cutaneous stimuli in a fashion that resembled the potentiating effects of S-DHPG, i.e., BRUSH > PRESS > PINCH. 4) Posttreatment with the mGluR1 antagonist AIDA and the mGluR1,5 antagonist CPCCOEt reversed the capsaicin-induced central sensitization. 5) The mGluR1 antagonist AIDA also antagonized the potentiation by the mGluR1,5 agonist S-DHPG. Taken together, these data suggest that it is the mGluR1 subtype that is activated endogenously during brief high-intensity cutaneous stimuli (PRESS and PINCH) and that this receptor is critically involved in capsaicin-induced central sensitization of STT neurons in primates.
The drug concentrations applied in this study by microdialysis (low
micromolar to low millimolar range) are well within in the
concentrations shown to be selective in in vitro studies (see Casabona et al. 1997; Conn and Pin 1997
;
Doherty et al. 1997
; Palmer et al. 1997
).
We have measured the amount of drug diffusing across the dialysis
membrane in vitro to be only 1-4% of the perfused concentration (see
METHODS). This concentration probably 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 (see Sluka et al. 1997b
;
Sorkin et al. 1988
). The "effective" concentrations
used in this study were estimated to be in the nanomolar to upper
micromolar range (see METHODS).
An increasing body of evidence supports a role for mGluRs in spinal
nociceptive transmission. Using intraspinal applications of
broad-spectrum mGluR antagonists, the first electrophysiological studies of the role of mGluRs in spinal nociception suggested that
mGluRs may be involved in prolonged nociceptive mechanisms but not in
the processing of innocuous information (Neugebauer et al.
1994; Young et al. 1994
, 1995
). Since then,
behavioral and electrophysiological studies have implicated in
particular group I mGluRs in spinal nociceptive responses evoked by
intraplantar formalin (Fisher and Coderre 1996a
) or
carrageenan (Young et al. 1997
), by repeated cutaneous
applications of mustard oil (Young et al. 1997
, 1998
),
and in a model of neuropathic pain (Fundytus et al.
1998
). Further, spontaneous nociceptive behaviors could be
produced by the mGluR1/5 agonist RS-DHPG but not by the relatively selective mGluR5 agonist trans-azetidine (Fisher and
Coderre 1996b
).
Analysis of the role of individual mGluR subtypes in spinal nociception
has been hampered, until now, by the lack of subtype-selective agents.
Using an alternative strategy, a recent study found that intrathecal
infusion of a mGluR1 antisense oligodeoxynucleotide reagent increased
tail-flick latencies. In addition, in treated rats the percentage of
dorsal horn neurons activated by the group I mGluR1,5 agonist DHPG, but
not the mGluR5 agonist trans-azetidine-2,4-dicarboxylic acid, was smaller than in control rats. Further, the mean number of
spikes evoked by repeated topical applications of mustard oil, but not
those evoked by innocuous brush, was lower in dorsal horn neurons in
antisense reagent-treated rats than in normal controls or in sense
reagent-treated and in mismatch reagent-treated rats (Young et
al. 1998). This approach did not, however, allow a direct comparison of the effects of mGluR1 blockade on the nociceptive responses of the same neuron recorded before and after treatment. This
was accomplished in the present study, which provides direct pharmacological evidence for the involvement of the mGluR1 subtype in
the responses of STT neurons to brief nociceptive stimuli and in
capsaicin-induced central sensitization.
One particularly interesting finding is that the mGluR1
antagonist AIDA reduced the enhanced responses to innocuous BRUSH following intradermal capsaicin but not under "normal" conditions where AIDA reduced only PRESS and PINCH responses. Different mechanisms may account for this change including increased glutamate release and
subsequently enhanced endogenous activation of the mGluR1, up-regulation of mGluR1 receptors, increased receptor sensitivity, alterations at the level of the second messengers that are activated by
mGluR1, and interaction with ionotropic GluRs such as NMDA and non-NMDA
GluRs. Intradermal capsaicin has been shown to increase the intraspinal
release of excitatory amino acids such as glutamate (Sluka and
Willis 1998; Sorkin and McAdoo 1993
) which may
result in the enhanced endogenous activation of mGluRs and stronger
inhibitory effects of mGluR antagonists on evoked responses of
sensitized STT cells. In vitro studies have provided evidence for the
up-regulation of mGluR subtypes following treatment with various growth
factors (Miller et al. 1995
; Nakahara et al.
1997
), in kindling-induced epileptogenesis (Holmes et
al. 1996
), in kainic acid induced status epilepticus
(Aronica et al. 1997
), and following chemical
deafferentiation (Casabona et al. 1998
). Increased mGluR
sensitivity has been shown in vitro following kindling-induced
epileptogenesis in vivo and chronic cocaine treatment in vivo
(Neugebauer et al. 1997a
,b
).
Second messenger systems play an important role in
capsaicin-induced central sensitization of STT cells. Recent
studies from this laboratory suggest that GTP-binding proteins
(G-proteins) and protein kinase C (PKC), cAMP-protein kinase A (PKA),
and the nitric oxide activated cGMP-protein kinase G (PKG) pathways are involved in the sensitization of STT neurons after intradermal capsaicin (Lin et al. 1996, 1997
, 1999
; Sluka et
al. 1997a
,b
; Wu et al. 1998
). Activation of
presynaptic group I mGluRs such as mGluR1 enhances glutamate release
via a mechanism involving protein kinase C-mediated inhibition of
presynaptic K+ channels, and this facilitation is
enhanced by arachidonic acid (Conn and Pin 1997
;
Pin and Duvoisin 1995
). Arachidonic acid release has
been shown to increase following co-activation of mGluRs and AMPA [(R,
S)-
-amino-3-hydroxy-5-methyl-4-isoxazole-propionate] receptors in
striatal neurons (see Conn and Pin 1997
). Postsynaptic group I mGluRs also mediate slow depolarization and an increase in cell
firing through a direct G-protein mediated depression of potassium
currents IK(AHP) and IK(M)
(Pin and Duvoisin 1995
). Further, activation of group I
mGluRs, especially mGluR1, can increase cAMP accumulation in the
hippocampus (Winder et al. 1993
; see also Conn
and Pin 1997
for other cell types) and mGluR1 activation can
increase cGMP levels in the cerebellum (Okada 1992
).
Therefore in capsaicin-induced central sensitization, a vicious cycle
may exist between enhanced activation of mGluR1 and mGluR1-activated second messenger systems resulting in increased glutamate release.
Finally, postsynaptic group I mGluRs can modulate both AMPA and NMDA
receptor-mediated currents in various neuronal populations (Aniksztejn et al. 1991; Aronica et al.
1993
; Fitzjohn et al. 1996
; Rahman and
Neuman 1996
) and in the spinal cord (Bleakman et al.
1992
; Cerne and Randic 1992
) in vitro.
Similarly, activation of mGluRs can potentiate the responses of dorsal
horn neurons to NMDA and AMPA receptor activation in vivo
(Neugebauer et al. 1994
) and this effect is mediated
through group I mGluRs (Bond and Lodge 1995
;
Jones and Headley 1995
). Behavioral studies suggest that
group I agonists interact with NMDA and/or non-NMDA receptors to
produce enhanced nociceptive responses and hyperalgesia (Coderre and Melzack 1992
; Fisher and Coderre 1996b
;
Meller et al. 1993
, 1996
).
In this study, the mGluR1,5 agonist S-DHPG, but not the mGluR5
selective agonist CHPG, potentiated the responses of STT neurons to
brief innocuous and noxious mechanical stimulation, suggesting the
involvement of the mGluR1 subtype. Interestingly, the capsaicin-induced potentiation of the responses was remarkably similar to the effects mediated through the activation of the mGluR1 subtype, i.e., the rank
order was BRUSH > PRESS > PINCH (cf. Fig. 2A and
Fig. 6). As outlined above, several mechanisms may underlie the
capsaicin-induced potentiation, and this may also be true for the
S-DHPG mediated effects. A somewhat surprising finding is the
inhibition of the BRUSH, PRESS, and PINCH responses by high
concentrations of S-DHPG as well as CHPG, which was not blocked by the
mGluR1 antagonist AIDA (Fig. 7), suggesting that this inhibitory effect
may be mediated through the mGluR5 subtype. Despite the heterogeneity
of mGluRs, it has not yet been shown that subtypes (mGluR1 and mGluR5)
of one mGluR subgroup (group I) exert opposite effects in the same brain area or synapse. It is well established however, that group I
mGluRs can inhibit synaptic transmission and decrease cell excitability through inhibition of glutamate release and L- and N-type calcium currents (see Conn and Pin 1997). Pharmacological and
immunocytochemical studies suggest that the mGluR5 subtype serves as an
autoreceptor to inhibit synaptic transmission in hippocampal area CA1
(Manzoni and Bockaert 1995
; Romano et al.
1995
). If in fact mGluR1 and mGluR5 subtypes exert opposite
effects on the same class of neurons (STT cells), then this finding may
add further evidence for a role of mGluRs in the fine-tuning of
synaptic transmission and neuronal excitability.
In conclusion, a variety of mechanisms at the synaptic, receptor and/or intracellular levels may account for the potentiating effects of the group I agonist S-DHPG and the (enhanced) inhibitory effects of the mGluR1 antagonist in capsaicin-induced sensitization. Because the mGluR1 antagonist did not affect the responses to brief innocuous BRUSH stimuli but reversed prolonged nociceptive processing, mGluR1s may be useful targets for the developing of drugs for the relief of persistent pain associated with central sensitization.
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ACKNOWLEDGMENTS |
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We thank K. Gondesen and G. Robak for excellent technical assistance.
This work was supported by National Institutes of Health Grants NS-09743 and MH-10322.
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FOOTNOTES |
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Address for reprint requests: W. D. Willis, Dept. of Anatomy and Neurosciences and Marine Biomedical Institute, The University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1069.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 22 January 1999; accepted in final form 17 March 1999.
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REFERENCES |
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