PROJECT SUMMARY Most corticospinal fibers cross the midline to form the contralateral corticospinal tract (cCST). However, approximately 10% of them project ipsilaterally to form the ipsilateral corticospinal tract (iCST). The iCST plays a role in recovery from unilateral stroke or other forms of motor cortical injury by allowing the undamaged cortical hemisphere to compensate for the damaged one. Despite the clinical importance of the iCST, its naturalistic function and detailed anatomy are unknown. Individuals recovering from unilateral hemispheric injury experience erroneous bilateral and symmetrical movements aptly termed “mirror movement disorders.” Transcranial magnetic stimulation studies have linked the relative strength of the iCST and the severity of these mirror movements. Furthermore, mutant animals with more robust iCSTs perform bilateral adaptive motor behaviors more than wild-type animals. This evidence led to the hypothesis that the iCST is involved in the coordination of bilateral behaviors. This project uses a combination of viral tracing methods, chemogenetic behavioral experiments, single nucleus sequencing, and sequencing-based neuronal reconstruction to test the hypothesis that the iCST coordinates bilateral behavior. In preliminary data, a novel strategy is developed combining monosynaptic anterograde tracing and single nucleus sequencing to map the postsynaptic targets of the iCST and cCST. Preliminary results from this method suggested top-down differences in the architecture of the two tracts, with the iCST projecting more so to interneurons involved in motor control and the cCST more so toward those involved in sensory modulation. This lead to the hypothesis that the iCST comprises bilaterally-projecting corticospinal neurons. Such bifurcated neurons would provide a structural justification for the link between the iCST and bilateral motor behavior: bilateral neurons are naturally well-situated to facilitate bilateral behavior. With MAPSeq, a method of anterograde viral tracing using molecular barcodes, as well as dual-fluorescence retrograde tracing, preliminary data indicated that the iCST comprises primarily of bilaterally-projecting neurons. In addition to ensuring these findings are robust, this project will directly investigate the role of the iCST in motor behavior by using chemogenetic silencing of the iCST and markerless pose estimation in a well-established behavioral paradigm of adaptive bilateral movement.