Brains were stored in 2 % paraformaldehyde in 10 mM phosphate-buffered saline (PBS) overnight and then cryoprotected in a 30 % sucrose/10 mM PBS solution until sectioned. The brains were sliced on a cryostat and floating sections, 30 microns thick, were collected and processed for c-Fos immunohistochemistry using standard protocols (Bachtell et al., 1999; Ryabinin et al., 2001). Briefly, the sections underwent a 0.3% hydrogen peroxide incubation to quench endogenous peroxidase activity and then blocking was carried out using goat serum (Vector Laboratories, Burlingame, CA). The c-Fos antibody used was obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and used at a 1:2000 dilution. A standard ABC kit (Vector Laboratories, Burlingame, CA) was used for the immunoreaction and DAB (Thermo Scientific, Rockford, IL) was used for visualization. Cells immunopositive for c-Fos were counted manually at 20× objective magnification by an experimenter blind to the group of animals, in the perioculomotor urocortin-containing neuronal population (pIIIu), the arcuate nucleus (Arc) and the ventral tegmental area (VTA). The same sections contained the VTA and the pIIIu and encompassed bregma levels −2.80 to −3.88.
