Neurogenetics of Stress Response and Reward Sensitivity The genetic factors that drive excessive alcohol consumption in humans can do so by acting on reward sensitivity, stress reactivity or impulsivity. I have had a long-standing interest in stress neurobiology and G x E interactions. Of particular interest for the study of these types of interactions are stress-responsive genes or genes that contribute to environmental sensitivity and stress vulnerability. Some of the genes that predict individual differences in reward sensitivity and temperament are activated or suppressed in response to stress or glucocorticoid exposure, and we have shown that others can moderate stress responding. Our studies further suggest some of these genes to be under evolutionary selection, meaning they are adaptive in certain contexts, and, of relevance to the NIH mission, that findings from the rhesus macaques often translate to the human condition. We have been performing both candidate gene based projects and whole genome sequencing using archived rhesus macaque and collaborative datasets in order to further these studies. Here, we describe several examples of these projects. CANDIDATE GENE BASED STUDIES: The Oxytocin Receptor (OXTR) gene is an obvious candidate for alcohol use and dependence because of its roles in reward, stress reactivity, sociality and ethanol response. We recently showed OXTR to be a substrate for epigenetic regulation in hippocampus from early stress exposed rhesus, which was valuable because of the species and tissue of interest, pointing to OXTR as a good candidate for looking at gene by environment interactions. We screened by sequencing the OXTR gene in rhesus macaques, identifying a genetic variant that causes an amino acid change in the arm of the receptor that binds to oxytocin. Animals carrying this variant exhibit increased affiliative behaviors in response to administration of oxytocin intranasally, a proposed therapeutic for autism spectrum disorders. We also find that this variant predicts whether or not exogenously administered oxytocin can reverse the asociality observed among some rhesus macaque infants. The coding regions within peptide genes tend to be under a high degree of purifying selection, such that much of the functional variation at these loci are is more likely to be observed in regulatory regions. However, one advantage of focusing on peptide genes is that they are relatively short in sequence and can be more easily be sequenced in their entirety by traditional methods. As a consequence of the small size and the high degree of selection, there are also fewer putatively functional variants to consider. Given these factors, I have had a long interest in sequencing neuropeptide genes, as it allows us to not only home in on functional variants with increased facility but also affords us greater power in performing genotype-phenotype correlation studies in the relatively small datasets to which we have access. We sequenced the rhAVP gene and its associated regulatory region, identifying a single nucleotide polymorphism (SNP) in the core promoter that disrupts a known regulatory element. This SNP results in lower central levels of AVP, and we see an effect of this variant on stress axis activity in response to both social stress and binge doses of alcohol. Of interest, this SNP also predicts sensitivity to Naltrexone-mediated downmodulation of ethanol seeking, suggesting that this system may be important when considering individualized treatment and therapy. WHOLE EXOME SEQUENCING: Among humans, while potentially rooted in their adaptive origins, excessive and inappropriate aggressive behavior is a feature of many psychiatric disorders, such as borderline personality disorder, antisocial personality disorder, post-traumatic stress disorder, and depression. It is also a trait observed among alcohol-dependent individuals. Aggressive tendencies can escalate under the influence of some drugs of abuse, including alcohol. Violent crime is frequently associated with alcohol intoxication, and alcohol consumption is widely held to be a causative factor in violent or aggressive behavior. Although these associations are not causal and may reflect the association between psychopathology or certain personality traits and higher levels and frequencies of alcohol drinking, controlled studies performed in a laboratory setting consistently demonstrate a causal relationship between alcohol consumption and enhanced aggressive responding in some individuals. Alcohol induces psychopharmacological effects that are proposed to contribute to alcohol-induced aggression through modulation of serotonin, endogenous opioid, dopamine, and GABA systems. Not only are impulse control deficits and reward-sensitivity instrumental in driving excessive alcohol consumption, but intoxication can impair information processing, and this may lead to misinterpretation of social cues and, thus, escalated aggressive responding. Although it said that genetic factors are likely to play a role in alcohol-related violence, genetic studies in this area are somewhat lacking. Moreover, whether genetic factors that predispose individuals to violence under the influence of alcohol are independent of genetic or trait-like factors that contribute to aggression in the non-intoxicated state has not formally been considered. We have previously demonstrated that the serotonin and opioid systems contribute to alcohol-facilitated aggression in primates, suggesting the behavioral disinhibition combined with alcohol-induced reward could increase alcohol-related violence in humans. Through whole exome sequencing using a human pull-down kit, we identified a damaging SNP in a Gaba-receptor (GABRA6) gene. This variant (Tyr28Phe) predicts increased sensitivity to motor-impairing effects of alcohol and protects against PR-induced increases in alcohol consumption. Consistent with what appeared to be a gain of function role based on its effects on intoxication and considering the potential for a role for the cerebellum in both motor function and social cognition, we queried GABRA6s link to neonatal attention to social cues and found that these behaviors were increased among 28Phe allele carriers. Given that rodent studies indicate that the GABA system is among those thought to be involved in alcohol-enhanced aggression and that misinterpretation of social cues is one proposed pathway through which alcohol promotes aggression in select human subjects, we cautiously postulated that we could examine whether GABRA6 Tyr28Phe predicted individual differences in alcohol-enhanced aggression following provocation by a human intruder in nursery/peer reared monkeys. Using alcohol response factors derived from analysis of aggressive behavior following IV administration of 2.2 g/kg ETOH, IV (Higley, archived data), we found that, among PR monkeys, GABRA6 genotype decreased high intensity aggression in response to threat from a human investigator. Another area for which primate models may be particularly useful are in examining the effects of prenatal stress and alcohol on development. In collaboration with the University of Wisconsin, we selected individual rhesus that had been characterized for sensitivity to fetal alcohol effects. Among the SNPs we identified was a stop-codon variant at a serotonin receptor (HT3RD), and we were able to show that there were interactive effects between this SNP and prenatal ethanol exposure on sensory processing-related phenotypes, a hallmark of developmental disorders. The HTR3D gene is also potentially of interest in that 5-HT3 blockade has been proposed as a treatment for alcohol dependence. We found that HT3RD3 genotype not only relates to sensitivity to prenatal ethanol exposure, but that it predicts alcohol intake in adulthood.
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