Extended Data Fig. 1. Mapping brainwide inputs to the spinal cord.

(A) Illustration of approach to visualize inputs to cervical spinal cord. (B) 3D reconstruction of inputs to spinal cord. Colors correspond to major brain divisions. (C) The top brain regions that project to spinal cord, determined by the relative fraction of total somata. Notable regions are indicated by colored bars. Insets illustrate exemplar brain regions with substantial labeling. Dashed boxes are colored to correspond to notable brain regions. The inset pie chart shows the major brain structures (“major divisions” as classified by the Allen Institute Mouse Common Coordinate Framework; see Methods) projecting to cervical spinal cord. N=3. (D–F) Micrographs of CSN axons expressing GFP (green) in transverse cross-sections of cervical (D), thoracic (E), and lumbar (F) spinal cord. The insets are high magnification images of GFP⁺ bulbous varicosities from different laminae of cervical (7Sp/8Sp), thoracic (7Sp/ICl), and lumbar (4Sp) segments. Neuronal processes expressing Cre.RFP are in red. Representative of N=3. (G) Quantification of cortical inputs to spinal cord (RFP⁺), divided by cortical region and laminae. The inset photomicrograph illustrates the L5b positioning of CSNs. Note that the Allen Brain Atlas classification did not subdivide L5 into L5a and L5b, and the position of corticospinal somata fell around the boundary between L5 and L6a. N=3. (H) Experimental strategy, same as Figure 1A. (I) The cortical regions giving rise to corticospinal somata (GFP⁺). Dark green bars represent the major regions; light green bars represent subdivisions of cortical regions. N=3. (J) Major brain regions containing GFP⁺ neurites. This includes dendritic processes in sensorimotor cortex. Grouping the many brain regions comprising these major structures reveals the intense innervation of several subcortical structures, including brainstem. (K) Photomicrograph of brainstem labeling by CSNs. N=3. (L) Experimental strategy to label synapses arising from CSNs. (M) Synaptophysin GFP (green) labeling in the brain. N=3. (N) Synaptophysin GFP (green) and FlpO (red) labeling in motor cortex. N=3. (O) Synaptophysin GFP (green) labeling in DLS. N=3. (P) Top brain regions to which CSNs project, excluding sensorimotor cortex and fiber tracts. N=2. Error bars are SEM.