Hippocampal network engagement in cue plus context-induced ethanol seeking in male mice

Exposure to environments associated with ethanol use can trigger craving and reinstatement of seeking behavior after extinction. The hippocampus integrates contextual information with motivational and emotional outputs, encoding drug-related memories, but intrahippocampal functional organization during ethanol context renewal remains poorly characterized. Here, we investigated the role of the dorsal and ventral hippocampus and its functional connectivity and neuronal composition during reinstatement of ethanol-seeking behavior. Adult male C57BL/6 mice self-administered Ethanol + Saccharin or Saccharin (control) in Context A, underwent extinction in Context B, and were tested in Context A versus B. Next, we quantified Fos (neuronal activation marker) expression in dorsal (CA1, CA2, CA3, and Dentate Gyrus - DG) and ventral (vSUB) hippocampus and assessed the coordinated activity among subregions by correlation-based network analysis. Finally, we determined the phenotype (co-labeling with GABA and glutamate markers) of Fos-labeled neuronal populations involved in the context-induced reinstatement of ethanol seeking. Reexposure to context A, but not context B, reinstated ethanol-seeking behavior and increased Fos expression in CA2. Reexposure to context B increased Fos expression in DG. Network topology identified a dynamic functional intrahippocampal connection, highlighting CA2 as a central hub during ethanol context renewal. Finally, despite low overall activation, we observed that activated neuronal populations were predominantly excitatory and a proportionally greater recruitment of GABAergic neurons. Our data indicate that hippocampal activation patterns and connectivity dynamically reorganize during context-induced ethanol seeking, supporting its role in relapse-related memory processes.