9% (m/v) saline (100 mL) followed by 4% (m/v) formaldehyde at pH 9.5 and 4 °C (800–1000 mL). The brains were removed from the skull, post-fixed for 4 h in the same fixative with the addition

of 20% sucrose and then transferred to 0.02 M potassium phosphate-buffered saline (KPBS) at Luminespib in vitro pH 7.4 with 20% (m/v) sucrose. The brains were sliced in four series of coronal sections (at bregma 2.70 mm, −0.30 mm, −1.80 mm, and −3.14 mm) at a thickness of 30 μm with the use of a freezing microtome and stored at −20 °C in buffered antifreeze solution (Sita et al., 2003). One series of each brain slice was stained by immunohistochemistry as follows: sections were treated in 0.3% (v/v) peroxide in KPBS + 0.3% (v/v)

Triton X-100 for 30 min and incubated in primary antiserum anti-c-Fos (PC38T IgG anti-c-Fos (Ab5) (4-17)) rabbit polyclonal antibody (Calbiochem, La Jolla, CA, USA) at 1:5000 and Roxadustat 3% (v/v) normal goat serum in KPBS + 0.3% (v/v) Triton X-100 for 18 h at room temperature. Sections were rinsed in KPBS and incubated for 1 h in biotinylated secondary antiserum made from goat anti-rabbit antibody (Jackson Labs 1:1000) for one additional hour in avidin–biotin complex (Vector, 1:500). Next, the sections were incubated in diaminobenzidine tetrahydrochloride (DAB; Sigma Chem Co.) and 0.01% (v/v) hydrogen peroxide dissolved in KPBS. The reaction was terminated after 2–3 min with repeated rinses in KPBS. Sections were mounted on slides and intensified with 0.005% (m/v) osmium tetroxide solution. To aid in the identification of brain regions presenting little or no c-Fos-immunoreactive neurons (mainly in the sections of control brain slices), Nissl method of counterstaining with thionin was used (Windle et al., 1943). Photomicrographs were acquired through a Spot RT digital camera (Diagnostics Instruments) adapted to a Leica DMR microscope

and an Apple Macintosh Power PC computer Fossariinae using the software Adobe Photoshop 5.0. Contrast, sharpness, colour balance and brightness were adjusted and images were combined in plates using Corel Draw 11 software. For the intravenous administration of nigriventrine, the rats were anaesthetised with chloral hydrate (7%, 350 mg/kg, ip) and submitted for venous catheterisation. A Silastic catheter containing heparinised saline (10 U/mL of pyrogen-free saline, Sigma, St. Louis, MO) was inserted into the femoral vein and sutured in place. The free end of the catheter was passed under the skin of the back, exteriorised between the scapulae, and plugged with a sterile wire stylet. A week later, nigriventrine (100 ng kg−1) was intravenously applied. For the quantitative analysis of c-Fos-ir and/or NMR1-ir cells, three representative slices of each brain region were chosen for each rat.