Profiling presynaptic scaffolds using split-GFP reconstitution reveals cell-type-specific spatial configurations in...

Characterization of intracellular synapse heterogeneity aids in understanding the intricate computational logic of neuronal circuits. Despite recent advances in connectomics, the spatial patterns of synapses and their inter-individual variability remain largely unknown. Using directed split-GFP reconstitution, we achieved visualization of endogenous Bruchpilot (Brp), a presynaptic active zone (AZ) scaffold protein, in a cell-type-specific manner. By developing a high-throughput quantification pipeline, we profiled AZ structures in identified neurons of the mushroom body circuit, where intracellular synaptic patterns are crucial due to compartmentalized connectivity. Quantitative characterization of the pattern of Brp clusters across multiple individuals revealed cell-type-dependent synaptic heterogeneity and stereotypy. Furthermore, we discovered previously unidentified sub-compartmental synapse configuration and its transient structural plasticity triggered by associative learning. These profiles reveal multilayered spatial configurations of AZs, from stereotyped overall AZ distribution patterns to local arrangements of neighboring synapses.