Accurate tracking of locomotory kinematics in mice moving freely in three-dimensional environments

Abstract

Marker-based motion capture (MBMC) is a powerful tool for precise, high speed, three-dimensional tracking of animal movements, enabling detailed study of behaviors ranging from subtle limb trajectories to broad spatial exploration. Despite its proven utility in larger animals, MBMC has remained underutilized in mice due to the difficulty of robust marker attachment during unrestricted behavior. In response to this challenge, markerless tracking methods, facilitated by machine learning, have become the standard in small animal studies due to their simpler experimental setup. However, trajectories obtained with markerless approaches at best approximate ground-truth kinematics, with accuracy strongly dependent on video resolution, training dataset quality, and computational resources for data processing.

Here, we overcome the primary limitation of MBMC in mice by implanting minimally invasive markers that remain securely attached over weeks of recordings. This technique produces high-resolution, artifact-free trajectories, eliminating the need for extensive post-processing. We demonstrate the advantages of MBMC by resolving subtle drug-induced kinematic changes that become apparent only within specific behavioral contexts, necessitating precise three-dimensional tracking beyond simple flat-surface locomotion. Furthermore, MBMC uniquely captures the detailed spatiotemporal dynamics of harmaline-induced tremors, revealing previously inaccessible correlations between body parts and thus significantly improving the translational value of preclinical tremor models. While markerless tracking remains optimal for many behavioral neuroscience studies in which general posture estimation suffices, MBMC removes barriers to investigations demanding greater precision, reliability, and low-noise trajectories. This capability significantly broadens the scope for inquiry into the neuroscience of movement and related fields.

Significance statement Studying fine-scale motor behaviors in mice demands data with precision and fidelity that markerless approaches often struggle to provide. While marker-based motion capture is the gold standard for high-resolution kinematic analysis, its use in freely moving mice has been limited by challenges in marker use. This work overcomes these barriers by introducing implantable markers with replaceable reflective heads, fundamentally transforming the feasibility of robust high-definition 3D tracking across a wide range of behaviors and experimental conditions. By enabling the detection of subtle phenomena, such as harmaline-induced tremors, with spatiotemporal detail unmatched by markerless tracking, this approach provides a powerful tool for advancing studies of motor control and sensorimotor integration in rodents.