Peripheral group I metabotropic glutamate receptors ... - Nature

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Peripheral group I metabotropic glutamate receptors modulate nociception in mice G. Bhave1, F. Karim1, S. M. Carlton2 and R. W. Gereau IV1 1 Division of Neuroscience, Baylor College of Medicine, 1 Baylor Plaza, Room S636, Houston, Texas 77030, USA 2 Department of Anatomy and Neurosciences, Marine Biomedical Institute, 301 University Boulevard, Galveston, Texas 77555, USA

The first two authors contributed equally to this work Correspondence should be addressed to R.W.G. ([email protected])

The metabotropic glutamate receptors (mGluRs) are found throughout the central nervous system, where they modulate neuronal excitability and synaptic transmission. Here we report the presence of phospholipase C-coupled group I mGluRs (mGluR1 and mGluR5) outside the central nervous system on peripheral unmyelinated sensory afferents. Given their localization on predominantly nociceptive afferents, we investigated whether these receptors modulate nociceptive signaling, and found that agonist-induced activation of peripheral group I mGluRs leads to increased sensitivity to noxious heat, a phenomenon termed thermal hyperalgesia. Furthermore, group I mGluR antagonists not only prevent, but also attenuate established formalin-induced pain. Taken together, these results suggest that peripheral mGluRs mediate a component of hyperalgesia and may be therapeutically targeted to prevent and treat inflammatory pain.

Glutamate is the predominant excitatory neurotransmitter in the central nervous system (CNS), and acts through ligandgated ion channels (ionotropic glutamate receptors, iGluRs) and G protein-coupled mGluRs. The function of glutamate in the peripheral nervous system is not well understood. iGluR subunits have been identified on thin, unmyelinated nociceptive fibers in the skin1,2. Subcutaneous injection of glutamate and iGluR agonists into the rat hind paw results in a reduction of thermal and mechanical thresholds1,3,4. Peripherally applied iGluR antagonists attenuate nociceptive scores during the formalin test, a model used to study inflammatory pain in rodents5. The concentration of glutamate rises in the skin with sciatic nerve stimulation and during the formalin test in rats6,7 and in synovial fluid samples from arthritis patients8. Taken together, these data suggest that glutamate is a true peripheral inflammatory mediator released after tissue injury, and suggest that peripheral glutamate activates iGluRs. The importance of mGluRs in the periphery has not been examined. mGluRs are widely expressed in the CNS, where they function to modulate neuronal excitability and synaptic transmission. They are divided into three groups based on pharmacology, signal transduction and sequence homology. Group I mGluRs consist of mGluR1 and mGluR5, and couple primarily to activation of phospholipase C (PLC), resulting in release of Ca2+ from intracellular stores and activation of protein kinase C (PKC)9. Thus far, most studies on the function of mGluRs in nociception have concentrated on the spinal cord dorsal horn. Physiological studies have found that group I mGluRs are critical in inflammation-induced hyperexcitability of spinal cord neurons10,11. Behavioral studies indicate that intrathecal group I mGluR agonists induce hyperalgesia and spontaneous nociceptive behaviors 12,13 , and intrathecal nature neuroscience • volume 4 no 4 • april 2001

group I mGluR antagonists reduce inflammatory and neuropathic pain (F.K., C. Wang & R.W.G., unpublished findConsistent with these findings, ings) 14–16 . immunocytochemical studies have localized group I mGluRs to the superficial dorsal horn17–20. These studies show that group I mGluRs are important in spinal central sensitization after tissue injury. However, given that glutamate functions as a peripheral inflammatory mediator, it is possible that mGluRs also modulate primary afferent activity in the periphery. mGluR5 mRNA expression has been found in neonatal rat dorsal root ganglia (DRG)21, and mGluR5 protein is expressed in all small and most medium diameter DRG neurons in adult rats19. In this study, we investigated the role of peripheral group I mGluRs in thermal nociception and inflammatory pain. First, we showed that mGluR1a and mGluR5 are expressed on unmyelinated nociceptive peripheral afferents. Second, peripheral injection of a selective group I mGluR agonist increased thermal sensitivity through activation of mGluR1 and mGluR5. Finally, peripherally applied group I mGluR antagonists blocked glutamate-induced thermal hypersensitivity and attenuated nociceptive scores in the formalin model of inflammatory pain.

RESULTS Localization of peripheral group I mGluRs To test our hypothesis that glutamate is an inflammatory mediator that exerts its actions via group I mGluRs, we first investigated whether mGluR1a and mGluR5 are expressed in the periphery on cutaneous primary afferents. Electron microscopy of the hind paw dermal–epidermal junction reveals bundles of unmyelinated axons, which are either primary afferent fibers or sympathetic efferents. Because previous work using tyrosine hydroxylase staining has 417


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found no evidence for sympathetic efferents in this preparation, these axons are mostly nociceptive primary afferent fibers22. Therefore, we conducted immunoelectron microscopy of the hind paw dermal–epidermal junction to examine mGluR1a and mGluR5 expression on predominantly nociceptive primary afferent fibers. Counts of labeled profiles demonstrated that 8.9 ± 0.9% and 16 ± 1.2% (mean ± s.d.; n = 4) of the unmyelinated axons at the dermal–epidermal junction were positively stained with the mGluR1a and mGluR5 antisera, respectively. Given that our counting method was designed to protect against double counting and the punctate localization of immunoreactivity (Fig. 1), it should be noted that these estimates are relatively conservative. Peripheral mGluR1 and 5 modulate thermal sensitivity Because mGluR1a and mGluR5 are expressed in a subset of predominantly nociceptive afferents, we examined whether activation of these receptors affects thermal nociception. We injected RS-DHPG, a selective group I mGluR agonist23,24, into the hind paw and monitored withdrawal latencies to thermal stimuli. Injection of RS-DHPG (50 nmol) into the mouse hindpaw resulted in a significant reduction in thermal withdrawal latency for 2 hours (Fig. 2a). This effect is dose dependent because RS-DHPG (10 nmol) caused a smaller, but significant reduction in thermal latency (Fig. 2b). Similar results were also obtained with S-DHPG, the enantiomer active at group I mGluRs25 (data not shown). Consistent with our hypothesis that a DHPG acts via group I mGluRs, selective mGluR1 and mGluR5 antagonists attenuated DHPG-induced thermal hypersensitivity. Subcutaneous injection of MPEP (30 nmol), a selective mGluR5 antagonist26, 15 minutes before DHPG injection, abolished DHPG-induced thermal hypersensitivity (Fig. 3a and b). To establish whether MPEP was acting locally or at a distant CNS site26, MPEP was injected in the contralateral hind paw 15 minutes before injection

Fig. 1. Localization of mGluR5 and mGluR1a on unmyelinated peripheral afferents. Electron micrographs represent cross-sections through axon fascicles at the dermal–epidermal junction of mouse hind paw. mGluR5 (a) and mGluR1a (b) immunoreactivity is localized at discrete sites along axonal membranes (arrows). Unlabeled axons can also be observed (*). Schwann cell processes do not completely envelop the mGluR1a- or mGluR5-labeled axons (between the arrowheads) exposing the axonal membrane directly to the surrounding basement membrane. Scale bar, 0.25 µm.

of DHPG. This contralateral injection had no effect, suggesting that MPEP acts at the site of injection (data not shown). Subcutaneous injection of CPCCOEt (100 nmol), a noncompetitive mGluR1 antagonist27, also reduced DHPG-induced thermal hypersensitivity (Fig. 3c and d). Unlike MPEP, CPCCOEt did not completely block DHPG-induced thermal hypersensitivity. Because CPCCOEt can block mGluR5 responses at higher concentrations28, we were concerned that CPCCOEt may be acting at mGluR5. To confirm the selectivity of CPCCOEt for mGluR1, we examined the effect of LY367385, a structurally unrelated, competitive mGluR1 selective antagonist29, on DHPG-induced thermal hypersensitivity. Injection of LY367385 (100 nmol) before DHPG injection also significantly decreased the effects of DHPG (Fig. 3d). Thus, our data suggest that both mGluR1 and mGluR5 are necessary to produce DHPG-induced thermal hypersensitivity. The group I mGluR antagonists did not affect baseline withdrawal latencies in the absence of DHPG (Fig. 3). To rule out a possible contribution of NMDA receptors to DHPG-induced thermal hypersensitivity30, we injected APV, an NMDA receptor antagonist. Subcutaneous injection of APV (50 nmol) before DHPG injection had no effect on DHPG-induced modulation of thermal latencies (Fig. 4a). The involvement of other mGluRs was tested using MPPG (500 nmol), a group II and III mGluR antagonist. MPPG had no effect on DHPG-induced thermal hypersensitivity (Fig. 4b). The APV and MPPG doses were maximized given pH, solubility and injection volume restrictions. Taken together, these data suggest that DHPG is most likely sensitizing thermal nociception via activation of group I mGluRs. Consistent with previous reports1,3, we found that peripherally applied glutamate, the endogenous agonist of mGluRs, also increases thermal sensitivity. Injection of MPEP (30 nmol) or CPCCOEt (100 nmol) 15 minutes before glutamate injection completely

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Fig. 2. The group I mGluR agonist, RS-DHPG, enhances thermal sensitivity. (a) 50 nmol RS-DHPG caused a significant reduction in thermal withdrawal latency for 150 min (n = 6, p < 0.01). Asterisks indicate time points at which DHPG was significantly different from vehicle treatment (p < 0.05). (b) RS-DHPG-induced thermal hyperalgesia was dose dependent. Thermal withdrawal latencies measured from 15–120 min after injection were averaged and expressed as a percent of the baseline response (vehicle, n = 6; 10 nmol DHPG, n = 8; 50 nmol DHPG, n = 6; *p < 0.05 when compared to zero dose).

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Fig. 3. DHPG-induced thermal hypersensitivity is attenuated by pretreatment with mGluR5 and mGluR1 antagonists. (a) MPEP (mGluR5 antagonist, 30 nmol) blocks thermal hypersensitivity induced by RS-DHPG (50 nmol, n = 8). MPEP/DHPG was significantly different from vehicle/DHPG (p < 0.01). Asterisks indicate time points at which MPEP/DHPG was significantly different from vehicle/DHPG (p < 0.05). (b) Thermal withdrawal latencies from 15–120 minutes after DHPG injection were averaged and expressed as a percent of the baseline response (Vehicle, n = 8; 3 nmol MPEP, n = 3; 30 nmol MPEP, n = 8; *p < 0.05 when compared to the zero dose). (c) CPCCOEt (mGluR1 antagonist, 100 nmol) reduced DHPG-induced thermal hypersensitivity (n = 8). A significant difference was found between CPCCOEt/DHPG and vehicle/DHPG (p < 0.01) as well as between CPCCOEt/vehicle and CPCCOEt/DHPG (p < 0.05). Asterisks represent time points at which CPCCOEt/DHPG was significantly different from vehicle/DHPG; cross denotes time point at which CPCCOEt/DHPG significantly differed from CPCCOEt/vehicle (p < 0.05). (d) Effects of mGluR1 antagonists on thermal withdrawal latencies after DHPG injection. Responses from 15–120 min were averaged and expressed as a percent of the baseline response (CPCCOEt, 0 nmol, n = 8; 10 nmol, n = 3; 100 nmol, n = 8; LY367385, 0 nmol, n = 8; 100 nmol n = 6; *p < 0.05 when compared to the corresponding zero dose).

blocked glutamate-induced thermal hypersensitivity (Fig. 5). This suggests that glutamate released during inflammation may activate group I mGluRs leading to thermal hyperalgesia. Group mGluR1 antagonists attenuate inflammatory pain To test whether group I mGluRs are involved in injury-induced hyperalgesia we examined the effect of group I mGluR antagonists on nociceptive scores during the formalin test, a model for inflammatory pain. Subcutaneous injection of 2% formalin resulted in a typical biphasic nociceptive response involving easily quantifiable spontaneous behaviors, such as lifting and licking of the injected paw. The first phase of the formalin test is commonly attributed to acute nociception occurring in response to primary afferent activity, whereas the second phase is attributed to tonic nociception probably resulting from a combination of peripheral activity, peripheral sensitization and central sensitization 31–34. Subcutaneous injection of either MPEP (30 nmol) or CPCCOEt (100 nmol) before formalin injection in the same paw resulted in a significant reduction in the second phase of the nociceptive response with no effect on the first phase (Fig. 6a and b). Injection of MPEP in the contralateral paw did not affect formalininduced nociceptive behavior, ruling out a central locus of action (data not shown). Co-injection of MPEP and CPCCOEt 15 minutes before formalin injection in the same paw diminished the second phase response as well as either drug injected alone (Fig. 6c). nature neuroscience • volume 4 no 4 • april 2001

The equal inhibition by MPEP, CPCCOEt and both antagonists together is consistent with the idea that either drug can completely inhibit peripheral glutamatergic signaling as shown in Figure 5. The fact that the mGluR antagonists do not completely eliminate formalin-induced behaviors likely reflects the involvement of other inflammatory mediators in the second phase response35. A selective reduction in the second phase of the formalin test suggests that peripheral group I mGluRs may be involved in the induction or maintenance of peripheral sensitization. To examine whether group I mGluR activation occurs during the second phase, we injected MPEP (30 nmol) or CPCCOEt (100 nmol) after the first phase. Both MPEP and CPCCOEt administered after the first phase significantly and equally attenuated nociceptive scores (Fig. 7). Co-injection of the antagonists also equally reduced the second phase, again suggesting that either drug can inhibit the mGluR-dependent component of the second phase response. Therefore, group I mGluRs contribute to the maintenance and, possibly, the induction of peripheral processes involved in the second phase of the formalin test.

DISCUSSION The present study provides anatomical and behavioral evidence that group I mGluRs on primary peripheral afferents can modulate thermal nociception and inflammatory pain in mice. Immunoelectron microscopy at the dermal–epidermal junction 419


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identified mGluR1a and mGluR5 expression on a subset of unmyelinated afferent fibers. Whereas these fibers are classically considered to be nociceptive unmyelinated C fibers, evidence suggests that some of these fibers may be myelinated A fibers, which have long unmyelinated branches innervating the skin36. Although most of these fibers are probably nociceptive A fibers, we cannot completely rule out that some of these fibers are nonnociceptive thermoreceptors or possibly even low-threshold mechanoreceptors. However, even after considering these alternate possibilities, the increases in thermal sensitivity observed with group I mGluR activation suggest that a significant portion of these receptors are localized on thermal nociceptors. Whereas the anatomical data corroborates the behavioral studies and vice versa, the behavioral studies rely on the specificity of the pharmacologic agents. Reduction of DHPG-induced thermal hypersensitivity by the mGluR5-selective antagonist MPEP and the mGluR1-selective antagonists CPCCOEt and LY367385 showed that DHPG activates group I mGluRs. Furthermore, NMDA and group II and III mGluR antagonists had no effect. MPEP, LY367385 and CPCCOEt do not significantly inhibit group II or III mGluRs or iGluRs at concentrations several orders of magnitude greater than their IC50 for mGluR1 and mGluR5 (refs. 26–29). The effective translation of the in vitro data to in vivo experiments requires one to define an in vivo dose, which correlates to the selective concentration range of the drug in vitro. To define a selective in vivo dose for MPEP and CPCCOEt, we determined the minimal dose required to significantly block DHPGinduced thermal hypersensitivity (Fig. 3b and d). Although the selectivity of MPEP for mGluR5 is clear26, we

Fig. 4. APV and MPPG did not reduce DHPG-induced thermal hypersensitivity. (a) Pretreatment with APV (NMDA receptor antagonist, 50 nmol), did not attenuate DHPG-induced thermal hypersensitivity (Vehicle/DHPG, n = 8; APV/DHPG, n = 6; p = 0.45). (b) MPPG (antagonist at group II and group III mGluRs, 500 nmol) did not alter DHPGinduced thermal hypersensitivity (n = 3; p = 0.59).

were initially less confident of CPCCOEt, for which in vitro studies showed only a single order of magnitude difference in IC50 between mGluR1 and mGluR5 (refs. 27, 28). However, LY367385, a structurally unrelated antagonist with a different mode of action and higher selectivity for mGluR1 (refs. 29), also inhibited DHPG-induced thermal hypersensitivity. Therefore, we are relatively confident as to the specificity of MPEP and CPCCOEt toward mGluR5 and mGluR1, respectively, at the doses used here. The finding that MPEP and CPCCOEt both completely block or significantly attenuate DHPG- and glutamate-induced thermal hypersensitivity suggests that both mGluR1 and mGluR5 are required for these effects. Furthermore, the antagonists, when given alone or in combination, equally reduce the second phase of the formalin test. However, previous studies involving peripheral injection of excitatory amino acids have also found that NMDA or AMPA receptor antagonists completely eliminate glutamate-induced hypersensitivity37,38. Therefore, it may be a general phenomenon of peripheral glutamatergic signaling that activation of AMPA, NMDA and group I mGluRs can each lead to increases in mechanical or thermal sensitivity, whereas blockade of any one of these receptor subtypes can completely prevent the effects of glutamate. This observation suggests that these receptors cannot act completely independently in a parallel fashion to mediate glutamate-induced hypersensitivity, but that they probably cross-talk and function serially in complicated ways at the molecular and neuronal circuit levels. Experiments examining group I mGluR signaling in peripheral afferents would have little broader significance unless periph-

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Fig. 5. MPEP and CPCCOEt blocked glutamate-induced thermal hypersensitivity. (a) Pretreatment with MPEP (30 nmol) prevented the thermal hypersensitivity induced by 100 nmol glutamate (vehicle, n = 4; MPEP, n = 5; *p < 0.01). (b) Pretreatment with CPCCOEt (100 nmol) also prevented glutamate-induced thermal hypersensitivity (n = 6; *p < 0.01).

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eral group I mGluRs could actually be targeted to treat persistent pain. Because a previous study has shown that glutamate is released intradermally after formalin injection into the hind paw7, we used the formalin model to test whether group I mGluR antagonists may reduce inflammatory pain. Either pre- or posttreatment with group I mGluR antagonists during the formalin test attenuated nociceptive scores during the second phase. As mentioned above, several studies including our own have shown that group I mGluRs in the spinal cord are also important for the expression of inflammatory hyperalgesia. However, the present results indicate that peripheral group I mGluR activation is necessary for full expression of inflammatory hyperalgesia, and blockade of these receptors is sufficient to completely eliminate the effects of increased glutamate levels in the periphery. In contrast to iGluR antagonists5, peripheral mGluR antagonists were also able to reverse established inflammatory

Fig. 7. CPCCOEt and MPEP reduced the formalin test second phase when administered after the first phase. (a) MPEP (30 nmol) caused a decrease in the time spent lifting or licking the injected paw (n = 8, p < 0.01). Asterisks indicate time points at which MPEP was significantly different from vehicle (p < 0.05). (b) CPCCOEt (100 nmol) caused a decrease in the time spent lifting or licking (n = 6, p < 0.01). Asterisks represent time points at which CPCCOEt was significantly different from vehicle (p < 0.05). (c) The total time spent lifting or licking in the second phase (> 10 min) is plotted. None of the vehicles or treatments was significantly different from each other (p > 0.05), and each treatment significantly reduced the time spent in the second phase when compared to its respective vehicle (MPEP vehicle and MPEP, n = 8; CPCCOEt and MPEP + CPCCOEt vehicle, n = 12; CPCCOEt, n = 6; MPEP + CPCCOEt, n = 6; *p < 0.01). Arrows indicate approximate time of antagonist injection. nature neuroscience • volume 4 no 4 • april 2001

Fig. 6. Pre-treatment with MPEP or CPCCOEt diminished the second phase of the formalin test. (a) When administered 15 min before formalin injection, MPEP (30 nmol) caused a significant decrease in the time spent lifting or licking the injected paw (n = 6, p < 0.01). Asterisks indicate time points at which MPEP is significantly different from vehicle (p < 0.05). (b) When administered 15 min before formalin injection, CPCCOEt (100 nmol) caused a significant decrease in the time spent lifting or licking (n = 6, p < 0.01). Asterisks represent time points at which CPCCOEt was significantly different from vehicle (p < 0.05). (c) Total time spent lifting or licking in the second phase (> 10 min). None of the vehicles or treatments was significantly different from each other (p > 0.05), whereas each treatment significantly reduced the time spent in the second phase when compared to its respective vehicle (MPEP vehicle and MPEP, n = 6; CPCCOEt and MPEP + CPCCOEt vehicle, n = 10; CPCCOEt, n = 6, MPEP + CPCCOEt, n = 4; *p < 0.01).

hypersensitivity. The therapeutic implications of this finding are potentially significant, as it suggests that peripheral application of mGluR antagonists may be effective in the treatment of inflammatory pain. Whereas administration of centrally acting mGluR antagonists could also be therapeutically beneficial, peripherally acting group I mGluR antagonists may be effective analgesics with little possibility of undesired central effects. While this manuscript was under review, another study was published that confirms our findings that MPEP reduces inflammatory hyperaglesia 39. In this study, the authors show that mGluR5 is expressed in DRG neurons, where it colocalizes with VR1, and that peripheral but not central administratrion of MPEP reduces hyperaglesia in response to inflammation in rat. In contrast to our findings, this study found no effect of an mGluR1 antagonist on peripheral mGluR-mediated mechanical hyper-

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sensitivity39. The reason for this discrepancy is not clear, but may involve species differences, pharmacologic differences in mGluR1 antagonist used or differences in the sensory modality tested.


METHODS Behavioral analysis. C57/BL6 mice were housed in cages with access to food and water ad libitum. Seven- to eleven-week-old male mice weighing 20–25 g were used for this study. The experimental procedures in this study were approved by the Baylor College of Medicine Animal Care and Use Committee. Mice were allowed to acclimate for at least three days before any behavioral tests. Behavioral tests began with a habituation period, in which mice were placed in plexiglass cubicles for at least one hour. All drugs were administered in a volume of 10 µl, subcutaneously, into the hind paw plantar surface using a 50-µl Hamilton syringe with a 30-gauge needle. The needle was inserted at the midpoint of the hind paw and advanced to the base of the third toe, where the drug solution was injected forming a bleb usually extending back to the initial point of needle entry. The bleb disappeared within five minutes after injection. In separate experiments, multiple injections were made in the same hindpaw, at similar sites with a bleb encompassing the same area. DHPG (R,S-3,5-dihydroxyphenylglycine), MPEP (2-methyl-6(phenylethynyl)pyridine hydrochloride), D-APV (D(–)-2-amino-5-phosphonopentanoic acid), and L -glutamate were dissolved in 100 mM HEPES-Na, pH 7.4 (HB). CPCCOEt (7-(hydroxyimino)cyclopropa[b] chromen-1a-carboxylate), LY367385 (S-+-a-amino-4-carboxy-2-methylbenzeneacetic acid) and MPPG (R,S-α-methyl-4-phosphonophenylglycine) were first prepared as stock solutions in 100 mM NaOH and then diluted to the final concentrations with HB. All drugs except for L-glutamate (Sigma, St. Louis, Missouri) were obtained from Tocris Cookson (Ballwin, Missouri). The final pH of all injected drugs was 7.4 to prevent any potential pH effects on peripheral nociception. Vehicles were prepared exactly as the corresponding drug solution. The first set of experiments aimed to establish the effects of DHPG on thermal nociception. Thermal sensitivity was measured using a method modified from previous work40. A thermal stimulus from a constant radiant heat source was delivered through the glass bottom of the chamber to the plantar surface of the hind paw (IITC Life Sciences, Woodland Hills, California) and the latency for foot withdrawal was measured. At time 0, three baseline responses were obtained about 10 min apart and averaged. DHPG or vehicle was then injected, and single responses were measured at 15-min intervals. In the second set of experiments, the effect of antagonists or vehicles on DHPG-induced thermal hypersensitivity was examined. Three baseline responses were measured and averaged (–15 time point), and the antagonist (MPEP, CPCCOEt, LY367385, APV or MPPG) was injected. Fifteen minutes later, a thermal response was measured (0 time point), DHPG was injected and single responses were measured at 15-min intervals. In the third set of experiments, the effect of antagonists and vehicles on glutamate-induced thermal hypersensitivity was examined in a similar way as described for the antagonist effects on DHPG-induced thermal hypersensitivity, except, following glutamate injection, only three responses were measured and averaged. The final set of experiments examined the effects of MPEP and CPCCOEt on nociceptive behavior resulting from formalin induced inflammation. The formalin test was conducted using a procedure modified from standard protocols35,41. Ten microliters of 2% formalin was injected subcutaneously into the hind paw, and the number of seconds spent lifting or licking the injected paw was summed and plotted in 5-min blocks. MPEP and/or CPCCOEt were injected either 15 min before formalin injection to examine the prophylactic effect of these antagonists or 5 min after formalin injection to determine the effect of the antagonists on existing inflammation. Thermal withdrawal latencies were expressed as a percent of baseline responses (latency at a given time point/mean baseline latency × 100%) and data points plotted as the mean ± s.e.m. Statistical analysis of the data was conducted using SAS (SAS Institute, Cary, North Carolina) in two complementary ways. Thermal withdrawal latency or time spent lifting or licking was treated as a function of treatment, time and experimental day using a repeated measures, mixed effects model. A compound symmetry covariance structure for the repeated measures and maximum 422

likelihood fitting were used. Post-hoc multiple comparisons of the least square means using Tukey’s method were conducted to examine differences at specific time points. A univariate statistical analysis was also used to compare treatments. For this analysis, withdrawal latencies were averaged across time points, or the time spent lifting and/or licking was summed across the second phase. One-way ANOVA followed by posthoc comparisons with Tukey’s method were conducted when comparing more than two treatments. An unpaired Student’s t-test was used when comparisons were restricted to two means. Immunohistochemistry and electron microscopy. Electron microscopy was used to localize mGluR1a and mGluR5 on peripheral primary afferent fibers and to quantitate percentages of labeled unmyelinated axons. Mice were anesthetized and perfused through the aorta with heparinized saline followed by a mixture of cold 2.5% glutaraldehyde, 1% paraformaldehyde and 0.1% picric acid in 0.1 M phosphate buffer, pH 7.4 (PB). Glabrous skin from the plantar surface of the hind toes was cut into approximately 1-mm blocks and placed in PB overnight. Tissues were rinsed in graded alcohols to increase antisera penetration then incubated for 1 h in sodium borohydride to remove excess glutaraldehyde. All tissue was immunostained using rabbit polyclonal antisera against mGluR1a (1:1000, DiaSorin, Stillwater, Minnesota) or mGluR5 (1:1000, Chemicon, Temecula, California) and the ABC detection method (Vector Laboratories, Burlingame, California) as previously described42. After incubation in diaminobenzidine (DAB) and a thorough rinsing in PB, the tissue samples were placed in 1% phosphate-buffered osmium tetroxide for 2 h, and then dehydrated and embedded in plastic. Ultrathin sections were cut at right angles to the dermal–epidermal junction in 2–3 blocks per mouse. The sections were mounted on slot grids and viewed with a JEOL 100CX electron microscope. To avoid repeated counting of labeled axons, only one thin section per block was analyzed. In each thin section analyzed, all axon bundles were photographed, and numbers of labeled and unlabeled axons were determined. This provided a random sample and conservative estimate of the percentage of immunostained axon profiles. The specificity of the antisera was confirmed by primary antibody preabsorption or omission, and examination of cross-reactivity by immunocytochemistry in transfected cell lines (data not shown).

ACKNOWLEDGEMENTS The authors thank L.S. Baggett (Rice University, Department of Statistics) for assistance with the statistical analysis and B. Nadin for cell line immunocytochemistry. This work was supported by grants from the National Institutes of Health (MH60230 to R.W.G. and NS11255 and NS27910 to S.M.C.) and the Spinal Cord Research Foundation (R.W.G.). G.B. is a McNair Scholar of the Baylor College of Medicine Medical Scientist Training Program.

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