Live Cell Imaging to Monitor Axonal Pruning in Drosophila Motor Neurons

Background

During the early stages of nervous system development, nerve branches and connections are excessively generated. This includes a number of branches of axons and dendrites far greater than what is needed in an adult individual. Neural pruning is the process by which unnecessary synaptic connections between neural cells are selectively eliminated during the development of the nervous system [1,2]. This process occurs primarily in childhood and adolescence and is an essential mechanism for the brain to efficiently and optimally function as it matures. It is worth noting that numerous neurodevelopmental disorders, like autism spectrum disorder (ASD) and schizophrenia, are considered to be correlated with aberrant neural pruning [3–7]. In autistic individuals, it was discovered that insufficient pruning of neuronal connections in certain areas of the brain may lead to abnormal functioning of neural circuits. Conversely, excessive pruning is a possible cause of schizophrenia [8]. By investigating the mechanisms of abnormal neural pruning, we can deeply explore the roots of the pathogenesis of these disorders.

During metamorphosis in the holometabolous insect Drosophila, early neurons undergo stereotypic pruning to re-establish adult-specific neural circuits. One widely recognized type IV dendritic arborization (C4da) sensory neuron that perceives pain in Drosophila undergoes dendrite-specific pruning during development [9–12]. In addition, the dendrites and axons of the mushroom body (MB) γ neuron in the central nervous system (CNS) of Drosophila are also known to undergo pruning [13–15]. Although MB γ neurons provide fundamental insights into the mechanisms of axonal pruning, they require fixed tissue analysis and present imaging challenges in dense central brains. Here, we describe a novel method to monitor the process of axonal pruning in Drosophila motor neurons. Using live-cell imaging, we found that axons in the middle part of Drosophila motoneurons complete their pruning by degradation during development, while their terminal neuromuscular junctions are pruned by retraction [16]. Thus, motor-neuron axon bundles project peripherally in a stereotyped array, allowing for real-time live imaging, providing characteristic temporal windows and mechanistic properties (fragmentation or degeneration), and supporting systematic genetic screening to find new regulators. On the other hand, the development of Drosophila motor neurons as a model for axon pruning complements the MB γ neuron system by offering a distinct neuronal context, technical advantages, and functional readouts. Overall, the establishment of this method provides an ideal platform for future investigations into the regulatory mechanisms of axon pruning.