Candidate Genes? 5-HTTLPR? In macaques, there is a length variant in the serotonin transporter linked polymorphic region (5-HTT-LPR) that is functionally equivalent to the HTT-LPR s allele in humans, an variant that increases risk of depression in the face of adversity. We found that carriers of this variant sensitize to repeated stress, exhibiting high levels of anxiety-like behaviors and behavioral pathology with repeated stress exposures. We also found that s allele carriers with prior histories of stress were more likely to exhibit anxiety-like and pathological behaviors during stress exposure.? ? OPRM1 C77G? In humans, a SNP in the Mu-Opioid receptor gene (OPRM1A118G) confers higher affinity for Endorphin, resulting in increased alcohol-induced euphoria. We demonstrated that a functionally equivalent SNP in rhesus macaques (OPRM1C77G) predicts increased alcohol-induced stimulation, a marker of euphorogenic alcohol actions. Male carriers of the G allele also consume more alcohol. This is of interest, given the fact that Type II alcoholism is more common among men and that, among addicted individuals, males are more responsive to Mu-Opioid receptor blockade. ? ? The fact that functionally equivalent variants of the OPRM1 gene have arisen in humans and rhesus suggests that they may have conferred selective advantage at some point in evolutionary history. Development of attachment is crucial for infant survival, and mother-infant attachment is mediated by endogenous opioids. Though factors controlling opioid release during alcohol exposure and maternal contact may be distinct, one would predict that subjects that are carriers of a genetic variant that make them more sensitive to the rewarding properties of alcohol would also experience more reward when in contact with their mothers. We found that infants carrying the G allele were more securely attached during early infancy and that they exhibited a more persistent distress response to maternal separation and increased preference for maternal contact upon reunion. This pattern of behavior shares some characteristics with the anxious form of insecure attachment in children. Insecure early attachment has been suggested as a risk factor for substance abuse and addiction. Our findings lend support to ethological arguments in favor of attachment theory and its association with alcohol use disorders, not necessarily in terms of causality, but by pointing to common underlying genetic or neurobiological substrates. ? ? MAOA-LPR? In a variety of species, highly aggressive behavior toward unfamiliar conspecifics may increase reproductive opportunity, social rank, and protection of self and offspring. In the regulatory region for the human MAOA gene, there is a repeat polymorphism (MAOA- LPR). The low-activity MAOA-LPR allele is associated with antisocial behavior in humans and, in rhesus macaque, an orthologous variant has been shown to predict various forms of aggression. We found that highly agonistic, risky aggressive behavior was higher in rhesus macaques with the low activity MAOA-LPR genotype. Our findings may have implications for the selection for traits that increase fitness, but at the same time increase vulnerability to psychopathology. ? ? Molecular Markers of GxE Interactions? Neuroimaging? The serotonin system may be particularly relevant to the intersection between early stress and subsequent vulnerability to psychopathology, as dysregulation of 5HT1A receptor expression and binding have been reported in mood and anxiety disorders. Using a PET ligand that selectively binds 5HT1A receptors (FPWAY), we performed a study in collaboration with NIDA, demonstrating that 5HT1A receptor density is increased among female macaques with histories of early adversity.? ? Gene Expression Studies? In humans, HTTLPR genotype interacts with stressful life events to increase depression vulnerability. We performed gene expression studies on post-mortem brain tissue (hippocampus and amygdala) from mother- and nursery-reared macaques. We demonstrated effects of peer-rearing in hippocampus, but not in amygdala. Using HTTLPR genotype as a predictor of gene expression within the peer reared group, we also found a significant effect on overall gene expression. ? ? Genetic Variation Within Systems Dysregulated in the Post-Dependent State? Corticotropin Releasing Hormone and Neuropeptide Y are opposing neuropeptides critically involved in stress responding and which are dysregulated in the post-dependent state. We have located polymorphisms within GREs in both the CRH and NPY regulatory regions. Whereas GREs are important to feedback regulation of the HPA axis via diminution in CRH transcription, they induce NPY expression. These variants would, therefore, be expected to result in changes in expression that are not unlike those observed in the post-dependent state.? ? CRH? We identified a SNP (-248C>T) that is present in a highly-conserved region of the proximal promoter. Using in vitro reporter assays, we found this variant to increase transcriptional activity following stimulation with TPA and to prevent corticosteroid-mediated downmodulation of CRH. EMSA studies performed using nuclear extract from hypothalamic cells support these observations. ? ? During stress exposure, infant macaques carrying the 248 T allele exhibited lower levels of environmental exploration, a species-typical behavior that reflects resiliency and which has been shown to be preserved during stress exposures in macaques treated with a CRHR1 antagonist. Carriers of the T allele also exhibited higher levels of HPA axis activation to stress. Early adversity in the form of peer rearing produces increases in CSF levels of CRH, and we found that stress responding and alcohol consumption were especially high among peer-reared subjects carrying the -248T allele. Our findings demonstrate the importance of the CRH system in influencing reactivity to stress and may further implicate over-activity of this system as a risk factor for alcohol problems.? ? NPY? Loss of a GRE half-site in the NPY regulatory region (-1002 T/G) is associated with lower CSF levels of NPY, and we found that peer-reared carriers of this variant exhibited increased behavioral responses to stress. We also found that peer-reared G/G subjects consume high levels of alcohol and exhibit an escalation in alcohol intake. This may be suggestive of increased vulnerability to allostatic breakdown of the NPY system in these subjects. ? ? The studies described above suggest a role for neuropeptide gene variation in the susceptibility to alcohol-related disorders and may further implicate the CRH and NPY systems as treatment targets in selected individuals. ? ? Genetic Factors, Addiction Vulnerability & Treatment Response? ? Intermittent Access ? Rodent studies have shown that intermittent access induces escalated alcohol intake. We found that, when given every-other-day access to ethanol, macaques will consume sufficient quantities of alcohol (5-6 g/kg/day) to produce blood ethanol consumption levels in the 150-200 mg% range. Current studies are underway to investigate whether functional variants in CRH or NPY system genes interact with intermittent access to produce high levels of alcohol consumption. ? ? Naltrexone Response as a Function of OPRM1 Genotype? Studies have shown that opiate receptors play a critical role in the rewarding effects of alcohol, and studies have demonstrated that the OPRM1 antagonist, naltrexone (NTX), is effective in the treatment of alcohol dependence. We addressed the hypothesis emerging from human studies, which predicts that the OPRM1C77G polymorphism would be associated with NTX response. We found that NTX treatment diminished alcohol intake and there was a trend for NTX to produce more marked reductions in consumption among G allele carriers.
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