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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Neurochem. Author manuscript; available in PMC 2011 January 1.
Published in final edited form as:
PMCID: PMC2900839
NIHMSID: NIHMS214394

Regulation of Increased Glutamatergic Input to Spinal Dorsal Horn Neurons by mGluR5 in Diabetic Neuropathic Pain

Abstract

Diabetic neuropathic pain is associated with increased glutamatergic input in the spinal dorsal horn. Group I metabotropic glutamate receptors (mGluRs) are involved in the control of neuronal excitability, but their role in the regulation of synaptic transmission in diabetic neuropathy remains poorly understood. Here we studied the role of spinal mGluR5 and mGluR1 in controlling glutamatergic input in a rat model of painful diabetic neuropathy induced by streptozotocin. Whole-cell patch-clamp recordings of lamina II neurons were performed in spinal cord slices. The amplitude of excitatory postsynaptic currents (EPSCs) evoked from the dorsal root and the frequency of spontaneous EPSCs (sEPSCs) were significantly higher in diabetic than in control rats. The mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) inhibited evoked EPSCs and sEPSCs more in diabetic than in control rats. Also, the percentage of neurons in which sEPSCs and evoked EPSCs were affected by MPEP or the group I mGluR agonist was significantly higher in diabetic than in control rats. However, blocking mGluR1 had no significant effect on evoked EPSCs and sEPSCs in either groups. The mGluR5 protein level in the dorsal root ganglion, but not in the dorsal spinal cord, was significantly increased in diabetic rats compared with that in control rats. Furthermore, intrathecal administration of MPEP significantly increased the nociceptive pressure threshold only in diabetic rats. These findings suggest that increased mGluR5 expression on primary afferent neurons contributes to increased glutamatergic input to spinal dorsal horn neurons and nociceptive transmission in diabetic neuropathic pain.

Keywords: diabetic neuropathy, neuropathic pain, mGluR5, synaptic transmission, spinal cord, primary afferents

Introduction

Diabetic neuropathy is one of the most common late complications of diabetes mellitus and is frequently painful, with pain occurring predominantly in the distal extremities. Pain associated with diabetic neuropathy can occur either spontaneously or as a result of exposure to mildly painful stimuli (hyperalgesia) or to stimuli not normally perceived as painful (allodynia) (Brown and Asbury 1984; Clark and Lee 1995; Chen and Pan 2003b). The development of diabetic neuropathic pain is associated with increased nociceptive input, neuronal hyperactivity, and sustained stimulation of certain glutamate receptors in the spinal cord (Calcutt and Chaplan 1997; Malcangio and Tomlinson 1998; Wang et al. 2007; Chen et al. 2009). Glutamate receptors include ionotropic glutamate receptors, such as α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors, and metabotropic glutamate receptors (mGluRs). The mGluRs are a family of G-protein linked receptors that have been classified into three groups (group I, II, and III) on the basis of similarities in their amino acid sequences, their linkage to second messenger systems, and their pharmacology (Conn and Pin 1997). In contrast to Group II and III mGluRs, which are coupled to inhibitory Gi/o proteins, Group I mGluRs (including the mGluR1 and mGluR5 subtypes) are coupled to the Gq protein family and generally increase neuronal firing and synaptic transmission through phospholipase C activation (Conn and Pin 1997; Schoepp et al. 1999; Pan et al. 2008). However, the role of Group I mGluRs in increased glutamatergic input to spinal dorsal horn neurons in diabetic neuropathic pain remains unknown.

mGluR5 is localized on small- and medium-diameter dorsal root ganglion (DRG) neurons, primary afferent central terminals, and the spinal dorsal horn neurons (Valerio et al. 1997; Berthele et al. 1999; Alvarez et al. 2000; Crawford et al. 2000; Walker et al. 2001a; Park et al. 2004). Low levels of mGluR1 are also present in the DRG and spinal lamina II (Alvarez et al. 2000; Zhou et al. 2001), although the mGluR1 mRNA is not detected in the DRG (Crawford et al. 2000). Stimulation of group I mGluRs in the spinal cord has been shown to potentiate the release of glutamate, in both in vitro and in vivo preparations (Lefebvre et al. 2000; Mills et al. 2001; Lorrain et al. 2002; Park et al. 2004). Administration of group I mGluR agonists stimulates dorsal horn neurons, whereas group I mGluR antagonists suppress neuronal hyperexcitation (Young et al. 1994; Neugebauer et al. 1999; Lorrain et al. 2002; Sotgiu et al. 2003). Several studies have shown that mGluR1 and mGluR5 are involved in neuropathic pain caused by traumatic nerve injury (Dogrul et al. 2000; Fundytus et al. 2001; Fisher et al. 2002; Osikowicz et al. 2008). However, the roles of mGluR1 and mGluR5 in the regulation of glutamatergic synaptic transmission in the spinal dorsal horn in diabetic neuropathic pain have not been investigated previously. Therefore, in this study, we sought to determine the function of mGluR1 and mGluR5 in the control of increased glutamatergic input to spinal dorsal horn neurons in a rat mode of painful diabetic neuropathy.

Methods and Materials

Animal model of diabetic neuropathic pain

Male Sprague-Dawley rats (Harlan Sprague–Dawley, Indianapolis, IN) initially weighing 220–250 g were used in this study. The experiments were performed according to NIH guidelines approved by the Animal Care and Use Committee of the University of Texas M. D. Anderson Cancer Center. All efforts were made to minimize both the suffering and number of animals used. Diabetes was induced by a single intraperitoneal (i.p.) injection of streptozotocin (STZ, 60 mg/kg; Sigma, St. Louis, MO) freshly dissolved in 0.9% sterile saline (Chen and Pan 2002). Diabetes was confirmed in STZ-injected rats by measuring the blood glucose concentration. Glucose levels in blood obtained from the tail vein were assayed using ACCU-CHEK test strips (Roche Diagnostics Corporation, Indianapolis, IN). The blood glucose level was measured 3 weeks after STZ administration, and only rats with high levels (> 300 mg/dl) were used for the diabetic groups. Age-matched vehicle-injected rats were used as controls. Neuropathic pain in diabetic rats was confirmed by examining nociceptive mechanical thresholds using an Ugo Basil Analgesimeter (see below). Only diabetic rats with evident hyperalgesia were included in the study. Final electrophysiological, biochemical, and behavioral experiments were performed on rats 3 weeks after STZ or vehicle injection.

Intrathecal cannulation and behavioral assessment of nociception

Intrathecal catheters (PE-10 polyethylene tubing) were inserted in diabetic and control rats during isoflurane-induced anesthesia. The catheter was advanced 8 cm caudally through an incision in the cisternal membrane and secured to the musculature at the incision site (Chen and Pan 2005, 2006). The rats were allowed to recover for at least 5 days before drug testing began. Only rats with no evidence of neurological deficit after catheter insertion were used in the study. Drugs for intrathecal injections were dissolved in normal saline and administered in a volume of 5 μl followed by a 10-μl flush with normal saline.

The nociceptive mechanical threshold was measured using an Ugo Basil Analgesimeter (Varese, Italy) to apply a noxious pressure to a hindpaw. By pressing a pedal that activated a motor, the force increased at a constant rate on the linear scale. When the rat responded by withdrawal of the paw or vocalization, the pedal was immediately released and the nociceptive threshold read on a scale. A cutoff of 400 g was used to avoid tissue injury (Chen and Pan 2006). Both hindpaws were tested in each rat, and the mean value was used as the nociceptive withdrawal threshold.

Spinal cord slice preparation

The rats were anesthetized with 2–3% isoflurane, and the lumbar segment of the spinal cord was removed by means of laminectomy. The spinal cord segment was immediately placed in ice-cold sucrose–artificial cerebrospinal fluid (aCSF) presaturated with 95% O2 and 5% CO2. The sucrose-aCSF contained 234 mM sucrose, 3.6 mM KCl, 1.2 mM MgCl2, 2.5 mM CaCl2, 1.2 mM NaH2 PO4, 12 mM glucose, and 25 mM NaHCO3. The tissue was then placed in a shallow groove formed in a gelatin block and glued to the stage of a vibratome (Technical Product International, St. Louis, MO). Transverse spinal cord slices (400 μm) were cut in the ice-cold sucrose-aCSF and then preincubated in Krebs solution oxygenated with 95% O2 and 5% CO2 at 34°C for at least 1 h before they were transferred to the recording chamber. The Krebs solution contained 117 mM NaCl, 3.6 mM KCl, 1.2 mM MgCl2, 2.5 mM CaCl2, 1.2 mM NaH2 PO4, 11 mM glucose, and 25 mM NaHCO3.

Electrophysiological recordings

Excitatory postsynaptic currents (EPSCs) were recorded using the whole-cell voltage-clamp method, as described previously (Li et al. 2002; Wang et al. 2007). Each spinal cord slice was placed in a glass-bottomed chamber (Warner Instruments, Hamden, CT) and fixed with parallel nylon threads supported by a U-shaped stainless steel weight. The slice was continuously perfused with Krebs solution at 5.0 ml/min at 34°C maintained by an inline solution heater and a temperature controller (TC-324; Warner Instruments). The lamina II was identified by light microscopy as a distinct translucent band across the superficial dorsal horn. Neurons in lamina II were selected because they primarily receive unmyelinated C-fiber input. Neurons in other laminae in the adult spinal cord slices cannot be visualized under the microscope because of heavy myelination. Neurons in the lamina II of the spinal cord slice were identified with differential interference contrast/infrared illumination on a fixed-stage microscope (BX50WI; Olympus, Tokyo, Japan).

Monosynaptic or polysynaptic EPSCs were evoked by electrical stimulation through a bipolar stimulation electrode placed on the attached dorsal root. We used a fixed stimulation intensity (0.2 ms and 0.6 mA) to evoke EPSCs from primary afferents in both diabetic and control rats. The evoked EPSCs were considered to be monosynaptic if (1) the latency was constant with repeated electrical stimulation at 0.1 Hz and (2) there was no conduction failure or changes in the latency when the stimulation frequency was increased to 20 Hz (Li et al. 2002; Wang et al. 2007). In contrast, evoked EPSCs were considered to be polysynaptic if the latency was variable and conduction failure occurred at a higher stimulation frequency (20 Hz). The electrode for the whole-cell recordings was pulled from borosilicate glass capillaries with a puller (P-97; Sutter Instruments, Novato, CA). The impedance of the pipette was 5–8 MΩ when filled with internal solution containing 110 mM Cs2SO4, 5 mM TEA, 2 mM MgCl2, 0.5 mM CaCl2, 5 mM HEPES, 5 mM EGTA, 5 mM ATP-Mg, 0.5 mM Na-GTP, 1 mM guanosine 5′-O-(2-thiodiphosphate), and 10 mM lidocaine N-ethyl bromide that had been adjusted to pH 7.2–7.3 with 1 M CsOH (290–300 mOsm).

Recordings of spontaneous EPSCs (sEPSCs) or evoked EPSCs began approximately 6 min after whole-cell access was established and the current reached a steady state. The input resistance was monitored, and the recording was abandoned if it changed by more than 15%. All signals were recorded using an amplifier (MultiClamp 700B; Molecular Devices, Sunnyvale, CA) at a holding potential of −60 mV, filtered at 1–2 kHz, digitized at 10 kHz, and stored in a computer using pCLAMP 9.2 software (Axon Instruments Inc.). The effects of different concentrations of 2-methyl-6-(phenylethynyl)-pyridine (MPEP), 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester (CPCCOEt), and (RS)-3,5-dihydroxyphenylglycine (DHPG) on sEPSCs and evoked EPSCs were studied. MPEP, DHPG, and CPCCOEt were obtained from Ascent Scientific LLC (Princeton, NJ). Drugs were dissolved in Krebs solution and perfused into the tissue chamber using syringe pumps.

Quantification of mGluR5 protein levels using Western blotting

To quantify the mGluR5 protein level in the DRG and dorsal spinal cord, rats were deeply anesthetized with sodium pentobarbital (60 mg/kg, i.p.) 3 weeks after receiving STZ or vehicle. The rats were then decapitated, and the L3–L6 DRGs and lumbar spinal cords were collected. All tissues were individually frozen in liquid nitrogen and stored at −70°C. Isolated DRG and spinal cord tissues were homogenized in ice-cold buffer containing 10 mM HEPES, 1 mM EGTA, 0.1 mM EDTA, 10% sucrose, and protease inhibitor cocktail (Sigma, St. Louis, MO). The homogenate was centrifuged at 1,000 g for 1 min at 4°C, and the supernatant was then centrifuged at 100,000 g for 1 h at 4°C. The resulting pellet was resuspended with 100 μl of lysis buffer containing 1% Triton X-100, 20 mM 4-morpholinepropanesulfonic acid, 4.5 mM Mg(CH3COO)2, 150 mM KCl, and protease inhibitor cocktail. The suspension was centrifuged at 1,000 g for 5 min at 4°C. The supernatant was collected and the protein concentration was determined using the Bradford protein assay. Thirty μg of proteins were mixed with an equal volume of sodium dodecyl sulfate (SDS) sample buffer (125 mM Tris, pH: 6.8; 4% SDS, 0.02% bromophenol blue, 20% glycerol, and 5% mercaptoethanol). The samples were heated for 10 min at 70°C. The samples were separated by 10% SDS–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Chen and Pan 2003a). The membrane was blocked for 2 h in 5% nonfat milk in phosphate-buffered saline and then incubated with rabbit anti-mGluR5 primary antibody (APC-021, Alomone Labs, Jerusalem, Israel; dilution: 1:1000) overnight at 4°C. For the protein loading control, some membranes were incubated with a rabbit anti-β-actin antibody (Sigma, St. Louis, MO; dilution: 1:2000). The membrane was then rinsed and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch, West Grove, PA) at 1:10000 dilution for 2 h at 22°C. The membrane was developed with an enhanced chemiluminescence kit according to the manufacturer's instructions. The intensity of bands was captured digitally and analyzed quantitatively with AIS software (Imaging Research Inc., London, ON, Canada).

Data Analysis and Statistics

Data are presented as means ± SEM. The effects of MPEP, DHPG, and CPCCOEt on the amplitude of evoked EPSCs were analyzed using Clampfit (Axon Instruments). The frequency and amplitude of sEPSCs were analyzed off-line using a peak detection program (Mini-Analysis; Synaptosoft, Leonia, NJ). Detection of events was accomplished by setting a threshold above the noise level. The sEPSCs were detected by the fast rise time of the signal over an amplitude threshold (typically 6–10 pA) above the background noise. We manually excluded the event when the background noise was erroneously identified as an sEPSC by the program. The background noise level was typically constant throughout the recording of a single neuron. Effects of MPEP and CPCCOEt on the amplitude of evoked EPSCs and the frequency of sEPSCs were determined using either two-way ANOVA with Bonferroni's post hoc test or repeated-measures ANOVA. Neurons were considered to be unresponsive to the mGluR1 or mGluR5 agonist and antagonist if the peak amplitude of evoked EPSCs or the frequency of sEPSCs was altered < 15%. The difference in the percentage of lamina II neurons responsive to MPEP and DHPG between control and diabetic groups was analyzed using Chi-square test. The effect of drug treatments on the paw withdrawal threshold was determined by using repeated-measures analysis of variance followed by Dunnett's post hoc test. A P value of less than 0.05 was considered to be statistically significant.

Results

Increased glutamatergic input to spinal dorsal horn neurons in diabetic rats and the effects of CPCCOEt on sEPSCs and evoked EPSCs

The baseline frequency, but not the amplitude, of glutamatergic sEPSCs of lamina II neurons was significantly higher in diabetic than in control rats (Fig. 1). Furthermore, the baseline amplitude of both monosynaptic and polysynaptic EPSCs evoked from the dorsal root before drug application was significantly greater in diabetic than in control rats (Fig. 2).

Figure 1
Effect of CPCCOEt on glutamatergic sEPSCs of spinal lamina II neurons in spinal cord slices from control and diabetic rats. A, original traces show sEPSCs during control; application of 10, 50, and 100 μM CPCCOEt; and washout in 1 lamina II neuron ...
Figure 2
Effects of CPCCOEt on monosynaptic and polysynaptic EPSCs of lamina II neurons evoked from primary afferents in spinal cord slices from control and diabetic rats. A, original recordings show evoked monosynaptic EPSCs during control; application of 10, ...

To examine the role of mGluR1 in the control of increased glutamatergic input to spinal dorsal horn neurons, we tested the effects of CPCCOEt, a highly selective mGluR1 antagonist (Park et al. 2004), on sEPSCs and EPSCs evoked from primary afferents in control and diabetic rats. Bath application of 10-100 μM CPCCOEt (each concentration was applied for 3 min) had no significant effect on the frequency and amplitude of sEPSCs of any lamina II neurons tested in diabetic (n = 9 neurons) or control rats (n = 7 neurons) (Fig. 1).

In addition, bath application of 10-100 μM CPCCOEt did not significantly alter the amplitude of evoked monosynaptic EPSCs of lamina II neurons in diabetic (n = 7 neurons) or control (n = 8 neurons) rats (Fig. 2). Furthermore, CPCCOEt had no significant effects on the amplitude of evoked polysynaptic EPSCs of any lamina II neurons examined in either control (n = 8 neurons) or diabetic (n = 10 neurons) rats (Fig. 2). Because blocking mGluR1 did not affect glutamatergic input to spinal dorsal horn neurons in either groups, we focused our subsequent experiments on mGluR5.

Effects of DHPG and MPEP on sEPSCs of lamina II neurons in the control and diabetic rats

We first tested the effect of DHPG, a selective group I mGluR agonist (Zhong et al. 2000), on sEPSCs of lamina II neurons. Highly specific agonists for mGluR1 and mGluR5 subtypes are currently not available. Bath application of 1-20 μM DHPG dose-dependently increased the frequency, but not the amplitude, of sEPSCs in both control and diabetic rats (Fig. 3A). The percentage of lamina II neurons in which the frequency of sEPSCs was increased by DHPG was significantly higher in diabetic (16/23, 69.57%) than in control rats (11/29, 37.93%; P < 0.05). We determined the concentrations of MPEP that can adequately block mGluR5 by testing the effect of 20 μM DHPG on sEPSCs before and during bath application of 5 μM MPEP. In both control and diabetic groups, the potentiating effect of DHPG on the frequency of sEPSCs was largely diminished by MPEP (Fig. 3B).

Figure 3
Effects of DHPG and MPEP on glutamatergic sEPSCs of spinal lamina II neurons in spinal cord slices from control and diabetic rats. A, summary data show the dose-response effect of DHPG on the frequency of sEPSCs in 11 of 29 neurons from control rats and ...

To determine the function of mGluR5 in the control of glutamatergic input to spinal dorsal horn neurons, we examined the effects of MPEP, which is the most potent and highly specific non-competitive antagonist for mGluR5 (Attucci et al. 2001; Anderson et al. 2002). We next compared the inhibitory effects of MPEP on the frequency of sEPSCs of lamina II neurons in diabetic and control rats. Bath application of 1-10 μM MPEP dose-dependently decreased the frequency, but not the amplitude, of sEPSCs in the majority of neurons (22/32, 68.8%) tested in diabetic rats (Fig. 4). The cumulative probability analysis of sEPSCs revealed that the distribution pattern of the inter-event interval of sEPSCs was shifted toward the right in response to MPEP. However, the distribution pattern of the amplitude of sEPSCs was not significantly changed (Fig. 4B). Notably, MPEP also inhibited the frequency of sEPSCs of lamina II neurons from control rats, but this effect was observed in only 14 of 36 (38.9%, P < 0.05) neurons examined in control rats. Because the frequency of the baseline sEPSCs was significantly different between diabetic and control rats, we normalized the effect of MPEP to the baseline of sEPSCs in each group. MPEP caused a significantly greater decrease in the frequency of sEPSCs in diabetic than in control rats (Fig. 4C).

Figure 4
Effects of MPEP on glutamatergic sEPSCs of spinal lamina II neurons in spinal cord slices from control and diabetic rats. A, original traces show sEPSCs during control; application of 1, 2, 5, and 10 μM MPEP; and washout in 1 lamina II neuron ...

Effects of MPEP on EPSCs evoked from primary afferents in the control and diabetic rats

To determine the role of mGluR5 on primary afferent terminals in the regulation of glutamatergic synaptic input to dorsal horn neurons in diabetic neuropathy, we examined the effects of MPEP on monosynaptic and polysynaptic EPSCs evoked from the dorsal root. Bath application of 1-10 μM MPEP dose-dependently reduced the peak amplitude of monosynaptic EPSCs in the majority of neurons (15/22, 68.18%) tested in diabetic rats (Fig. 5, A and B). By contrast, MPEP inhibited the amplitude of monosynaptic EPSCs in only 13 of 35 neurons (37.1%, P < 0.05) tested in the control group. Because the baseline amplitude of the evoked EPSCs was significantly different between the two groups, we normalized the effect of MPEP to the baseline control of evoked EPSCs in each group. At concentrations of 5 and 10 μM, MPEP produced a significantly greater effect on the amplitude of monosynaptic EPSCs in diabetic than in control rats (Fig. 5C).

Figure 5
Effects of MPEP on monosynaptic EPSCs of lamina II neurons evoked from primary afferents in spinal cord slices from control and diabetic rats. A, original recordings show monosynaptic EPSCs during control; application of 1, 2, 5, and 10 μM MPEP; ...

Furthermore, MPEP dose-dependently inhibited the peak amplitude of evoked polysynaptic EPSCs in both groups (Fig. 6, A and B). When the effect of MPEP on evoked EPSCs was normalized to the baseline control, MPEP produced a significantly greater effect on the amplitude of polysynaptic EPSCs in diabetic than in control rats (Fig. 6C). The percentage of lamina II neurons in which the amplitude of polysynaptic EPSCs was inhibited by MPEP was also significantly higher in diabetic (25/35, 71.4%) than in control rats (15/41, 36.6%; P < 0.05).

Figure 6
Effects of MPEP on polysynaptic EPSCs of lamina II neurons evoked from primary afferents in spinal cord slices from control and diabetic rats. A, original recordings show polysynaptic EPSCs during control; application of 1, 2, 5, and 10 μM MPEP; ...

The mGluR5 protein levels in the DRG and dorsal spinal cord in control and diabetic rats

To determine diabetes-induced changes in the protein level of mGluR5 in primary sensory neurons and the spinal cord, we quantified mGluR5 protein levels in the DRG and dorsal spinal cord from diabetic and control rats (n = 6 in each group) using Western immunoblotting. The mGluR5 protein level in the DRG was significantly higher in diabetic than in control rats (Fig. 7). However, there was no significant difference in mGluR5 protein levels in the dorsal spinal cord between the two groups (Fig. 7).

Figure 7
Changes in the mGluR5 protein levels in the DRG and dorsal spinal cord in control and diabetic rats. A, representative gel images show the density of mGluR5 protein band in the dorsal spinal cord and DRG. Note that β-actin was used as a loading ...

Effect of MPEP on mechanical nociception in diabetic and control rats

To further determine the function of mGluR5 in the regulation of nociceptive transmission at the spinal level in diabetic neuropathy, we tested the dose-response effects of intrathecal injection of MPEP on the nociceptive withdrawal threshold of the rat hindpaw in both control and diabetic rats. The baseline pressure withdrawal threshold was significantly lower in diabetic than in control rats, suggestive of hyperalgesia (Fig. 8). Intrathecal administration of 30 μg (n = 8 rats) or 60 μg (n = 9 rats), but not 10 μg (n = 8 rats), of MPEP significantly increased the paw withdrawal threshold in response to noxious mechanical stimulus in diabetic rats (Fig. 8). The MPEP effect reached its maximum within 30 min after intrathecal injection, and the effect lasted for about 120 min. By contrast, intrathecal injection of up to 60 μg of MPEP had no significant effect on the nociceptive withdrawal threshold in control rats (n = 8 rats, Fig. 8).

Figure 8
Effects of intrathecal injection of MPEP on the nociceptive withdrawal threshold in diabetic and control rats. Intrathecal administration of 10–60 μg MPEP dose-dependently increased the paw withdrawal threshold in response to noxious mechanical ...

Discussion

In this study, we determined the contribution of mGluR5 to increased glutamatergic input to spinal dorsal horn neurons in a rat model of diabetic neuropathic pain. We found that the frequency of glutamatergic sEPSCs and the amplitude of evoked monosynaptic and polysynaptic EPSCs were significantly higher in diabetic than in control rats. The inhibitory effects of the mGluR5 antagonist MPEP on sEPSCs and on evoked monosynaptic and polysynaptic EPSCs were significantly greater in diabetic than in control rats. The percentage of lamina II neurons in which sEPSCs and evoked EPSCs affected by MPEP and the group I mGluR agonist DHPG was also significantly higher in diabetic than in control rats. However, the mGluR1 antagonist CPCCOEt had no significant effect on evoked EPSCs and sEPSCs in either diabetic or control rats. Western blotting analysis showed that the protein level of mGluR5 in the DRG, but not in the dorsal spinal cord, was significantly higher in the diabetic group than in the control group. Furthermore, intrathecal administration of MPEP significantly increased the nociceptive withdrawal threshold in diabetic but not in control rats. Collectively, our results provide new evidence that mGluR5 is up-regulated on primary afferent neurons and that increased mGluR5 activation contributes to augmented glutamatergic input and nociceptive transmission at the spinal level in diabetic neuropathic pain.

The spinal dorsal horn is a critical site for the transmission and modulation of nociception. Glutamate released from the central terminals of nociceptive primary afferents is an important neurotransmitter in the spinal dorsal horn. In our study, the frequency of glutamatergic sEPSCs and the amplitude of monosynaptic and polysynaptic evoked EPSCs were much higher in diabetic than in control rats. These differences likely reflect increased glutamate release from the primary afferent terminals to the spinal dorsal horn neurons in diabetic neuropathy. This enhanced glutamate release contributes to the hyperexcitability of dorsal horn neurons and the maintenance of diabetic neuropathic pain (Chen and Pan 2002; Chen et al. 2009). It has been shown that blocking mGluR1 at the spinal level has no effect on nociception in rats (Tambeli et al. 2003). However, treatment with the mGluR1 antisense oligonucleotide reduces neuropathic pain induced by sciatic nerve constriction injury (Fundytus et al. 2001). In the present study, we found that the specific mGluR1 antagonist CPCCOEt had no significant effects on sEPSCs or EPSCs evoked from primary afferents in either group. Therefore, our results suggest that mGluR1 in the spinal cord does not play an important role in the control of increased glutamatergic input in diabetic neuropathy.

mGluR5 is widely expressed on C-fiber afferent terminals and only sparsely expressed on A-fiber afferents (Bhave et al. 2001; Walker et al. 2001b). In the spinal cord, mGluR5 is strongly expressed in laminae I and II and gradually decreases toward the deeper layers of the spinal dorsal horn (Valerio et al. 1997; Alvarez et al. 2000). We reasoned that mGluR5 is involved in the potentiated glutamatergic input to the spinal dorsal horn neurons in diabetic neuropathic pain. In support of this hypothesis, we found that the inhibitory effects of MPEP on the amplitude of monosynaptic and polysynaptic EPSCs evoked from primary afferents and sEPSCs were significantly greater in diabetic than in control rats. Also, the percentage of lamina II neurons in which evoked EPSCs and the frequency of sEPSCs affected by MPEP or DHPG was significantly higher in the diabetic group. MPEP is a highly selective and potent antagonist of mGluR5, with no appreciable agonist or antagonist activity at mGluR1 (Gasparini et al. 1999). Our results suggest that the expression of mGluR5 on primary afferents is increased and plays an important role in augmenting the presynaptic glutamate release to spinal dorsal horn neurons in diabetic neuropathic pain.

Consistent with our electrophysiological results, we found that the protein level of mGluR5 in the DRG was significantly higher in diabetic than in control rats. Thus, the electrophysiological and biochemical data suggest that mGluR5 is up-regulated on primary afferent neurons and terminals and that it mediates increased glutamatergic input in painful diabetic neuropathy. Because diabetic neuropathy primarily affects the peripheral nervous system, it could explain why we detected a significant change in the mGluR5 protein level only in the DRG, but not in the dorsal spinal cord, from diabetic rats. It has been reported that mGluR5 protein levels are increased in the DRG after spinal nerve section in rats (Hudson et al. 2002). Previous studies suggest that there is an active axonal transport of mRNA and proteins between the DRG soma and the afferent central terminals in the superficial dorsal horn (Bi et al. 2006; Price et al. 2006). Because the mRNA and protein of mGluR5 are present in the DRG (Crawford et al. 2000), it is possible that the mGluR5 protein is synthesized in DRG neurons and transported to the afferent central terminals in the spinal cord. On the other hand, the spinal dorsal horn is very heterogenous and the protein from the central afferent terminals is only a small portion of the total proteins obtained from the dorsal spinal cord. Thus, using Western blotting is perhaps not sensitive to detect regional changes, such as increased expression of mGluR5 on afferent central terminals in diabetic rats.

We found that intrathecal injection of MPEP dose-dependently increased the paw withdrawal threshold in response to noxious mechanical stimulus in diabetic rats. Interestingly, although MPEP inhibited glutamatergic input in a small population of lamina II neurons in control rats, intrathecal administration of MPEP had little effect on nociception in these animals. These data suggest that blocking mGluR5 alone is not sufficient to affect normal nociceptive transmission in the spinal dorsal horn. Similar to our results, it has been shown that although MPEP can reverse hyperalgesia caused by inflammation, it has no effect on normal nociception (Walker et al. 2001b; Tambeli et al. 2003). Intrathecal pretreatment with MPEP has also been shown to reduce the development of mechanical hypersensitivity induced by nerve injury in rats (Fisher et al. 2002). Blocking mGluR5 could therefore reduce increased glutamatergic input from primary afferents to second-order dorsal horn neurons, leading to inhibition of central sensitization and nociceptive transmission at the spinal level in diabetic neuropathic pain. Because activation of mGluR5 also potentiates the NMDA receptor activity (Awad et al. 2000; Pisani et al. 2001), blocking mGluR5 could reduce both presynaptic glutamate release and NMDA receptor activation in the spinal cord in neuropathic pain. These mechanisms of actions could account for the profound inhibitory effects of MPEP on mechanical hyperalgesia in diabetic rats and provide further evidence that the up-regulation of mGluR5 is involved in nociceptive transmission at the spinal level in painful diabetic neuropathy.

In summary, our findings suggest that diabetic neuropathy up-regulates mGluR5 on primary afferent neurons and that mGluR5 contributes to the augmented glutamatergic input to spinal dorsal horn neurons in diabetic neuropathic pain. Our study provides new evidence for the important role of mGluR5 in the control of nociceptive transmission at the spinal level in diabetic neuropathic pain. Thus, mGluR5 may represent a potential new target for development of new treatments for painful diabetic neuropathy.

Acknowledgments

This work was supported by Grants GM64830 and NS45602 from the National Institutes of Health and the Hawkins Endowment to H.L.P.

List of abbreviations

mGluR5
metabotropic glutamate receptor 5
mGluR1
metabotropic glutamate receptor 1
STZ
streptozotocin
MPEP
2-methyl-6-(phenylethynyl)-pyridine
DHPG
3, 5-dihydroxyphenylglycine
CPCCOEt
7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester
EPSCs
excitatory postsynaptic currents

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