After the training and the test sessions, the rats were dried and placed back in their home cages. The EPM test Alectinib chemical structure was used to assess anxiety-like and exploratory behaviors, and consisted of two opposite open arms and two opposite closed arms (45 × 15 cm) connected by a central area (15 × 15 cm), elevated 70 cm above the floor. The test was
performed under dim light conditions. The rat was placed in the central square, and its behavior was observed for 5 min. During that time, the number of entries into and the time spent in the open and closed arms were measured. After each rat had been tested, the EPM was cleaned with a 10% ethanol solution. This test was performed as previously described (Ennaceur et al., 2005), and consisted of two phases. On the first day, two identical objects were placed in the back corners of an open Smad inhibitor box made of PVC (width, 80 cm; length, 80 cm; height, 50 cm), 10 cm away from the sidewall, and the rats
were placed facing away from the objects. The rat was allowed to explore the box for 3 min, and placed back in its home cage. After a 15-min delay, it was replaced in the box and allowed to explore it for another 3 min. This process was repeated five times (3 min each), with a 15-min interval between exposures. The second phase, performed 24 h later, consisted of placing the rat in the box for 3 min, and, after a 15-min interval, placing one of the objects in a different location (diagonally), and analysing the frequency and total duration of approaches to each object. A discrimination index was also used to evaluate possible memory deficits, calculated with the following equation – [(TNP − TOP)/(TNP + TOP) × 100], where TNP is the time spent in the new position, and TOP is the time spent in the old position. The rats were decapitated, their brains were removed, and the hippocampi were dissected on a cold surface. The tissue samples were weighed individually, and homogenised by sonication in 500 mL of extraction solution Etofibrate (0.1 m perchloric acid containing
0.4 mm sodium metabisulfite and 0.2 mm EDTA) (Machado et al., 2008). The mobile phase was filtered through a 0.2-mm filter membrane, degassed under vacuum, and delivered at a flow rate of 1.2 mL/min (HITACHI Pump System L-7100; LaChrom Elite, USA). Each sample was analysed in duplicate for the concentrations of 5-HT and the metabolite 5-HIAA. Recovery of the analytes was determined by adding a fixed concentration of the internal standard dihydroxybenzylamine before tissue homogenisation. An automatic injector (HITACHI L-7250, cut injection method) was utilised to improve the reproducibility of injections. All standards and salts were purchased from Sigma (USA), and the solvents (high-performance liquid chromatography grade) were purchased from J. T. Baker (USA).