, 2010). The open field arena consisted of a polypropylene box (37.6 cm × 30.4 cm × 17 cm) in which the floor was divided into 16 same-sized rectangles (7.6 cm × 9.4 cm), 12 peripheral and 4 central. The experiments were conducted under bright white light illumination during the dark part of the daily cycle, 1–2 h after Pexidartinib solubility dmso its onset. Each mouse was individually placed in the center of the arena. Behaviors in the open field were recorded for 10 min (divided into 1 min

intervals) with an overhead video camera. At the end of the session, the floor and walls were washed with odorless liquid soap, rinsed thoroughly with tap water and dried with a disposable paper towel. Recorded images of the tests were used to analyze behavior. The observer was blind regarding the experimental treatment of the animals. The ambulation was quantified on the basis of the number of rectangles crossed by the animals (Filgueiras et al., 2009). Mice

had to place all four legs on a given rectangle for a crossing to be counted. The following ambulation variables see more were evaluated: ambulation in the center (C), ambulation in the periphery (Pe), C/Pe ratio and total ambulation (C + Pe). In addition, considering that direct comparisons between the activity in the center and in periphery can be influenced by the fact that the number of rectangles in the periphery is greater than that in the center, the rectangles crossed in the center and in the periphery were respectively divided by 4 (C/4) and 12 (Pe/12). A separate group of mice was injected with ethanol or saline as described above. One or 2 h after the second injection (at P4), animals were decapitated and blood was collected (ethanol – 1 h: n = 13, 2 h: n = 9; saline – 1 h: n = 10, 2 h: n = 6). Blood was centrifuged at 2000 rpm for 5 min and the PDK4 supernatant stored at 4 °C until assayed. BEC was assessed using an enzymatic kit (Alcohol Reagent Set, Pointe Scientific Inc., Michigan, USA) in accordance with the manufacturer’s recommendations. After the test, 60 animals (at least 7 per group) were

sacrificed by cervical dislocation. Frontal cerebral cortices (approximately the rostral third of the cerebral wall) and hippocampi were immediately dissected and incubated for 1 h at 37 °C in minimum essential medium (MEM) buffered with 20 mM HEPES at pH 7.3 and containing 100 mM ascorbic acid, 100 mM pargyline and 0.5 mM Rolipram (Sigma Chemical Co., St. Louis, MO, USA). After incubation, the reaction was interrupted by the addition of TCA to 10% (final concentration). cAMP was purified by removing trichloroacetic acid and endogenous interfering compounds from supernatant solution, using an ion exchange column of AGSOW-X4 (200–400 mesh, hydrogen form, Bio-Rad, Rio de Janeiro, Brazil), previously washed and equilibrated with H2O (Matsuzawa and Nirenberg, 1975). Cyclic AMP concentrations of purified samples were determined by a protein binding assay described previously (de Mello et al., 1982 and Gilman, 1970).