Automated quantification of synaptic boutons reveals their 3D distribution in the insect mushroom body

Abstract

Synaptic boutons are highly plastic structures undergoing experience-dependent changes in their number, volume, and shape. Their plasticity has been intensively studied in the insect mushroom bodies by counting manually the number of boutons within small regions of interest and extrapolating this number to the volume of the mushroom body neuropil. Here we extend the analysis of synaptic bouton distributions within the mushroom body neuropil of honey bees (Apis mellifera) beyond sampling small volumes. This study, based on large 3D two-photon imaging combined with advanced image post-processing and multiple thresholds segmentation, suggests that previously reported results overestimated the number of synaptic boutons significantly. As a reason for that, we identified boundaries effects in the small volume samples. The application of the automated analysis to large volumes of the mushroom body neuropil revealed a corrected average density of synaptic boutons and, for the first time, their 3D spatial distribution. This distribution exhibited a considerable heterogeneity. This additional information on synaptic bouton distribution provides new dimensions to future studies on brain development, symmetry, and plasticity.