Targeted stimulation of motor cortex neural ensembles drives learned movements

During the execution of learned motor skills, the neural population in the layer 2/3 (L2/3) of the primary motor cortex (M1) expresses a reproducible spatiotemporal activity pattern. It is debated whether M1 actively participates in generating this activity pattern and whether this learned pattern causally drives the learned movement. To address these questions, we utilized in vivo two-photon calcium imaging combined with holographic optogenetic stimulation of functionally defined M1 L2/3 neuronal ensembles in mice performing a skilled lever-pressing task. A brief and synchronous stimulation of [~]20 neurons whose activity onset in voluntary trials precedes movement onsets induced movements that resembled the learned movement, while producing spatiotemporal activity patterns in other M1 neurons that resembled those during the voluntary learned movement. Moreover, trial-by-trial variability of optogenetically triggered population activity correlated with the variability in the induced movements. These trial-by-trial variabilities were predicted by the initial state of M1 population activity immediately preceding the stimulation. In some trials, the stimulation induced the learned activity without inducing overt movements, indicating that the learned activity is not simply a reflection of movements and instead can be induced internally. The stimulation failed to generate movements or learned activity when mice were disengaged from the task. Stimulation of the neurons whose activity followed voluntary movement onsets failed to induce the learned movement in task-engaged mice. Taken together, the learned activity pattern in M1 L2/3 can be generated when the M1 network is prepared at the optimal initial state and receives precise triggering inputs, supporting the active role of M1 in the generation of learned activity and learned movements.