Dissociable dorsal medial prefrontal cortex ensembles are necessary for cocaine seeking and fear conditioning in mice

The dorsal medial prefrontal cortex (dmPFC) plays a dual role in modulating drug seeking and fear-related behaviors. Learned associations between cues and drug seeking are encoded by a specific ensemble of neurons. This study explored the stability of a dmPFC cocaine seeking ensemble over 2 weeks and its influence on persistent cocaine seeking and fear memory retrieval. In the first series of experiments, we trained TetTag c-fos-driven-EGFP mice in cocaine self-administration and tagged strongly activated neurons with EGFP during the initial day 7 cocaine seeking session. Subsequently, a follow-up seeking test was conducted 2 weeks later to examine ensemble reactivation between two seeking sessions via c-Fos immunostaining. In the second series of experiments, we co-injected viruses expressing TRE-cre and a cre-dependent inhibitory PSAM-GlyR into the dmPFC of male and female c-fos-tTA mice to enable "tagging" of cocaine seeking ensemble or cued fear ensemble neurons with inhibitory chemogenetic receptors. These c-fos-tTA mice have the c-fos promoter that drives expression of the tetracycline transactivator (tTA). The tTA can bind to the tetracycline response element (TRE) site on the viral construct, resulting in the expression of cre-recombinase, which enables the expression of cre-dependent inhibitory chemogenetic receptors and fluorescent reporters. Then we investigated ensemble contribution to subsequent cocaine seeking and fear recall during inhibition of the tagged ensemble by administering uPSEM792s (0.3 mg/kg), a selective ligand for PSAM-GlyR. In both sexes, there was a positive association between the persistence of cocaine seeking and the proportion of reactivated EGFP+ neurons within the dmPFC. More importantly, inhibition of the cocaine seeking ensemble suppressed cocaine seeking, but not recall of fear memory, while inhibition of the fear ensemble reduced conditioned freezing but not cocaine seeking. The results demonstrate that cocaine and fear recall ensembles in the dmPFC are stable, but largely exclusive from one another.