5 illustrates alcohol and water self-administration behavior before and after dependence induction via chronic intermittent alcohol vapor exposure

5 illustrates alcohol and water self-administration behavior before and after dependence induction via chronic intermittent alcohol vapor exposure. treatment of alcohol dependence and stress-related disorders. and for 20 min at 4C. The pellet was resuspended and spun at 45,000 for 20 min at 4C. The final pellet was resuspended in assay buffer (homogenizing buffer supplemented with protease inhibitor; 1 tablet/10 ml; Sigma CAT#S8829-20TAB, St. Louis, MO, pH 7.4) using a Polytron. The reaction was initiated by adding 0.05 ml of [125I]Tyr0-sauvagine to 1 1.5 ml polypropylene tubes containing 0.1 ml of membrane preparation (~2 mg protein/ml) and 0.05 ml of a CRF1 antagonist at logarithmic interval concentrations from 10?6 to 10?11 M. MPZP binding affinity was compared to that of DMP904, a structurally related reference compound that exhibits high, selective affinity for CRF1 receptors (Gilligan et al, 2000) and which dose-dependently occupies brain CRF1 receptors, reduces anxiety-like behavior, and prevents stress-induced increases in circulating corticosterone levels following oral dosing (Lelas et al., 2004). Total binding was determined using assay buffer in lieu of a CRF1 antagonist, and nonspecific binding was determined in the EBI-1051 presence of 1 M of the unlabeled homologous ligand (Hoare et al., 2003, 2004, 2005; Gross et al., 2005). The final radioligand concentration was 0.2 nM, and the reaction was incubated at room temperature for 2 h. The reaction tubes were centrifuged at 12,000 rpm for 5 min to terminate the reaction. The supernatant was removed and the pellets washed twice with ice-cold washing buffer (DPBS with 0.01% Triton-X100). Tubes then were centrifuged at 12,000 rpm, and the supernatant was removed. The pellet-containing tip was cut off and counted in an automated 10-detector gamma counter (MicroMedic Apex, ICN Biomedical, Costa Mesa, CA) at 80% efficiency. Six independent radioligand displacement assays, each involving a freshly prepared membrane preparation from a unique brain and freshly prepared solutions, were performed on different days using duplicate replicates for each data point. In each assay, the total radioligand bound was less than 10% of the total amount of radioligand added to the tube. Specificity of MPZP for other receptor, transporter, ion channel, or enzyme targets was determined in duplicate at a 1 M concentration via the NovaScreen commercial screening service (GEN SEP I panel, Hanover, MD). For CRF receptor autoradiography, brain tissue was sectioned coronally (20 m) using a cryostat (?17C). Sections were mounted EBI-1051 on Superfrost Plus + charged glass slides (Fisher Scientific, Pittsburgh, PA), allowed to dry completely, and stored in airtight boxes at ?80C until the day of autoradiography. Autoradiography was performed using standard procedures based on the previous characterization of [125I]Tyr0-sauvagine (Grigoriadis et al., 1996). Slides containing triplicate adjacent brain sections were thawed to room temperature and allowed to dry completely. Each section then was outlined using a PAP pen (Calbiochem, San Diego, CA). Sections were incubated in assay buffer (DPBS with 10 mM MgCl2, 2 mM EGTA, 1 tablet/100 ml protease inhibitor, 0.15% bovine serum albumin) for 15 min to remove endogenous ligand. Slides then were incubated under one of four conditions: (1) 0.2 nM [125I]Tyr0-sauvagine to determine total binding; (2) 0.2 nM radiolabeled sauvagine + 1 M R121919 (3-[6-(dimethylamino)-4-methyl-pyrid-3-yl]-2,5-dimethyl-N,N-dipropyl-pyrazolo[2,3-a]pyrimidin-7-amine, also referred to as NBI-30775) to Mouse monoclonal to CD80 determine non-CRF1 (e.g., CRF2) receptor binding; (3) 0.2 nM radiolabeled sauvagine + 3 M MPZP to determine non-CRF1 (e.g., CRF2) receptor binding using the experimental compound under study; (4) 0.2 nM radiolabeled sauvagine + 0.3 M unlabeled D-Phe-CRF12C41, a subtype-nonspecific CRF receptor antagonist, to determine non-CRF1/CRF2 (e.g., nonspecific binding). After 2 EBI-1051 h incubation at room temperature, unbound radioligand was removed via a brief dip in ice-cold assay buffer,.