A Semi‐Isolation Housing Paradigm Affects Anxiety, Ethanol Intake, and the Gut Microbiota in Adult Male C57BL/6JRj Mice

Background There is a complex link between environmental factors, anxiety levels, and ethanol consumption. Social conditions such as loneliness, peer influence, and housing environment shape drinking behaviors and mental health outcomes. The gut microbiota, in the context of the gut‐brain axis, may act as a biomarker. Objectives This study investigates how different housing conditions impact anxiety‐like behavior, ethanol consumption, and gut microbiota in adult male mice. Methods Adult male C57BL/6JRj mice were housed under isolated, semi‐isolated, or group‐housed conditions. Anxiety‐like behavior was assessed using the open field (OF), elevated plus maze (EPM), and novelty‐suppressed feeding (NSF) tests. Ethanol intake was evaluated using a 4‐week intermittent two‐bottle choice paradigm. Fecal samples were collected and analyzed by 16S rRNA sequencing to characterize gut microbiota. Behavioral, drinking, and microbiota data were analyzed using ANOVA‐based statistics and QIIME2 pipelines. Results Semi‐isolated mice displayed anxiety‐like behavior only in the NSF test. Gut microbiota analysis revealed no significant changes in global diversity. Semi‐isolated mice exhibited a specific reduction in the Prevotellaceae, particularly Prevotella UCG_001 . Housing conditions also influenced ethanol intake: isolated mice exhibited a low initial preference that increased over time, whereas semi‐isolated mice showed a high initial preference followed by avoidance. Conclusion Housing conditions strongly shape voluntary ethanol consumption. Semi‐isolation was associated with elevated initial alcohol preference and anxiety‐like behavior only in the NSF test, while isolation led to a gradual escalation of ethanol intake. These findings highlight the importance of housing environment and anxiety‐related factors in alcohol‐related preclinical research.