NOT UPDATED - PI LEFT 5/31/06. Over the past year we have continued with our examination of the serotonin transporter and MAOa genotypes as candidate genes for alcohol intake and alcohol-related phenotypes and expanded our range of candidate genes that are potentially related to excessive alcohol intake and its associated phenotypes.? New Genotypes? NPY? Neuropeptide Y (NPY) is an anxiolytic peptide that is involved in the modulation of stress response. Although studies have produced variable results, the NPY system has also been associated with alterations in alcohol intake in both animal models and human subjects. Dr. Barr and Stephen Lindell sequenced the rhesus NPY gene exons, exon-intron boundaries and 2.5 kB of the 5?flanking region to screen for functional NPY variants. They identified sixteen polymorphisms, and one of these, located within a region conserved between rodents and primates (-1002T>G), disrupts an androgen response element. Consistent with the demonstrated role for androgens in induction of NPY, animals with the ?1002 G-containing haplotype exhibited lower levels of NPY in cerebrospinal fluid. Carriers of the G allele also exhibit higher ACTH response to an acute novel stressor. They extended this work, finding a gene dosage effect with regard to separation-induced behaviors. Though not the expected outcome, they found that this NPY promoter variant is also associated with decreased alcohol intake in animals without a history of dependence, with the effect being more marked among animals exposed to early adversity in the form of peer rearing. ? Mu Opiate Receptor ? In humans, a variant in the Mu opiod receptor gene (OPRM1A118G) produces a 3-fold increase in opiate receptor binding affinity and has been associated with increased subjective euphoria following alcohol administration in human subjects. Last year, Dr. Barr found that an orthologous polymorphism in rhesus macaques (OPRM1C77G) was associated with increased alcohol preference, particularly among males. She furthered these analyses, tripling the number of animals included in the study. She also found that this variant was associated with increased stimulation following ethanol administration in alcohol-naive rhesus. This year?s studies on alcohol consumption verify the preliminary results reported last year and also show that male carriers of the G allele consume more alcohol and more frequently consume alcohol to levels sufficient to produce signs of intoxication. These animals drank to intoxication on 40% of testing days, whereas other study animals consumed to this level on fewer than 8% of testing days. This is of interest, given that, among alcoholics, both carriers of the OPRM1118G allele and males are more likely to respond to mu opiod receptor blockade than are females. ? TPH2 and DRD4? In a collaboration with Dennis Murphy, Peter Lesch, and Jens Wendland, Dr. Newman began a series of studies to characterize TPH2 and DRD4. Dr. Newman and his collaborators sequenced the rhesus TPH2 gene including 2 kb of the promoter, all exons plus flanking sequences, and 1 kb of 3' UTR. They identified 19 new SNPs: 5 in the promoter, 11 intronic/exonic sites, and three in the 3' UTR, as well as a 152bp insertion deletion in the 3' UTR. Using the same approach, he sequenced 2 kb of the rhesus DRD4 promoter, characterizing 5 SNPs. These SNPs form two major haplotypes, one of which is strongly associated with a measure of impulsivity, the latency to approach a stranger.? Arrays? Dr. Barr continued her work microarrays with assessing gene systems dysregulated in response to early rearing experiences in mother-reared (MR) and peer-reared (PR) rhesus macaques. She performed gene microarray studies in collaboration with Stan Watson, Robert Thompson and Juan Lopez at the University of Michigan. A number of putative genes were differentially expressed (1.5+-Fold) in cerebral cortices (Area 46) of these differentially subjects. Among the functions of genes that were differentially expressed were those involved in: cell adhesion/migration/intracellular transport (44), apoptosis (13), cell division (13), energy/mitochondria (19), lipid/fatty acid/steroid metabolism (15), neurotransmission (34), nucleic acid binding (70), protein synthesis (12), protein metabolism/modification (15), cell signaling (58), stress/immune (40), unknown (51) or other (29). These molecular changes may underlie the behavioral differences observed in monkeys exposed to early adversity in the form of peer-rearing.? Serotonin transporter (5HTTLPR) and monoamine oxidase A (MAOA-LPR) gene promoter polymorphisms ? Several 5HITTPR studies directly related to alcohol intake are outlined in the other 3 Annual reports. In a study by Dr. Newman and his staff, MAO was investigated as a potential candidate gene in impulsivity, a phenotype shown to be related to excessive alcohol intake in the monkey. Males living in social groups were assessed for their latency to approach a stranger using an Intruder Challenge Test. Genotypes were clustered based on inferred high or low MAOA enzymatic activity (MAOA-HA/MAOA-LA). Younger males with the MAOA-LA genotype were much faster to approach an intruder and were equivalent in approach time to older subjects with either genotype, suggesting an age by genotype interaction on impulsivity and its associated risk for excessive alcohol intake.? Self-Injurious Behavior, Genotype X Rearing Interactions and Potential Treatment? Self-injurious behavior (SIB) is a major public health problem that thousands of children and adult suffer from. In a study with Melinda Novak and colleagues at Harvard, we assessed potential gene by environment interactive effects on SIB using a nonhuman primate model. We investigated the role of the 5HTTLPR and MAOA-LPR gene promoter polymorphisms on the expression of self-biting in the largest population of SIB monkeys that is known. Rates of self-biting were collected over a 5-year period. Subjects homozygous for the long-allele of 5HTTLPR and/or the low-activity MAOA-LPR allele demonstrated significantly higher levels of self-biting, regardless of early rearing experience (i.e., MR/PR), or the presence/absence of wounding history. In addition, MR/PR and/or carrying a veterinary record of self-wounding were associated with higher rates of self-biting for both loci. Significant genotype by rearing interactions were found for each genotype, implicating that the short-allele of 5HTTLPR and the high activity allele of MAOA-LPR may offer some protection against the adverse effects of MR/PR-rearing on self-biting. These data suggest an important role for 5HTTLPR and MAOA-LPR gene polymorphisms and rearing, and their interaction, on the expression of SIB in monkeys and suggest potential candidate genes for SIB in humans. In a second study with Babette Fontenot and colleagues at the New Iberia Research Primate Center we investigated the effects of two serotonergic treatment agents--fluoxetine, an SSRI, and buspirone, a 5-HT 1a agonist--on rates of self-injurious and stereotypic behavior. Fluoxetine and buspirone were significantly effective in reducing rates of self-biting during treatment weeks 1 to 8 and self-directed stereotypic behavior during weeks 5 to 12 and post-treatment. Rates of anxiety-like scratching and yawning decreased in the buspirone-treated condition. In the fluoxetine-treatment condition, rates of anxiety-like yawning, scratching, and self-directed grooming were higher overall when compared with those of buspirone-treated animals, and rates of scratching increased. Such findings suggest that animals in the fluoxetine-treated condition experienced higher levels of anxiety throughout the study. Such findings suggest that fluoxetine and buspirone may be efficacious for treatment of self-injurious and self-directed stereotypic behavior in macaques.
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