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Involvement of mGluR5 on acute nociceptive transmission
Brain Research 871 (2000) 223–233 www.elsevier.com / locate / bres
Research report
Involvement of mGluR 5 on acute nociceptive transmission Fabio Bordi*, Annarosa Ugolini Pharmacology Department, GlaxoWellcome Medicine Research Centre, Via Fleming 4, 37100 Verona, Italy Accepted 2 May 2000
Abstract The effect of the mGluR 5 antagonist, MPEP (2-Methyl-6-(phenylethynyl)-pyridine), and of the mGluR 1 antagonist, AIDA((RS)-1Aminoindan-1,5-dicarboxylic acid), were examined on nociceptive neurons in the ventroposterolateral (VPL) nucleus of the thalamus in response to pressure stimuli to the contralateral hindpaw of rats under urethane anesthesia. Intravenous (i.v.) injection of MPEP (0.1, 1, and 10 mg / kg) blocked responses to noxious stimulation in a dose-dependent and reversible manner. AIDA (3 and 15 mg / kg, i.v.), in contrast, had no effect on these cells. MPEP action was selective to noxious stimulation because even when tested at the highest dose (10 mg / kg, i.v.) it did not alter the responses of non-nociceptive neurons to brush stimulation. To investigate the site of action of MPEP, intra-thalamic injections were made during electrophysiological recordings. Using this method, the mGluR 5 antagonist did not affect nociceptive responses, suggesting that thalamic receptors were not involved in this action. On the other hand, the NMDA thalamic receptors seem to be involved because the NMDA receptor antagonist, MK801, successfully blocked responses to noxious pressure stimulation following intra-thalamic injections. In the spinal cord in vitro model, MPEP (30 mM, 60 min) was also able to attenuate ventral root potentials after single shock electrical stimulation of the dorsal root and inhibit wind-up response evoked by repetitive stimulation. Taken together, these findings suggest that blockade of the mGluR 5 , but not mGluR 1 decreases nociceptive transmission in the thalamus and that these effects may be mediated by spinal cord receptors. 2000 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Excitatory amino acid receptors: physiology, pharmacology and modulation Keywords: Metabotropic glutamate; mGluR 5 ; NMDA; Thalamus; Nociception; Sensory transmission; Spinal cord
1. Introduction It is widely accepted that the excitatory amino acids, glutamate and aspartate, are implicated the in transmission of both acute and chronic pain [3,13,48]. In particular, the N-methyl-D-aspartate (NMDA) glutamate receptor subtype has an important role in mediating nociceptive processing. Several results from behavioral studies suggest that NMDA receptor antagonists have antinociceptive effects in a number of models of nociception, both in animals and man [23,24,31,32,37,38]. Only recently, however, data have indicated the involvement of metabotropic glutamate receptors (mGluRs) in nociceptive transmission, in particular of Group I mGluRs, which include the mGluR 1 and mGluR 5 subtypes [10]. *Corresponding author. Tel.: 139-045-921-8845; fax: 139-045-9218047. E-mail address: [email protected] (F. Bordi)
Behavioral studies using the hot-plate model to study the nociceptive reflex showed that Group I mGluR antagonists injected intracerebroventricularly in mice induce a doserelated increase in paw-licking latency [11,30]. Rats pretreated with Group I mGluR antagonists injected intrathecally have reduced nociceptive scores in the formalin test, a widely used animal model of persistent pain, while the agonists 3,5-dihydroxyphenylglycine (DHPG) or 1S, 3R-1-amino-1,3-cyclopentanedicarboxylate (ACPD) enhanced these nociceptive responses in a dose-dependent manner [15]. Furthermore, antibodies selective for mGluR 1 or mGluR 5 injected intrathecally in rats, reduced the Group I mGluR selective agonist DHPG-induced spontaneous nociceptive behavior, and significantly attenuated hypersensitivity following chronic constriction injury (CCI) of the sciatic nerve [18]. Like NMDA receptor antagonists, Group I mGluR antagonists are also effective in inhibiting wind-up in spinal cord neurons [7], or reducing single shock electrical
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stimulation of the spinal cord dorsal root [6,7], an in vitro model used to study response to acute noxious stimulation. It is still not clear, however, whether Group I mGluR blockade is important primarily in chronic pain, as suggested by some studies [17,18,34,51], or it may be also effective in reducing nociceptive responses to acute pain. In addition, the relative contribution of the two Group I mGluR subtypes in nociceptive processes has not been evaluated yet. The availability of more potent and selective Group I mGluR antagonists is making now possible to examine the effects of the two receptor subtypes separately [19,40]. The aim of this study was to examine the role of the Group I mGluR subtypes in the processing of nociceptive stimuli, using the novel mGluR 5 antagonist 2-Methyl-6(phenylethynyl)-pyridine (MPEP) and the mGluR 1 -selective antagonist (RS)-1-Aminoindan-1,5-dicarboxylic acid (AIDA). Electrophysiological recordings were made of nociceptive responses of wide dynamic range (WDR) neurons in the ventroposterolateral (VPL) nucleus of the thalamus of anesthetized rats [5]. The spinothalamic tract originates in the dorsal horn of the spinal cord and terminates in VPL. Here, neurons have receptive fields on a relatively restricted area of the contralateral skin, making them well suited for the sensory–discriminative aspects of pain [25,36,48,50]. The effects of MPEP on nociceptive transmission were further investigated in the spinal cord using two in vitro models, single shock stimulation and wind-up evoked by repetitive stimulation.
2. Materials and methods
2.1. Thalamic in vivo recording 2.1.1. Animals and surgical preparation Male Sprague–Dawley rats (n542) weighing 300–400 g were anesthetized with urethane (1.5 g / kg, i.p.) and placed in a Kopf stereotaxic frame adjusted so that the surface of the skull was level between lambda and bregma. Body temperature was regulated at 37618C by means of a heating pad. Depth of anesthesia was monitored during the experiment by assessing responsivity to tail pinch and urethane was supplemented as needed. In most cases no extra anesthetic was necessary as the period of anesthesia lasted 4–5 h, determined by previous experiments. Stereotaxic coordinates were measured from bregma and calculated using a brain atlas [35]. A small hole was made over the cortex above VPL, and a stainless steel electrode (3–4 MV impedance at 1000 Hz; Frederick Haer & Co., Bowdoinham, ME) was inserted into VPL using an electronic micropositioner (Kopf Inst., Tujunga, CA). Neurons were identified by spontaneous activity or by a light brush stimulus of the contralateral hindpaw using a wooden probe or brush. Neurons responding to the search stimulus were selected and their responses tested to a graded
pressure stimulus applied to the receptive field. Data were collected for 2 s before the stimulus onset, during the 5-sec stimulus presentation, and for an additional 5 s at the end of stimulus offset. A tail vein was cannulated for intravenous (i.v.) injections. Drugs were tested once only in each animal, at a single dose. In some experiments (n59) drugs were administered directly into the VPL through a 33gauge injection needle connected to Hamilton syringes and assembled together with the recording electrode, allowing drug injection to be at a distance of 0.3–0.5 mm from the electrode tip. Injections were carried out over one min time.
2.1.2. Pain stimulation and recording A computer-controlled air cylinder was used to administer the pressure stimulus, as previously shown (Fig. 1A) [5]. Briefly, air pressure was controlled by a computerdriven Picospritzer II (General Valve, Fairfield, NJ) and was measured on-line in the air cylinder with a pressure transducer (Spectramed BV, Bithoven, the Netherlands). The stimulus was 5 s in duration, rising continuously from zero to a peak of about 4.2 kg / cm 2 , applied to the probe itself, which was about 2 mm 2 in cross section. The relation between air pressure and stimulus force was determined empirically. To determine the threshold to pain, a behavioral experiment was carried out. At a dose of urethane (1 g / kg, i.p.) that reduced motor tone but did not suppress the animal’s reflexes, pressure stimuli were applied to the animal’s hind paw and the mean pressure at which the behavioral response occurred was calculated for each rat. The mean pressure eliciting limb withdrawal was 3.160.15 kg / cm 2 (n58), as shown by dotted line in Fig. 1B. Once a neuron was isolated and found responsive to light brushing, its baseline responses to the noxious pressure stimulus were determined by applying the stimulus 10 times at 1-min intervals. The drug or vehicle was then injected intravenously (or intra-thalamically in some cases), and the stimulus was applied again to examine the effects of the treatment. Recovery from the effects of the drug were measured for the next h, during which responses were tested every 2–3 min. Neuronal potentials were amplified by an a.c. amplifier (Fintronic model WDR 420, Derby, CT), band pass filtered (400–8000 Hz), and displayed on a dual-trace storage oscilloscope (Tektronix 5111A, Beaverton, OR). A window discriminator was used to convert single action potentials to logic pulses, which were recorded (Digidata 1200, Axon Inst., Foster City, CA), displayed and stored to disk using customized software. Peristimulus histograms (PSTHs) and raster plots of each response were displayed at the end of each stimulus presentation on the computer screen. To obtain stimulus firing rates before and after drug treatment, firing rates for each neuron were measured at 9 levels of pressure (from 0 to 4.2 kg / cm 2 ). These mean
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Fig. 1. (A) Diagram of the pressure device. (B) Mean pre-drug stimulus–response function for all nociceptive wide dynamic range neurons before intravenous (i.v.) drug testing (n530). Dotted lines indicate mean pressure eliciting limb withdrawal in behavioral experiments (see Methods). (C) Evoked firing of a representative neuron before and after administration of morphine (0.5 mg / kg, i.v.). (D) Example of a typical response of a VPL neuron before and after administration of the vehicle. Top, level of applied pressure in register with the PSTHs below; Middle, average of pre-drug firing rate histograms and raster plot of the 10 individual trials; Bottom, PSTHs and raster plots after treatment. A single action potential is shown in the inset at the top left of each example.
firing rates and pressures were subjected to linear regression analysis. Only neurons that exhibited stimulus–response functions with a correlation coefficient of at least 0.5 were classified as wide dynamic range (WDR) neurons and included in the study [26]. Non-nociceptive mechanoresponsive neurons (n56) were also recorded in VPL. Because these neurons are
silent or low rates or spontaneous activity, as described [5,26], a search stimulus (light brush) was used regularly during the electrode penetration. Once isolated, the neuron’s response to light touch stimulation was examined. Tapping the skin within the receptive field of the neuron produced a reliable activation of mechanoresponsive neurons. These neurons were defined as non-nociceptive
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because they did not respond to noxious pressure stimulus with a greater response than to the light brush stimulus. Usually they also habituated rapidly to the pressure stimulus. Responses were measured to light taps every 1–2 min for 6–10 times before drug infusion and the average spikes calculated. The effect of the drug was examined 5–15 min post-injection to monitor recovery of the response. The average of the post-injection response was compared to pre-injection control. One cell only was tested in a single animal.
2.1.3. Data analysis For each unit, the mean spontaneous firing rate was calculated during the 2-s interval immediately before each cutaneous stimulation. Mean evoked activity was calculated during the 5 s of pressure stimulus. The average of all units for each drug condition was used to construct the time course of the effect of the drug as fraction of the pre-drug response. The effects of drug treatment on stimulus–response functions of nociceptive neurons were determined by calculating the average rate of firing of the cell at nine pressure levels before and after drug injection. A custom software program was used to construct PSTHs and individual raster plots, as shown in Fig. 1C and D. Responses were compared using ANOVA measurements followed by Newman–Keuls post-hoc comparisons. Statistical differences for non-nociceptive cells were established using the Student’s t-test. 2.1.4. Histology At the end of the experiment a small lesion was made at the recording site by applying an anodal current of 40–50 mA for 5 s Animals were then perfused with 10% buffered formalin, 5% potassium ferrocyanide, and 5% potassium ferricyanide [4]. The brains were frozen and cut on a sliding microtone (50-mm sections), and mounted sections were stained with cresyl violet. The position of the recording location was confirmed microscopically. 2.1.5. Drug treatment 2-Methyl-6-(phenylethynyl)-pyridine (MPEP), purchased from Tocris Cookson, UK, was dissolved in dimethyl sulphoxide (DMSO) (final concentration 0.5– 2%) and diluted in saline (0.9% NaCl). (RS)-1-Aminoindan-1,5-dicarboxylic acid (AIDA, Tocris Cookson, UK) was dissolved in equimolar NaOH (1 M) and diluted to final volume with saline. MK801 (RBI, Natick, MA) was dissolved in saline. The vehicle control was 2% DMSO. Drugs were administered in a volume of 1 ml / kg through the lateral tail vein. In the intra-thalamic experiments, MPEP (2 mg), MK801 (0.1 mg), or vehicle (DMSO 1%) were administered at a volume of 2 ml. MPEP was dissolved in DMSO and diluted in saline (1% final DMSO concentration). MK801 was dissolved in saline. For both drugs pH was adjusted to 7.660.2.
2.2. Spinal cord in vitro recording Spinal cords were prepared from 4 to 10 day old Sprague Dawley rat pups [43,44]. Under ether anesthesia animals were killed by decapitation, spinal cord removed and placed into aerated (95% O 2 , 5% CO 2 ) and cooled artificial cerebrospinal fluid (ACSF) (in mM: 126 NaCl, 2 KCl, 26 NaHCO 3 , 1.25 NaH 2 PO 4 , 10 glucose, 2 MgSO 4 , 3 CaCl 2 , pH 7.4). Spinal cords were hemisected, transferred to a recording chamber and superfused at 3 ml / min with aerated Krebs solution at room temperature (20– 238C). Single shock electrical stimulation of the spinal cord dorsal root, sufficient to recruit both A- and C-fibers, is used to study in vitro the response to acute noxious stimulation [42]. Spinal reflex activity was evoked by dorsal root stimulation and measured as a ventral root potential (VRP). VRPs were recorded with close-fitting suction electrodes from the L 4 or L 5 ventral roots following electrical stimulation of the ipsilateral L 4 or L 5 dorsal roots. The stimulus intensity used was 50 V, 1 ms which evoked a compound action potential on the ventral root which corresponded to recruitment of afferent fibers conducting within the C-group IV afferent fiber range [41]. In different experiments, short-duration (20 s) trains at 1 Hz were used to evoked a cumulative ventral root response (‘wind-up’). The wind-up response reproduces a phenomenon of central sensitization and it is used as a model to study the antinociceptive effects of drugs [49]. Responses were amplified (Axon Instruments), digitized and recorded by a PC using a custom software. MPEP was dissolved in 100% DMSO, diluted in ACSF from stock solution (final DMSO concentration 0.3%), and superfused into the recording chamber at the same flow rate as control ACSF.
3. Results
3.1. Effects of drug treatment on VPL cells A total of 36 cells isolated in the VPL responded to noxious stimulation of the contralateral hindpaw and an additional 6 cells were activated by innocuous tactile stimulation. Cells were identified on the basis of spontaneous activity or in response to cutaneous search stimuli (light brushing of the skin). The recording sites of all units were located in the VPL region, as previously reported [1,5,12,26].
3.1.1. Effects of intravenous drug treatment on VPL nociceptive neurons The VPL nociceptive neurons had an average spontaneous activity of 4.360.5 Hz. Their firing rate increased up to 22.361.0 Hz at the maximum pressure strength of the graded mechanical stimulus applied to their receptive field (Fig. 1B). In some experiments (n53), the effects of intravenous
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morphine (0.5 mg / kg) on the responses of these neurons to the pressure stimulus was investigated. Responses of a representative VPL neuron are shown in Fig. 1C. Morphine produced a marked depression of the stimulus-evoked response, as previously shown [5], and in agreement with earlier findings [2,20,21]. Injection of the vehicle control (DMSO 2%), on the other hand, was without effect on nociceptive responses (Fig. 1D). The effect of mGluR 5 blockade on nociceptive function was tested using the specific blocker MPEP. As shown in Fig. 2, MPEP has a potent antinociceptive action. All three doses employed significantly reduced the response at all pressures above |1.5 kg / cm 2 . There were dose-dependent differences in the length of time that MPEP was effective (Fig. 2B). At 10 mg / kg (n55), MPEP was effective for the entire period of recording following the injection (Fig. 2D). At 0.1 mg / kg (n55), MPEP action on nociceptive responses of WDR neurons was different from control up to 15 min post-injection time (P,0.05), but undistinguishable from control 30 and 60 min post-injection (Fig. 2C). At 1 mg / kg (n55), MPEP significantly reduced the responsiveness of nociceptive cells for 15 min (P,0.01). The response looked still different from control 30 and 60 min after i.v. injection, but there was no statistical difference (Fig. 2B). The mGluR 1 -preferring antagonist AIDA [30] was also tested on VPL neurons. In contrast to the effects of MPEP, AIDA did not reduce the responsiveness of nociceptive cells at 3 or 15 mg / kg (n55 and n53, respectively, Fig. 3). Responses of cells after treatment with AIDA were not different from control at all times after injection.
3.1.2. Effects of MPEP on non-nociceptive neurons A total of 6 non-nociceptive neurons were identified by search stimulation. They all had no spontaneous activity and responded to light touch of the contralateral hindpaw. After intravenous injection of MPEP (10 mg / kg, n56) the response to non-noxious stimulation was examined again. MPEP did not reduce the response to cutaneous stimulation as determined by counting the number of spikes elicited by brushing the skin (a light touch) (Fig. 4). Responses after treatment were 88% of the pre-drug response (t52.2, ns). 3.1.3. Effects of intra-thalamic treatment on nociceptive neurons To determine the place of action of MPEP effects, in some experiments injections were made directly into the VPL nucleus near the recording site. Fig. 5 shows that MPEP injection (2 mg) did not modify evoked responses of WDR neurons to noxious pressure stimulation. On average, response of the cells treated with MPEP was 92.7% of pre-drug period (n53). In contrast, the NMDA blocker MK801 markedly reduced firing evoked by noxious stimulation. After MK801 intra-thalamic injections
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(0.1 mg), responses were 26.7% of pre-injection time (n53; t56.9, P,0.05).
3.2. Effects of MPEP on spinal cord in vitro preparation MPEP was able to attenuate the late prolonged component of ventral root potentials after single shock electrical stimulation of the spinal cord dorsal root (Fig. 6A). On average, MPEP (30 mM, 60 min) depressed the late phase of the response, measured as the area under the curve 8–20 s post-stimulation, by 3761.8% (n55, P,0.05). Likewise, MPEP showed a significant inhibition of wind-up evoked by repetitive stimulation (Fig. 6B). At 30-mM dose (n57, 60 min) MPEP depressed the response, measured as the area under the curve, by 2661.4% (P,0.05). At a dose of 3 mM (n57, 60 min), MPEP was able to reduce wind-up by 1062.6% (not shown).
4. Discussion The present study shows that the mGluR 5 -specific antagonist MPEP inhibits stimulus-evoked activity of nociceptive responses in VPL neurons of the rat thalamus. The effects were potent, dose-dependent, and reversible. MPEP was selective for noxious stimulation, because it did not alter the firing of non-nociceptive neurons responding to touch. In contrast, the mGluR 1 antagonist AIDA did not change the firing of nociceptive neurons in the VPL nucleus of the thalamus, suggesting that mGluR 1 may not be involved in acute pain processing. Earlier findings have showed evidence for an involvement of Group I mGluRs, and in particular mGluR 1 , in nociceptive transmission. The mGluR 1 -preferring antagonists (S)-4-carboxyphenylglycine (4CPG) or (S)-4-carboxy-3-hydroxyphenylglycine (4C3HPG) were shown to significantly reduce sensitization induced by carrageenan [52], or by intraplantar formalin application [16]. Recently, intrathecal injection of mGluR 1 antisense reduced responses of dorsal horn neurons to repeated cutaneous mustard oil applications and attenuated behavioral nociceptive responses to noxious heat applied to the tail of rats [51]. The more selective and potent mGluR 1 antagonist AIDA was also found to inhibit noxious responses after capsaicin-induced central sensitization of primate spinal cells in vivo [33] and to increase pain thresholds in mice [30]. All these findings seem to strongly suggest a role for mGluR 1 in chronic pain, and in some cases in acute pain processing associated with thermal stimulation. Our study, using AIDA to obtain blockade of mGluR 1 , rules out an involvement of these receptors in the mediation of responses to noxious mechanical stimuli under normal conditions. Given the efficacy of AIDA in learning and memory behavioral tests even at doses well below those used in the present study [8,9], we are confident that
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Fig. 2. Effects of MPEP on nociceptive VPL neurons. (A) Mean stimulus–response function after the i.v. administration of vehicle (filled circles, n54), 0.1 mg / kg MPEP (filled diamonds, n55), 1 mg / kg MPEP (filled squares, n55), or 10 mg / kg MPEP (filled triangles, n55). (B) Evoked firing over time before and after administration of the vehicle (filled circles, n54) or MPEP (0.1 mg / kg, n55; 1 mg / kg, n55; 10 mg / kg, n55). (C) Example of a response to pressure stimulus of a VPL neuron before, immediately after, and 60 min after 0.1 mg / kg MPEP i.v. injection. (D) Response of a VPL neuron before, immediately after, and 60 min after 10 mg / kg MPEP i.v. injection. Top, level of applied pressure in register with the PSTHs below. Middle, average of pre-drug firing rate histograms and raster plot of the 10 individual trials. Bottom, PSTHs and raster plots after drug injection. A single action potential is shown for each example. Asterisks indicate statistical difference from vehicle (**P,0.01, *P,0.05, ANOVA followed by Newman–Keuls tests).
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Fig. 3. Effects of AIDA on nociceptive VPL neurons. (A) Mean stimulus–response function after injection of vehicle (filled circles, n54), 3 mg / kg AIDA (open triangles, n55), or 15 mg / kg AIDA (open squares, n53). (B) Effect of AIDA in a representative neuron. Top, pressure applied to the paw. Average PSTHs and individual raster plots before (middle) and after (bottom) injection of AIDA (15 mg / kg, i.v.).
the drug reached the appropriate brain areas. It is still possible, however, that AIDA may be efficacious in blocking acute pain responses in spinal cord cells. The lack of effect of the mGluR 5 antagonist on nonnociceptive mechanosensitive neurons in VPL implies that the inhibiting effect of MPEP on noxious stimulus-evoked activity does not represent an anesthetic effect of the drug
and suggests that the effect of mGluR 5 on nociceptive cells is selective. MPEP, however, did not modify neuronal activity when the drug was injected directly into the thalamus near the site of the electrophysiological recordings, excluding an involvement of thalamic mGluR 5 in the antagonist’s antinociceptive effects and hinting at an effect at the spinal
Fig. 4. Effects of MPEP on non-nociceptive VPL neurons. (A) Activity of a representative non-nociceptive neuron before and after the intravenous administration of MPEP (10 mg / kg). The response of the neuron following brushing stimulation of the hind paw is shown at the top. (B) Average response of non-nociceptive neurons after MPEP (10 mg / kg, n56), expressed as percent of pre-drug control value.
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Fig. 5. Intra-thalamic drug treatment. (A) Responses of a nociceptive VPL neuron after MPEP injection (2 mg, 2 ml). (B) Example of a neuron treated with the NMDA antagonist MK801 (0.1 mg, 2 ml). Top, level of applied pressure in register with the PSTHs below. Middle, average of pre-drug firing rate histograms and raster plot of the ten individual trials. Bottom, PSTHs and raster plots after drug injection. A single action potential is shown for each example.
level. In contrast, recent data have shown antinociceptive actions of MPEP to noxious heat stimulation when administered iontophoretically into the thalamus [39]. Thus, a direct intra-thalamic injection of MPEP in our data seems to have a less focal effect. Alternatively, thalamic mGluR 5 may be involved in non-mechanical nociceptive stimulation. More studies are needed to ascertain the site of action of the mGluR 5 antagonist. Our in vitro spinal cord recordings seem to suggest an
involvement of spinal mGluR 5 in nociceptive processing because MPEP was effective against nociceptive responses, both in the single shock stimulus of dorsal root and after the wind-up evoked by repetitive stimulation. While the wind-up model is suggested to be a central mechanism for hyperalgesia [29], single shock electrical stimulation of the spinal cord dorsal root, sufficient to recruit both A- and C-fibers, is used to study in vitro the response to acute noxious stimulation [42]. The late phase
Fig. 6. (A) Effect of MPEP (30 mM) on a spinal cord preparation after single stimulation of the dorsal root (50 V, 1 ms). One h perfusion of 30 mM MPEP reduced the evoked response. (B) Representative trace showing the inhibitory effect of MPEP on cumulative depolarization (‘wind-up’) of the ventral root evoked by 20 s of 1 Hz repetitive high-intensity (50 V, 1 ms) electrical stimulation of the dorsal root.
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of this response, mostly attributed to C-fiber activation and defined as ‘peptidergic’ because it is also affected by neurokinin receptor antagonists [41,42], was attenuated by MPEP. The early, monosynaptic component of the response, dependent on AMPA and kainate receptors [14], instead was not affected by MPEP in our study. The less marked effects obtained with MPEP in the spinal cord preparation could be due to the younger age of the animals compared to those used for the thalamic recordings. Since the expression of mGluRs is developmentally regulated, it is possible that our findings were influenced by the development changes in the level of mGluR 1 and mGluR 5 receptors. The NMDA receptor antagonist MK801, on the other hand, inhibited noxious-evoked responses in VPL neurons after intra-thalamic injections. This finding extends our earlier demonstration that MK801 blocked acute nociceptive activity in the thalamus after intravenous treatment [5], and suggests that NMDA receptors located in the thalamus are relevant in the modulation of pain transmission either via ascending or descending pain pathways. Recent behavioral results are also supporting the view that thalamic NMDA receptors may participate in the response to noxious stimulation [22], whereas spinal NMDA receptors play a minor role on acute processing (see [5], for a discussion). Our earlier evidence also showed that MK801 is less selective than MPEP in its action because it also significantly depressed firing of non-nociceptive mechanosensitive VPL neurons [5]. Activation of Group I mGluRs can modulate NMDA and AMPA receptor-mediated responses in a number of brain areas, including the spinal cord [43]. Recent data have shown that both mGluR 5 and mGluR 1 act to enhance ionotropic glutamate responses in spinal cord, but the two types of mGluRs may have different intracellular mechanisms of action [44]. Some behavioral data have also suggested some interaction between Group I mGluR agonists and ionotropic glutamate receptors to produce enhanced nociceptive responses and hyperalgesia [15,27,28]. It is possible that interaction with NMDA and / or AMPA receptors with mGluR 1 may be more efficacious in modulating nociceptive transmission associated with chronic, sensitized pain, while interaction with mGluR 5 may be relevant also for modulation of acute pain. Recently, MPEP has been reported also efficacious in an inflammation-induced pain model [47]. More studies are needed to clarify this point. Group I mGluRs have been implicated in nociception (reviewed in [6]), but particularly in persistent pain associated with central sensitization [17,34,52,53]. The present study shows, for the first time, that a subtype of this Group, mGluR 5 , may be also important in mediating mechanical acute nociceptive responses, without affecting normal sensory perception. We suggest a possible involvement of spinal receptors in this action, in accord with immunohistochemical distribution studies showing the
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presence of mGluR 5 in layers I and II of the dorsal horn [45,46]. Blockade of these receptors may be a promising new route for treatment of pain disorders.
Acknowledgements The authors wish to thank Mr. Giorgio Tarter for technical assistance and Prof. Eric Frank (University of Pittsburgh) for providing the software analysis and for his valuable comments on the manuscript.
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Research report
Involvement of mGluR 5 on acute nociceptive transmission Fabio Bordi*, Annarosa Ugolini Pharmacology Department, GlaxoWellcome Medicine Research Centre, Via Fleming 4, 37100 Verona, Italy Accepted 2 May 2000
Abstract The effect of the mGluR 5 antagonist, MPEP (2-Methyl-6-(phenylethynyl)-pyridine), and of the mGluR 1 antagonist, AIDA((RS)-1Aminoindan-1,5-dicarboxylic acid), were examined on nociceptive neurons in the ventroposterolateral (VPL) nucleus of the thalamus in response to pressure stimuli to the contralateral hindpaw of rats under urethane anesthesia. Intravenous (i.v.) injection of MPEP (0.1, 1, and 10 mg / kg) blocked responses to noxious stimulation in a dose-dependent and reversible manner. AIDA (3 and 15 mg / kg, i.v.), in contrast, had no effect on these cells. MPEP action was selective to noxious stimulation because even when tested at the highest dose (10 mg / kg, i.v.) it did not alter the responses of non-nociceptive neurons to brush stimulation. To investigate the site of action of MPEP, intra-thalamic injections were made during electrophysiological recordings. Using this method, the mGluR 5 antagonist did not affect nociceptive responses, suggesting that thalamic receptors were not involved in this action. On the other hand, the NMDA thalamic receptors seem to be involved because the NMDA receptor antagonist, MK801, successfully blocked responses to noxious pressure stimulation following intra-thalamic injections. In the spinal cord in vitro model, MPEP (30 mM, 60 min) was also able to attenuate ventral root potentials after single shock electrical stimulation of the dorsal root and inhibit wind-up response evoked by repetitive stimulation. Taken together, these findings suggest that blockade of the mGluR 5 , but not mGluR 1 decreases nociceptive transmission in the thalamus and that these effects may be mediated by spinal cord receptors. 2000 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Excitatory amino acid receptors: physiology, pharmacology and modulation Keywords: Metabotropic glutamate; mGluR 5 ; NMDA; Thalamus; Nociception; Sensory transmission; Spinal cord
1. Introduction It is widely accepted that the excitatory amino acids, glutamate and aspartate, are implicated the in transmission of both acute and chronic pain [3,13,48]. In particular, the N-methyl-D-aspartate (NMDA) glutamate receptor subtype has an important role in mediating nociceptive processing. Several results from behavioral studies suggest that NMDA receptor antagonists have antinociceptive effects in a number of models of nociception, both in animals and man [23,24,31,32,37,38]. Only recently, however, data have indicated the involvement of metabotropic glutamate receptors (mGluRs) in nociceptive transmission, in particular of Group I mGluRs, which include the mGluR 1 and mGluR 5 subtypes [10]. *Corresponding author. Tel.: 139-045-921-8845; fax: 139-045-9218047. E-mail address: [email protected] (F. Bordi)
Behavioral studies using the hot-plate model to study the nociceptive reflex showed that Group I mGluR antagonists injected intracerebroventricularly in mice induce a doserelated increase in paw-licking latency [11,30]. Rats pretreated with Group I mGluR antagonists injected intrathecally have reduced nociceptive scores in the formalin test, a widely used animal model of persistent pain, while the agonists 3,5-dihydroxyphenylglycine (DHPG) or 1S, 3R-1-amino-1,3-cyclopentanedicarboxylate (ACPD) enhanced these nociceptive responses in a dose-dependent manner [15]. Furthermore, antibodies selective for mGluR 1 or mGluR 5 injected intrathecally in rats, reduced the Group I mGluR selective agonist DHPG-induced spontaneous nociceptive behavior, and significantly attenuated hypersensitivity following chronic constriction injury (CCI) of the sciatic nerve [18]. Like NMDA receptor antagonists, Group I mGluR antagonists are also effective in inhibiting wind-up in spinal cord neurons [7], or reducing single shock electrical
0006-8993 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )02467-7
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stimulation of the spinal cord dorsal root [6,7], an in vitro model used to study response to acute noxious stimulation. It is still not clear, however, whether Group I mGluR blockade is important primarily in chronic pain, as suggested by some studies [17,18,34,51], or it may be also effective in reducing nociceptive responses to acute pain. In addition, the relative contribution of the two Group I mGluR subtypes in nociceptive processes has not been evaluated yet. The availability of more potent and selective Group I mGluR antagonists is making now possible to examine the effects of the two receptor subtypes separately [19,40]. The aim of this study was to examine the role of the Group I mGluR subtypes in the processing of nociceptive stimuli, using the novel mGluR 5 antagonist 2-Methyl-6(phenylethynyl)-pyridine (MPEP) and the mGluR 1 -selective antagonist (RS)-1-Aminoindan-1,5-dicarboxylic acid (AIDA). Electrophysiological recordings were made of nociceptive responses of wide dynamic range (WDR) neurons in the ventroposterolateral (VPL) nucleus of the thalamus of anesthetized rats [5]. The spinothalamic tract originates in the dorsal horn of the spinal cord and terminates in VPL. Here, neurons have receptive fields on a relatively restricted area of the contralateral skin, making them well suited for the sensory–discriminative aspects of pain [25,36,48,50]. The effects of MPEP on nociceptive transmission were further investigated in the spinal cord using two in vitro models, single shock stimulation and wind-up evoked by repetitive stimulation.
2. Materials and methods
2.1. Thalamic in vivo recording 2.1.1. Animals and surgical preparation Male Sprague–Dawley rats (n542) weighing 300–400 g were anesthetized with urethane (1.5 g / kg, i.p.) and placed in a Kopf stereotaxic frame adjusted so that the surface of the skull was level between lambda and bregma. Body temperature was regulated at 37618C by means of a heating pad. Depth of anesthesia was monitored during the experiment by assessing responsivity to tail pinch and urethane was supplemented as needed. In most cases no extra anesthetic was necessary as the period of anesthesia lasted 4–5 h, determined by previous experiments. Stereotaxic coordinates were measured from bregma and calculated using a brain atlas [35]. A small hole was made over the cortex above VPL, and a stainless steel electrode (3–4 MV impedance at 1000 Hz; Frederick Haer & Co., Bowdoinham, ME) was inserted into VPL using an electronic micropositioner (Kopf Inst., Tujunga, CA). Neurons were identified by spontaneous activity or by a light brush stimulus of the contralateral hindpaw using a wooden probe or brush. Neurons responding to the search stimulus were selected and their responses tested to a graded
pressure stimulus applied to the receptive field. Data were collected for 2 s before the stimulus onset, during the 5-sec stimulus presentation, and for an additional 5 s at the end of stimulus offset. A tail vein was cannulated for intravenous (i.v.) injections. Drugs were tested once only in each animal, at a single dose. In some experiments (n59) drugs were administered directly into the VPL through a 33gauge injection needle connected to Hamilton syringes and assembled together with the recording electrode, allowing drug injection to be at a distance of 0.3–0.5 mm from the electrode tip. Injections were carried out over one min time.
2.1.2. Pain stimulation and recording A computer-controlled air cylinder was used to administer the pressure stimulus, as previously shown (Fig. 1A) [5]. Briefly, air pressure was controlled by a computerdriven Picospritzer II (General Valve, Fairfield, NJ) and was measured on-line in the air cylinder with a pressure transducer (Spectramed BV, Bithoven, the Netherlands). The stimulus was 5 s in duration, rising continuously from zero to a peak of about 4.2 kg / cm 2 , applied to the probe itself, which was about 2 mm 2 in cross section. The relation between air pressure and stimulus force was determined empirically. To determine the threshold to pain, a behavioral experiment was carried out. At a dose of urethane (1 g / kg, i.p.) that reduced motor tone but did not suppress the animal’s reflexes, pressure stimuli were applied to the animal’s hind paw and the mean pressure at which the behavioral response occurred was calculated for each rat. The mean pressure eliciting limb withdrawal was 3.160.15 kg / cm 2 (n58), as shown by dotted line in Fig. 1B. Once a neuron was isolated and found responsive to light brushing, its baseline responses to the noxious pressure stimulus were determined by applying the stimulus 10 times at 1-min intervals. The drug or vehicle was then injected intravenously (or intra-thalamically in some cases), and the stimulus was applied again to examine the effects of the treatment. Recovery from the effects of the drug were measured for the next h, during which responses were tested every 2–3 min. Neuronal potentials were amplified by an a.c. amplifier (Fintronic model WDR 420, Derby, CT), band pass filtered (400–8000 Hz), and displayed on a dual-trace storage oscilloscope (Tektronix 5111A, Beaverton, OR). A window discriminator was used to convert single action potentials to logic pulses, which were recorded (Digidata 1200, Axon Inst., Foster City, CA), displayed and stored to disk using customized software. Peristimulus histograms (PSTHs) and raster plots of each response were displayed at the end of each stimulus presentation on the computer screen. To obtain stimulus firing rates before and after drug treatment, firing rates for each neuron were measured at 9 levels of pressure (from 0 to 4.2 kg / cm 2 ). These mean
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Fig. 1. (A) Diagram of the pressure device. (B) Mean pre-drug stimulus–response function for all nociceptive wide dynamic range neurons before intravenous (i.v.) drug testing (n530). Dotted lines indicate mean pressure eliciting limb withdrawal in behavioral experiments (see Methods). (C) Evoked firing of a representative neuron before and after administration of morphine (0.5 mg / kg, i.v.). (D) Example of a typical response of a VPL neuron before and after administration of the vehicle. Top, level of applied pressure in register with the PSTHs below; Middle, average of pre-drug firing rate histograms and raster plot of the 10 individual trials; Bottom, PSTHs and raster plots after treatment. A single action potential is shown in the inset at the top left of each example.
firing rates and pressures were subjected to linear regression analysis. Only neurons that exhibited stimulus–response functions with a correlation coefficient of at least 0.5 were classified as wide dynamic range (WDR) neurons and included in the study [26]. Non-nociceptive mechanoresponsive neurons (n56) were also recorded in VPL. Because these neurons are
silent or low rates or spontaneous activity, as described [5,26], a search stimulus (light brush) was used regularly during the electrode penetration. Once isolated, the neuron’s response to light touch stimulation was examined. Tapping the skin within the receptive field of the neuron produced a reliable activation of mechanoresponsive neurons. These neurons were defined as non-nociceptive
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because they did not respond to noxious pressure stimulus with a greater response than to the light brush stimulus. Usually they also habituated rapidly to the pressure stimulus. Responses were measured to light taps every 1–2 min for 6–10 times before drug infusion and the average spikes calculated. The effect of the drug was examined 5–15 min post-injection to monitor recovery of the response. The average of the post-injection response was compared to pre-injection control. One cell only was tested in a single animal.
2.1.3. Data analysis For each unit, the mean spontaneous firing rate was calculated during the 2-s interval immediately before each cutaneous stimulation. Mean evoked activity was calculated during the 5 s of pressure stimulus. The average of all units for each drug condition was used to construct the time course of the effect of the drug as fraction of the pre-drug response. The effects of drug treatment on stimulus–response functions of nociceptive neurons were determined by calculating the average rate of firing of the cell at nine pressure levels before and after drug injection. A custom software program was used to construct PSTHs and individual raster plots, as shown in Fig. 1C and D. Responses were compared using ANOVA measurements followed by Newman–Keuls post-hoc comparisons. Statistical differences for non-nociceptive cells were established using the Student’s t-test. 2.1.4. Histology At the end of the experiment a small lesion was made at the recording site by applying an anodal current of 40–50 mA for 5 s Animals were then perfused with 10% buffered formalin, 5% potassium ferrocyanide, and 5% potassium ferricyanide [4]. The brains were frozen and cut on a sliding microtone (50-mm sections), and mounted sections were stained with cresyl violet. The position of the recording location was confirmed microscopically. 2.1.5. Drug treatment 2-Methyl-6-(phenylethynyl)-pyridine (MPEP), purchased from Tocris Cookson, UK, was dissolved in dimethyl sulphoxide (DMSO) (final concentration 0.5– 2%) and diluted in saline (0.9% NaCl). (RS)-1-Aminoindan-1,5-dicarboxylic acid (AIDA, Tocris Cookson, UK) was dissolved in equimolar NaOH (1 M) and diluted to final volume with saline. MK801 (RBI, Natick, MA) was dissolved in saline. The vehicle control was 2% DMSO. Drugs were administered in a volume of 1 ml / kg through the lateral tail vein. In the intra-thalamic experiments, MPEP (2 mg), MK801 (0.1 mg), or vehicle (DMSO 1%) were administered at a volume of 2 ml. MPEP was dissolved in DMSO and diluted in saline (1% final DMSO concentration). MK801 was dissolved in saline. For both drugs pH was adjusted to 7.660.2.
2.2. Spinal cord in vitro recording Spinal cords were prepared from 4 to 10 day old Sprague Dawley rat pups [43,44]. Under ether anesthesia animals were killed by decapitation, spinal cord removed and placed into aerated (95% O 2 , 5% CO 2 ) and cooled artificial cerebrospinal fluid (ACSF) (in mM: 126 NaCl, 2 KCl, 26 NaHCO 3 , 1.25 NaH 2 PO 4 , 10 glucose, 2 MgSO 4 , 3 CaCl 2 , pH 7.4). Spinal cords were hemisected, transferred to a recording chamber and superfused at 3 ml / min with aerated Krebs solution at room temperature (20– 238C). Single shock electrical stimulation of the spinal cord dorsal root, sufficient to recruit both A- and C-fibers, is used to study in vitro the response to acute noxious stimulation [42]. Spinal reflex activity was evoked by dorsal root stimulation and measured as a ventral root potential (VRP). VRPs were recorded with close-fitting suction electrodes from the L 4 or L 5 ventral roots following electrical stimulation of the ipsilateral L 4 or L 5 dorsal roots. The stimulus intensity used was 50 V, 1 ms which evoked a compound action potential on the ventral root which corresponded to recruitment of afferent fibers conducting within the C-group IV afferent fiber range [41]. In different experiments, short-duration (20 s) trains at 1 Hz were used to evoked a cumulative ventral root response (‘wind-up’). The wind-up response reproduces a phenomenon of central sensitization and it is used as a model to study the antinociceptive effects of drugs [49]. Responses were amplified (Axon Instruments), digitized and recorded by a PC using a custom software. MPEP was dissolved in 100% DMSO, diluted in ACSF from stock solution (final DMSO concentration 0.3%), and superfused into the recording chamber at the same flow rate as control ACSF.
3. Results
3.1. Effects of drug treatment on VPL cells A total of 36 cells isolated in the VPL responded to noxious stimulation of the contralateral hindpaw and an additional 6 cells were activated by innocuous tactile stimulation. Cells were identified on the basis of spontaneous activity or in response to cutaneous search stimuli (light brushing of the skin). The recording sites of all units were located in the VPL region, as previously reported [1,5,12,26].
3.1.1. Effects of intravenous drug treatment on VPL nociceptive neurons The VPL nociceptive neurons had an average spontaneous activity of 4.360.5 Hz. Their firing rate increased up to 22.361.0 Hz at the maximum pressure strength of the graded mechanical stimulus applied to their receptive field (Fig. 1B). In some experiments (n53), the effects of intravenous
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morphine (0.5 mg / kg) on the responses of these neurons to the pressure stimulus was investigated. Responses of a representative VPL neuron are shown in Fig. 1C. Morphine produced a marked depression of the stimulus-evoked response, as previously shown [5], and in agreement with earlier findings [2,20,21]. Injection of the vehicle control (DMSO 2%), on the other hand, was without effect on nociceptive responses (Fig. 1D). The effect of mGluR 5 blockade on nociceptive function was tested using the specific blocker MPEP. As shown in Fig. 2, MPEP has a potent antinociceptive action. All three doses employed significantly reduced the response at all pressures above |1.5 kg / cm 2 . There were dose-dependent differences in the length of time that MPEP was effective (Fig. 2B). At 10 mg / kg (n55), MPEP was effective for the entire period of recording following the injection (Fig. 2D). At 0.1 mg / kg (n55), MPEP action on nociceptive responses of WDR neurons was different from control up to 15 min post-injection time (P,0.05), but undistinguishable from control 30 and 60 min post-injection (Fig. 2C). At 1 mg / kg (n55), MPEP significantly reduced the responsiveness of nociceptive cells for 15 min (P,0.01). The response looked still different from control 30 and 60 min after i.v. injection, but there was no statistical difference (Fig. 2B). The mGluR 1 -preferring antagonist AIDA [30] was also tested on VPL neurons. In contrast to the effects of MPEP, AIDA did not reduce the responsiveness of nociceptive cells at 3 or 15 mg / kg (n55 and n53, respectively, Fig. 3). Responses of cells after treatment with AIDA were not different from control at all times after injection.
3.1.2. Effects of MPEP on non-nociceptive neurons A total of 6 non-nociceptive neurons were identified by search stimulation. They all had no spontaneous activity and responded to light touch of the contralateral hindpaw. After intravenous injection of MPEP (10 mg / kg, n56) the response to non-noxious stimulation was examined again. MPEP did not reduce the response to cutaneous stimulation as determined by counting the number of spikes elicited by brushing the skin (a light touch) (Fig. 4). Responses after treatment were 88% of the pre-drug response (t52.2, ns). 3.1.3. Effects of intra-thalamic treatment on nociceptive neurons To determine the place of action of MPEP effects, in some experiments injections were made directly into the VPL nucleus near the recording site. Fig. 5 shows that MPEP injection (2 mg) did not modify evoked responses of WDR neurons to noxious pressure stimulation. On average, response of the cells treated with MPEP was 92.7% of pre-drug period (n53). In contrast, the NMDA blocker MK801 markedly reduced firing evoked by noxious stimulation. After MK801 intra-thalamic injections
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(0.1 mg), responses were 26.7% of pre-injection time (n53; t56.9, P,0.05).
3.2. Effects of MPEP on spinal cord in vitro preparation MPEP was able to attenuate the late prolonged component of ventral root potentials after single shock electrical stimulation of the spinal cord dorsal root (Fig. 6A). On average, MPEP (30 mM, 60 min) depressed the late phase of the response, measured as the area under the curve 8–20 s post-stimulation, by 3761.8% (n55, P,0.05). Likewise, MPEP showed a significant inhibition of wind-up evoked by repetitive stimulation (Fig. 6B). At 30-mM dose (n57, 60 min) MPEP depressed the response, measured as the area under the curve, by 2661.4% (P,0.05). At a dose of 3 mM (n57, 60 min), MPEP was able to reduce wind-up by 1062.6% (not shown).
4. Discussion The present study shows that the mGluR 5 -specific antagonist MPEP inhibits stimulus-evoked activity of nociceptive responses in VPL neurons of the rat thalamus. The effects were potent, dose-dependent, and reversible. MPEP was selective for noxious stimulation, because it did not alter the firing of non-nociceptive neurons responding to touch. In contrast, the mGluR 1 antagonist AIDA did not change the firing of nociceptive neurons in the VPL nucleus of the thalamus, suggesting that mGluR 1 may not be involved in acute pain processing. Earlier findings have showed evidence for an involvement of Group I mGluRs, and in particular mGluR 1 , in nociceptive transmission. The mGluR 1 -preferring antagonists (S)-4-carboxyphenylglycine (4CPG) or (S)-4-carboxy-3-hydroxyphenylglycine (4C3HPG) were shown to significantly reduce sensitization induced by carrageenan [52], or by intraplantar formalin application [16]. Recently, intrathecal injection of mGluR 1 antisense reduced responses of dorsal horn neurons to repeated cutaneous mustard oil applications and attenuated behavioral nociceptive responses to noxious heat applied to the tail of rats [51]. The more selective and potent mGluR 1 antagonist AIDA was also found to inhibit noxious responses after capsaicin-induced central sensitization of primate spinal cells in vivo [33] and to increase pain thresholds in mice [30]. All these findings seem to strongly suggest a role for mGluR 1 in chronic pain, and in some cases in acute pain processing associated with thermal stimulation. Our study, using AIDA to obtain blockade of mGluR 1 , rules out an involvement of these receptors in the mediation of responses to noxious mechanical stimuli under normal conditions. Given the efficacy of AIDA in learning and memory behavioral tests even at doses well below those used in the present study [8,9], we are confident that
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Fig. 2. Effects of MPEP on nociceptive VPL neurons. (A) Mean stimulus–response function after the i.v. administration of vehicle (filled circles, n54), 0.1 mg / kg MPEP (filled diamonds, n55), 1 mg / kg MPEP (filled squares, n55), or 10 mg / kg MPEP (filled triangles, n55). (B) Evoked firing over time before and after administration of the vehicle (filled circles, n54) or MPEP (0.1 mg / kg, n55; 1 mg / kg, n55; 10 mg / kg, n55). (C) Example of a response to pressure stimulus of a VPL neuron before, immediately after, and 60 min after 0.1 mg / kg MPEP i.v. injection. (D) Response of a VPL neuron before, immediately after, and 60 min after 10 mg / kg MPEP i.v. injection. Top, level of applied pressure in register with the PSTHs below. Middle, average of pre-drug firing rate histograms and raster plot of the 10 individual trials. Bottom, PSTHs and raster plots after drug injection. A single action potential is shown for each example. Asterisks indicate statistical difference from vehicle (**P,0.01, *P,0.05, ANOVA followed by Newman–Keuls tests).
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Fig. 3. Effects of AIDA on nociceptive VPL neurons. (A) Mean stimulus–response function after injection of vehicle (filled circles, n54), 3 mg / kg AIDA (open triangles, n55), or 15 mg / kg AIDA (open squares, n53). (B) Effect of AIDA in a representative neuron. Top, pressure applied to the paw. Average PSTHs and individual raster plots before (middle) and after (bottom) injection of AIDA (15 mg / kg, i.v.).
the drug reached the appropriate brain areas. It is still possible, however, that AIDA may be efficacious in blocking acute pain responses in spinal cord cells. The lack of effect of the mGluR 5 antagonist on nonnociceptive mechanosensitive neurons in VPL implies that the inhibiting effect of MPEP on noxious stimulus-evoked activity does not represent an anesthetic effect of the drug
and suggests that the effect of mGluR 5 on nociceptive cells is selective. MPEP, however, did not modify neuronal activity when the drug was injected directly into the thalamus near the site of the electrophysiological recordings, excluding an involvement of thalamic mGluR 5 in the antagonist’s antinociceptive effects and hinting at an effect at the spinal
Fig. 4. Effects of MPEP on non-nociceptive VPL neurons. (A) Activity of a representative non-nociceptive neuron before and after the intravenous administration of MPEP (10 mg / kg). The response of the neuron following brushing stimulation of the hind paw is shown at the top. (B) Average response of non-nociceptive neurons after MPEP (10 mg / kg, n56), expressed as percent of pre-drug control value.
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Fig. 5. Intra-thalamic drug treatment. (A) Responses of a nociceptive VPL neuron after MPEP injection (2 mg, 2 ml). (B) Example of a neuron treated with the NMDA antagonist MK801 (0.1 mg, 2 ml). Top, level of applied pressure in register with the PSTHs below. Middle, average of pre-drug firing rate histograms and raster plot of the ten individual trials. Bottom, PSTHs and raster plots after drug injection. A single action potential is shown for each example.
level. In contrast, recent data have shown antinociceptive actions of MPEP to noxious heat stimulation when administered iontophoretically into the thalamus [39]. Thus, a direct intra-thalamic injection of MPEP in our data seems to have a less focal effect. Alternatively, thalamic mGluR 5 may be involved in non-mechanical nociceptive stimulation. More studies are needed to ascertain the site of action of the mGluR 5 antagonist. Our in vitro spinal cord recordings seem to suggest an
involvement of spinal mGluR 5 in nociceptive processing because MPEP was effective against nociceptive responses, both in the single shock stimulus of dorsal root and after the wind-up evoked by repetitive stimulation. While the wind-up model is suggested to be a central mechanism for hyperalgesia [29], single shock electrical stimulation of the spinal cord dorsal root, sufficient to recruit both A- and C-fibers, is used to study in vitro the response to acute noxious stimulation [42]. The late phase
Fig. 6. (A) Effect of MPEP (30 mM) on a spinal cord preparation after single stimulation of the dorsal root (50 V, 1 ms). One h perfusion of 30 mM MPEP reduced the evoked response. (B) Representative trace showing the inhibitory effect of MPEP on cumulative depolarization (‘wind-up’) of the ventral root evoked by 20 s of 1 Hz repetitive high-intensity (50 V, 1 ms) electrical stimulation of the dorsal root.
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of this response, mostly attributed to C-fiber activation and defined as ‘peptidergic’ because it is also affected by neurokinin receptor antagonists [41,42], was attenuated by MPEP. The early, monosynaptic component of the response, dependent on AMPA and kainate receptors [14], instead was not affected by MPEP in our study. The less marked effects obtained with MPEP in the spinal cord preparation could be due to the younger age of the animals compared to those used for the thalamic recordings. Since the expression of mGluRs is developmentally regulated, it is possible that our findings were influenced by the development changes in the level of mGluR 1 and mGluR 5 receptors. The NMDA receptor antagonist MK801, on the other hand, inhibited noxious-evoked responses in VPL neurons after intra-thalamic injections. This finding extends our earlier demonstration that MK801 blocked acute nociceptive activity in the thalamus after intravenous treatment [5], and suggests that NMDA receptors located in the thalamus are relevant in the modulation of pain transmission either via ascending or descending pain pathways. Recent behavioral results are also supporting the view that thalamic NMDA receptors may participate in the response to noxious stimulation [22], whereas spinal NMDA receptors play a minor role on acute processing (see [5], for a discussion). Our earlier evidence also showed that MK801 is less selective than MPEP in its action because it also significantly depressed firing of non-nociceptive mechanosensitive VPL neurons [5]. Activation of Group I mGluRs can modulate NMDA and AMPA receptor-mediated responses in a number of brain areas, including the spinal cord [43]. Recent data have shown that both mGluR 5 and mGluR 1 act to enhance ionotropic glutamate responses in spinal cord, but the two types of mGluRs may have different intracellular mechanisms of action [44]. Some behavioral data have also suggested some interaction between Group I mGluR agonists and ionotropic glutamate receptors to produce enhanced nociceptive responses and hyperalgesia [15,27,28]. It is possible that interaction with NMDA and / or AMPA receptors with mGluR 1 may be more efficacious in modulating nociceptive transmission associated with chronic, sensitized pain, while interaction with mGluR 5 may be relevant also for modulation of acute pain. Recently, MPEP has been reported also efficacious in an inflammation-induced pain model [47]. More studies are needed to clarify this point. Group I mGluRs have been implicated in nociception (reviewed in [6]), but particularly in persistent pain associated with central sensitization [17,34,52,53]. The present study shows, for the first time, that a subtype of this Group, mGluR 5 , may be also important in mediating mechanical acute nociceptive responses, without affecting normal sensory perception. We suggest a possible involvement of spinal receptors in this action, in accord with immunohistochemical distribution studies showing the
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presence of mGluR 5 in layers I and II of the dorsal horn [45,46]. Blockade of these receptors may be a promising new route for treatment of pain disorders.
Acknowledgements The authors wish to thank Mr. Giorgio Tarter for technical assistance and Prof. Eric Frank (University of Pittsburgh) for providing the software analysis and for his valuable comments on the manuscript.
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