Three-dimensional quantification of mossy-fiber presynaptic boutons in living hippocampal slices using confocal...

Confocal laser scanning microscopy (CLSM) was used to visualize presynaptic elements of mossy-fiber synapses in living rat hippocampal slices. Mossy fiber (mf) axons and their boutons were labeled in transverse hippocampal slices by injecting one of three fluorescent dyes (diI, diA, or fast diI) into stratum granulosum of the dentate gyrus and/or stratum lucidum of CA3. Three-dimensional (3D) images of the mf boutons were obtained from serial optical sections in stratum lucidum. The 3D data were used to quantify and characterize the size and shape of the boutons based on their volumes (V) and surface areas (A), and the latter were compared with conventional 2D analyses. Various geometric models were fitted to the V-A relationship, one of which provided a reasonable approximation to the data. The results demonstrate that this approach is useful for quantifying and characterizing the size and shape of mf expansions and raise the possibility of detecting and analyzing in real time activity-dependent modifications in presynaptic structure.