Classifying Drosophila olfactory projection neuron boutons by quantitative analysis of electron microscopic reconstruction

In Drosophila melanogaster, olfactory projection neurons (PNs) convey odor information from the antenna lobe to higher brain regions. Recent transcriptomic studies reveal a large diversity of transcription factors, cell-surface molecules, neurotransmitter-coding, and neuropeptide-coding genes in PNs; however, their structural diversity remains unknown. Herein, we achieved a volumetric reconstruction of 89 PN boutons under Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) and quantitatively analyzed the internal presynaptic active zones (PAZs) and dense-core vesicles (DCVs). The ultrastructure-based cluster analysis reveals three morphological distinct bouton subtypes: complex boutons, unilobed boutons, and simple boutons. The complex boutons contain the most PAZs and DCVs, which suggests that they are of the highest capability of releasing neurotransmitters and neuromodulators. By labeling a subset of boutons under FIB-SEM, we found that DCVs are preferentially distributed in certain GH146-positive subtypes. Our study demonstrates that PN boutons display distinct morphology, which may determine their capacity of releasing neurotransmitters and neuromodulators.