Neuroscience

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. Drosophila 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 Drosophila motor neurons using live cell imaging.

In this protocol, we focused on analyzing internal branches of Drosophila class IV neurons. These neurons are characterized by their highly branched axons and dendrites and intricately tile the larval body. As Drosophila larvae progress through developmental stages, the dendritic arbors of Class IV neurons undergo notable transformations. As Drosophila larvae develop, their Class IV dendritic arbors grow. In the initial 24 h after egg laying (AEL), the dendrites are smaller than segments. During the subsequent 24 h of the first instar larval stage, dendritic arbors outpace segment growth, achieving tiling. After 48 h, arbors and segments grow concurrently. Epidermal cells near Class IV dendrites expand in proportion to segment growth. This observation suggested that Class IV cells might grow via branch dilation—uniformly elongating branches, akin to Class I cells [1,2]. To understand whether the class IV complex arbor structure is formed by dilation or simply from growing tips, we developed this protocol to introduce a systematic approach for quantitatively assessing the growth dynamics of internal branches.