Characterization of neuronal ensembles in a model of dual reward conditioned place preference

Abstract

Substance use disorder (SUD) is associated with maladaptive alterations in behavior. Drug-seeking behavior has been associated with neuronal ensembles, defined here as a small group of neurons exhibiting coordinated activity patterns, in the prelimbic prefrontal cortex (PL) and nucleus accumbens core (NAcore). Most SUD preclinical research focuses on the effects of single-reward exposure on the general population of neurons within the reward pathway, rather than on poly-reward exposure. Here, we seek to characterize and compare the ensembles linked to cocaine and chocolate using a within-subject approach. We used Ai14xFos^2A-iCreER (c-Fos-TRAP2) transgenic mice to tag neuronal ensembles in a novel dual cocaine and chocolate conditioned place preference (CPP) model, where each chamber was associated with a different reward, either cocaine or chocolate. We found that, after successful dual conditioning and in the absence of the rewards, mice preferred cocaine over chocolate. Additionally, in mice exposed to both cocaine and chocolate, cortical and accumbal ensembles linked to each reward were comparable in size to reward ensembles in mice exposed only to one reward. However, reward-seeking ensembles were larger than ensembles tagged in homecage control mice across groups. We also found that ensemble size does not correlate with the level of reward seeking across single and dual CPP models. These results offer a new paradigm for studying drug-related neuronal ensembles in comparison to natural rewards in non-contingent behavioral models.

Keywords: c-Fos-TRAP2; chocolate; cocaine; dual-reward; neuronal ensembles; nucleus accumbens core; prefrontal cortex.

Figure 1.

Behavior of Cocaine CPP. (A) Timeline of cocaine CPP. Different colors denote different contexts. (B) Movement counts during conditioning sessions for the cocaine-paired chamber and the vehicle-saline chamber. * p < 0.05 Comparing movement counts during conditioning. (C) Percent time in each chamber on test day. The dotted line signals 50% time, or no preference. *** p < 0.001 Comparing time in the cocaine-paired chamber to the vehicle-saline chamber, paired t-test. $ p < 0.05 Cocaine compared to 50%, one-sample t-test. (D) No sex differences in cocaine CPP. $ p < 0.05, Males and females compared to 50%, one-sample t-test. Numbers at the bottom of the bars refer to the number of animals.

Figure 2.

Behavior of Chocolate CPP. (A) Timeline of chocolate CPP. Different colors denote different contexts. (B) Movement counts during conditioning sessions for the chocolate-paired chamber and the vehicle-empty chamber. (C) Percent time in each chamber on test day. The dotted line signals 50% time, or no preference. ** p < 0.01 Comparing time in the chocolate-paired chamber to the empty-paired chamber, paired t-test. $ p < 0.05 Chocolate chamber compared to 50% one-sample t-test. (D) Sex differences in chocolate preference showed that male mice preferred chocolate more than females. * p < 0.05 Comparing chocolate chamber time between sexes, unpaired t-test. $ p < 0.05, Males compared to 50% one-sample t-test. Numbers at the bottom of the bars refer to the number of animals.

Figure 3.

Behavior of dual CPP. (A) Timeline of dual CPP. Different colors denote different contexts. (B) Movement counts during conditioning sessions for the cocaine-paired chamber and the chocolate-paired chamber. **** p < 0.0001 Comparing movement counts during conditioning. (C) Percent time in each chamber on test day. The dotted line signals 50% time, or no preference. * p < 0.05 Comparing the cocaine-paired chamber and the chocolate-paired chamber, paired t-test. $ p < 0.05 Chamber preference compared to 50% one-sample t-test. (D) Male and female preferences for cocaine were not different from each other. $ p < 0.05 Female cocaine preference compared to 50% one-sample t-test. Numbers at the bottom of the bars refer to the number of animals.

Figure 4.

Ensemble sizes following single- and dual-reward CPP. (A) Representative image of the nucleus accumbens core (NAcore), Red TdTomato+, Blue NeuN+. (B) Representative image of the prelimbic cortex (PL). (C) Size of the reward-seeking ensemble in the NAcore and Prelimbic cortex across reward groups. Anterior commissure (AC), cocaine (Coc), chocolate (Choc), **** p < 0.0001. Numbers refer to the number of image acquisitions per animal.

Figure 5.

Comparison of ensemble sizes between groups. (A) Comparing the size of the ensemble in the different treatment groups in the NAcore. (B) Comparing the size of the ensemble in the PL. Nucleus Accumbens Core (NAcore), Prelimbic cortex (PL), homecage (HC), saline (Sal), cocaine (Coc), chocolate (Choc), dual cocaine (Dual), $ p < 0.05 compared to saline group in PL, # p < 0.0001 all groups compared to homecage group. Numbers refer to the number of image acquisitions per animal.