An immunohistochemical analysis was conducted to evaluate c-Fos and NGF expression in the MPA, vlPAG, PMC, and spinal cord L4–5 regions, following a previously described method [10 (link),12 (link)]. Free-floating tissue sections were incubated overnight with rabbit anti-c-Fos and mouse anti-NGF antibodies (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and the sections were incubated for 1 hour with biotinylated anti-rabbit c-Fos and anti-mouse NGF secondary antibodies (1:200; Vector Laboratories, Burlingame, CA, USA). Next, the sections were incubated with avidin-biotin-peroxidase complex (Vector Laboratories) for 1 hour at room temperature. For staining, the sections were incubated in 0.03% DAB and 0.03% hydrogen peroxide for 5 minutes. The sections were mounted onto gelatincoated slides. The slides were air-dried overnight at room temperature, and coverslips were mounted using Permount (Fisher Scientific, Waltham, MA, USA).