The selective reduction of c-Fos–immunoreactivity after D-Lys3-GHRP-6 observed in Experiment 2 could be due to direct actions of this drug on GHSR 1a in the pIIIu, or may be due to actions of this drug elsewhere leading to a reduction of ethanol intake and the resulting BECs, which in turn would result in lower activation in the pIIIu. To distinguish between these two possibilities, we tested whether D-Lys3-GHRP-6 could suppress ethanol injection-induced c-Fos in pIIIu. Saline or D-Lys3-GHRP-6 were injected followed 30 min later by either 2.5 g/kg ethanol or saline in male C57BL/6J mice. As published previously (Bachtell et al., 2002a; Hill et al., 2007; Spangler et al., 2009; Turek and Ryabinin, 2005), i.p. ethanol dramatically induced c-Fos in the pIIIu [F(3,15) = 18.86, p < 0.0001, ANOVA]. The number of c-Fos immunopositive cells seen after systemic ethanol in the pIIIu was dramatically reduced by D-Lys3-GHRP-6 as demonstrated by significant interaction between the effects of two injections [F(1,15) = 13.59, p = 0.002; Fisher’s PLSD post-hoc p < 0.0001 for the saline pre-treated ethanol-injected animals versus all other groups]
