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