3-DIMENSIONAL QUANTIFICATION OF MOSSY-FIBER PRESYNAPTIC BOUTONS IN LIVING HIPPOCAMPAL SLICES USING CONFOCAL MICROSCOPY

Confocal laser scanning microscopy (CLSM) was used to visualize presyn aptic elements of messy-fiber synapses in living rat hippocampal slice s. Messy fiber (mf) axons and their boutons were labeled in transverse hippocampal slices by injecting one of three fluorescent dyes (diI, d iA, or fast diI) into stratum granulosum of the dentate gyrus and/or s tratum lucidum of CA3. Three-dimensional (3D) images of the mf boutons were obtained hom 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 latt er were compared with conventional 2D analyses. Various geometric mode ls were fitted to the V-A relationship, one of which provided a reason able approximation to the data. The results demonstrate that this appr oach is useful for quantifying and characterizing the size and shape o f mf expansions and raise the possibility of detecting and analyzing i n real time activity-dependent modifications in presynaptic structure. (C) 1994 Wiley-Liss, Inc.