IKK protein levels were analyzed using immunohistochemistry. to selectively knock down IKK in mice genetically designed with a conditional deletion (gene on a C57BL/6J background (i.e., C57BL/6J mice with flanked by LoxP sites, denoted as access to water and rodent chow (Prolab RMH 180 5LL2 chow, TestDiet) in heat- and humidity-controlled rooms. Behavioral testing began when the mice were at least 2 months old. Mice were individually housed at least 2 weeks before beginning Rabbit polyclonal to PDK4 the drinking assessments. Experiments were Cyclamic Acid conducted in isolated behavioral testing rooms in the Animal Resources Center at The University of Texas at Austin. All experiments were approved by The University of Texas Institutional Animal Care and Use Committee and were conducted in accordance with National Institutes of Health guidelines Cyclamic Acid with regard to the use of animals in research. Pharmacological inhibitors Cyclamic Acid of IKK Cyclamic Acid Sulfasalazine (Sigma-Aldrich) was injected intraperitoneally, and TPCA-1 (Tocris Bioscience) was administered by mouth. Both drugs were freshly prepared as suspensions in saline answer, with four to five drops of Tween-80, and were injected in a volume of 0.1 ml/10 g of body weight for intraperitoneal administration, and 0.05 ml/10 g of body weight for oral administration. Drugs were administered Cyclamic Acid 30 min prior to ethanol presentation occasions (see below). Doses of drugs and routes of administration were based on published data that showed anti-inflammatory activity mice were injected bilaterally (into the NAc or CeA) with either a vesicular stomatitis computer virus glycoprotein (VSV-G) pseudotyped lentivirus (LV) expressing recombinase fused to enhanced green fluorescent protein (EGFP) under the control of a cytomegalovirus (CMV) promoter (LV-Cre-EGFP) or an empty VSV-G pseudotyped lentiviral vector expressing only the EGFP transgene under a CMV promoter. Mice were anesthetized by isoflurane inhalation, were placed in a stereotaxic apparatus (model 1900, David Kopf Devices), and were administered a preoperative analgesic (Rimadyl 5 mg/kg). The skull was uncovered, and bregma and lambda were visualized with a dissecting microscope. A digitizer attached to the micromanipulator of the stereotaxic apparatus was used to locate coordinates relative to bregma. Burr holes were drilled bilaterally above the injection sites in the skull using a drill equipped with a #75 carbide bit (David Kopf Devices). The injection sites targeted either the NAc [using the following coordinates relative to bregma: anteroposterior (AP) +1.49 mm, mediolateral (ML) 0.9 mm, dorsoventral (DV) ?4.8 mm] or the CeA (using the following coordinates: AP ?1.14 mm, ML 2.84 mm, DV ?4.8 mm). Injections were performed using a 10 l microsyringe (model #1701, Hamilton) and a 30 gauge needle. The needle of the syringe was lowered to the DV coordinate and retracted 0.2 mm. Computer virus solutions (1.0 l with a titer of 1 1.8 108 viral particles/ml in PBS) were injected into each site at a rate of 200 nl/min. After each injection, the syringe was left in place for 5 min before being retracted over a period of 3 min. Incisions were closed with tissue adhesive (Vetbond, 3M). Mice were individually housed after surgery and given a 4 week recovery period before starting the ethanol drinking tests. Behavioral testing The following three different ethanol-drinking models were used in this study: (1) continuous 24 h 2BC with access to water and ethanol (15%, v/v); (2) 2BC drinking-in-the-dark (DID) with limited 3 h access to 15% ethanol (2BC-DID); and (3) 2BC using ascending concentrations of ethanol solutions (3C16%; see below). Pharmacological inhibitors of IKK The effects IKK antagonists on ethanol intake were measured in adult male C57BL/6J mice in two different drinking paradigms: 2BC with 15% ethanol and 2BC-DID per the protocols previously described (Blednov et al., 2003, 2014). For both assessments, mice were pretrained to consume 15% ethanol for at least 3 weeks to provide stable consumption. Ethanol intake was measured after saline injection (intraperitoneally or by mouth, corresponding to the route of administration for the antagonists) for 2 d, and mice were grouped to provide comparable levels of ethanol intake and preference. In.