Abstract
Synapses, as specialised cell-cell contacts, allow for a faithful and controlled signal transmission between a neuron and a target cell. Presynapses, the sites of neurotransmitter release, form de novo throughout the development of an organism. Although this process is fundamental to the development and function of synaptic circuits, how developing neurons control number and distribution of individual synapses remains poorly understood. In-vivo imaging analysis of synapse formation at the neuromuscular junction of anaesthetised Drosophila third instar larvae allows for spatial and temporal resolution of the underlying molecular processes. However, high-throughput, comprehensive analysis are hampered by the manual and time-consuming imaging analysis methods applied hitherto. Here, we focus on the early presynaptic formation steps, that is, the presynaptic seeding, initiated by the formation of transient Liprin-α/SYD1 seeding sites, either stabilised or disintegrated over a time span of 30-90 min. To investigate the dynamics of the Liprin-α/SYD1 seeding sites, we developed an automated analysis pipeline for 3D confocal images from in-vivo imaging at distinct time points to analyse fluorescently labelled presynaptic protein dynamics during early synapse formation. The workflow is realised in the data analysis software Amira, utilising the hierarchical watershed algorithm, and was designed for automatic processing with an option for manual proofreading. Compared to the previous 2D manual quantification, this automated approach provides a higher sensitivity in single Liprin-α seeding site detection in low-intensity areas and in regions of dense seeding sites. In addition, it substantially reduces the work time. To account for possible errors occurring in the automated processing, we implemented an additional proofreading step allowing for a manual correction of Liprin-α seeding site segmentation and assignment, thus greatly improving the analysis while only marginally increasing work time by 10% to a total work time reduction of 70% compared to the 2D manual analysis paradigm. The process of synaptogenesis underlies the general principles of locomotion, learning and memory formation. The developed fast and accurate semi-automated 3D workflow will provide a substantial progress in the analysis of this molecular process.
