Targeted stimulation of motor cortex ensembles drives learned movements

During the execution of learned motor skills, the neural population in layer 2/3 (L2/3) of primary motor cortex (M1) expresses a reproducible spatiotemporal activity pattern¹. It is debated whether M1 actively participates in generating this learned pattern and whether this pattern causally drives the learned movement^2-5. Here we combined in vivo two-photon calcium imaging and holographic optogenetic stimulation of functionally defined M1 L2/3 ensembles in mice performing a skilled lever-pressing task. Brief synchronous stimulation of about 20 neurons whose activity onset precedes voluntary movement onset induced movements resembling the learned movement, while producing activity patterns in other M1 neurons resembling the learned activity. Moreover, trial-by-trial variability of optogenetically triggered population activity correlated with variability in the induced movements. These variabilities were predicted by the initial state of M1 activity preceding stimulation. In some trials, stimulation induced the learned activity without inducing movements, indicating that the learned activity is not simply a reflection of movements but can arise internally. Stimulation failed to generate movements or learned activity in task-disengaged mice. Stimulation of neurons whose activity followed movement onsets failed to induce the learned movement in task-engaged mice. Together, the learned activity in M1 L2/3 can be generated when M1 is prepared at the optimal initial state and receives precise triggering inputs, supporting the active role of M1 in generating learned activity and learned movements.