Abstract Within the spinal cord, interneurons shape motor neuron activity. These interneurons can project over long distances in the longitudinal axis, but systematic mapping of their connectivity has been limited. In this study, using larval zebrafish, we mapped local and long-range connectivity of a cardinal spinal population, the Gata3 + V2b class. V2b neurons are inhibitory and project ipsilateral, descending axons. We show that V2b neurons are active during fictive swimming, slightly leading the motor burst. Via optogenetic mapping of output in the rostrocaudal axis, we demonstrate that V2b neurons robustly inhibit motor neurons and other major spinal interneuron classes, including V2a, V1, commissural neurons and other V2b neurons. V2b inhibition is patterned along the rostrocaudal axis, providing long-range inhibition to motor and V2a neurons but more localized innervation of the V1 class. Furthermore, these results provide the first demonstration of reciprocal V1/V2b inhibition in axial circuits, potentially representing an ancestral motif of the limb control network.

V2b neurons act via multiple targets in spinal motor networks