ALL-OPTICAL DISSECTION OF A SPATIALLY ORGANIZED RETICULOSPINAL CIRCUIT FOR LOCOMOTOR INITIATION AND MAINTENANCE

Locomotion requires precise coordination of descending motor commands conveyed by reticulospinal neurons (RSNs). How these commands are transformed within the brainstem into spinal instructions remains unclear. Here, we leverage the transparent, genetically accessible larval zebrafish to record and optogenetically manipulate a major RSN population expressing the transcription factor vsx2. Volumetric calcium imaging identifies two clusters of vsx2⁺ neurons in the retropontine region and caudal medulla that are recruited at movement onset and are here termed Start/Maintain neurons. Optical backfilling reveals that neurons in both clusters project beyond the mid-spinal cord. To probe function, we combined visible holographic optogenetics with behavioral recordings to selectively stimulate a thin column of RSNs within these clusters. Brief unilateral activation within the retropontine Start/Maintain cluster produced rapid large-amplitude ipsilateral tail bends, sometimes followed by tail oscillations. In contrast, the same unilateral stimulation in the caudal Start/Maintain cluster is sufficient to evoke sustained symmetric tail beats resembling forward swimming. Ongoing voltage imaging enables parallel recordings from over 100 vsx2⁺ neurons during fictive locomotion, revealing distinct firing patterns between Start and Maintain neurons that are organized along the rostrocaudal axis. Single-cell morphological reconstructions support a model where retropontine Start neurons deliver descending signals to caudal medullary Maintain neurons, which in turn sustain rhythmic activity through recurrent connectivity and spinal inputs. Together, these findings support a spatially organized reticulospinal circuit that transforms rostral initiation and steering signals into sustained motor drive in the caudal hindbrain.