d), For CPP for 0.5mg/kg of nicotine, 12.0mg/kg PHA blocked preference; this was reversed by pretreatment with 10.0mg/kg of methyllycacontinine (MLA). nucleus accumbens tissue, followed by confirmation with quantitative PCR and immunoblotting. In the BXD panel, we found a putative eQTL for in nucleus accumbens that correlated inversely to nicotine CPP. We observed that gain-of-function 7 mice did not display nicotine preference at any dose tested, while conversely, 7 KO mice showed nicotine place preference at a dose below that routinely required to produce preference. In B6 mice, the 7 nAChR-selective agonist, PHA-543613, dose-dependently blocked nicotine CPP, which was restored using the 7 nAChR-selective antagonist, MLA. Our genomic studies implicated an mRNA co-expression network regulated by in nucleus accumbens. Mice lacking demonstrate increased insulin signaling in the nucleus accumbens, which may modulate nicotine place preference. Our studies provide novel targets for future work on development of more effective therapeutic approaches to counteract the rewarding properties of nicotine for smoking cessation. mRNA expression and its potential regulation of insulin signaling as modulators of nicotine conditioned place preference. These studies may have important implications for understanding and treating nicotine dependence in humans. Methods and Materials Mice For all those studies, male mice were housed 3-5 per cage and allowed at least a BMS 626529 one-week acclimation period to the vivarium following shipment to Virginia Commonwealth University or college (VCU). Mice were managed on a 12-hour light/dark cycle with access to food and water. Adult mice were tested or experienced tissues harvested between 7-12 weeks of age during their light phase. C57BL/6J (B6, Stock No. 000664), DBA/2J (D2, Stock No. 000671), and BXD (B6 D2) recombinant inbred mice were obtained from Jackson Laboratories (Bar Harbor, ME). knock-in, gain-of-function (7 KI) mice (L250T +/?), bred from heterozygous breeding pairs, were obtained from Baylor College of Medicine (Houston, TX) (Broide et al. 2002). homozygous knock-out (7 KO) mice (B6.129S7-Chrna7tm1Bay/J, Stock BMS 626529 No. 003232) were either obtained from Jackson Laboratories or heterozygote breeding pairs were obtained from which WT and KO mice were bred and genotyped at VCU. Both 7 KI and 7 KO mice were backcrossed to the background strain, C57BL/6J, for an additional 8-10 generations and wild-type littermates (7 WT) were used as controls. The animal facility was approved by the Association for Assessment and Accreditation of Laboratory Animal Care. Experiments were performed during the light cycle and approved by the Institutional Animal Care and Use Committee of VCU. Drugs and Chemicals (?)-Nicotine hydrogen tartrate salt and Rabbit Polyclonal to WEE2 methyllycaconitine citrate (MLA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). PHA-543613 [N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]furo[2,3-c]pyridine-5-carboxamide] and cocaine hydrochloride were obtained from the Drug Supply Program of the National Institute on Drug Abuse (Rockville, MD). All drugs were dissolved in a vehicle of physiological saline (0.9% sodium chloride), filter sterilized, and administered at a volume of 0.1mL per 10g of mouse mass. Nicotine, PHA-543613, and MLA were administered subcutaneously (s.c.), BMS 626529 while cocaine was given intraperitoneally (i.p.). All doses are expressed as the free base of the drug. Place Conditioning Experiments For all those place conditioning experiments with BXD strains, 7 KO, KI, and WT mice, a five-day paradigm was performed as explained previously (Kota et al. 2007). Each animal received cage enrichment, and on Wednesday, Thursday, and Friday of the week prior to place conditioning screening, the experimenter dealt with each mouse for approximately two moments. The experimental apparatus (Med-Associates, St. Albans, VT, ENV3013) consisted of white and black chambers (20 20 20 cm each), which differed in floor texture (white mesh and black rod). The chambers were separated by a smaller grey chamber with a easy PVC floor and partitions that allowed access to the black and white chambers. Briefly, on Day 1 (pre-conditioning day), mice were placed in the center chamber for 5 minutes, partitions were lifted, and mice were allowed to roam freely for 15 minutes. The times spent in the white and black chambers were used to establish baseline chamber preferences, if any. Mice were separated into vehicle and drug groups such that initial chamber biases in each group BMS 626529 were approximately balanced. On days 2-4 (conditioning days), twice per.