V2b neurons act via multiple targets to produce in phase inhibition during locomotion

Spinal interneurons shape motor neuron activity. Gata3⁺ V2b neurons are a major inhibitory spinal population. These neurons are present at multiple spinal levels in mice, suggesting an important function in motor control. In zebrafish, our previous work showed that V2b neurons are evenly distributed along the spinal cord, where they act to slow down locomotion. However, the timing of V2b activity during locomotion, their postsynaptic targets other than motor neurons, and their recruitment across different behaviors remain unknown. In this study, we address these questions using larval zebrafish. First, via optogenetic mapping of output in the rostrocaudal axis, we demonstrate that V2b neurons robustly inhibit motor neurons and other major spinal populations, 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 inhibition of V1 neurons. Next, by recording V2b activity during different visually and electrically evoked movements, we show that V2b neurons are specifically recruited for forward swims and turns, but not for fast escape movements. Furthermore, a subset of V2b neurons also exhibited short-latency sensory-evoked activity preceding motor initiation. Finally, we show that V2b inhibition occurs in phase with the leading edge of the motor burst, in contrast to V1 inhibition which occurs in phase with the falling edge of the motor burst. Taken together, these data show that in axial motor networks, V2b neurons act via multiple targets to produce in phase, leading inhibition during locomotion.Significance statement Spinal interneurons are critical for executing and regulating movements. However, it has been challenging to understand their functions and interconnections because the spinal cord circuit is complex, with many long-range connections that are challenging to map. Using optogenetics in the larval zebrafish, we mapped the connectivity and activity of an inhibitory spinal population: V2b neurons. We show that V2b neurons not only inhibit motor neurons but also other major excitatory and inhibitory populations. With electrophysiology and calcium imaging, we recorded V2b activity during different behaviors and found that V2b neurons inhibit their targets on the rising phase of motor bursts, preferentially during slow locomotion. These results suggest that V2b neurons have a distinctive role in motor control.