Whole-brain mapping of spinal-projecting neurons in larval zebrafish

To elicit voluntary movements and integrative reflexes underlying behavior, the brain sends command signals to the spinal cord via specialized long-range descending neurons, known as spinal-projecting neurons (SPNs). The vast and widespread distribution of SPNs, combined with their complex long-distance connectivity, poses a significant challenge for mapping their anatomical organization and associating specific populations with distinct functions. Here, we took advantage of the transparency and genetic accessibility of larval zebrafish to uncover the fundamental principles of SPN anatomical organization in a teleost. Using an optical backfilling method relying on photoactivable GFP, we generate a whole-brain map of all neurons sending axons toward the spinal cord. This approach reveals far more SPNs than previously described through conventional strategies, offering an unparalleled opportunity to revisit distinct spinal-projecting nuclei distributed across the hindbrain, midbrain, and diencephalic structures. Combining information on cell location, morphology, and projection patterns, we propose tentative homological designations for zebrafish of SPN nuclei based on established descriptions in mammals and other vertebrates.