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We previously reported that SNPs near TSPAN5 were associated with plasma serotonin (5-HT) concentrations which were themselves associated with selective serotonin reuptake inhibitor treatment outcomes in patients with major depressive disorder (MDD). TSPAN5 SNPs were also associated with alcohol consumption and alcohol use disorder (AUD) risk. The present study was designed to explore the biological function of TSPAN5 with a focus on 5-HT and kynurenine concentrations in the tryptophan pathway. Ethanol treatment resulted in decreased 5-