Role of Metabotropic Glutamate Receptor Subtype mGluR1 in Brief Nociception and Central Sensitization of 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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 MOmega ). 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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1. S-DHPG, a group I G-protein coupled metabotropic glutamate receptor (mGluR1 and mGluR5) agonist, potentiates the responses of a primate spinothalamic tract (STT) neuron to brief mechanical stimulation of the cutaneous receptive field (bottom right). Extracellular recordings from a STT neuron of the wide dynamic range (WDR) type in the dorsal horn (1388 µM) of the lumbar enlargement. The STT cell could be activated antidromically by electrical stimulation (T = 450 µA) in the VPL nucleus of the thalamus, i.e., the unit followed a high-frequency (333 Hz) stimulus (bottom left) and showed collision of an orthodromic action potential with the antidromic spike (down-arrow , bottom trace). Bin width of the histograms in A-C is 0.1 s. A: responses to brief (15 s) graded mechanical stimuli (BRUSH, PRESS, and PINCH) applied to the receptive field in the presence of normal ACSF administered by microdialysis into the dorsal horn. B: administration of S-DHPG (100 µM) by microdialysis potentiated the responses to mechanical stimuli. C: effects of DHPG were partially reversible after wash-out with normal ACSF by microdialysis.

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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Fig. 2. S-DHPG (agonist at mGluR1 and mGluR5), but not CHPG (agonist at mGluR5), potentiates brief nonnociceptive and nociceptive responses of STT cells. A: low concentrations of S-DHPG administered into the dorsal horn by microdialysis, enhance the BRUSH, PRESS, and PINCH responses whereas higher concentrations have inhibitory effects. Cumulative concentration-response relationships were obtained from 5 WDR STT neurons in the lumbar spinal dorsal horn (depths 828-1,388 µM). B: low concentrations of CHPG have no effect whereas higher concentrations inhibit the responses to BRUSH, PRESS, and PINCH stimuli. Cumulative concentration-response relations were measured in 5 STT neurons in the lumbar spinal dorsal horn (depths 930-1,560 µM). A and B: cumulative concentration-response relations were constructed by averaging the mean frequency of the responses for each concentration of S-DHPG and CHPG, respectively; the averages were then expressed as percentage of predrug control values (set to 100%); the EC50s were calculated using the formula given in METHODS.

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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Fig. 3. The mGluR1 antagonist AIDA inhibits the PINCH and PRESS responses. Extracellular recordings from one STT neuron in the lumbar spinal dorsal horn (depth 1,134 µM). The STT cell could be activated antidromically by electrical stimulation (T = 250 µA) in the VPL nucleus of the thalamus (bottom left for high-frequency activation and collision test). Brief (15 s) graded mechanical stimuli were applied to the skin of the neuron's receptive field (bottom right). 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 AIDA (1 mM dissolved in ACSF) by microdialysis inhibited the responses to PINCH and PRESS but not to BRUSH. C: effects of AIDA were partially reversible after wash-out with normal ACSF by microdialysis.

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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Fig. 4. AIDA (A, mGluR1 antagonist) and CPCCOEt (B, antagonist at mGluR1 and mGluR5) inhibit the responses to PINCH and PRESS but not BRUSH in a concentration-dependent manner. Cumulative concentration-response relations were obtained from 6 WDR STT cells recorded at 924-1,476 µM in the dorsal horn (A) and from 6 WDR STT cells recorded at 950-1,546 µM in the dorsal horn (B). A and B: cumulative concentration-response relations were constructed by averaging the mean frequency of the responses for each concentration of AIDA and CPCCOEt, respectively; the averages were then expressed as percentage of predrug control values (set to 100%); the EC50s were calculated using the formula given in METHODS. The similar maximum inhibition obtained with each antagonist indicates that the observed effects may be mediated through the mGluR1 subtype.

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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Fig. 5. The mGluR1 antagonist AIDA reverses capsaicin-induced sensitization of an STT cell. Extracellular recordings from one WDR 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. Intradermal injection of capsaicin (3%) strongly enhanced the responses to BRUSH and PRESS. Administration of AIDA into the dorsal horn by microdialysis reversed the capsaicin-induced potentiation. The responses increased again after washout of drug with normal ACSF and returned to baseline at ~ 2 h after capsaicin. Each symbol represents the number of spikes per stimulus (15 s). Ongoing activity has been subtracted from the evoked response.

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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Fig. 6. Both AIDA (mGluR1 antagonist) and CPCCOEt (antagonist at mGluR1 and mGluR5) reverse capsaicin-induced sensitization of STT cells. A: AIDA (1 mM, n = 7) administered into the dorsal horn by microdialysis 10-25 min after capsaicin injection, reduced the capsaicin-induced potentiation of the responses to cutaneous mechanical stimuli. B: CPCCOEt (1 mM, n = 5) administered by microdialysis 10-25 min after capsaicin injection, also reversed the capsaicin-induced increase in the responses to mechanical stimuli. The similarity of the inhibitory effects obtained with each antagonist suggests that the inhibition of capsaicin-induced spinal sensitization involves the mGluR1 subtype. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with control; +P < 0.05, ++P < 0.01, and +++P < 0.001 compared with 15 min postcapsaicin (repeated measures analysis of variance (ANOVA) followed by Newman-Keuls multiple comparison test).

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).



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Fig. 7. The mGluR1 antagonist AIDA blocks the potentiating effects of S-DHPG (agonist at mGluR1 and mGluR5) but not the inhibition by CHPG (mGluR5 agonist). A: extracellular recordings from one WDR STT neuron in the lumbar spinal dorsal horn (depth 925 µM). Brief (15 s) graded mechanical stimuli (BRUSH, PRESS, and PINCH) were applied to the skin of the neuron's receptive field. S-DHPG (10 µM, 20 min) administered into the dorsal horn by microdialysis reversibly enhanced the responses but had no effect in the presence of AIDA (1 mM, 20 min). CHPG (10 mM, 20 min) reversibly reduced the responses. AIDA (10 mM, 20 min) did not block the effects of CHPG. Each symbol represents the number of spikes per stimulus (15 s). Ongoing activity has been subtracted from the evoked response. B: the potentiation of BRUSH, PRESS, and PINCH responses by S-DHPG (10 µM, n = 5) was blocked when the mGluR1 antagonist AIDA (1 mM) was administered together with DHPG (n = 5). Drugs were administered into the dorsal horn by microdialysis for 20 min. *P < 0.05, paired two-tailed t-test.

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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)-alpha -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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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