Cerebellar interneurons encode single steps in locomotion

Locomotion in complex environments depends on the precise timing and active control of single paw movements in order to adapt steps to surface structure and coordinate between paws. Control of motor timing crucially depends on the cerebellum, which has been shown to provide signals necessary for movement initiation and termination. While recent work found single-paw kinematics during locomotion to be encoded in cerebellar Purkinje cells, precise, event-related activity in the cerebellum in more complex environments is less well understood. Moreover, whether precise action timing in complex environments is controlled upstream in the cerebellar circuit is currently unknown. To address this question, we studied step-related activity in cerebellar molecular layer interneurons (MLIs) which provide direct, fast control of Purkinje cell activity. We trained mice to walk on a motorized, runged treadmill while simultaneously acquiring behavioral videos and electrophysiological recordings from MLIs. Paw trajectories were extracted from videos and divided into swing and stance phases. Over several days of learning, mice became increasingly proficient at walking on the runged treadmill, so that they made fewer, longer strides with faster swings and fewer missteps. When analyzing behaviorally-evoked responses in MLIs, we found sharp changes in activity in relation to paw swing and stance onsets. To uniquely attribute neural activity patterns to single paw movements, we fitted sparsity-constrained linear models with swing and stance event kernels. While most MLIs in the left simplex preferentially encoded front left paw variables, a large proportion of cells additionally showed activity variations related to multiple paws. Throughout learning, we found increases in swing and stance encoding on the timescale of a single session as well as over multiple sessions. Together, these results demonstrate that single-paw movement initiation and termination during complex locomotor behavior is encoded in cerebellar MLIs across different stages of locomotor proficiency.