Tonically active interneurons gate motor output in <i>Drosophila</i> larvae

Tonic inhibition and its transient removal are fundamental neural mechanisms for gating activity, suppressing out-of-phase excitation, and enhancing the temporal precision of behavior. However, their roles within locomotor central pattern generators (CPGs) remain poorly understood. Here, we identify A02l neurons-segmentally repeated excitatory interneurons in the <i>Drosophila</i> larval ventral nerve cord (VNC)-that regulate crawling behavior through tonic inhibition. A02l neurons exhibit high baseline activity and are transiently suppressed during motor output. They provide excitatory input to inhibitory premotor networks, enabling widespread silencing of motor neurons to maintain muscle relaxation. Optogenetic activation of A02l neurons reduced crawling frequency, whereas their inhibition induced transient whole-body contractions. Together, these findings reveal a cellular-resolution mechanism for tonic inhibition within the nerve cord and highlight its importance in gating motor output and maintaining motor quiescence.