Byline: Barbara Ruggeri, Christine Macare, Serena Stopponi, Tianye Jia, Fabiana M. Carvalho, Gabriel Robert, Tobias Banaschewski, Arun L.W. Bokde, Uli Bromberg, Christian Buchel, Anna Cattrell, Patricia J. Conrod, Sylvane Desrivieres, Herta Flor, Vincent Frouin, Jurgen Gallinat, Hugh Garavan, Penny Gowland, Andreas Heinz, Bernd Ittermann, Jean Luc Martinot, Marie-Laure Paillere Martinot, Frauke Nees, Dimitri Papadopoulos-Orfanos, TomaA Paus, Luise Poustka, Michael N. Smolka, Nora C. Vetter, Henrik Walter, Robert Whelan, Wolfgang H. Sommer, Georgy Bakalkin, Roberto Ciccocioppo, Gunter Schumann, Keywords: OPRL1 methylation; stressful life events; adolescence; binge drinking; nucleus accumbens Background Nociceptin is a key regulator linking environmental stress and alcohol drinking. In a genome-wide methylation analysis, we recently identified an association of a methylated region in the OPRL1 gene with alcohol-use disorders. Methods Here, we investigate the biological basis of this observation by analysing psychosocial stressors, methylation of the OPRL1 gene, brain response during reward anticipation and alcohol drinking in 660 fourteen-year-old adolescents of the IMAGEN study. We validate our findings in marchigian sardinian (msP) alcohol-preferring rats that are genetically selected for increased alcohol drinking and stress sensitivity. Results We found that low methylation levels in intron 1 of OPRL1 are associated with higher psychosocial stress and higher frequency of binge drinking, an effect mediated by OPRL1 methylation. In individuals with low methylation of OPRL1, frequency of binge drinking is associated with stronger BOLD response in the ventral striatum during reward anticipation. In msP rats, we found that stress results in increased alcohol intake and decreased methylation of OPRL1 in the nucleus accumbens. Conclusions Our findings describe an epigenetic mechanism that helps to explain how psychosocial stress influences risky alcohol consumption and reward processing, thus contributing to the elucidation of biological mechanisms underlying risk for substance abuse. Article Note: www.imagen-europe.com Conflict of interest statement: See acknowledgements for disclosures. CAPTION(S): Table S1. Frequency of stressful life events. Table S2. Human and rat OPRL1 methylation and rat oprl1 gene expression assessment. Table S3. Association of OPRL1 genetic variants and OPRL1 methylation or lifetime binge drinking. Table S4. List of OPRL1 methyl-eQTL in the blood of 14-year-old adolescents. Table S5. Enriched disease categories (p Table S6. Enriched GO biological processes categories (p Table S7. PCA loadings of 39 CpG sites of the rat oprl1 gene (N = 16). Table S8. Water consumption (in g/kg) of stressed versus naive rats during intermittent 10% alcohol exposure. Table S9. Food consumption (in g/kg) of stressed versus naive rats during intermittent 10% alcohol exposure. Figure S1. The scatter plot of rat Oprl1 methylation factor and Oprl1 gene expression conditioned on the status of stress in the nucleus accumbens.