The Institutional Animal Care and Use Committee at the University of Alabama at Birmingham approved the experimental protocol using mice. Adult male C57BL/6 mice (8-10 weeks of age, Frederick Cancer Research, MD) were used for pharmacological treatments. After a one-week accommodation in the university animal facility, mice were treated with d-fenfluramine or imipramine dissolved in saline and injected intraperitoneally (i.p.). For intra-cerebroventricular (i.c.v.) injection, mice were anesthetized with ketamine and xylazine (100 mg/kg:10 mg/kg) to placed a guide cannula (2.2 mm) stereotaxically (posterior 0.8 mm and left 1.6 mm to Bregma). Five days later, the PI3K inhibitor LY294002 (5 nM) or vehicle (10% DMSO) was infused into the left ventricle via an internal cannula. Animals were given d-fenfluramine (i.p.) 90 min after LY294002. At the end of treatment, mice were rapidly decapitated and brain regions (cerebral cortex, hippocampus, and striatum) were immediately dissected in ice-cold saline and used for immunoassays.
