Decoding the Role of Secondary Motor Cortex Neuronal Ensembles during Cocaine Self-Administration: Insights from Longitudinalin vivoCalcium Imaging via Miniscopes

ABSTRACT Background We recently reported that the risk of cocaine relapse is linked to hyperexcitability in the secondary motor cortex (M2) after prolonged withdrawal following intravenous self-administration (IVSA). However, the neuronal mechanisms underlying drug-taking behaviors and the response of M2 neurons to contingent drug delivery remain poorly understood. Methods Mice received cocaine as reinforcement (RNF) following active lever presses (ALP) but not inactive lever presses (ILP). Using in vivo calcium imaging during cocaine IVSA, we tracked M2 neuronal activity with single-cell resolution. We then analyzed Ca 2+ transients in M2 at the early vs . late stages during the 1-hr daily sessions on IVSA Day 1 and Day 5. Results M2 neurons adapted to both operant behaviors and drug exposure history. Specifically, saline mice showed a reduction in both saline taking behaviors and Ca 2+ transient frequency with the 1-hr session. In contrast, cocaine mice maintained high ALP and RNF counts, with increased Ca 2+ transient frequency and amplitude on Day 1, persisting through Day 5. Compared to saline controls, cocaine mice exhibited a lower % of positively responsive neurons (Pos) and higher % of negatively responsive neurons (Neg) before ALP and after RNF, a difference not seen before ILP. Furthermore, as drug-taking behaviors progressed during the daily session, cocaine mice showed greater neuronal engagement with a larger population, particularly linked to ALP and RNF, with reduced overlap in neurons associated with ILP. Conclusion The M2 undergoes dynamic neuronal adaptations during early drug-taking behaviors, indicating its role as a potential substrate mediating the persistence of drug-seeking behaviors in cocaine relapse.