Pharmacological and Anatomical Evidence for an InteractionBetween mGluR5- and GABAA α1-Containing Receptors in theDiscriminative Stimulus Effects of EthanolJoyce Besheer1,* and Clyde W Hodge11Department of Psychiatry, Bowles Center for Alcohol Studies, University of North Carolina atChapel Hill, Chapel Hill, NC, USAAbstractThe discriminative stimulus properties of ethanol are mediated in part by positive modulation ofGABAA receptors. Recent evidence indicates that metabotropic glutamate receptor subtype 5(mGluR5) activity can influence GABAA receptor function. Therefore, the purpose of this work wasto examine the potential involvement of mGluR5 in the discriminative stimulus effects of ethanol.In rats trained to discriminate ethanol (1 g/kg, intragastric gavage (i.g.)) from water, 2-methyl-6-(phenylethyl)-pyridine (MPEP) (1–50 mg/kg, i.p.) a selective noncompetitive antagonist of themGlu5 receptor did not produce ethanol-like stimulus properties. However, pretreatment with MPEP(30 mg/kg) reduced the stimulus properties of ethanol as indicated by significant reductions inethanol-appropriate responding, specifically at 0.5 and 1 g/kg ethanol, and a failure of ethanol testdoses (1 and 2 g/kg) to fully substitute for the ethanol training dose. To test whether mGluR5antagonism altered the GABAA receptor component of the ethanol stimulus, the ability of MPEP tomodulate pentobarbital and diazepam substitution for ethanol was assessed. Pentobarbitalsubstitution (1–10 mg/kg, i.p.) for ethanol was not altered by MPEP pretreatment. However, MPEPpretreatment inhibited the ethanol-like stimulus properties of diazepam (5 mg/kg, i.p.). To examinea potential anatomical basis for these pharmacological findings, expression patterns of mGluR5- andbenzodiazepine-sensitive GABAA α1-containing receptors were examined by dual-label fluorescentimmunohistochemistry with visualization by confocal microscopy. Results indicated that mGluR5-and GABAA α1-containing receptors were both coexpressed in limbic brain regions and colocalizedon the same cells in specific brain regions including the amygdala, hippocampus, globus pallidus,and ventral pallidum. Together, these findings suggest an interaction between mGluR5- andbenzodiazepine-sensitive GABAA receptors in mediating ethanol discrimination.Keywordsethanol; discriminative stimulus; mGluR5; MPEP; GABAA; benzodiazepine; diazepam;immunohistochemistry; confocalINTRODUCTIONThe amino-acid glutamate is the primary excitatory transmitter in the mammalian CNS. Fastexcitatory actions of glutamate are mediated by ionotropic glutamate receptor (iGluR) N-© 2005 Nature Publishing Group All rights reserved*Correspondence: Dr J Besheer, Department of Psychiatry, Bowles Center for Alcohol Studies, Thurston-Bowles Building, CB#7178,University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA, Tel: +1 919 843 3509, Fax: +1 919 966 5679,jbesheer@med.unc.edu.NIH Public AccessAuthor ManuscriptNeuropsychopharmacology. Author manuscript; available in PMC 2010 June 25.Published in final edited form as:Neuropsychopharmacology. 2005 April ; 30(4): 747–757. doi:10.1038/sj.npp.1300616.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptmethyl-D-aspartate (NMDA), α-amino-3-hydroxi-5-methyl-ioxyzole-4-propionic acid, andkainate receptors. Metabotropic glutamate receptors (mGluRs) mediate slower glutamateresponses through G-protein coupling to various intracellular signaling cascades that canmodulate, or fine-tune, iGluR function (Benquet et al, 2002). Eight mGluR subtypes (mGluR1–8) have been cloned. These receptors have been classified into three groups based on amino-acid sequence similarity, agonist pharmacology, and the signal transduction pathways to whichthey couple. Group I mGluRs (mGluR1 and 5) upregulate Ca2+ cascades, whereas Group II(mGluR2 and 3) and Group III (mGluR4 and 6–8) receptors are both negatively coupled to thecAMP cascade but are distinguished by their differing agonist–antagonist profiles and sequencehomologies (Gereau and Conn, 1995).The availability of selective pharmacological agents has quickly begun to reveal basicfunctional roles of mGluR5. MGluR5 activation with a selective agonist (CHPG) elevatesglutamate release (Pintor et al, 2000) and augments NMDA-induced membrane depolarizationin striatal slices (Pisani et al, 2001). Other evidence indicates that mGluR5 contributes tostriatal synaptic plasticity since the mGluR5 selective antagonist, 2-methyl-6-(phenylethyl)-pyridine (MPEP), decreased long-term depression at corticostriatal synapses (Sung et al,2001). Other studies have found beneficial effects of mGluR5 antagonists in animal models ofanxiety, pain, and neurodegeneration (Bordi and Ugolini, 1999; Spooren et al, 2001).Moreover, genetic and pharmacological data have started to reveal a role for mGluR5 inaddiction. For instance, morphine (10 mg/kg) conditioned reward is inhibited by MPEP (30mg/kg) (Popik and Wrobel, 2002) but lower doses of MPEP do not alter morphine conditionedplace preference (McGeehan and Olive, 2003). Mice lacking the mGluR5 gene do not self-administer cocaine and show no cocaine-induced increase in locomotor activity (Chiamuleraet al, 2001), indicating a significant role of mGluR5 in the behavioral effects of psychomotorstimulants.Many of the physiological, biochemical, and behavioral effects of ethanol are known to involveiGluR function (Dodd et al, 2000; Mihic, 1999; Aschner et al, 2001; Woodward, 1999;Tabakoff and Hoffman, 1993; Littleton et al, 2001; Weight et al, 1993; Costa et al, 2000). Inaddition to its effects on iGluRs, however, ethanol also modulates general metabotropicreceptor activity as evidenced by its ability to reduce basal and stimulated phosphoinositidehydrolysis (Gonzales et al, 1986). Low concentrations of ethanol selectively alter neuronalfiring rates (Netzeband and Gruol, 1995) and Ca2+ levels (Gruol et al, 1997) mediated bymGluRs in vitro. Chronic exposure to ethanol reduces mGluR1 mRNA levels in cerebellarPurkinje neurons of mice (Simonyi et al, 1996) and early withdrawal from ethanol leads toalterations in mGluR-evoked Ca2+ signaling in cerebellar neurons (Netzeband et al, 2002).Moreover, pharmacologically relevant concentrations of ethanol inhibit glutamate-inducedCa2+-dependent Cl− currents in Xenopus oocytes expressing mGluR5, but have no effect oncurrents in oocytes expressing mGluR1 (Minami et al, 1998), suggesting that ethanol mayselectively alter mGluR5 function. In addition, the mGluR5 antagonist MPEP reduces thereinforcing properties of ethanol (Sharko et al, 2002), and reduces alcohol relapse to alcoholself-administration in two animal models (Bäckström et al, 2004).In addition to their reinforcing properties, drugs of abuse produce distinctive subjective (iediscriminative stimulus) effects that are an important determinant of abuse liability (Stolerman,1992). Using differential reinforcement methods, drug discrimination experiments involvetraining animals (eg pigeons, mice, rats, gerbils, monkeys, humans) to emit one response (ie alever press) after being injected with one dose of drug (ie called the training drug or trainingdose). Thus, reinforcement (food or sucrose) is contingent on selecting the ‘correct’ or ‘drug-appropriate’ lever. An alternative response (ie pressing a different lever) results inreinforcement after vehicle injection. Once this discriminated performance is established, otherdoses of the training drug, or other drugs, are administered to determine if they engenderBesheer and Hodge Page 2Neuropsychopharmacology. Author manuscript; available in PMC 2010 June 25.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptresponding on the drug-appropriate lever. The extent to which a novel dose, or novel drug,produces responding on the lever associated with the training drug is called drug ‘substitution’or ‘generalization.’ The interpretation of these results is that a drug that produces high levelsof responding on the lever associated with the training drug (ie ethanol) has similar subjectiveproperties and acts by similar neurobiological mechanism(s) (Grant and Colombo, 1993;Kostowski and Bienkowski, 1999). Novel drugs can also be tested for pharmacologicalpotentiation or antagonism of the discriminative stimulus effects of the training drug byconducting pretreatment experiments (Besheer et al, 2003).The discriminative stimulus effects of ethanol are mediated at least by GABAA receptors. Forexample, positive modulators of GABAA receptors, such as barbiturates, benzodiazepines, andneurosteroids, produce ethanol-like stimulus effects (Ator et al, 1993; Barry, 1991; Evans andBalster, 1991; Grant et al, 1996, 1997; Jarbe and McMillan, 1983; Shelton and Grant, 2002)in multiple species. MGluR5 activity has the potential to influence ethanol discrimination basedon evidence that mGluR5 can influence GABA neurotransmission (de Novellis et al, 2003;Hoffpauir and Gleason, 2002; Diaz-Cabiale et al, 2002). For example, the mGluR5 agonistCHPG has been shown to increase GABA levels in vivo and antagonism of mGlu5 receptorsblocks the CHPG-induced increases in GABA (de Novellis et al, 2003). In cultured amacrinecells, GABA-gated currents are enhanced by CHPG administration, an enhancement that islikely due to postsynaptic GABAA receptors (Hoffpauir and Gleason, 2002). MGlu5 receptorsare abundant in limbic brain regions such as the nucleus accumbens, cortex, and hippocampus(Bordi and Ugolini, 1999; Spooren et al, 2001; Romano et al, 1995), where GABAA receptorsare known to modulate ethanol discrimination. For example, site-specific infusion ofGABAA-positive modulators such as muscimol, pentobarbital, or allopregnanolone in thenucleus accumbens or amygdala substitute fully for systemic ethanol (Besheer et al, 2003;Hodge and Cox, 1998; Hodge et al, 2001). It is plausible, therefore, that mGlu5 receptors mightregulate ethanol discrimination via modulation of GABAA receptor function.Thus, the purpose of this study was to determine the potential involvement of mGluR5 in themodulation of the discriminative stimulus effects of ethanol. In rats trained to discriminate (1g/kg) ethanol from water, MPEP a noncompetitive antagonist of the mGlu5 receptor (Gaspariniet al, 1999) was assessed for its ability to substitute for ethanol and to modulate ethanoldiscrimination. Further, the ability of MPEP to modulate pentobarbital and diazepamsubstitution for ethanol was assessed. Finally, to examine the potential anatomical basis for aninteraction between mGlu5 and GABAA receptors, expression patterns of these receptors wereexamined using dual-label florescent immunohistochemistry with confocal visualization.METHODAnimalsIn all, 12 male Long Evans rats (Harlan Sprague–Dawley, Indianapolis, IN) weighing 150–200 g upon arrival to the colony were individually housed in Plexiglas cages. Rats were handledand weighed daily for 2 weeks before lever press training began. Once rats reached a bodyweight of approximately 300 g, rats were fed 16 g of food daily for the remainder of the 12-month study (weights maintained at approximately 325 g). Water was available continuouslyin the home cage. The colony room was maintained on a 12-h light/dark cycle and experimentswere conducted during the light portion of the cycle. Animals were under continuous care andmonitoring by veterinary staff from the Division of Laboratory Animal Medicine (DLAM) atUNC-Chapel Hill, which is accredited by the Association for the Assessment and Accreditationof Laboratory Animal Care International (AAALAC). DLAM staff considered all of the ratsto be healthy throughout the course of the experiment, indicating that the procedures producedno adverse affects on animal health or well-being. All procedures were also carried out inBesheer and Hodge Page 3Neuropsychopharmacology. Author manuscript; available in PMC 2010 June 25.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptaccordance with the NIH Guide to Care and Use of Laboratory Animals and institutionalguidelines.ApparatusOperant chambers (Med Associates, Georgia, VT) measuring 31 × 32 × 24 cm3 were used fordiscrimination sessions. The chambers were located within sound-attenuating cubicles andequipped with an exhaust fan that provided ventilation and masked external sounds. Tworesponse levers were located on the right wall of each chamber. Responses on the leversactivated a liquid dispenser centered between the levers that presented fluid in a 0.1 ml dippercup for 4 s during each operation. A stimulus light was located above each response lever andwas activated each time a reinforcer was delivered. The chambers were illuminated by an 8-W light located on the left wall 28 cm above the floor. The chambers were interfaced (MedAssociates) to a computer that was programmed to control sessions and record data.ProcedureLever press training—Rats were trained to lever press on a fixed ratio 1 (FR1) schedule ofreinforcement in overnight sessions. That is, a single lever press on either of the two leversresulted in presentation of 0.1 ml of a liquid sucrose solution (10% (w/v)). After three overnightsessions, 1 h lever press training began. During these sessions, rats were placed in the chamberfor 10 min before the house light was illuminated and either the right or the left lever wasextended into the chamber and signaled the beginning of the session. The presentation of theright and left lever was alternated each day and the schedule of reinforcement was graduallyincreased to FR10. That is, once 10 lever presses occurred, the sucrose was presented for 4 s.Rats received equal experience with both levers at each reinforcement schedule. Discriminationtraining began once responding on the FR10 schedule was stable (<10% daily variation in thetotal number of responses).Discrimination training—Training sessions were conducted 5 days per week (M–F) duringwhich ethanol (1 g/kg) or water was administered by intragastric gavage (i.g.) prior to the startof the 15-min sessions. Immediately following the ethanol or water administration, the ratswere placed in the chambers. Similar to lever press training sessions, after 10 min the houselight was illuminated and both levers were introduced into the chamber signaling the beginningof the session. Following ethanol administration, completion of 10 responses on the ethanol-appropriate lever resulted in the presentation of the sucrose solution. Similarly, following wateradministration, completion of 10 responses on the water-appropriate lever resulted in sucrosedelivery. During both the ethanol and water sessions, responses on the inappropriate lever wererecorded but produced no programmed consequences. The lever associated with ethanol orwater administration was randomly assigned and counterbalanced across animals. Water andethanol training days varied on a double alternation schedule (W, W, E, E, etc.). The trainingsessions continued until the percentage of ethanol- and water-appropriate lever press responsesemitted prior to the first reinforcer, and during the entire session was >80% for 10 consecutivedays. Once these criteria were met, testing began.Testing procedures—Test sessions were identical to the training sessions, except that theywere 2 min in duration, and completion of an FR10 on either lever resulted in sucrose delivery.Reinforcement was delivered during test sessions to assess the possible effects of testtreatments on overall response rates. These test sessions were interspersed with trainingsessions only if performance during the previous five training sessions met the accuracycriteria. If the criteria were not met, training continued until response accuracy was >80% for5 consecutive days, consequently, not all rats were used in each test condition.Besheer and Hodge Page 4Neuropsychopharmacology. Author manuscript; available in PMC 2010 June 25.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptSubstitution TestingEthanol substitution—In four different test sessions, various ethanol doses (0.1, 0.5, 1, and2 g/kg, i.g.) were administered to determine an ethanol substitution curve. Rats (n = 12) receivedeach ethanol dose in a random order.MPEP substitution—To test whether MPEP would substitute for ethanol, rats (n = 6) wereadministered MPEP (0, 1, 10, 30, and 50 mg/kg, i.p.) 20 min before receiving a water gavageand were then placed in the chambers. Each MPEP dose was administered once in randomorder.Ethanol substitution with MPEP pretreatment—In order to evaluate the effects ofMPEP pretreatment on the stimulus effects of multiple ethanol doses, rats (n = 8) wereadministered MPEP (30 mg/kg, i.p.) or saline 20 min before ethanol (0.1, 0.5, 1, and 2 g/kg,i.g.). Rats were tested once on each pretreatment combination and the combinations were testedrandomly.GABAA-positive modulator substitution with MPEP pretreatment—Saline orMPEP (30 mg/kg, i.p.) was administered 20 min before pentobarbital injection (1, 3, and 10mg/kg, i.p.). Rats (n = 10) were tested once on each pretreatment combination and thecombinations were tested randomly. After pentobarbital testing, saline or MPEP (30 mg/kg,i.p.) was administered 20 min before diazepam injection (1, 3, and 5 mg/kg, i.p.). Rats (n = 11)were tested once on each pretreatment combination and the combinations were testedrandomly. Gavage was withheld during these tests.DrugsEthanol (95% (w/v)) was diluted in distilled water to a concentration of 20% (v/v) and wasadministered i.g. in various volumes to obtain doses of 0.1, 0.5, 1, and 2 g/kg. A correspondingvolume of water to the 1 g/kg ethanol training dose was used. Pentobarbital (Sigma-Aldrich,St Louis, MO) was dissolved in saline, and injected i.p. at a volume of 1 ml/kg. MPEP (Sigma)was also dissolved in saline by gently heating the solution and injected i.p. at a volume of 1ml/kg, except for the 50 mg/kg dose, which was injected at a volume of 2 ml/kg. Diazepam(Sigma) in a suspension of 1% carboxymethylcellulose was injected i.p. at a volume of 2 ml/kg.Data AnalysisResponse accuracy was expressed as the percentage of ethanol-appropriate lever presses upondelivery of the first reinforcer. Response rate (responses/min) was analyzed for the entiresession and provided an index of locomotor ability. Complete substitution for the ethanolstimulus was defined as >80% choice of the ethanol lever upon completion of the first FR10during test sessions. If an animal did not complete an FR10 during these test sessions, then thatanimal was not included in the response accuracy analysis, but was included in the responserate analysis. The left-most panels of each figure show the percentage of ethanol-appropriatelever responding on completion of the first FR10 and response rate during the ethanol andwater session that preceded testing of that drug combination. One or two-way repeatedmeasures analysis of variance (ANOVA) were used to analyze response accuracy and responserate data. Planned comparisons were made within each pretreatment condition (eg eachdiazepam dose compared to lowest diazepam dose in the saline pretreatment condition), andbetween the pretreatment conditions at each test drug dose (eg responses after saline and MPEPcompared at each diazepam dose). ED50 values for the dose effects were determined by log-dose probit analysis when possible, and paired t-tests were used to compare the ED50’s for theethanol, pentobarbital, and diazepam tests.Besheer and Hodge Page 5Neuropsychopharmacology. Author manuscript; available in PMC 2010 June 25.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptImmunohistochemistryAt the conclusion of the ethanol discrimination study, the animals (n = 10) were deeplyanesthetized with pentobarbital (100 mg/kg, i.p.) and perfused transcardially with 0.1 Mphosphate-buffered saline (PBS) followed by 4% paraformaldehyde. The brains were removedfrom the skull and placed in the same fixative solution for at least 24 h before being washedwith PBS and sliced on a vibratome into 40 µm sections. Primary antibodies used in the studywere a rabbit polyclonal antibody raised against mGluR5 (Neuromics, Northfield, MN; dilution1:300) and a guinea-pig polyclonal antibody raised against GABAA α1-subunit (courtesy ofDr Jean-Marc Fritschy; dilution 1:300). The fluorescent-coupled secondary antibodies wereAlexa Fluor® 488 goat anti-rabbit IgG (1:200 dilution) for mGluR5 and Alexa Fluor® 594 goatanti-guinea pig IgG (1:200 dilution) for GABAA α1 (Molecular Probes, Eugene, OR, USA).Visualization was performed using a Zeiss 410 laser-scanning confocal microscope (Carl Zeiss,Oberkochen, Germany). Images were saved in tagged image format and exported to AdobePhotoshop CS (Adobe Systems, Mountain View, CA) for analysis.RESULTSEthanol SubstitutionThe left-most panels of Figure 1a and b show the percentage of ethanol-appropriate leverresponding on completion of the first FR10 and response rate during the ethanol and watersession that preceded testing of the ethanol dose response. Responding on the ethanol-appropriate lever during the ethanol session was approximately 96% and less than 2% on thewater session. The percentage of ethanol-appropriate responses increased significantly as afunction of ethanol test dose (F(3,47) = 52.31, p<0.001), indicating that the proceduresestablished reliable stimulus control. Both the 1 and 2 g/kg test doses of ethanol substitutedfully for the 1 g/kg training dose (Figure 1a), with an ED50 value of 0.56 g/kg (±0.06 SEM).The training dose (1 g/kg) did not affect response rate (Figure 1b). However, a significantreduction in lever press responding was observed (F(3,47) = 5.85, p = 0.003), with the highestdose (2 g/kg) suppressing responding relative to the lowest dose tested.MPEP SubstitutionAs shown in Figure 2a, MPEP did not substitute for the stimulus effects of 1 g/kg ethanol (F(4,20) = 1.58, p = 0.22). All MPEP doses tested failed to substitute for ethanol. The highestMPEP dose tested (50 mg/kg) reduced response rate relative to saline (F(4,20) = 2.91, p =0.047).Ethanol Substitution with MPEP PretreatmentFigure 3a shows the ethanol dose–response curve following saline and MPEP pretreatment.MPEP pretreatment at the 1 and 2 g/kg ethanol doses includes seven animals due to the deathof one animal before testing was completed. Similar to the previous ethanol dose–responsecurve presented in Figure 1a, dose-dependent substitution for the ethanol training dose (1 g/kg) was observed. This dose-dependent substitution was inhibited by MPEP pretreatment. Thetwo-way repeated measures ANOVA revealed a main effect of ethanol dose (F(3,21) = 20.57,p<0.001). After saline pre-treatment, ethanol-appropriate responding was greater at everyethanol dose (0.5, 1, and 2 g/kg) relative to the 0.1 g/kg ethanol dose, p’s <0.03. MPEPpretreatment significantly altered this pattern of responding, as only the highest ethanol dose(2 g/kg) produced significantly greater ethanol-appropriate responding than the 0.1 g/kgethanol dose, p = 0.002. There was a significant main effect of pretreatment injection (F(1,7)= 15.37, p = 0.005). MPEP pretreatment significantly reduced ethanol-appropriate respondingat the 0.5 and 1 g/kg ethanol doses, p’s <0.001. The ethanol dose by pretreatment injectioninteraction was also significant, (F(3,19) = 4.62, p = 0.01). The mean ED50±SEM for ethanolBesheer and Hodge Page 6Neuropsychopharmacology. Author manuscript; available in PMC 2010 June 25.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptsubstitution was 0.39±0.08 g/kg following saline pretreatment and 1.33±0.19 g/kg followingMPEP pretreatment. This 3.4-fold rightward shift in the ethanol dose–response curve wassignificant, (t(3) = 5.4 p = 0.01).As shown in Figure 3b, response rate was dose dependently reduced by ethanol (F(3,21) =6.38, p = 0.003), with significant rate reductions observed at the highest ethanol dose (2 g/kg)relative to the lowest dose (0.1 g/kg) after saline and MPEP pretreatment, p’s <0.02. The maineffect of pretreatment injection was also significant (F(1,7) = 7.58, p = 0.03), but no significantinteraction was observed. At the lowest ethanol dose tested (0.1 g/kg), response rate wassignificantly lower after MPEP pretreatment, p = 0.02. MPEP did not induce a rate reductionat any other ethanol dose. Taken together, MPEP pretreatment reduced ethanol-appropriateresponding, at doses that were not affected by response rate reductions (0.5 and 1 g/kg).Pentobarbital Substitution for Ethanol with MPEP PretreatmentPentobarbital dose dependently substituted for ethanol as shown in Figure 4a. Two rats did notcomplete a first FR when MPEP was administered before the highest pentobarbital dose (10mg/kg). Consequently, those animals are not included in the response accuracy analysis (Figure4a), but are included in the response rate analyses (Figure 4b). The two-way repeated measuresANOVA revealed a main effect of pentobarbital dose (F(2,18) = 23.13, p<0.001), with thehighest pentobarbital dose (10 mg/kg) producing significantly greater responding on theethanol-appropriate lever than the lowest dose (1 mg/kg) after saline and MPEP pretreatment,p’s<0.002. Pentobarbital substitution was not altered by MPEP pretreatment, as confirmed bythe lack of a significant main effect of pretreatment injection (F<1). The pentobarbital dose bypretreatment injection interaction was also not significant (F<1). The mean±SEM ED50 forpentobarbital substitution was 5.26±0.81 mg/kg following saline pretreatment and 4.89±0.85mg/kg following MPEP pretreatment.Response rate was significantly altered by pentobarbital dose (F(2,18) = 17.05, p<0.001), witha significant reduction observed at the 10 mg/kg dose after saline and MPEP pretreatment,p’s<0.001 (Figure 4b). Response rate was also reduced by MPEP pretreatment (F(1,9) = 15.46,p = 0.003), and at each pentobarbital dose MPEP decreased responding relative to salinepretreatment, p’s<0.03. The pentobarbital dose by pretreatment injection interaction was notsignificant (F<1).Diazepam Substitution for Ethanol with MPEP PretreatmentDiazepam dose dependently substituted for ethanol as shown in Figure 5a. One animal diedbefore completion of the tests, and accordingly, the 1 mg/kg diazepam dose following MPEPpretreatment includes data from 10 animals. The two-way repeated measures ANOVA revealeda significant main effect of diazepam dose (F(2,20) = 4.94, p = 0.02). After saline pretreatment,significantly greater responding on the ethanol-appropriate lever at the highest diazepam dose(5 mg/kg) relative to the 1 mg/kg diazepam dose was evident, p = 0.002. This effect wasprevented by MPEP pretreatment. The main effect of pretreatment injection was notsignificant; however, the diazepam dose by pretreatment injection interaction was significant(F(2,19) = 3.72, p = 0.04). MPEP pretreatment reduced ethanol-appropriate responding at the5 mg/kg diazepam dose relative to saline pretreatment, p = 0.01. The mean± SEM ED50 fordiazepam substitution was 2.23±0.21 mg/kg and was 2.03±70.26 mg/kg following MPEPpretreatment. Given that MPEP pretreatment reduced diazepam substitution, the ED50following MPEP pretreatment was determined from the data of five animals.Figure 5b illustrates response rate for the diazepam substitution tests. There was a significantmain effect of diazepam dose (F(2,20) = 4.69, p = 0.02), with a response reduction observedafter saline pretreatment at the highest diazepam dose (5 mg/kg) relative to the lowest diazepamBesheer and Hodge Page 7Neuropsychopharmacology. Author manuscript; available in PMC 2010 June 25.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptdose (1 mg/kg), p = 0.006. MPEP pretreatment also significantly reduced response rate (F(1,10)= 11.14, p = 0.007); a significant reduction was observed at the 1 and 3 mg/kg diazepam doses.The diazepam dose by pretreatment drug interaction was not significant. Thus, MPEPpretreatment significantly reduced ethanol-appropriate responses at the 5 mg/kg diazepamdose. A response rate reduction was observed at this dose after saline pretreatment; however,MPEP pretreatment did not enhance that response rate reduction.Immunohistochemistry with Confocal MicroscopyIn order to determine if there is an anatomical basis for the pharmacological interaction betweenBZ-sensitive GABAA receptors and mGlu5 receptors, dual-label fluorescentimmunohistochemistry with confocal microscopy was used to examine both the individual andjoint expression patterns of mGluR5 and GABAA receptors expressing the α1 molecularsubunit, which constitutes the majority of BZ-sensitive GABAA receptors (Mohler et al,1997). Previous work has shown that GABAA α1 receptors and mGlu5 receptors are bothexpressed in brain regions known to modulate ethanol discrimination such as the nucleusaccumbens, amygdala, and hippocampus (Romano et al, 1995; Hodge and Cox, 1998; Pirkeret al, 2000; Schwarzer et al, 2001; Besheer et al, 2003). In the shell and core (Figure 6a) ofthe nucleus accumbens, relatively few cell bodies showed GABAA α1 immunoreactivity,consistent with Härtig et al, (1995). Several GABAA α1-labeled processes were evident withsome processes visibly extending into the ventral pallidum. Consistent with the existingliterature, diffuse mGluR5 staining was observed throughout the striatum including the core(Figure 6b) and shell regions of the nucleus accumbens. In the basolateral amygdala, moderatemGluR5 and GABAA α1 immunoreactivity was found with labeled cell bodies and processes.In the CA1, CA2, CA3, and dentate gyrus of the hippocampus, intense mGluR5 staining ofcell bodies and processes was evident (Figure 6e). GABAA α1 staining was also found on thecell bodies and processes in the same regions of the hippocampal formation (Figure 6d). Thepallidal regions (ventral pallidum and globus pallidus) were also examined given that mGluR5and GABAA α1 are expressed in those regions (Lu et al, 1999; Pirker et al, 2000; Schwarzeret al, 2001; Poisik et al, 2003) and that activation of mGluR5 in the nucleus accumbens hasbeen shown to increase GABA levels in the ventral pallidum (Diaz-Cabiale et al, 2002),suggesting a potential interaction between the two receptor systems in this brain area. In thepresent work, intense staining of both GABAA α1 and mGluR5 was found on cell bodies andprocesses in the ventral pallidum (Figure 6g and h) and the globus pallidus (Figure 6j and k).The coexpression of mGluR5 and GABAA α1 on neurons within these brain regions has notbeen examined previously. As illustrated in Figure 6f, i and l, mGluR5 and GABAA α1 werecoexpressed on cells in all of the limbic brain regions examined, except for the nucleusaccumbens (Figure 6c). Interestingly, the coexpression pattern was relatively consistent acrossthe other brain regions. That is, intense mGluR5 immunoreactivity appeared to be on thenuclear membrane, whereas intense GABAA α1 staining was localized on cell surfacemembrane receptors and processes.DISCUSSIONThe discriminative stimulus effects of ethanol are mediated in part by positive modulation ofGABAA receptor activity (Ator et al, 1993; Grant and Lovinger, 1995; Hodge and Cox,1998; Kostowski and Bienkowski, 1999; Hodge et al, 2001; Shelton and Grant, 2002).Evidence indicates that mGlu5 receptors can modulate GABAA receptor function (Awad etal, 2000; Hoffpauir and Gleason, 2002) and extracellular GABA levels. Moreover, ethanolinhibits glutamate-induced Ca2+-dependent Cl− currents in Xenopus oocytes expressingmGluR5 (Minami et al, 1998). Together, these findings suggest that the GABAA receptorcomponent of ethanol’s stimulus effects may be modulated by mGluR5 activity.Besheer and Hodge Page 8Neuropsychopharmacology. Author manuscript; available in PMC 2010 June 25.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptThe purpose of this work was to examine the involvement of mGlu5 receptors in thediscriminative stimulus properties of ethanol. Long Evans rats were trained by differentialreinforcement to discriminate ethanol (1 g/kg, i.g.) from water vehicle using standard two-leveroperant drug discrimination procedures (Hodge and Cox, 1998; Hodge et al, 2001; Besheer etal, 2003). The discrimination training procedures established reliable stimulus control asethanol dose dependently substituted for the training dose of ethanol (1 g/kg). Administrationof the selective mGluR5 antagonist MPEP (0–50 mg/kg) did not produce ethanol-like stimuluseffects when administered alone, as indicated by low ethanol-appropriate responding. Thehighest dose of MPEP (50 mg/kg) significantly decreased response rate, which indicates thatthe compound was behaviorally active in the dose range tested and supports previous studiesthat found locomotor suppression by high doses of MPEP (Spooren et al, 2000). These datademonstrate that antagonism of mGluR5 function alone was not sufficient to producediscriminative stimulus properties that correspond to ethanol (1 g/kg).A primary finding of the present study is that mGluR5 antagonism inhibited the discriminativestimulus properties of ethanol. Under control conditions, test doses of ethanol (1 and 2 g/kg)substituted fully (ie >80% ethanol-lever selection) for the discriminative stimulus propertiesof the ethanol (1 g/kg) training dose. Following MPEP (30 mg/kg) pretreatment, these testdoses failed to fully substitute for the training dose. In addition, administration of MPEP priorto test doses of ethanol shifted the ethanol substitution curve more than three-fold to the right.This MPEP-induced shift in the ED50 for ethanol substitution was attributable to significantreductions in ethanol-appropriate responding following ethanol (0.5 and 1 g/kg) test doses.Reductions in response rate were seen only after combination of MPEP with the highest doseof ethanol (2 g/kg) tested, indicating that the reduction in the discriminative stimulus propertiesof ethanol was not associated with locomotor impairment. These results suggest that thediscriminative stimulus effects of ethanol may be mediated in part by mGluR5 function.In order to determine if the mGluR5 antagonist reduced ethanol discrimination by altering theGABAA receptor component of the ethanol stimulus, the effect of MPEP pretreatment onpentobarbital and diazepam substitution for ethanol (1 g/kg) was examined. Consistent withprevious reports, pentobarbital (Ator et al, 1993; Grant et al, 1996; Hodge et al, 2001; Sheltonand Grant, 2002) and diazepam (Jarbe and McMillan, 1983; Ator et al, 1993; Kostowski andBienkowski, 1999; Green-Jordan and Grant, 2000; Shelton and Grant, 2002) dose dependentlysubstituted for the stimulus effects of ethanol. MPEP pretreatment did not alter pentobarbital(1–10 mg/kg) substitution for ethanol. However, MPEP enhanced the response rate-reducingeffects of pentobarbital, indicating that the mGluR5 antagonist was behaviorally active andable to modulate the motor effects of pentobarbital. Although a higher MPEP dose would mostlikely further enhance the locomotor depressant effects of pentobarbital, rendering effects onethanol-appropriate responding difficult to interpret, it remains possible that a lower MPEPdose may have altered pentobarbital substitution. Conversely, the ethanol-like stimulusproperties of diazepam (5 mg/kg) were significantly inhibited by 30 mg/kg MPEP pretreatment.The ED50 for ethanol substitution by diazepam was not altered by MPEP administration. Thiswas due to the selective inhibition of ethanol-appropriate responding following only the highestdose of diazepam (5 mg/kg) tested. MPEP did not alter response rate when coadministeredwith diazepam, indicating that inhibition of the ethanol-like stimulus effects of diazepam wasnot associated with motor impairment. Both pentobarbital and diazepam are positivemodulators of GABAA receptors, and consequently share a similar mechanism of action. Thatis, both compounds potentiate the action of GABA at the GABAA receptor (Study and Barker,1981; Steinbach and Akk, 2001; Skerritt and Macdonald, 1984). However, pentobarbital canalso directly gate the GABAA receptor in the absence of GABA (Serafini et al, 2000; Akk andSteinbach, 2000; Bormann, 1988). Another difference between the two compounds is thatbenzodiazepines, like diazepam, have specific binding sites on GABAA receptors (see later),while a specific binding site(s) for the barbiturates, like pentobarbital, has not yet beenBesheer and Hodge Page 9Neuropsychopharmacology. Author manuscript; available in PMC 2010 June 25.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptdetermined (Serafini et al, 2000). Further, barbiturates have pharmacological actions on otherligand-gated ion channel receptors such as neuronal nicotinic acetylcholine receptors andinhibitory glycine receptors (for a review see Krasowski and Harrison, 1999). Thus, the lackof an effect of MPEP pretreatment (30 mg/kg) on pentobarbital substitution for ethanol couldbe due to nonspecific effects of pentobarbital. These findings suggest that the mGluR5antagonist MPEP inhibited the discriminative stimulus effects of ethanol specifically byreducing, or otherwise interfering with, the component of ethanol discrimination that ismediated by benzodiazepine-sensitive GABAA receptors.Evidence of an interaction between GABAA and mGlu5 receptors has been reported (Hoffpauirand Gleason, 2002). In that study, the activity of GABAA receptors in retinal amacrine cellswas enhanced by mGluR5 activation, as evidenced by an increase in GABA-gated currentsafter administration of CHPG, an mGluR5 agonist. The mGlu5 receptor has also been shownto influence GABA levels. For example, using microdialysis techniques, de Novellis et al,(2003) found that activation of mGluR5 by perfusion of CHPG through the dialysis probesignificantly enhanced extracellular levels of GABA in the periaquaductal gray of rats.Similarly, intra-accumbens administration of CHPG increased GABA levels in the ventralpallidum (Diaz-Cabiale et al, 2002). In both studies, MPEP pretreatment blocked the agonist-induced increases in GABA levels. Clearly, mGlu5 receptors can influence GABA functioning,a major mediating factor in the discriminative stimulus effects of ethanol. The MPEP-inducedreduction in the stimulus properties of ethanol, and the ethanol-like properties of diazepam,may have been a result of altered GABAergic functioning by MPEP pretreatment. Given thatpentobarbital substitution for ethanol was not affected, BZ-sensitive GABAA receptors mayhave been differentially affected by MPEP-induced alterations. To our knowledge, interactionbetween BZ-sensitive GABAA and mGlu5 receptors has not been examined previously.A majority of BZ-sensitive GABAA receptors with high affinity for diazepam include theα1β2γ2-subunit combination. The α1β2γ2- and α1β2γ2-subunit combinations also express highaffinity for diazepam but are less abundant throughout the brain (McKernan and Whiting,1996). The BZ-binding site is generally thought to exist on the α-subunit (Fuchs et al, 1988;Stephenson et al, 1990). The GABAA α1-subunit is distributed in brain regions known tomodulate ethanol discrimination (eg nucleus accumbens, amygdala, hippocampus; Pirker etal, 2000), as is the mGlu5 receptor (Romano et al, 1995), and these distribution patterns werealso found in the immunohistochemical studies of the present work. Further, we found thatmGluR5 and GABAA α1 were coexpressed on cell bodies in all of the limbic brain regionsexamined, with the exception of the nucleus accumbens. The lack of coexpression in thenucleus accumbens may be due to the relatively few GABAA α1 immunoreactive cell bodies,and the diffuse nature of the mGluR5 staining in that brain region. Regardless, the consistentpattern of coexpression on cells suggests that mGluR5- and GABAA α1-containing receptorsmight interact at the brain regional level.We also observed coexpression of mGlu5 and GABAA α1 receptors on the same cells in brainregions that modulate the discriminative stimulus effects of ethanol such as the amygdala andhippocampus (Hodge and Cox, 1998; Hodge et al, 2001; Besheer et al, 2003). Representativecells from the hippocampus (CA3) are shown in Figure 6 (panel f). In those cells showingcoexpression, intense mGluR5 immunoreactivity appeared to be on the nuclear membrane,whereas GABAA α1 was localized on the cell surface, or plasma membrane. This pattern ofmGluR5 nuclear membrane distribution is consistent with O’Malley et al (2003). In that study,using confocal microscopy mGluR5 was colocalized with lamin B2, a nuclear membranemarker. Accordingly, mGlu5 receptors expressed on nuclear membranes can produce sustainedoscillatory Ca2+ responses (O’Malley et al, 2003), which indicates that these receptors canmodulate intracellular signaling. This suggests that mGlu5 receptors might modulateGABAA receptor function on specific cells via alterations in Ca2+ -linked second messengerBesheer and Hodge Page 10Neuropsychopharmacology. Author manuscript; available in PMC 2010 June 25.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptsystems such as PKC (eg Hodge et al, 1999), which is a possible basis for the pharmacologicalinteraction between mGlu5 and GABAA receptors seen in the present work.In addition to finding mGluR5 and GABAA α1 coexpression in brain regions known tomodulate ethanol discrimination, the same cellular coexpression pattern was also observed inthe ventral pallidum and the globus pallidus. Interestingly, GABAA α1 receptors in the ventralpallidum are known to mediate ethanol self-administration (June et al, 2003), but the role ofpallidal regions in ethanol discrimination has not been examined. In the present study, both theventral pallidum and globus pallidus showed intense immunoreactivity for the GABAA α1-subunit, which is consistent with previous findings that showed prominent expression of theα1-subunit along with β2- and γ2-subunits (Schwarzer et al, 2001), and suggests that thesebrain regions mediate pharmacological sensitivity to diazepam. Thus, the mGluR5 antagonisttested in the present study may have inhibited both ethanol discrimination, and diazepamsubstitution for ethanol, by modulating GABAA receptor function within pallidal regions.Other evidence suggests that mGlu5 and GABAA receptors may interactively modulate neuralcircuits among brain regions. For example, a majority of projection neurons from the nucleusaccumbens to the ventral pallidum express mGluR5 (Lu et al, 1999), suggesting thatGABAergic projections from the accumbens can be modulated by mGluR5 activity. Indeed,infusion of the mGluR5 agonist CHPG into the nucleus accumbens increased GABA levels inthe ventral pallidum (Diaz-Cabiale et al, 2002). The nucleus accumbens plays a major role inmediating the discriminative stimulus effects of ethanol. GABAA-positive modulators such asmuscimol and allopregnanolone, microinjected into the nucleus accumbens produce ethanol-like stimulus properties (Hodge and Cox, 1998; Hodge et al, 2001; Besheer et al, 2003).Therefore, there exists the possibility that the discriminative stimulus properties of ethanol aremediated in part by GABAergic projection neurons from the nucleus accumbens to the pallidalregions. Moreover, antagonism of mGluR5 in this pathway may have inhibited ethanoldiscrimination and diazepam substitution for ethanol by altering GABA neurotransmission inthe ventral pallidum. Clearly, the potential role of nucleus accumbens projections and thecellular interactions between mGluR5 and GABAA α1 in modulating the stimulus effects ofethanol will need to be further examined. However, given the behavioral andimmunohistochemical findings of the present work and the existing literature, the foundationexists to predict an interaction between mGluR5 and GABAA α1 receptors within limbiccircuits.In conclusion, drugs of abuse produce distinctive subjective effects in humans that are oftendescribed as ‘high’ or ‘euphoria’. These subjective effects play a major role in the onset andmaintenance of drug-taking behaviors (Stolerman, 1992). Thus, examination of thediscriminative stimulus effects of drugs is essential for the effective development oftherapeutics designed to aide in drug abuse-related disorders. Indeed, MPEP pretreatment hasbeen found to reduce the acute reinforcing effects of ethanol (Sharko et al, 2002) and relapseto ethanol-seeking behavior (Bäckström et al, 2004), suggesting that targeting the mGlu5receptor system may be a potential therapeutic strategy for the treatment of alcohol-relateddisorders. The findings from the present work suggest a specific behavioral mechanism bywhich MPEP may reduce ethanol reinforcement and relapse. That is, MPEP appears to reducethe subjective (ie discriminative stimulus) properties of ethanol, which may contribute toreductions in chronic drinking or excessive consumption during relapse. Moreover, the presentdata indicate that MPEP does not substitute for the stimulus effects of ethanol, indicatingminimal coabuse liability. Further examination of potential interactions between ethanol’sphysiological and behavioral effects and the mGlu5 receptor system may indeed reveal themGluR5 system to be a novel and effective therapeutic target in the treatment of alcoholism.Besheer and Hodge Page 11Neuropsychopharmacology. Author manuscript; available in PMC 2010 June 25.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptAcknowledgmentsThis work was supported by Grants AA09981 and AA011605 from the National Institute on Alcohol Abuse andAlcoholism to CWH. We would like to thank Dr Jean-Marc Fritschy for the gift of the GABAA α1 antibody, Drs JohnJ Lemasters and Jae Sung Kim from the Bowles Center for Alcohol Studies Imaging core, and Mary Beth Wilkie fortechnical assistance.REFERENCESAkk G, Steinbach JH. Activation and block of recombinant GABA(A) receptors by pentobarbitone: asingle-channel study. Br J Pharmacol 2000;130:249–258. [PubMed: 10807661]Aschner M, Mutkus L, Allen JW. 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[PubMed: 10405994]Besheer and Hodge Page 15Neuropsychopharmacology. Author manuscript; available in PMC 2010 June 25.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 1.(a) Mean (±SEM) percentage of ethanol-appropriate responding upon completion of the firstFR10 at each ethanol dose tested (n = 12). (b) Mean (±SEM) test session response rate at eachethanol dose tested. Data points to the left of the x-axis break represent performance on thewater (W) and ethanol (E) training session prior to testing. Data points to the right of the x-axis break represent test session performance following IG ethanol administration. Thehorizontal dashed line (>80%) represents full substitution for the discriminative stimuluseffects of ethanol (1 g/kg, i.g.). *Significant difference from 0.1 g/kg ethanol (Tukey’s,p<0.05).Besheer and Hodge Page 16Neuropsychopharmacology. Author manuscript; available in PMC 2010 June 25.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 2.(a) Mean (±SEM) percentage of ethanol-appropriate responding upon completion of the firstFR10 at each MPEP dose tested (n = 6). (b) Mean (±SEM) test session response rate at eachMPEP dose tested. Data points to the left of the x-axis break represent performance on thewater (W) and ethanol (E) training session prior to testing. Data points to the right of the x-axis break represent test session performance following i.p. MPEP administration. Thehorizontal dashed line (>80%) represents full substitution for the discriminative stimuluseffects of ethanol (1 g/kg, i.g.). *Significant difference from saline (Tukey’s, p<0.05).Besheer and Hodge Page 17Neuropsychopharmacology. Author manuscript; available in PMC 2010 June 25.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 3.(a) Mean (±SEM) percentage of ethanol-appropriate responding upon completion of the firstFR10 after saline or MPEP pretreatment at each ethanol dose (n = 8). (b) Mean (±SEM) testsession response rate after saline or MPEP pretreatment at each ethanol dose tested. Data pointsto the left of the x-axis break represent performance on the water (W) and ethanol (E) trainingsession prior to testing. The horizontal dashed line (>80%) represents full substitution for thediscriminative stimulus effects of ethanol (1 g/kg, i.g.). *Significantly different from therespective 0.1 g/kg ethanol dose within pretreatment condition. †Significant difference betweensaline and MPEP pretreatment (Tukey’s, p>0.05).Besheer and Hodge Page 18Neuropsychopharmacology. Author manuscript; available in PMC 2010 June 25.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 4.(a) Mean (±SEM) percentage of ethanol-appropriate responding upon completion of the firstFR10 after saline or MPEP pretreatment at each pentobarbital dose tested (n = 10). (b) Mean(±SEM) test session response rate after saline or MPEP pretreatment at each pentobarbital dosetested. Data points to the left of the x-axis break represent performance on the water (W) andethanol (E) training session prior to testing. The horizontal dashed line (>80%) represents fullsubstitution for the discriminative stimulus effects of ethanol (1 g/kg, i.g.). *Significantlydifferent from the respective 1 mg/kg pentobarbital dose within pretreatmentcondition. †Significant difference between saline and MPEP pretreatment (Tukey’s, p<0.05).Besheer and Hodge Page 19Neuropsychopharmacology. Author manuscript; available in PMC 2010 June 25.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 5.(a) Mean (±SEM) percentage of ethanol-appropriate responding upon completion of the firstFR10 after saline or MPEP pretreatment at each diazepam dose tested (n = 11). (b) Mean(±SEM) test session response rate after saline or MPEP pretreatment at each diazepam dosetested. Data points to the left of the x-axis break represent performance on the water (W) andethanol (E) training session prior to testing. The horizontal dashed line (>80%) represents fullsubstitution for the discriminative stimulus effects of ethanol (1 g/kg, i.g.). *Significantlydifferent from the respective 1 mg/kg diazepam dose within pretreatmentcondition. †Significant difference between saline and MPEP pretreatment (Tukey’s, p<0.05).Besheer and Hodge Page 20Neuropsychopharmacology. Author manuscript; available in PMC 2010 June 25.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 6.Confocal images from a representative rat showing the α1-subunit of the GABAA receptor andmGluR5 in the core of the nucleus accumbens (AcbC; a–c), the CA3 region of the hippocampus(CA3; d–f), the ventral pallidum (VP; g–i), and the globus pallidus (GP; j–l). GABAA α1labeling appears in red, and mGluR5 labeling appears in green. Images a–i taken at × 63magnification; images j–l taken at × 40 magnification.Besheer and Hodge Page 21Neuropsychopharmacology. Author manuscript; available in PMC 2010 June 25.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript

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