Animals and treatments
Animal maintenance and experimentation were conducted in strict compliance with the European and National Law for Laboratory Animal use (Directive 2010/63/EU and Greek Law 161/91), the FELASA recommendations and the ethical and practical guidelines for the care and use of laboratory animals set by the competent veterinary services of Athens. All experiments were carried out in wild-type C57BL/6J mice. Three-month-old male mice were divided into two groups (n=4/ group) and injected intraperitoneally once per day with saline or 5 mg/kg U50,488H for 6 consecutive days. The mice were sacrificed 3 h after the last U50,488H/saline injection and the hippocampus, cortex, and striatum were rapidly dissected on ice. Isolated regions were placed in cold PBS and immediately homogenized by sonication in RIPA buffer containing 50 mM Tris-HCl pH 7.2, 150 mM NaCl, 2 mM EDTA, 1% Triton 100-X, 1 % sodium deoxycholate, 0.5 % SDS and 1 mM dithiothreitol in the presence of protease inhibitors and incubated for 1 h at 4 οC. The resulting supernatant was collected after centrifugation at 8,000 x g for 20 min.