Genotype and Rearing Low level of response to alcohol is considered an important risk for alcohol abuse and alcoholism. Last year Dr. Christina Barr has performed analyses in the macaques, demonstrating an interaction between early rearing condition and serotonin transporter genotype, such that animals homozygous for the l allele exhibited lower levels of response to alcohol. When animals were segregated according to rearing condition, however, serotonin transporter gene variation predicted intoxication scores only among parentally-deprived peer-reared (PR) subjects, suggesting that the risk for excessive alcohol intake and possibly alcoholism may be regulated in part by the interacting influences of early rearing and 5-HTTLPR genotypes. Last year Dr Newman reported that low HVA was predictive of high voluntary alcohol consumption, finding that independent of rearing, subjects with high HVA levels consumed more alcohol than those with low CSF HVA. As a follow-up of that study, Dr Barr and Rachel Dvoskin have genotyped 270 animals for a single nucleotide polymorphism (SNP) of the dopamine transporter gene. They found that the PR, but not the mother-reared (MR) animals, possessing the SNP had increased CSF HVA and decreased sensitivity to the intoxicating effects of alcohol, suggesting a risk for high alcohol intake in PR subjects with this genotype. Both the polymorphisms were associated with decreased sensitivity to the intoxicating effects of alcohol, respectively. When thethe l/l rh5-HTTLPR genotype and Arg509/Gln DAT polymorphisms were considered simultaneously, their effects on intoxication scores were additive. Comparison of rh5-HTTLPR l/l-Arg509/Gln animals to those with the rh5-HTTLPR l/s ?w/t DAT genotype demonstrated even more marked differences in intoxication ratings. In parallel with the recent paper in the journal Science by Caspi et al, (in which there was a strong correlation with a low activity variant of the MAOA gene, but only in males with conduct disorder that had experienced childhood abuse) we found that rates of competitive aggression were higher in MR males with the low activity allele. Dr. Newman recently investigated the hypothesis that variation in the MAOA gene promoter region VNTR is associated with variation in alcohol consumption in adolescent macaque males. He found that one of the three MAOA alleles present in macaques conferring reduced transcriptional activity was strongly associated with decreased alcohol consumption, but only in MR males. Thus, as with the previously described study, there is a clear gene by environment interaction. In this case, the low activity allele may provide a mild buffering effect against the stress of early deleterious rearing. It is thought that chronic activation of the neuroendocrine stress axis contributes to allostatic load (or, wear and tear) at the level of the brain. Various factors, including genetic or sex differences, experience, and lifestyle choices are also thought to contribute to allostatic load via their influences on physiologic pathways. We have been interested in determining individual differences in vulnerability to stress as well as to dysregulated activity of the hypothalamic pituitary adrenal (HPA) -axis, as such differences may be markers for predisposition to stress-related disorders and may contribute to allostatic load in the brain. Dr. Barr found that macaques with genetic variation in the rh5-HTTLPR that have been exposed to early-life stress have exaggerated HPA-axis responses to stress, particularly among females. Females exposed to early-life stress also have augmented HPA-axis responses to alcohol. When given access to alcohol, these females, especially those carrying the rh5-HTTLPR s allele, show marked increases in their levels of consumption with successive exposures to alcohol. Working with a graduate student in our laboratory, Rachel Dvoskin, Dr. Barr has just completed characterization of the CRH gene and promoter, identifying multiple polymorphic regions. A number of these polymorphisms are located within putative transcription factor binding sites. Electrophoretic mobility shift assays show that many of these are functional polymorphsims. We are also in the process of characterizing a neuropeptide Y gene and have identified multiple single nucleotide polymorphisms. In addition, we are initiating projects that entail screening for polymorphisms in the glucocorticoid (GR) and corticotrophic hormone (CRHR1/CRHR2) receptors in macaques. Like the serotonin transporter gene, the expression of these genes are under the control of a glucocorticoid response element, and therefore may also interact with early-life stress to influence gene expression, physiologic markers, and behavior. Examination of Novel Neurotransmitters Given the potent role for Ghrelin in appetite regulation, investigation of its potential role in alcohol intake was initiated. In a collaboration with Drs. Barbara Hansen and Stephen Angeloni at the University of Maryland, Baltimore, School of Medicine, Obesity and Diabetes Research Center we characterized the ghrelin gene in the rhesus monkey and analyzed the association of plasma ghrelin levels with various metabolic and endocrine markers. The nucleotide sequence of rhesus ghrelin cDNA is 97% homologous to the human ghrelin cDNA resulting in five amino acid substitutions in the prepropeptide. Comparison of ghrelin expression in tissues of lean versus obese monkeys showed down regulation of ghrelin in muscle and duodenum of obese monkeys, while expression in the stomach did not change. Fasting plasma ghrelin concentrations were compared to markers such as age, body weight, BMI, leptin, glucose and insulin levels. Age and fasting plasma ghrelin exhibited a non-linear association, exemplified by a significant reverse relationship in younger ages and a positive trend in older monkeys. Fasting plasma ghrelin concentrations were inversely correlated with BMI, but not with leptin. Glucose administration did not significantly alter ghrelin levels. A significant inverse correlation between fasting plasma ghrelin and fasting insulin levels was found, however plasma ghrelin levels were not affected by acute insulin during a euglycemic-hyperinsulinemic clamp. These studies show that ghrelin expression and secretion are age-dependent and affected by adiposity in the rhesus monkey. Studies are planned to assess the role of Ghrelin in alcohol consumption.