Neurochemical heterogeneity of rats predicted by different measures to be high ethanol consumers.

Alcohol use disorders may appear similar phenotypically; however, they have considerable heterogeneity (Heilig et al., 2011). This may be due to genetic heterogeneity, with alcohol use disorders having a large degree of genetic susceptibility, suggesting that individuals require different interventions. This is supported by findings with the alcoholism medication and opioid antagonist, naltrexone, which is effective only in a subgroup of patients (Heilig et al., 2011), such as those with a specific allele of the mu-opioid receptor (Heilig et al., 2011; Oslin et al., 2003). While pharmacogenetic tests will be beneficial in implementing personalized medication, behavioral and physiological phenotypes that correspond to these genetic variations may also help to identify subjects who are not only more likely to be responsive to a drug treatment but also at greater risk for developing alcohol use disorders. A number of measures can identify subjects at risk for drinking excess alcohol. In prospective clinical studies, the development of alcohol use disorders can be predicted by personality factors such as anxiety or novelty-seeking, and also metabolic factors such as low level of response to alcohol that reflects poor ethanol oxidation (Schmidt et al., 2007; Schuckit et al., 2011). Similarly, in outbred rats, novelty-seeking assessed by high locomotor activity in a novel environment and also circulating triglyceride (TG) levels after a high-fat meal can identify at-risk animals (Karatayev et al., 2010a; Nadal et al., 2002). This latter measure, reflecting less efficient hydrolysis of meal-induced TGs by lipoprotein lipase, may be related to the poor ethanol oxidation of certain at-risk patients (Schuckit et al., 2011) as well as the inhibition of lipoprotein lipase by alcohol (Liu et al., 2011). While both measures detect at-risk rats, they show little relationship to each other and thus may identify phenotypically different subgroups (Karatayev et al., 2010a). These subgroups, due to biological heterogeneity, may also differ in their responsiveness to pharmacological treatment. Indeed, rats with high compared to low novelty-induced activity are more responsive to naltrexone’s ability to reduce both activity levels (Radcliffe and Erwin, 1998) and ingestion of palatable food (White et al., 2004). In determining biological mechanisms that drive subgroups to drink alcohol, it is preferable to study subjects in the absence of ethanol, as it alters neurochemicals (Vengeliene et al., 2008). Studies in ethanol-naive rats bred to consume ethanol suggest that at-risk subjects already show dysregulation of specific systems. Selectively-bred P and HAD compared to NP and LAD rats exhibit altered levels of monoamines (Strother et al., 2005), and AA compared to ANA rats show elevated expression of the opioid enkephalin (ENK) in corticolimbic regions (Marinelli et al., 2000). While these reports provide information on neurochemicals that may contribute generally to ethanol intake, they do not distinguish between subgroups that may drink for different reasons. Humans with the novelty-seeking personality trait also have altered levels of monoamines (Heck et al., 2009). In addition, a recent study in our laboratory comparing high-risk subjects showed that novelty-seeking rats have elevated expression of ENK in the hypothalamic paraventricular nucleus (PVN) and neuropeptide Y (NPY) in the arcuate nucleus (ARC), whereas those with high fat-induced TGs exhibit elevated galanin (GAL) and ENK in the PVN but not NPY in the ARC (Karatayev et al., 2010a). Whereas this provides some evidence that high-risk subgroups differ in their hypothalamic neurochemical profile, it is unclear whether the peptides, assessed immediately after the predictor tests, were differentially affected by the tests themselves. Neurochemicals may not only drive ethanol intake but may also contribute to the predictor measures themselves. The hypothalamic orexigenic peptides that may serve these functions include GAL and ENK in the PVN, orexin/hypocretin (OX) and melanin-concentrating hormone (MCH) in the perifornical lateral hypothalamus (PFLH), and NPY in the ARC. When injected, GAL, ENK, OX and MCH each promote ethanol intake (Barson et al., 2010; Morganstern et al., 2010b; Schneider et al., 2007), while NPY decreases drinking, at least in Myers' high ethanol-preferring rats (Karatayev et al., 2010b). Also, both GAL and ENK stimulate locomotor activity (Lyudyno et al., 2008; Vujic-Redzic et al., 2000), but only GAL decreases fat oxidation that determines TG levels (Gunion et al., 1991; Yun et al., 2005). With neurochemicals affecting both risk for drinking and the associated behavioral or metabolic profile, assessment of these latter measures could give insight into the neurochemical phenotypes of different high-risk subgroups. With subjects prone to drinking ethanol for different reasons, the present study was designed to determine whether at-risk animals show peptide disturbances prior to exposure and whether this varies as a function of the specific predictor measure. In outbred rats with variability in their responses, we used novelty-induced locomotor activity and fat-induced TG levels to identify high-risk subjects, examining these measures first in the same set of rats to determine their relationship to each other. We then examined different subgroups for their expression of hypothalamic peptides and responsiveness to naltrexone. For peptide measurements, we kept subjects ethanol-naive and gave them a one-week rest period following the predictor tests, to ensure that baseline expression was evaluated. We hypothesized that predicted high ethanol consumers would show disturbances in their peptide systems and responsiveness to medication, but that these disturbances would differ as a function of the predictor employed and thus subgroup identified.