The films were scanned and bands intensities were analyzed using Image J software (developed at the US National Institutes of Health and available on the web site (http://rsb.info.nih.gov/nih-image/).

In order to determine the adequate amount of protein to be assayed, different protein concentrations were carried out in the same gel for each antibody tested. Perfusion and fixation of the brain from 4 animals/group were performed 24 h after the end of seizures period through transcardiac perfusion with 4% paraformaldehyde and 0.25% glutaraldehyde, followed by cryoprotection ON-01910 cost in 30% sucrose solution overnight. Brain was sectioned (50 μm coronal sections) using a Leica VT1000S microtome (Leica Microsystems, São Paulo, Brazil). Coronal sections were separated in 4 series throughout the dorsal hippocampus with 300 μm interval between

each section and collected in PBS. Free-floating sections of rat brain were processed for immunohistochemistry against the neuronal specific protein neuronal nuclei (NeuN) and glial fibrillary acidic protein (GFAP), using a primary mouse anti-NeuN (1: 500, Chemicon International, São Paulo/SP, Brazil) as well as rabbit anti-GFAP antibodies (1:500, Dako, Denmark A/S). Antibodies were diluted in Tris buffer saline (TBS, 0.5 M NaCl and 30 mM Tris, Romidepsin cost pH 7.4) containing 0.2% Triton X-100 and 10% normal goat serum and incubated for 48 h at 4 °C. After incubation, sections were rinsed 4 times for 10 min in TBS and subsequently incubated with secondary fluorescent antibodies overnight: Alexa fluor anti-rabbit 488 and anti-mouse 594 (1:500, Invitrogen, Porto Alegre/RS), in 0.1 M TBS containing 0.2% Triton

X-100 and 10% normal goat serum for 24 h at 4 °C. After rinsing 4 times for 10 min in TBS, the sections were mounted on slides coated with 2% gelatin with chromium and potassium sulfate. The slices were mounted in a Vectashield mounting medium containing the nuclear marker DAPI (4′-6-diamidino-2-phenylindole dilactate) (Vector Laboratories, São Astemizole Paulo/SP, Brazil). The CA1, CA3 and dentate gyrus (DG) subfields of each hippocampus were examined in the Olympus FluorView 1000 system and the fluorescence was quantified using ImageJ software. The images were captured and a square region of interest (ROI) was created considering the pyramidal layer size. The ROI square of 8019 μm2 was overlaid on the analyzed subfields with blood vessels and other artifacts being avoided, using a magnification of 20x. Six ROI were analyzed per subfield. Rats (60-day-old) were exposed to the elevated plus-maze apparatus that consisted of a central platform (10 cm × 10 cm) with 2 open and 2 closed arms (45 cm × 10 cm), arranged in such a way that the 2 arms of each type were opposite to each other.