Characterization of Neuronal Ensembles in a Model of Dual-Reward Conditioned Place Preference

Substance use disorder is characterized by maladaptive patterns of reward-seeking behavior. Such behavior has been linked to neuronal ensembles, typically identified through activity-dependent expression. These ensembles have been studied in regions such as the prelimbic prefrontal cortex and the nucleus accumbens core. In this study, we characterized ensembles tagged during concurrent exposure to cocaine- and chocolate-associated contexts. We used 33 male and 33 female Ai14xFos^2A-iCreERT2 (c-Fos-TRAP2) transgenic mice to tag neuronal ensembles in a dual cocaine-chocolate conditioned place preference (CPP) paradigm, in which each chamber was associated with a different reward, either cocaine or chocolate. We found that after successful dual conditioning and in the absence of rewards, mice preferred the cocaine-paired chamber to the chocolate-paired chamber. Additionally, in mice exposed to both cocaine and chocolate, cortical and accumbal ensembles (measured as tdTomato⁺ cell density) tagged during the test session were comparable in size to those in single-reward conditions. However, TRAP2 neurons labeled during exposure to reward-paired contexts were larger than those in home cage control mice. We also found that the TRAP2-labeled ensemble did not correlate with the degree of chamber preference in either single- or dual-CPP models. These results introduce a dual-reward paradigm for studying context- and activity-dependent neuronal ensembles, in contrast to natural rewards used in noncontingent behavioral models.