2 μL) was unilaterally microinjected into the dH. The upper incisor bar was set at 3.3 mm below the interaural line, such that the skull was horizontal between bregma and lambda. The microneedle (Injex, Ourinhos, São Paulo, Brazil) was vertically introduced into the hippocampus according to the coordinates from Paxinos and Watson’s atlas (1997), as mentioned above. After the injection

was completed, a protective layer of sterile gelatin foam was laid in the bone cavity above the pons; the bone was then closed with cyanoacrylate glue thickened with dental cement powder. Seven days after microinjection Inhibitors,research,lifescience,medical of the neurotracer, the Inhibitors,research,lifescience,medical animal was deeply anesthetized with sodium pentobarbital (45 mg/kg, IP) and transcardially perfused at a rate of 3.5 mL/min with a 20-mL saline followed by 100–200 mL of 2% paraformaldehyde and 2% glutaraldehyde in 0.1 mol/L phosphate buffer, pH 7.4. After fixation, the brains were removed and the mesencephalon was separated from diencephalon, and the pons and medulla oblongata were separated from the spinal cord. The mesencephalon and the prosencephalon were immersed in 10% and 20% Inhibitors,research,lifescience,medical sucrose dissolved in 0.1 mol/L phosphate buffer, pH 7.4, at 8°C, for at least 12 h in each solution. Tissue fragments were immerse in 2-methylbutane (Sigma, London, UK), Inhibitors,research,lifescience,medical frozen on dry ice

(30 sec), embedded in Tissue Tek O.C.T. compound (Sakura Finetek Europe B.V., the Netherlands), and cut with a cryostat (Leica CM1950, Wetzlar, Germany). All sections were immediately mounted on gelatin-coated slides and stained with hematoxylin–eosin in a robotized autostainer (CV 5030 Leica Autostainer XL, Wetzlar, Germany) to locate the positions of the microinjections

sites in a bright-field photomicroscope (AxioImager ZI, Zeiss). The location of the injection site of the Texas Red-labeled biodextran and fluorescent bidirectionally labeled cells were visualized using fluorescence microscopy (AxioImager ZI Inhibitors,research,lifescience,medical with APOTOME, Etomidate Carl-Zeiss-Straße, Oberkochen, Germany). Behavioral tests and seizure intensity evaluation Behavioral tests were performed by GSK J4 supplier placing the rats in the interior of a circular arena, in which the transparent acrylic walls measured 60 cm in diameter and 50 cm in height. The arena was located in an experimental compartment illuminated by a fluorescent lamp (350 lux at the arena floor level). The effects of drug administration (PTZ, atropine, mecamylamine, and physiological saline) were evaluated with the rats inside the arena. The motor parameters used to evaluate the effect of the Cl− influx and GABA-mediated blockade were the tonic–clonic convulsive reactions induced by IP administration of PTZ at 64 mg/kg.