The mGluR5 Antagonist 6-Methyl-2-(phenylethynyl)pyridine Decreases Ethanol Consumption via a Protein Kinase C - Dependent Mechanism

Glutamatergic neurotransmission plays a critical role in addictive behaviors, and recent evidence indicates that genetic or pharmacological inactivation of the type 5 metabotropic glutamate receptor (mGluR5) reduces the self-administration of cocaine, nicotine, and alcohol. Because mGluR5 is coupled to activation of protein kinase C (PKC), and targeted deletion of the epsilon isoform (PKC ) in mice reduces ethanol self-administration, we investigated whether there is a functional link between mGluR5 and PKC . Here, we show that acute administration of the mGluR5 agonist (R,S)-2-chloro-5-hydroxyphenylglycine to mice increases phosphorylation of PKC in its activation loop (T566) as well as in its C-terminal region (S729). Increases in phospho-PKC are dependent not only on mGluR5 stimulation but also on phosphatidylinositol-3 kinase (PI3K). In addition, the selective mGluR5 antagonist 6-methyl-2-(phenylethynyl)pyridine (MPEP) reduced basal levels of phosphorylation of PKC at S729. We also show that MPEP dose dependently reduced ethanol consumption in wild-type but not in PKC -null mice, suggesting that PKC is an important signaling target for modulation of ethanol consumption by mGluR5 antagonists. Radioligand binding experiments using [H]MPEP revealed that these genotypic differences in response to MPEP were not a result of altered mGluR5 levels or binding in PKC null mice. Our data indicate that mGluR5 is coupled to PKC via a PI3K-dependent pathway and that PKC is required for the ability of the mGluR5 antagonist MPEP to reduce ethanol consumption. Glutamatergic neurotransmission plays an important role in the behavioral and neurochemical effects of drugs of abuse as well as in drug self-administration. Although most studies have examined the role of ionotropic glutamate receptor subtypes (Tzschentke and Schmidt, 2003; Kalivas, 2004), there is increasing evidence that metabotropic glutamate receptors (mGluRs) also play an important role in drug-related behaviors (Kenny and Markou, 2004; Olive, 2005). For example, mice lacking the type 5 mGluR (mGluR5) show dramatically reduced levels of cocaine self-administration and cocaineinduced hyperactivity (Chiamulera et al., 2001). In addition, studies using selective mGluR5 antagonists have confirmed a role for mGluR5 in cocaine-related behaviors such as conditioned place preference (Mcgeehan and Olive, 2003), hyperlocomotion (Mcgeehan et al., 2004), and self-administration (Chiamulera et al., 2001; Kenny et al., 2003b; Tessari et al., 2004). Self-administration of nicotine (Kenny et al., 2003b; Paterson et al., 2003; Tessari et al., 2004) and ethanol (Sharko et al., 2002; Backstrom et al., 2004) are also reduced This work was supported by Public Health Service Grants AA13276 and AA013852 (to M.F.O.), AA08117 and AA13588 (to R.O.M.), the Bowles Center for Alcohol Studies, and funds provided by the State of California for medical research on alcohol and substance abuse through the University of California at San Francisco. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.104.003319. ABBREVIATIONS: mGluR5, type 5 metabotropic glutamate receptor; DAG, diacylglycerol; PKC , protein kinase C ; CHPG, (R,S)-2-chloro-5hydroxyphenylglycine; MPEP, 6-methyl-2-(phenylethynyl)pyridine; ANOVA, analysis of variance; PDK1, phospholipids-dependent kinase 1; PI3K, phosphatidylinositol-3 kinase; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride. 0026-895X/05/6702-349–355$20.00 MOLECULAR PHARMACOLOGY Vol. 67, No. 2 Copyright © 2005 The American Society for Pharmacology and Experimental Therapeutics 3319/1193675 Mol Pharmacol 67:349–355, 2005 Printed in U.S.A. 349 at A PE T Jornals on O cber 3, 2017 m oharm .aspeurnals.org D ow nladed from by mGluR5 antagonists. Finally, blockade of mGluR5 increases threshold current levels for intracranial self-stimulation (Harrison et al., 2002; Kenny et al., 2003a), suggesting that these receptors are centrally involved in primary reward mechanisms. Activation of group I mGluRs, which include mGluR1 and mGluR5, stimulates the activity of phospholipase C, thereby generating diacylglycerol (DAG), which in turn can activate PKC (Hermans and Challiss, 2001). Nine different PKC genes have been identified, seven of which encode isozymes activated by DAG. It is currently not known which PKC isozyme(s) is activated by mGluR5. One candidate is PKC , which, like mGluR5, is enriched in regions of the brain involved in drug self-administration and addiction, including the frontal cortex, striatum, nucleus accumbens, and hippocampus (Saito et al., 1993; Minami et al., 2001). Moreover, there is considerable anatomical overlap in the expression of mGluR5 and PKC in the brain (Saito et al., 1993; Shigemoto et al., 1993; Romano et al., 1995; Minami et al., 2001), suggesting that this PKC may be a downstream signaling target of mGluR5. It is interesting to note that our research has shown that similar to pharmacological blockade of mGluR5, genetic deletion of PKC reduces ethanol consumption in mice (Hodge et al., 1999; Olive et al., 2000; Choi et al., 2002). Here, we sought to establish biochemical link between mGluR5 and PKC and to determine whether PKC is necessary for the ability of mGluR5 antagonists to reduce ethanol consumption. Materials and Methods Animals. Male mice (age 2–4 months) were used for all experiments and were housed in temperatureand humidity-controlled Plexiglas cages (BioZone, Fort Mill, SC) under a 12-h/12-h light/dark cycle with lights on at 6:00 AM. Food and water were available ad libitum except where noted. Male wild-type C57BL/6J 129SvJae littermates (F1 or F2 generation) were used for all studies except ethanol consumption experiments (see below). All procedures were conducted in accordance with Institutional Animal Care and Use Committee procedures and the Society for Neuroscience’s Policy on the Use of Animals in Neuroscience Research. Drugs and Reagents. (R,S)-2-Chloro-5-hydroxyphenylglycine (CHPG, Tocris Cookson Inc., Ellisville, MO) was dissolved in 110 mM NaOH, and LY294002 (Calbiochem, San Diego, CA) was suspended in dimethyl sulfoxide before dilution in artificial cerebrospinal fluid (Harvard Apparatus Inc., Holliston, MA) for intracerebroventricular (i.c.v.) administration. 6-Methyl-2-(phenylethynyl)pyridine (MPEP, Sigma-Aldrich, St. Louis, MO) was dissolved in physiological saline before intraperitoneal (i.p.) administration. All other chemical reagents were obtained from Fisher Scientific (Santa Clara, CA) or Sigma-Aldrich. Intracerebroventricular Cannula Implantation and Drug Treatments. For experiments involving administration of CHPG, mice were anesthetized with ketamine (100 mg/kg i.p.) and xylazine (7 mg/kg i.p.) and placed in a stereotaxic frame equipped with a mouse adapter (Harvard Apparatus Inc.). A plastic guide cannula (26-gauge outer diameter; Plastics One, Roanoke, VA) aimed at the lateral ventricle was unilaterally implanted using the following coordinates with bregma and skull surface as reference points (Franklin and Paxinos, 2001): posterior, 0.3 mm; lateral, 1.0 mm; and ventral, 2.0 mm. The cannula was secured with skull screws and dental cement, and mice were allowed at least 5 days of postsurgical recovery before experiments. A 33-gauge outside diameter microinjection needle (Plastics One) that extended 1 mm beyond the ventral tip of the guide cannula was used for i.c.v. drug administration. Microinjection needles were connected to gastight Hamilton syringes via FEP tubing (CMA/Microdialysis, North Chelmsford, MA). Syringes were driven by a Harvard syringe pump at a flow rate of 1 l/min for a total infusion of 1 l of fluid. The microinjection needle was left in place for an additional 1 min after each infusion to allow for diffusion of the drug into the