Synapse profiling of identified Drosophila neurons revealed cell-type-specific spatial configurations of presynaptic active zones

Synapses are highly heterogeneous even within a single motor neuron in Drosophila melanogaster . In the central nervous system (CNS), the degree of stereotypy in the synaptic structure of specific neurons across individuals remains largely unexplored, due to the significant effort required to analyze multiple brain samples. Utilizing the split-GFP fluorescence-tagging, we achieved endogenous labeling of the presynaptic active zone (AZ) scaffold protein Bruchpilot (Brp) in a cell-type-specific manner. By developing a high-throughput quantification pipeline, we resolved single Brp clusters and characterized cell-type-specific AZ structures across individuals. We found that multiple structural parameters such as Brp localization and AZ size revealed significant intracellular synaptic heterogeneity and cell-type-dependent stereotypy in the mushroom body (MB) circuit. Furthermore, we found previously unidentified spatial configuration composed of neighboring AZs with similar properties. These findings therefore suggest multi-scale organizations of AZs, from neighboring synapses to across individuals.