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Pain 137 (2008) 295–305

Involvement of metabotropic glutamate 5 receptor in visceral pain

Erik Lindstrom a,*, Mikael Brusberg a, Patrick A. Hughes b,d, Christopher M. Martin b,Stuart M. Brierley b,d, Benjamin D. Phillis b,d, Rakel Martinsson a,

Christina Abrahamsson a, Hakan Larsson a, Vicente Martinez a, L. Ashley Blackshaw b,c,d

a AstraZeneca R&D, Molndal, Swedenb Nerve Gut Research Laboratory, Hanson Institute, Royal Adelaide Hospital, University of Adelaide, Australia

c Discipline of Medicine, School of Molecular and Biomedical Sciences, University of Adelaide, Australiad Discipline of Physiology, School of Molecular and Biomedical Sciences, University of Adelaide, Australia

Received 27 April 2007; received in revised form 30 August 2007; accepted 10 September 2007

Abstract

Metabotropic glutamate 5 receptor (mGluR5) antagonists are effective in animal models of inflammatory and neuropathic pain.The involvement of mGluR5 in visceral pain pathways from the gastrointestinal tract is as yet unknown. We evaluated effects ofmGluR5 antagonists on the colorectal distension (CRD)-evoked visceromotor (VMR) and cardiovascular responses in consciousrats, and on mechanosensory responses of mouse colorectal afferents in vitro. Sprague–Dawley rats were subjected to repeated, iso-baric CRD (12 · 80 mmHg, for 30 s with 5 min intervals). The VMR and cardiovascular responses to CRD were monitored. ThemGluR5 antagonists MPEP (1–10 lmol/kg, i.v.) and MTEP (1–3 lmol/kg, i.v.) reduced the VMR to CRD dose-dependently withmaximal inhibition of 52 ± 8% (p < 0.01) and 25 ± 11% (p < 0.05), respectively, without affecting colonic compliance. MPEP(10 lmol/kg, i.v.) reduced CRD-evoked increases in blood pressure and heart rate by 33 ± 9% (p < 0.01) and 35 ± 8% (p < 0.05),respectively. Single afferent recordings were made from mouse pelvic and splanchnic nerves of colorectal mechanoreceptors. Circum-ferential stretch (0–5 g force) elicited slowly-adapting excitation of action potentials in pelvic distension-sensitive afferents. Thisresponse was reduced 55–78% by 10 lM MTEP (p < 0.05). Colonic probing (2 g von Frey hair) activated serosal splanchnic affer-ents; their responses were reduced 50% by 10 lM MTEP (p < 0.01). We conclude that mGluR5 antagonists inhibit CRD-evokedVMR and cardiovascular changes in conscious rats, through an effect, at least in part, at peripheral afferent endings. Thus, mGluR5participates in mediating mechanically evoked visceral nociception in the gastrointestinal tract.� 2007 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

Keywords: Colorectal distension; Visceral pain; Visceromotor response; Colonic afferents; mGluR5; Glutamate

1. Introduction

The metabotropic glutamate receptors (mGluRs) areG-protein coupled receptors activated by the excitatoryamino acid glutamate [8]. Eight mGluRs (mGluR1–8)have been identified so far. They are divided into threegroups based on receptor pharmacology, signal trans-duction coupling and primary sequence homology [8].

0304-3959/$34.00 � 2007 International Association for the Study of Pain. P

doi:10.1016/j.pain.2007.09.008

* Corresponding author. Present address: Medivir AB, Lunastigen 7,S-14122 Huddinge, Sweden. Tel.: +46 8 5468 3232.

E-mail address: Erik.Lindstrom@medivir.se (E. Lindstrom).

Group I mGluRs consist of mGluR1 and mGluR5which are positively coupled to phospholipase C. GroupI receptor antagonists, particularly antagonists formGluR5, have received a great deal of interest due totheir potential as analgesics and anxiolytics [38].mGluR5 is expressed in various regions along thesomatosensory pathway, including the dorsal horn ofthe spinal cord (laminae I and II) and pain-relatedCNS centers [30,40,41]. In addition, selective mGluR5antagonists, such as 2-methyl-6-(phenylethynyl)-pyri-dine (MPEP) and 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP), have shown analgesic properties in

ublished by Elsevier B.V. All rights reserved.

296 E. Lindstrom et al. / Pain 137 (2008) 295–305

several animal models of inflammatory and neuropathicsomatic pain [10,13,14,18,21,42–44,46]. Interestingly,mGluR5 antagonists appear to modulate somatic noci-ceptive processing at various levels of the nervous sys-tem such as peripheral afferent endings, spinal dorsalhorn and supraspinal sites [12,31,47]. However, thereis limited data on the role of mGluR5 in visceral pain,a condition which represents a major clinical problem.Acetic acid-evoked writhing behaviour and c-fos expres-sion in the spinal cord are reported to be inhibited byMPEP [5,46]. However, administration of acetic acidinto the peritoneal cavity is not regarded as a selectivevisceral stimulus since the peritoneum, a partly somaticstructure, and probably other somatic structures areinvolved in the responses observed [22,25].

Afferent signals from the colon reach the central ner-vous system via the splanchnic and pelvic nerves, whichterminate in the thoracolumbar and lumbosacral spinalcord, respectively [27]. Afferent fibres in each pathwayare highly specialized according to their adequate stim-ulus, threshold, and target organ. Those responding tocolorectal distension at low threshold are found mainlyin the pelvic pathway, whilst high-threshold afferentspredominate in the splanchnic pathway [7].

The aim of the present study was to investigate howthe selective mGluR5 antagonists MPEP and MTEPaffect the response to visceral pain emanating from thegastrointestinal tract. This was examined by assessingthe visceromotor response (VMR) and the cardiovascu-lar autonomic response to noxious colorectal distension(CRD) in conscious rats (described by [27,39]). We alsoinvestigated if mGluR5 antagonists could have a periph-eral site of action by recording the effects of MTEP onthe responses of distension-sensitive mechanoreceptorsin the mouse pelvic nerve and serosal mechanoreceptors,which are responsive to noxious mechanical stimuli, inmouse splanchnic nerve in mice.

2. Methods

2.1. Animals

Adult female Sprague–Dawley rats (Harlan, The Nether-lands, 250–300 g), and adult male and female C57BL/6 mice(Institute of Medical and Veterinary Sciences (IMVS), Ade-laide, South Australia, 20–30 g) were used. Prior to CRD stud-ies in conscious rats, they were allowed to acclimatize to theanimal facility for at least one week after arrival. Rats werehoused in groups of 5 in an enriched environment with freeaccess to food (standard pellets) and water on a 12:12 hlight–dark cycle. The estrous stage of the rats was notaccounted for in the current study. All conscious animal exper-iments were approved by the local animal ethics review com-mittee in Goteborg, Sweden, and afferent recordingexperiments by the animal ethics committees of the IMVSand University of Adelaide.

2.2. Colorectal distension

The colorectal distension (CRD) procedure used has beenpreviously described in detail elsewhere [39]. Rats were habit-uated to Bollmann cages (Plexi-glass tubes, length 18 cm,diameter 6 cm, AstraZeneca, Molndal, Sweden) 30 min perday for three consecutive days prior to experiments to reducemotion artefacts due to restraint stress. A 3 cm polyethyleneballoon (made in-house) with connecting catheter was insertedin the distal colon, 2 cm from the base of the balloon to theanus, during light isoflurane anesthesia (Forene�, AbbottScandinavia AB, Solna, Sweden). The catheter was fixed tothe tail with tape. An intravenous catheter (Neoflon�, BectonDickinson AB, Helsingborg, Sweden) was inserted in the tailvein for vehicle or compound administration. The intravenouscatheter was flushed with 0.2 ml of heparin 50 IE/KY/ml (LeoPharma, Ballerup, Denmark). The balloons were connected topressure transducers (P-602, CFM-k33, 100 mmHg, Bronk-horst HI-TEC, Veenendal, The Netherlands) to control intra-balloon pressure during the CRD procedure. Rats wereallowed to recover from sedation in the Bollmann cages forat least 15 min before the start of experiments.

A customized barostat (AstraZeneca, Molndal, Sweden)was used to manage air inflation and balloon pressure control.A customized computer software (PharmLab on-line 4.0,AstraZeneca), running on a standard computer, was used tocontrol the barostat and to collect data. The distension para-digms generated by the barostat were achieved by generatingpulse patterns on an analog output channel. The CRD para-digm used consisted of repeated phasic distensions, 12 pulsesat 80 mmHg, with a pulse duration of 30 s at 5 min intervals.

MPEP, MTEP or respective vehicles were administered asintravenous (i.v.) bolus injections (1 ml/kg) between pulsesthree and four during the repeated phasic distension paradigm.Each rat received both vehicle and compound on differentoccasions with at least two days between experiments. Hence,each rat served as its own vehicle control. All rats receivedvehicle in the first experiment followed by compound in thesecond experiment.

2.3. Acquisition of data and analysis of visceromotor responses

Rapid pressure changes in the distending balloon, reflectingcontractions of the abdominal muscles, were used to assessVMRs [39]. This modified read-out has been shown to be moresensitive in recording hypersensitivity during, for instance,repeated noxious distensions and in detecting analgesic effectsof analgesic drugs (e.g. the l opioid receptor agonist fentanyl)than traditionally used EMG recordings [39]. Recently, thismethod was used to detect analgesic effects exerted by cannab-inoids [3] and lidocaine [20].

The analog input channels were sampled with individualsampling rates, and digital filtering was performed on the sig-nals. The balloon pressure signals were sampled at 50 sam-ples/s. A highpass filter at 1 Hz was used to separate thecontraction-induced pressure changes from the slow varyingpressure generated by the barostat. A resistance in the airflowbetween the pressure generator and the pressure transducer fur-ther enhanced the pressure variations induced by abdominalcontractions of the animal. A customized software (PharmLab

E. Lindstrom et al. / Pain 137 (2008) 295–305 297

off-line 4.0, AstraZeneca) was used to quantify the magnitudeof the highpass-filtered balloon pressure signals. The averagerectified value (ARV) of the highpass-filtered balloon pressuresignals was calculated for 30 s before the pulse (i.e. baselineactivity) and for the duration of the pulse. When calculatingthe magnitude of the highpass-filtered balloon pressure signals,the first and last second of each pulse were excluded since thesereflect artefact signals produced by the barostat during inflationand deflation and do not originate from the animal.

2.4. Acquisition of data and analysis of cardiovascular responses

A telemetric system was used to assess cardiovascularresponses during CRD. Rats were anesthetized with a mixture(2 ml/kg) of ketamine (88 mg/kg; Ketalar� Vet, Pfizer AB,Taby, Sweden) and xylazine (5 mg/kg; Rompun� Vet, BayerAG, Leverkusen, Germany) and were surgically equipped withintraperitoneal radio transmitters (PhysioTel� PXT-C50, DSI,St. Paul, MN, USA). One of the electrodes from the radiotransmitter was placed across the chest to measure the electro-cardiogram (ECG) while the catheter was inserted into theabdominal aorta and fixed with tissue adhesive (Vetbond�,3M, St. Paul, MN, USA) for blood pressure measurement.The surgery was performed under semi-sterile conditions andthe animals were pre-treated with antibiotics (Bactrim�,Roche, Basel, Switzerland) before the surgical procedure andreceived antibiotics and analgesics (Finadyne� vet., Schering-Plough, Kenilworth, NJ, USA) for 2 days after surgery. A 7-day recovery period was allowed before starting any experi-mental procedures. After recovery, the rats were habituatedto Bollman cages, before starting the CRD procedures asdescribed above.

Animals were treated with vehicle (saline) or MPEP(10 lmol/kg) in separate experiments, with a 5–7-day periodbetween treatments. Four rats received saline in the first exper-iment while three rats received MPEP. The treatments werereversed during the second experiment (i.e. crossover fashion).For CRD, the same procedures as described above were fol-lowed. In this case, in addition to the changes in intracolonicballoon pressure, telemetric data (heart rate (beats per minute,bpm) and arterial blood pressure (mmHg)) were sampled fromthe DSI receivers (PhysioTel� RPC-1, DSI, St. Paul, MN,USA) using custom-made acquisition software (PharmLabon-line 5.0.1; AstraZeneca).

Cardiovascular parameters were analyzed using custom-made analysis software (PharmLab off-line 5.0.1; AstraZene-ca). Heart rate (bpm) and blood pressure (mmHg) were deter-mined for the 30 s period before each distension, taken asbaseline, and for the duration of the distension (30 s). Dataare presented as D heart rate (D bpm) and D blood pressure(D mmHg) for each distension, calculated as the mean heartrate or mean blood pressure values during each distensionminus their respective mean baseline values.

2.5. Measurements of colorectal compliance

As described above, the animals were equipped with a 3 cmpolyethylene balloon inserted in the distal colon and an intra-venous catheter in the tail vein for vehicle or compoundadministration. In this set of experiments, the barostat system

was used to inflate the balloon to a pre-determined pressurebut also to simultaneously measure the volume changes inthe balloon. The system was controlled by a custom-made soft-ware (Pharmlab 4.0, AstraZeneca), which was also used fordata acquisition and evaluation. The pressure-dependentchanges in volume during isotonic CRD were taken as a mea-sure of colorectal compliance.

The CRD protocol consisted of 10 phasic distensions at 2,4, 6, 8, 10, 12, 14, 16, 18 and 20 mmHg. Each distension lastedfor 1 min, with a 5 min interval between consecutive disten-sions. MPEP (10 lmol/kg) or vehicle (saline) was administered10 min before the start of the compliance paradigm. Each ratreceived both vehicle and compound on different occasionswith at least two days between experiments. Hence, each ratserved as its own vehicle control. All rats received vehicle inthe first experiment followed by MPEP in the second experi-ment. Low distension pressures, which are below the thresh-olds typically required to induce a significant VMR (see[39]), were used to avoid interferences due to fluctuations inthe balloon volume caused by abdominal contractions.

2.6. In vitro mouse colonic primary afferent preparation

Dissections were carried out in male and female miceaccording to protocols described in detail previously [7].Briefly, the colon (5–6 cm) and mesentery were removedintact along with the attached neurovascular bundle contain-ing either the major pelvic ganglion and pelvic nerves or theinferior mesenteric ganglion and lumbar splanchnic nerves.The tissue was transferred to ice cold Krebs solution and, fol-lowing further dissection, the distal colon was opened longi-tudinally along the anti-mesenteric border to orientate nerveinsertions to lie along the edge of the open preparation.The tissue was pinned flat, mucosal side up, in a specializedorgan bath consisting of two adjacent compartmentsmachined from clear acrylic (Danz Instrument Service, Ade-laide, South Australia), the floors of which were lined withSylgard� (Dow Corning Corp., Midland, MI, USA). Theextrinsic nerves were extended from the tissue compartmentinto the recording compartment where they were laid ontoa mirror. A movable wall with a small ‘‘mouse hole’’ (toallow passage of the nerves) was lowered into position andthe recording chamber filled with paraffin oil. The coloniccompartment was superfused with a modified Krebs solution(in mM: 117.9 NaCl, 4.7 KCl, 25 NaHCO3, 1.3 NaH2PO4,1.2 MgSO4(H2O)7, 2.5 CaCl2, 11.1 D-glucose, 2 sodium buty-rate, and 20 sodium acetate), bubbled with carbogen (95%O2/5% CO2) at a temperature of 34 �C. All preparations con-tained the L-type calcium channel antagonist nifedipine(1 lM) to suppress smooth muscle activity and the prosta-glandin synthesis inhibitor indomethacin (3 lM) to suppresspotential inhibitory actions of endogenous prostaglandins[7,23]. Under a dissecting microscope, the pelvic or splanch-nic nerves were dissected away from the neurovascular bun-dle and the nerve sheath surrounding the nerves peeledgently back exposing the nerve trunk. Using fine forceps,the nerve trunk was teased apart into 6–10 bundles whichwere individually placed onto a platinum recording electrode.A platinum reference electrode rested on the mirror in a smallpool of Krebs solution adjacent to the recording electrode.

298 E. Lindstrom et al. / Pain 137 (2008) 295–305

2.7. Measurement of colonic afferent responses

Receptive fields were first identified by systematically strok-ing the mucosal surface with a brush of sufficient stiffness toactivate all types of mechanosensitive afferents. Colonic affer-ents were then characterized using the classification systempreviously applied in mouse and rat colon [4,7,17,23]. Thus,serosal afferents are classified as having receptive fields onthe gut wall, and responding to a blunt probe, but not tostretch of the muscle (0.5–5 g) or fine (10 mg) stroking of themucosa. Muscular afferents respond to probing and stretch,but not to stroking. Muscular/mucosal afferents respond toall three stimuli. In the present study we focused on these threepopulations, pelvic muscular and muscular/mucosal afferentsbecause they are the main populations responsive to low levelsof distension [7] and splanchnic serosal afferents because theyare high-threshold nociceptive afferents [7]. Pelvic afferentswere assessed for their graded response to circular stretch (3and 5 g stimuli, 60 s each), which we have shown to give thehalf maximal and maximal responses, respectively. Stretchwas applied using a claw made from bent dissection pins con-nected to a pulled system via thread. Weights were applied tothe opposite side of the cantilever system to initiate gradedcolonic stretch. Splanchnic afferents were assessed for theirresponses to probing with calibrated von Frey hairs (70, 160,1000 mg, 2 and 4 g force) for a period of 3 s each. A submax-imal stimulus (2 g) was repeated three times and the meanresponse for each fibre entered into statistical analysis [7].Stimuli were repeated after pre-incubation for 10 min withMTEP (10 lM) – a concentration we previously showed tobe maximal for inhibition of mouse gastro-oesophageal vagalafferents [36]. The drug was added into a small chamber placedaround the receptive field which was removed immediatelyprior to testing so as not to interfere with delivery of themechanical stimulus.

2.8. Electrophysiological data recording and analysis

Electrical signals generated by nerve fibres placed on theplatinum recording electrode were fed into a differential ampli-fier, filtered, sampled at a rate of 20 kHz using a 1401 interface(Cambridge Electronic Design, Cambridge, UK) and stored ona PC. The amplified signal was also used for online audio mon-itoring. Action potentials were analysed off-line using theSpike 2 wavemark function and discriminated as single unitson the basis of distinguishable waveform, amplitude and dura-tion. Responses to circumferential stretch and von Frey probeswere assessed as the number of action potentials counted overthe whole period of the stimulus, expressed as the averagespikes/s over this period.

2.9. Drugs

MPEP hydrochloride and MTEP were synthesized atAstraZeneca, Molndal. MPEP was dissolved in saline andadministered intravenously (i.v.) at 1, 3, 6 and 10 lmol/kg(equivalent to 0.23, 0.69, 1.38, and 2.3 mg/kg, respectively).Amorphous nanosuspensions of MTEP were prepared bysuspension in 2% dimethylacetamide and injection into anaqueous stabiliser solution (containing 0.06% (w/w) polyvinyl-

pyrrolidine and 0.075 mM sodiumdodecylsulphate) followedby dilution with 5% mannitol to reach isotonicity. MTEPwas administered intravenously (i.v.) at 1 and 3 lmol/kg(equivalent to 0.2 and 0.6 mg/kg, respectively). For in vitro

studies MTEP was dissolved in distilled water at 10 mM anddiluted to final concentration on the day of the experimentin Krebs solution.

2.10. Statistical analysis

All data were analysed using Prism 4 software (GraphPadSoftware, San Diego, CA, USA). Repeated measures ANOVAfollowed by Dunnett’s posthoc test was used to compareresponses to repeated distensions with the response to the firstdistension in vehicle-treated rats in CRD experiments. Stu-dent’s paired t test was used to assess statistical differencesbetween compound-treated and vehicle-treated animals foreach distension in CRD experiments. The degree of inhibitionby MPEP or MTEP was calculated by dividing cumulativeresponses to distension after compound administration (i.e.distensions 4–12) with respective responses after vehicle treat-ment. In afferent experiments, a two-way analysis of variance(ANOVA) with Bonferroni posthoc tests was used to deter-mine significant differences at comparable loads. All data areexpressed as mean ± SEM. In CRD experiments, n reflectsthe number of individual rats. In electrophysiology experi-ments, n reflects the number of individual afferents. A p valueless than 0.05 was considered statistically significant.

3. Results

3.1. Effect of mGluR5 receptor antagonists on noxious

CRD-evoked VMRs

In vehicle-treated animals, CRD evoked a VMRobserved as significant oscillations in the intraballoonmanometric recordings, as compared with baselineactivity. Moreover, the VMR increased over the CRDprotocol. From the first to the 12th pulse, the responseto CRD increased by 89 ± 20% (n = 32, p < 0.05 for dis-tension 6–7, p < 0.01 for distension 8–12).

At 6 lmol/kg (i.v.), the selective mGluR5 antago-nist MPEP significantly reduced the VMR to CRDcompared to the vehicle-treated group (Fig. 1a). Over-all, MPEP (6 lmol/kg) inhibited the response to CRDby 39 ± 9% (p < 0.01 vs. vehicle, as integrated overdistensions 4–12). Fig. 1b illustrates the dose–responserelationship for MPEP showing that lower doses (1and 3 lmol/kg) were without significant effect whiletreatment with 10 lmol/kg resulted in further inhibi-tion (52 ± 6%, p < 0.01 vs. vehicle). The selectivemGluR5 antagonist MTEP (3 lmol/kg, i.v.) alsoattenuated the VMR to CRD during the first 5–6 dis-tensions after administration (i.e. for about 25–30 min,Fig. 1c). Overall, MTEP inhibited the response toCRD by 25 ± 11% (p < 0.05 vs. vehicle, as integratedover distensions 4–12). A lower dose of MTEP

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ig. 2. Effect of MPEP (10 lmol/kg (2.3 mg/kg), i.v.) on the (a) CRD-voked VMR, (b) CRD-evoked increase in blood pressure and (c)RD-evoked change in heart rate. The CRD paradigm consisted of 12istensions at 80 mmHg. All parameters were monitored simulta-eously in the same group of telemetrized rats. Open circles representesponses to CRD in rats receiving vehicle between distensions 3 and 4hile closed circles represent the effects of MPEP. The arrows indicatehen vehicle or compound is given. The same rats were used in vehiclend MPEP experiments on different occasions. Mean ± SEM, n = 7.p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle-treated rats.

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Fig. 1. (a) Effect of MPEP on the VMR to CRD. The CRD paradigmconsisted of 12 distensions at 80 mmHg. Open circles representresponses to CRD in rats receiving vehicle between distensions 3 and4 while closed circles represent the effects of MPEP (6 lmol/kg(1.38 mg/kg), i.v.). The arrow indicates when vehicle or compound isgiven. The same rats were used in vehicle and MPEP experiments ondifferent occasions. Mean ± SEM, n = 8. (b) Demonstrates the dose–response relationship of MPEP on the VMR to CRD integrated overdistensions 4–12. The VMR to CRD during vehicle treatment were setto 100%. (c) Effect of MTEP on the VMR to CRD. Open circlesrepresent responses to CRD in rats receiving vehicle between disten-sions 3 and 4 while closed circles represent the effects of MTEP(3 lmol/kg (0.6 mg/kg), i.v.). The arrow indicates when vehicle orcompound is given. The same rats were used in vehicle and MTEPexperiments on different occasions. Mean ± SEM, n = 8. *p < 0.05,**p < 0.01, ***p < 0.001 vs. vehicle-treated rats.

E. Lindstrom et al. / Pain 137 (2008) 295–305 299

(1 lmol/kg, i.v.) transiently reduced the VMR for10 min after administration (data not shown). Nosigns of side effects, such as deficits in motor coordi-nation, were seen at the doses used. However a higherdose of MTEP (10 lmol/kg, i.v.) was associated withslight, but visible, motor-related side effects and wasthus not evaluated in the CRD paradigm.

3.2. Effect of MPEP on noxious CRD-evoked

cardiovascular responses

In vehicle-treated animals (n = 7), noxious CRD(80 mmHg · 12 pulses) elicited a VMR, similar to thatdescribed above in the dose–response studies (Fig. 2a).In addition, CRD evoked significant rises in blood pres-sure and heart rate (Fig. 2b and c). Over the CRD pro-tocol, the VMR response increased in magnitude by73 ± 27%, from the first to the last pulse (p < 0.05 fordistensions 9–10, p < 0.01 during 11th distension). Sim-ilar responses were also observed for blood pressure

FeCdnrwwa*

300 E. Lindstrom et al. / Pain 137 (2008) 295–305

and heart rate, which increased by 43 ± 22% and63 ± 26%, respectively (for both parameters: p < 0.05at 7th distension and p < 0.01 between distensions 8and 11). After each pulse, blood pressure and heart ratereturned to their baseline control values, which were sta-ble over the experiment (�2 ± 1% and �7 ± 2% changeover the complete CRD protocol, respectively).

MPEP (10 lmol/kg, i.v., n = 7) attenuated the overall(distensions 4–12) VMR response to CRD by 26 ± 5%(Fig. 2a, p < 0.01 vs. vehicle). Similar effects wereobserved for blood pressure and heart rate changes,which were attenuated by 33 ± 9% (Fig. 2b, p < 0.01vs. vehicle) and 35 ± 8% (Fig. 2c, p < 0.05 vs. vehicle),respectively. Effects of MPEP were transitory and lastedbetween distensions 4 and 9 (i.e. for about 30 min).Thereafter, responses to CRD increased, and by theend of the CRD protocol reached values similar to thosein the vehicle-treated group.

3.3. Effect of MPEP on colorectal compliance

MPEP (10 lmol/kg, i.v.) did not affect the pressure–volume relationship during phasic (2–20 mmHg) CRDcompared to vehicle, indicating that MPEP does notaffect colorectal compliance in rats (Fig. 3).

3.4. Pelvic mechanoreceptor responses to distension

A total of 11 distension-sensitive fibres were testedwith MTEP. Their receptive fields to probing werelocated in the distal colon and rectum, correspondingto the same area that was distended in the in vivo studiesin rats. Under control conditions they showed a rapidonset, slowly adapting increase in action potential dis-charge to isotonic circumferential stretch of the prepara-tion (3 and 5 g, Fig. 4a). After removal of the stimulus,the response immediately ceased and the fibres becamesilent once again. This response was reproducible on

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repeated application of the stimulus. Following a10 min incubation with MTEP (10 lM), this responsewas dramatically attenuated by 55% at 3 g, and by78% at 5 g (p < 0.05 and p < 0.001, respectively,Fig. 4b and c). Control experiments with no compoundadministration over a similar timecourse showed noattenuation of the response over a similar timecourse.

3.5. Splanchnic mechanoreceptor responses to von Frey

probes

A total of 5 probe-sensitive splanchnic serosal fibreswere tested with MTEP. Their receptive fields werelocated in the distal colon as shown previously [7], cor-responding to the proximal part of the area that was dis-tended in the in vivo studies in rats. Under controlconditions they showed a rapidly adapting burst ofaction potentials in response to application of the vonFrey probe, which was graded according to stimulusintensity (Fig. 5a). These fibres did not respond to cir-cumferential stretch as did the pelvic afferents describedabove. Responses to probing were reproducible onrepeated application of the stimulus. Previous findingsindicate that these afferents respond to distension ofthe whole colon only at intraluminal pressures well intothe noxious range. Following a 10 min incubation withMTEP (10 lM), the splanchnic afferent stimulusresponse function to a 2 g von Frey hair was attenuatedby more than 50% (p < 0.01, Fig. 5c). Two experimentswere carried out on rat tissue to allow translation offindings from single afferents to the whole animal. Thesedid not allow examination of responses to a graded stim-ulus (or comparison with pelvic data), but these experi-ments demonstrated a similar degree of inhibition byMTEP as our data on mouse splanchnic afferents (datanot shown).

4. Discussion

The major findings of this study are that mGluR5receptor antagonists inhibit the visceromotor and auto-nomic responses to colorectal distension (CRD) in con-scious rats. This correlates with the observation in vitro

that a mGluR5 antagonist inhibits responses to colorec-tal distension in pelvic mechanoreceptor afferents to acomparable extent. Together these findings indicatefirstly that endogenous activation of mGluR5 contrib-utes to visceral pain, and secondly that a possible siteof action of mGluR5 antagonism is in the periphery,on the endings of colorectal afferents.

Inhibitory effects of MPEP on the VMR and the car-diovascular response to CRD indicate an involvement ofmGluR5 in visceral sensory mechanisms, as previouslysuggested for somatic pain [10,13,14,18,21,42–44,46].At high concentrations, MPEP has been claimed to actthrough non-mGluR5 mechanisms, such as via NMDA

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-0.01

-0.01

-0.01-0.01

5 g

10 sec

400μV

10spikes/sec

-0.01-0.01

5 g

a.Control

5

b.MTEP

c

Fig. 4. MTEP (10 lM) inhibits responses to circumferential tension in mouse pelvic mechanoreceptors. (a) Response to a 60 s application of 5 g forceacross the receptive field of a muscular afferent: upper trace – integrated record of action potential discharge in 1 s bins; lower trace – raw record ofaction potentials. (b): Effect of 10 min pre-incubation with MTEP (10 lM) on the response of the same fibre in (a) to colonic distension. (c) Compileddata from 11 distension-sensitive colorectal afferents, showing responses at 3 and 5 g force before and after MTEP treatment. Data shown asmean ± SEM, *p < 0.05, ***p < 0.001 vs. pre-drug response.

E. Lindstrom et al. / Pain 137 (2008) 295–305 301

receptors or the norepinephrine transporter (NET)[16,32]. Inhibition of NMDA receptors or the NETcould conceivably affect the VMR to CRD in rats[11,37]. However, the mGluR5 antagonist MTEP, whichhas improved selectivity compared to MPEP [10], wasalso effective at attenuating the VMR to CRD suggest-ing that the effects of the compounds are indeed medi-ated via inhibiton of mGluR5.

Anti-nociceptive effects of mGluR5 antagonists inmodels of somatic pain are mediated at multiple sitesof action; peripheral tissues, dorsal horn or at supraspi-

nal sites [47]. Central mGluR5-mediated analgesiceffects have been extensively reported in different mod-els, however, contribution of mGluR5 in specific centralregions was not assessed in the current study. Since afterperipheral administration the brain penetrance ofMPEP and MTEP is relatively high [1], central sites ofaction mediating the analgesic effects on visceral paincannot be ruled out. Nevertheless, several reportsshowed also a significant analgesic activity of peripheralmGluR5 in models of somatic pain [6,44]. Therefore, anaction at peripheral sites mediating the analgesic effects

40

200μV

4 sec

a.Control

spikes/sec

b.MTEP

**

2 g probe

2 g probe

control MTEP 10μM

2g von Frey hair probe

**

0

5

10

15

20

25

spik

es p

er s

eco

nd

c

Fig. 5. MTEP (10 lM) inhibits responses to von Frey probing in mouse splanchnic mechanoreceptors. (a) Response to a 3 s application of 2 g forceonto the receptive field of a serosal afferent: upper trace – integrated record of action potential discharge in 1 s bins; lower trace – raw record of actionpotentials. (b) Effect of 10 min pre-incubation with MTEP (10 lM) on the response of the same fibre in (a) to probing. (c) Compiled data from 5serosal splanchnic afferents, showing responses at 2 g before and after MTEP treatment. Data shown as mean ± SEM, **p < 0.01 vs. pre-drugresponse.

302 E. Lindstrom et al. / Pain 137 (2008) 295–305

was considered a possibility in the current study. Thiswas tested by recording from two major populationsof colonic mechanoreceptors – pelvic muscular and mus-cular/mucosal afferents, and splanchnic serosal afferents[7]. These studies demonstrated that MTEP was able toinhibit mechanosensitivity in both populations of affer-ents, corresponding to those responding at low and highthreshold to distension in situ. This provides the first evi-dence for a peripheral site of action of mGluR5 antago-nists contributing to the inhibition of the responses toCRD. It does not, however, rule out an additionalaction in the central nervous system, at either spinal orsupraspinal sites as mentioned above.

A peripheral site of action is perhaps not surprisingsince mGluR5 is strongly expressed in small diameterdorsal root ganglion neurons [41,44] and peripheralaxons [6,44], although these studies did not discriminatebetween ganglion neurons innervating the colon andothers. Recent data showed that mGluR5 is expressedin nodose ganglia neurons and that functional receptors

can be localized in gastric vagal afferents [45]. In addi-tion, MTEP and MPEP were effective at reducingmechanosensory input from the stomach, through anaction on vagal afferents [36,45]. These observationstogether with those of the current investigation indicatethat mGlu5R expressed in extrinsic nerves is importantin modulating afferent information from the gastrointes-tinal tract, in both spinal and vagal pathways. Our pre-vious investigations of glutamatergic actions on vagalafferents indicated that although a mGluR5 antagonistcaused potent afferent inhibition [36,45], a group I ago-nist had no significant effect [33]. This may be attribut-able to the high levels of intrinsic activation oftenfound in group I mGluR. This is mediated by intracellu-lar proteins maintaining the receptor in an active state,without the need for extracellular agonist binding [2].mGluR5 antagonists are in fact negative modulators,and so act at a site on the receptor independent of ago-nist binding or intracellular modulation [24]. Endoge-nous glutamate is another likely source of ongoing

E. Lindstrom et al. / Pain 137 (2008) 295–305 303

mGluR5 activation, as glutamate levels in various tis-sues may rise into the micromolar range in vivo [15],and similar levels of glutamate release may be evokedfrom the gut in vitro [35]. Therefore, the actions ofmGluR5 antagonists on afferent mechanosensitivity inthe present study could be via three possible mecha-nisms: first, inverse agonism of intrinsic mGluR5 activ-ity; second, blockade of the actions of endogenousglutamate; and third, an action via other non-mGluR5mechanisms. We consider the last option unlikely forreasons discussed earlier, but the first two mechanismsare both likely to operate in the colon. Although sub-stantial activation of mGluR5 may take place via thesemechanisms under normal conditions, neither of themare likely to saturate activation of the receptor, allowingscope for regulation and tuning of the system by evokedglutamate release from intrinsic gut neurons (see [35]).

Recently, we have shown that phasic bursts ofabdominal activity induce rapid, transient pressurechanges in the distending balloon in rats [39], and canbe used as an alternative method to characterize theVMR to CRD in rodents. Indeed, this read-out is partic-ularly sensitive to compounds having analgesic-likeeffects like the l opioid receptor agonist fentanyl [39],cannabinoid receptor agonists [3] or intracolonicallyapplied lidocaine [20]. In the current study, in vehicle-treated controls, the same stimulus (repeated 80 mmHgdistensions) evoked progressively larger VMR and car-diovascular responses as previously shown by others[29,39]. These two reflex responses (viscero-somaticand viscero-visceral) increase to a similar degree in thesame group of rats further suggesting that the elevatedresponse probably reflects a common hyperalgesic phe-nomenon and not a time-related increased efferent out-put to the abdominal muscles or the cardiovascularsystem independently. The development of hyperalgesiaafter repeated rectal distensions in man lend further sup-port to this view [9,26,28]. The fact that MPEP inhibitedboth the VMR and the cardiovascular response to CRDsuggests inhibition of a common mechanism elicited bypain stimulation. Thus, MPEP, at the doses used,appears to be acting selectively in pain-related responsesrather than in the control of cardiovascular or motormechanisms. In addition, MPEP did not modify thecolonic pressure–volume relationship during CRD, sug-gesting that the analgesic effects of MPEP are likely dueto inhibition of nociceptive pathways rather than sec-ondary effects due to an improvement of colorectalcompliance.

MPEP and MTEP are not only claimed to possessanalgesic properties but have also demonstrated efficacyin rat anxiety models [34]. Experimental models of painin animals can be expected to increase levels of anxiety-like behaviour and potential analgesic effects of com-pounds can be difficult to discriminate from possibleanxiolytic effects. Conceivably, the CRD protocol used

in the current study most likely causes an increaseddegree of anxiety that may contribute to the enhancedVMR and cardiovascular responses during the experi-ment. Therefore, it cannot be ruled out that potentialanxiolytic effects exerted by MPEP and MTEP may con-tribute to the reduced responses to CRD demonstratedin the present study.

Both MPEP and MTEP are reported to induce cen-tral side effects such as hypothermia and decreases inlocomotor activity at high doses [42,46]. Doses of 10and 30 mg/kg (�50 lmol/kg MTEP and �150 lmol/kg MPEP, respectively) were associated with decreasesin exploratory behaviour and rotarod performance,which could confound VMR data. Although the dosesrequired in the present study to achieve efficacy arewell below those reported to decrease motor perfor-mance, different routes of administration (i.p. vs. i.v.)have been used rendering direct comparisons difficult.Importantly, no gross side effects were seen with thedoses providing efficacy in the current study, althoughMTEP was visually associated with deficits in motorcoordination at higher doses which were not tested fur-ther. In addition, cardiovascular responses to noxiousCRD were also decreased by MPEP at a dose whichdid not affect basal heart rate or blood pressure. Fur-thermore, similar doses of MPEP, given intravenouslyto conscious dogs, inhibited transient lower esophagealsphincter relaxations without obvious side effects [19].Taken together, the data suggest that mGluR5 receptorantagonists tested are acting in a manner reflectinganalgesia.

In summary, the present study shows, for the firsttime, that the mGluR5 antagonists MPEP and MTEPattenuate pseudoaffective responses elicited by noxiousCRD in conscious rats. These observations suggest thatmGlu5 receptors participate in mediating visceral noci-ception and might represent a target for the treatmentof visceral pain-related states.

Acknowledgements

L.A.B. is supported by a National Health and Medi-cal Research Council of Australia Senior Research Fel-lowship, and P.A.H. by a University of Adelaide/RoyalAdelaide Hospital Dawes Scholarship.

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