All experimental protocols were consistent with guidelines issued by the National Institutes of Health and were approved by The Scripps Research Institute’s Institutional Animal Care and Use Committee. We prepared CeA slices as previously described (Roberto et al., 2003) from male Sprague-Dawley rats (120–200 g) that were anesthetized with halothane (3%) and decapitated. In brief, the brains were rapidly removed and placed into ice-cold artificial cerebrospinal fluid (ACSF) gassed with 95% O2 and 5% CO2. Transverse slices 400 μm thick were cut on a Vibratome Series 3000 (Technical Products International, St. Louis, MO), incubated in an interface configuration for about 30 min, and then completely submerged and superfused at a constant flow rate of 2–4 ml/min with warm (31° C), gassed ACSF of the following composition in mM: NaCl, 130; KCl, 3.5; NaH2PO4, 1.25; MgSO4·7H2O, 1.5; CaCl2, 2.0; NaHCO3, 24; glucose, 10. We added drugs to the ACSF from stock solutions to obtain known concentrations in the superfusate. The inner recording chamber had a total volume of 0.8 ml, so drug concentrations reach 90% of the nominal concentration within 2 min.
