3D pose estimation enables virtual head fixation in freely moving rats

The impact of spontaneous movements on neuronal activity has created the need to quantify behavior. We present a versatile framework to directly capture the 3D motion of freely definable body points in a marker-free manner with high precision and reliability. Combining the tracking with neural recordings revealed multiplexing of information in the motor cortex neurons of freely moving rats. By integrating multiple behavioral variables into a model of the neural response, we derived a virtual head fixation for which the influence of specific body movements was removed. This strategy enabled us to analyze the behavior of interest (e.g., front paw movements). Thus, we unveiled an unexpectedly large fraction of neurons in the motor cortex with tuning to the paw movements, which was previously masked by body posture tuning. Once established, our framework can be efficiently applied to large datasets while minimizing the experimental workload caused by animal training and manual labeling. [Display omitted] •Virtual head fixation reveals clear neural tuning to paw movements in unfixed rats•FreiPose enables holistic 3D reconstruction of animals’ poses and limb trajectories•FreiPose allows for assignments of optogenetic effects to individual body parts•The approach works across species and enables automated behavioral classification Schneider et al. develop a marker-free movement-tracking system for holistic 3D reconstruction. They combine this approach with virtual head fixation, which enables the removal of disturbing effects of movements on rat neuronal activity. This reveals a much larger fraction of paw-tuned neurons, which were previously masked by body posture tuning.