With our study, we aimed to determine whether mPFC neurons that are activated during cue-paired alcohol consumption function as a lasting physical trace for alcohol cue associations that maintain cue reactivity after prolonged abstinence. For this, we applied a viral-TRAP method to selectively tag and manipulate mPFC neurons that are activated during alcohol SA in mice. In this study, a total of N = 162 C57BL/6J mice were trained to self-administer alcohol (N = 144) or sucrose (N = 18) in operant chambers, and activated mPFC neurons were tagged with an inhibitory DREADD during a last SA session or after exposure to non–alcohol-related context. Relapse tests were performed 3 to 4 weeks after cessation of alcohol/sucrose intake, and the effect of chemogenetic suppression of tagged mPFC ensembles on alcohol/sucrose seeking (measured by resumption of active lever pressing) in the absence and presence of an alcohol-paired cue was examined. At the end of behavioral experiments, mice were euthanized and their brains were isolated. After immunohistochemical stainings, confocal microscopy was used to quantify the number of tagged neurons (mCherry+), GABAergic neurons (GAD67+), activated neurons (Fos+), and overlap between markers, as well as for analysis of ensemble-derived axonal fibers (mCherry+) in the BLA and NAcc.

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