Pharmacological activation of group II metabotropic glutamate receptors (mGluR2/3) inhibits cocaine self-administration and reinstatement of drug-seeking behavior suggesting a feasible use of mGluR2/3 agonists in the treatment of cocaine dependence. cocaine-induced reinstatement of drug-seeking behavior likely by attenuating cocaine-induced increases in NAc DA and glutamate via presynaptic mGluR2/3s. microdialysis was used to study the effects of 2-PMPA on basal or cocaine-enhanced NAc DA and glutamate in rats during reinstatement testing in the presence or absence of LY341495 a selective mGluR2/3 antagonist. MATERIALS AND METHODS Animals Male Long-Evans rats (Charles River Laboratories Raleigh NC USA) weighing 250 to 300 g were used. Rats were housed individually in a climate-controlled room on a reversed light-dark cycle (lights on at 7:00 PM lights off at 7:00 AM) with free access to food and water. The animal facility was fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. All experimental procedures were conducted in accordance with the of the U.S. National Academy of Sciences Rabbit Polyclonal to Guanylate Cyclase beta. and were approved by the Animal Care and Use Committee of the National Institute on Drug Abuse of the U.S. National Institutes of Health. Experiment 1: microdialysis with HPLC microdialysis protocols were as reported previously (Xi et al. 2006 Briefly rats were anesthetized with sodium pentobarbital and guide cannulae (20 gauge Plastics One Roanoke VA) were surgically implanted into the NAc (AP +1.6 mm ML ± 2.0 mm DV ?4.0 mm 6 from vertical) according to the rat brain atlas Ferrostatin-1 (Fer-1) of Paxinos and Watson (1998). The guide cannulae were fixed to Ferrostatin-1 (Fer-1) the skull with 4 stainless steel jeweler’s screws (Small Parts Inc. Miami Lakes FL USA) and dental acrylic. After 7 days of recovery from surgery rats were divided into two groups. One group of rats (drug na?ve rats) were used directly for microdialysis while another group of rats were trained for cocaine self-administration first and then used for microdialysis beginning at 24 hrs after the last cocaine self-administration. Microdialysis probes were inserted into the NAc 12 hr before the onset of microdialysis to minimize damage-induced neurotransmitter release. Microdialysis samples were collected every 20 min into 10 μl 0.5 M perchloric acid to prevent DA degradation. After collection samples were frozen at ?80°C. Dialysate DA and glutamate were measured Ferrostatin-1 (Fer-1) using high pressure liquid chromatography (HPLC) with electrochemical and flourometric detection respectively as reported previously (Xi et al. 2006 DA and glutamate values were quantified with external standard curves (DA 0.1-1.0 nM; glutamate 10-1000 nM). The limits of detection for DA and glutamate were 0.01-10 nM and 1 nM-10 μM respectively. Effects of 2-PMPA or LY341495 on basal Ferrostatin-1 (Fer-1) or cocaine-enhanced extracellular DA and glutamate in the NAc To determine the neurochemical mechanisms underlying the antagonism of 2-PMPA on cocaine-induced reinstatement of drug seeking we further observed the effects of 2-PMPA (0 30 100 mg/kg i.p.) and/or LY341495 (1 mg/kg i.p.) on basal extracellular DA and glutamate and then observed the effects of 2-PMPA pretreatment on cocaine-enhanced NAc DA and glutamate in rats during reinstatement test. After microdialysis experiments were completed rats were anesthetized with a high dose of pentobarbital (>100mg/kg i.p.) and perfused transcardially with 0.9% saline followed by 10% formalin. Brains were removed and placed in 10% formalin for histological verification of microdialysis probe locations in rat brain. Drugs Cocaine HCl was provided by the National Institute on Drug Abuse (NIDA Baltimore MD) and dissolved in physiological saline. 2-PMPA (2-(phosphonomethyl)pentanedioic acid) was provided by Guilford Pharmaceuticals Inc. (Baltimore MD USA). LY341495 was purchased from Tocris Bioscience (Ellisville MO USA). 2-PMPA was dissolved in 0.5 M HEPES buffer (vehicle) purchased from MP Biomedicals Inc (Solon Ohio USA) for sustemic (i.p.) administration or artificial cerebrospinal fluid (aCSF) for intracranial microinjections or microdialysis. The pretreatment time (30 min prior to cocaine) of 2-PMPA was chosen on the basis of our preliminary pilot studies and an microdialysis finding that a significant reduction in extracellular DA and glutamate occurs at 20 min after 2-PMPA administration. Data analyses All data are presented as means (± S.E.M.). One-way analysis of variance (ANOVA) was used to analyze the effects of 2-PMPA or NAAG on cocaine self-administration or cocaine-induced reinstatement of drug-seeking behavior. Two-way ANOVA with repeated measures were used to.