Decoding the Role of Secondary Motor Cortex Neuronal Ensembles during Cocaine Self-Administration: Insights from Longitudinal in vivo Calcium Imaging via Miniscopes.

Recent findings in our lab demonstrated that the risk of cocaine relapse is closely linked to the hyperexcitability of cortical pyramidal neurons in the secondary motor cortex (M2), noticeable 45 days after cocaine intravenous self-administration (IVSA). The present study was designed to explore the underlying mechanisms of neuronal alterations in M2. Our hypothesis was that M2 neurons were affected directly by cocaine taking behaviors. This hypothesis was tested by monitoring individual neuronal activity in M2 using MiniScopes for in vivo Ca ²⁺ imaging in C57BL/6J mice when they had access to cocaine IVSA as a reinforcement (RNF) contingent to active lever press (ALP) but not to inactive lever press (ILP). With support of our established pipeline to processing Ca ²⁺ imaging data, the current study was designed to monitor M2 neuronal ensembles at the single-neuron level in real time with high temporal resolution and high throughput in each IVSA session and longitudinally among multiple IVSA sessions. Specifically, five consecutive 1-hr daily IVSA sessions were used to model the initial cocaine taking behaviors. Besides detailed analyses of IVSA events (ALP, ILP, and RNF), the data from Ca ²⁺ imaging recordings in M2 were analyzed by (1) comparing neuronal activation within a daily IVSA session (i.e., the first vs. the last 15 min) and between different daily sessions (i.e., the first vs. the last IVSA day), (2) associating Ca ²⁺ transients with individual IVSA events, and (3) correlating Ca ²⁺ transients with the cumulative effects of IVSA events. Our data demonstrated that M2 neurons are exquisitely sensitive to and significantly affected by concurrent operant behaviors and the history of drug exposure, which in turn sculpt the upcoming operant behaviors and the response to drugs. As critical nodes of the reward loop, M2 neurons appear to be the governing center orchestrating the establishment of addiction-like behaviors.