MULTI-LIMB CODING IN PRIMARY MOTOR CORTEX DURING FREELY MOVING CLIMBING IN MICE

Climbing is an ethologically relevant mammalian behaviour that requires continuous integration of sensory feedback with dynamic whole-body motor coordination. These features make climbing a powerful paradigm for investigating higher-order motor control; however, existing behavioural setups limit precise quantification of multi-limb kinematics and, consequently, the study of cortical contributions to vertical locomotion.
We developed a custom climbing apparatus enabling high-resolution extraction of forelimb and hindlimb kinematics in freely moving mice using markerless pose estimation. To investigate neural representations during climbing, we implanted a lightweight 64-channel laminar silicon probe (Cambridge Neurotech), coated in DiI, into the left primary motor cortex (M1). Probe placement was confirmed with serial two-photon microscopy post hoc. Neural activity was recorded during freely moving climbing bouts and aligned to discrete reach-and-grasp epochs across all four limbs.
Analysis of single-unit and multi-unit activity revealed distinct modulation patterns associated with movement initiation, peak velocity, and movement cessation. Notably, a subset of multi-unit clusters exhibited bilateral tuning, showing increased firing for movements of the ipsilateral limbs and decreased firing for the contralateral limbs. These patterns indicate that during complex whole-body behaviours, M1 integrates information across the body axis rather than encoding movements in a strictly lateralised manner.
Together, these findings demonstrate that M1 represents multi-limb dynamics during vertical locomotion and support the idea that cortical circuits contribute to coordinating distributed motor actions during naturalistic behaviour.