When threatened by dangerous or harmful stimuli, animals engage in diverse forms of rapid escape behavior. In Drosophila larvae, escape behavior is characterized by C-shaped bending and lateral rolling, followed by rapid forward crawling. The sensory circuitry that promotes escape has been extensively characterized, but the motor programs underlying escape are unknown. Here, we characterize the neuromuscular basis of escape. We use high-speed, volumetric, Swept Confocally-Aligned Planar Excitation (SCAPE) microscopy to image muscle activity during larval rolling. Unlike the sequential peristaltic muscle contractions from segment to segment that underlie crawling, muscle activity progresses in a circumferential sequence during bending and rolling. Certain muscle subgroups show functional antagonism during bending and rolling. We use EM connectome data to identify premotor to motor connectivity patterns that could drive rolling behavior and test the necessity of specific groups of motor neurons in rolling using neural silencing approaches. Our data reveal body-wide muscle activity patterns and putative premotor circuit organization for escape.

Animals can use a common set of muscles and motor neurons (MNs) to generate diverse locomotor behaviors, but how this is accomplished remains poorly understood. Previously, we characterized the muscle activity patterns for Drosophila larval forward and backward locomotion and found that ventral oblique (VO) muscles become active earlier in backward than in forward locomotion (Zarin et al. 2019). Here, we describe how premotor circuits generate differential activation timing of VO muscles. We identify inhibitory (A06c) and excitatory (A27h) premotor neurons (PMNs) with the greatest number of synapses with VO MNs. Strikingly, A06c is a bi-modal PMN that fires before and after VO MNs in forward locomotion but fires only after MNs in backward locomotion. Further, A27h is a forward-dedicated PMN active only in forward locomotion. These two PMNs interconnect with another forward-dedicated excitatory PMN (A18b3), to create feedforward inhibitory microcircuits that define the activity window for VO MNs/muscles, producing precise VO muscle patterns underlying forward locomotion. Silencing A06c, A27h or A18b3 PMN results in premature VO muscle activation in forward locomotion, resembling early VO activation in backward locomotion. Our results identify PMN micro-circuits that produce unique MN/muscle activity patterns to create behavior-specific motor output.