Reconfiguration of Premotor Excitation and Inhibition Drives Behavior-Specific Protopodium Dynamics in Drosophila Larvae

Abstract Animals use the same muscles to generate distinct locomotor behaviors, raising the question of how shared motor systems produce behavior-specific outputs. In Drosophila larvae, forward and backward crawling recruit largely overlapping motor pools but differ in the timing of ventral oblique (VO) muscle activation, which occurs late in forward crawling and early in backward crawling. Although this timing difference has been described, the circuit mechanisms that impose it—and its functional significance—have remained unclear. Here, we show that VO muscles are primary drivers of protopodium movement, a limb-like structure involved in substrate engagement during crawling, and that their behavior-specific activation timing is controlled by a tri-segmental premotor circuit. Using muscle calcium imaging, optogenetic and connectome-based circuit analysis, and biomechanical modeling, we identify a feedforward inhibitory motif composed of two previously characterized excitatory premotor neurons (A27h and A18b3) and the inhibitory premotor neuron A06c. During forward crawling, A06c exhibits a biphasic activity pattern that defines a narrow temporal window for VO contraction and prevents premature, multi-segmental VO activation. During backward crawling, this premotor configuration is disengaged: A27h and A18b3 are inactive, and the initial inhibitory peak of A06c is absent, permitting earlier VO activation. Disrupting this circuit causes premature VO activation, impaired protopodium folding, compressed intersegmental coordination, and reduced forward crawling efficiency. Together, our findings show how reconfiguration of premotor excitation and inhibition generates behavior-specific muscle dynamics within a shared motor system. Competing Interest Statement The authors have declared no competing interest.