Live Cell Imaging to Monitor Axonal Pruning in <em>Drosophila</em> Motor Neurons

Over the lifespan of an individual, brain function requires adjustments in response to environmental changes and learning experiences. During early development, neurons overproduce neurite branches, and neuronal pruning removes the unnecessary neurite branches to make a more accurate neural circuit. <i>Drosophila</i> motoneurons prune their intermediate axon bundles rather than the terminal neuromuscular junction (NMJ) by degeneration, which provides a unique advantage for studying axon pruning. The pruning process of motor axon bundles can be directly analyzed by real-time imaging, and this protocol provides a straightforward method for monitoring the developmental process of <i>Drosophila</i> motor neurons using live cell imaging. Key features • Long-range projecting axon bundles of <i>Drosophila</i> motor neurons extending from soma on the ventral nerve cord (VNC) undergo degeneration rather than retraction during metamorphosis. • The pruning process of motor axon bundles can be directly observed by real-time live-cell imaging. • The complete clearance of axon bundles occurs approximately 22 h after pupal formation (22 h APF). • Mushroom body (MB) γ neuron axon pruning regulatory genes are conserved for motor neurons.