Zarin, A. | bioRxiv Science

bioRxiv2026

Quantification of compensatory motor neuron sprouting at the Drosophila larval neuromuscular junction

Following nervous system injury, surviving bystander neurons may rewire and form synapses with new postsynaptic targets to restore circuit function. This protocol quantifies compensatory motor neuron sprouting at the Drosophila larval neuromuscular junction. We describe larval dissection, immunostaining, Airyscan imaging, image masking, muscle-specific region-of-interest selection, and Fiji-based quantification of Dlg-positive area, bouton number, mean Dlg-positive area per bouton, NMJ arbor length, axonal versus NMJ branch points, ectopic muscle acquisition, and sprouting penetrance. For complete details on the use and execution of this protocol, please refer to Olsen et al.1

neuroscience↗

bioRxiv2026

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

Animals use shared muscles and motor neurons to generate distinct movements, but how premotor circuits differentially recruit these neuromuscular components remains unclear. Drosophila larvae crawl forward and backward using overlapping motor pools, yet the timing of ventral oblique (VO) muscle activity differs between these behaviors. Here, we show that VO muscles are major contributors to the movement of protopodia, limb-like structures that help larvae interact with the crawling surface. Muscle imaging, perturbation, and modeling indicate that VO activity is particularly important for protopodium folding, whereas contractions of both VO and ventral longitudinal (VL) muscles contribute to protopodium stride length and crawling efficiency. We identify a tri-segmental premotor motif--comprising excitatory (A27h, A18b3) and inhibitory (A06c) neurons--that shapes VO timing during forward crawling. A06c exhibits two activity peaks during forward fictive locomotion, defining a constrained window of VO MN activation, whereas during backward locomotion the early A06c peak is absent and the excitatory PMNs A27h and A18b3 are inactive. Disrupting this motif shifts VO activity earlier, reduces intersegmental timing delays, increases temporal overlap of VO activity, and impairs forward protopodium folding, stride, and crawling efficiency. These findings show how premotor excitation and inhibition coordinate muscle timing to shape locomotor mechanics.

neuroscience↗