Ghrelin receptor antagonism decreases alcohol consumption and activation of perioculomotor urocortin-containing neurons

Recent reports showing an overlap in the neural pathways responsible for obesity and those involved in excessive alcohol use (for review see Thiele et al., 2003) suggest that orexigenic peptides are promising targets for pharmacotherapy of alcoholism. The orexigenic peptide ghrelin, produced in the gastric fundic mucosa (Korbonits et al., 2004; Nakazato et al., 2001), causes the secretion of growth hormone, and acts to stimulate appetite and food intake (Locke et al., 1995; Wren et al., 2000). Ghrelin is also expressed in the hypothalamus, namely in the lateral hypothalamus, the arcuate nucleus (Arc), the ventromedial nucleus, dorsomedial nucleus and the paraventricular nucleus (Cowley et al., 2003; Hou et al., 2006; Kageyama et al., 2008; Kojima et al., 1999). The receptor at which ghrelin acts to stimulate the release of growth hormone is the growth hormone secretagogue receptor 1a (GHSR 1a) (Kojima et al., 1999; van der Lely et al., 2004). Zigman et al. (2006) showed that in the mouse, high levels of GHSR 1a mRNA occur in the perioculomotor urocortin-containing neurons (pIIIu, formerly known as the Edinger-Westphal nucleus; Horn et al., 2008; May et al., 2008). GHSR 1a mRNA is also abundant in the Arc (Tannenbaum et al., 1998; Willesen et al., 1999; Zigman et al. 2006) where the main targets of ghrelin are the neuropeptide Y/Agouti-related protein neurons and the leptin-responsive neurons (Traebert et al., 2002). Other regions of the brain where GHS 1a receptor mRNA is expressed, albeit in substantially lesser amounts than the Arc and pIIIu, include the ventral tegmental area (VTA), the laterodorsal tegmental area and the hippocampus (Guan et al., 1997; Zigman et al., 2006). Ghrelin has been studied extensively with regards to food intake and obesity (for review see Neary et al., 2003). Ghrelin is secreted in anticipation of a meal, and ghrelin levels decrease after food has been consumed (Cummings et al., 2001; Drazen et al., 2006). Central or systemic ghrelin increases food intake, and the effects of ghrelin on feeding and weight gain involve the Arc (Nakazato et al., 2001; Ruter et al., 2003; Wren et al., 2000). The peripheral administration of a GHSR antagonist, D-Lys3-GHRP-6, in contrast, reduced food intake as well as weight gain (Asakawa et al., 2003). Recent studies have also started to implicate ghrelin in alcohol drinking. Increased plasma levels of ghrelin have been reported in active drinkers, alcohol-dependent female patients, in alcohol withdrawal groups and in alcohol abstainers (Kim et al., 2005; Kraus et al., 2005; Wurst et al., 2007). A single nucleotide polymorphism in the GHSR 1a gene was found to be associated with heavy alcohol drinking in a Spanish population (Landgren et al., 2008). In mice, centrally administered ghrelin increased alcohol drinking while the GHSR 1a antagonists, BIM28163 and JMV2959, reduced alcohol consumption. The increased alcohol intake in response to centrally administered ghrelin was also found to be absent in GHSR 1a knock-out mice (Jerlhag et al., 2009b), showing that ghrelin acts at the GHSR 1a to affect alcohol-seeking behavior. However, conflicting findings showing reduced plasma and stomach ghrelin levels in alcohol-dependent patients have also been reported (Badaoui et al., 2008). In normal volunteers, plasma ghrelin levels were reduced after acute alcohol (Calissendorff et al., 2005; Zimmermann et al., 2007). Addolorato et al. (2006) showed decreased ghrelin levels in alcohol-dependent patients compared to healthy subjects, but the authors also found a positive correlation between ghrelin levels and craving for alcohol. Lyons et al. (2008) reported that systemic ghrelin does not affect alcohol consumption in a “drinking in the dark” (DID) procedure in food-deprived C57BL/6J mice. Thus, the relationship between ghrelin and alcohol consumption and alcoholism still needs to be clarified. It may be important to understand the neural substrates of ghrelin to clarify this relationship. Jerlhag et al. (2009b) showed that administering ghrelin, either intracerebroventricularly or into the VTA increased alcohol intake in mice whereas administering GHSR 1a antagonists, centrally or systemically, reduced alcohol intake. Thus, the authors concluded that ghrelin acting in the tegmental regions was responsible for the rewarding effects of alcohol. However, the VTA is located physically close to the pIIIu, and as observed in the Allen Brain Atlas (http://www.brain-map.org) and reported by Zigman et al. (2006), it contains substantially lower levels of GHSR1a mRNA than pIIIu. Several studies, using c-Fos expression mapping, electrolytic lesion of the pIIIu and rodents selectively bred for differences in alcohol intakes, have shown that the pIIIu is involved in the regulation of alcohol-related phenotypes, including high alcohol intake (Bachtell et al., 1999; Bachtell et al., 2002a; Bachtell et al., 2002b; Bachtell et al., 2003; Bachtell et al., 2004; Fonareva et al., 2009; Ryabinin et al., 2001; Ryabinin et al., 2003; Sharpe et al., 2005; Topple et al., 1998; Turek et al., 2005; Weitemier et al., 2001; Weitemier and Ryabinin, 2005). The Arc contains GHSR 1a receptors at levels comparable to the pIIIu (Zigman et al., 2006). Lesion and microinjection studies have implicated the Arc in ethanol-induced locomotor activation (Pastor and Aragon, 2008; Sanchis-Segura et al., 2000). It is, therefore, important to understand whether ghrelin acts via the VTA, pIIIu or Arc to regulate alcohol-related behaviors. In the current study, we investigated the neuroanatomical locus/loci through which ghrelin acts to regulate alcohol consumption. At first we confirmed that the ghrelin receptor antagonist that we used, D-Lys3-GHRP-6 acts to decrease alcohol preference in C57BL/6J mice in a similar fashion to the GHSR antagonists used by Jerlhag and colleagues (2009b). Following this confirmation, we investigated the effects of D-Lys3-GHRP-6 on c-Fos immunoreactivity induced in pIIIu, VTA and Arc by alcohol intake in the drinking in the dark procedure and by systemic administration of ethanol. We also determined if D-Lys3-GHRP-6 affects blood ethanol concentrations after systemic ethanol administration.