Axonal Barcode Analysis of Pyramidal Tract Projections from Mouse Forelimb M1 and M2.

Forelimb-related areas of the motor cortex communicate directly to downstream areas in the brainstem and spinal cord via axons that project to and through the pyramidal tract (PT). To better understand the diversity of the brainstem branching patterns of these pyramidal tract projections, we used MAPseq, a molecular barcode technique for population-scale sampling with single-axon resolution. In experiments using mice of both sexes, we first confirmed prior results demonstrating the basic efficacy of axonal barcode identification of primary motor cortex (M1) PT-type axons, including corticobulbar (CBULB) and corticospinal (CSPI) subclasses. We then used multiplexed MAPseq to analyze projections from M1 and M2 (caudal and rostral forelimb areas). The four basic axon subclasses comprising these projections (M1-CSPI, M1-CBULB, M2-CSPI, M2-CBULB) showed a complex mix of differences and similarities in their brainstem projection profiles. This included relatively abundant branching by all classes in the dorsal midbrain, by M2 subclasses in the pons, and by CSPI subclasses in the dorsal medulla. Cluster analysis showed graded distributions of the basic subclasses within the PT class. Clusters were of diversely mixed subclass composition and showed distinct rostrocaudal and/or dorsomedial projection biases. Exemplifying these patterns was a subcluster likely enriched in corticocuneate branches. Overall, the results indicate high yet systematic PT axon diversity at the level of brainstem branching patterns; projections of M1 and M2 appear qualitatively similar, yet with quantitative differences in subclasses and clusters. Axons of the PT class of cortical projection neurons, which includes corticospinal and corticobulbar neurons, anatomically link motor cortex to brainstem and spinal cord circuits. Both of these subclasses can form branches to brainstem destinations along the way, but the extent and diversity of these branching patterns is incompletely understood. Here, we used MAPseq to tag PT axons with individual molecular barcodes for high-throughput quantification of branching patterns across the brainstem. The results reveal diverse, complex, yet systematic branching patterns of corticospinal and corticobulbar neurons arising from two motor cortex areas, M1 and M2.