REACHING INTO SPACE: PROBING THE CAUSAL LINK BETWEEN MOTOR CORTEX ACTIVITY AND DIRECTIONAL MOVEMENTS

Goal-directed voluntary movements require the motor cortex to integrate internal cortical dynamics with external sensory information. However, how behaviorally relevant motor representations emerge from local circuit activity—and how they causally contribute to movement execution in space—remains poorly understood.

Here, we combined a directional reaching task in mice with two-photon calcium imaging and targeted optogenetic photostimulation to causally interrogate motor cortical ensembles during behavior. Head-fixed mice performed directional reaching movements toward a waterspout presented at three distinct spatial locations (left, right, or center relative to the snout). Functional imaging revealed that layer 2/3 neurons in medial anterior cortex exhibit highly selective spatial tuning, forming largely non-overlapping neuronal ensembles specific to one of the three target locations.

To test the causal role of these ensembles, we developed a dual-light-path two-photon microscope enabling simultaneous imaging and optogenetic single-cell–resolved photostimulation during ongoing behavior. Selective manipulation of small groups of neurons (<10) tuned to a given reach direction was sufficient to selectively impair reaches toward that specific target. These deficits manifested as misdirected paw trajectories, truncated forward extension, and failures to complete the reach-to-grasp-to-drink sequence, with mice frequently failing to contact the water droplet or initiate drinking. Importantly, photostimulation had no detectable effect on reaches toward the other spatial targets, which remained indistinguishable from control trials.

This selective disruption of movements toward a single spatial location demonstrates that motor cortical ensembles are causally involved in guiding voluntary actions in a space-specific manner, linking local circuit activity to the directional control of behavior.