In Experiment 2, we observed that the c-Fos immunoreactivity in the pIIIu of mice consuming alcohol was dramatically reduced following administration of systemic D-Lys3-GHRP-6, and that there was no effect in the VTA or Arc. This result suggested a specific effect of this antagonist in the pIIIu, which is in agreement with the ability of this nucleus to regulate alcohol intake (reviewed in Ryabinin and Weitemier, 2006) and the high levels of GHSR 1a receptor mRNA in this brain area. However, since activation of c-Fos in pIIIu is dose-dependently sensitive to the amount of administered or self-administered ethanol (Bachtell et al., 2002a; Sharpe et al., 2005), we needed to eliminate the potential confound that lower numbers of c-Fos positive cells after D-Lys3-GHRP-6 could be secondary to lower intakes of alcohol. Theoretically, the antagonist could have acted elsewhere in the brain leading lower intakes of alcohol, and hence less c-Fos immunoreactivity in pIIIu. To eliminate this possibility, we investigated the effects of D-Lys3-GHRP-6 on c-Fos immunoreactivity induced following systemic injections of 2.5 g/kg ethanol in Experiment 3. Systemic administration results in
