To visualize Fos and TH, residual aldehydes were removed with 0.1% sodium borohydride after the first series Selumetinib of Trizma-buffered saline rinses, and endogenous peroxidase activity was quenched with 1% hydrogen peroxide. Tissue was blocked and made permeable with 20% goat serum and 0.3% Triton-X Trizma-buffered saline, followed by incubation in the Fos primary antibody for 48 h at 4°C. Tissue was then incubated consecutively in the Fos secondary antibody and avidin-biotin complex for 1 h each. Lastly, sections

were reacted for approximately 2 min with 10 mg 3,3′-diaminobenzideine tetrahydrochloride in 50 mL Trizma-buffered saline and 45 μL of 30% hydrogen peroxide to produce a dark brown reaction product in the nucleus of Fos-immunoreactive (ir) cells. After rinsing, tissue phosphatase inhibitor library was again blocked and made permeable and then incubated overnight in TH primary antibody. TH secondary antibody and avidin–biotin complex were then each applied consecutively for 1 h. Finally, sections were reacted for approximately 2 min with one drop of Vector SG enzyme substrate in 7 mL Trizma-buffered saline and 50 μL 30% hydrogen peroxide to produce a cytoplasmic

blue reaction product in TH-ir cells. To visualize Fos and orexin, a similar immunohistochemistry protocol was used, but with the appropriate reagents (see Table 1). Primary and secondary antibody deletion control studies were run on separate sections. Non-specific background staining was low or absent in these sections. Tissue sections were mounted onto glass slides and dehydrated with

a series of ethanols before coverslipping. Regions of interest included the nucleus accumbens (Acb), medial prefrontal cortex (mPFC) and ventral tegmental area (VTA) because they are primary components of the mesocorticolimbic dopamine circuitry (Fibiger & Phillips, 1988); the lateral hypothalamus (LH) because of its orexinergic cell population (Aston-Jones et al., 2009); the ventromedial hypothalamus (VMH) because of its role in gating reproductive and defensive behaviors (Choi et al., 2005); and the posterior medial amygdala (MeP) as a positive control region known to express Fos in response to VS in both juvenile and adult male hamsters (Romeo et al., 1998). Regions were subdivided according to the hamster brain atlas (Morin & Wood, 2001), ID-8 as indicated by previous research demonstrating distinct functional and anatomical characteristics of the subregions (Groenewegen et al., 1999; Bradley & Meisel, 2001; Heidbreder & Groenewegen, 2003; Balfour et al., 2006; Ikemoto, 2007). The mPFC included the anterior cingulate (Cg1), prelimbic (PrL) and infralimbic (IL) subregions; the Acb included the core (AcbC) and medial portion of the shell (AcbSh); the MeP included the dorsal (MePD) and ventral (MePV) subregions; the VMH included medial (VMHM) and lateral (VMHL) portions; and the VTA included interfasicular (IF), paranigral (PN), parabrachial pigmented (PBP) and Tail nuclei (Fig. 1).