Three‐dimensional quantification of mossy‐fiber. Presynaptic boutons in living hippocampal slices using confocal microscopy

Abstract

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.

Dual mechanism for presynaptic modulation by axonal metabotropic glutamate receptor at the mouse mossy fibre‐CA3 synapse

“…1). The fluorescent band was considered to represent labelled mossy fibres, since the labelled structure had the characteristic appearance of mossy fibres (Claiborne et al 1986; Regehr & Tank, 1991; Yu & Brown, 1994) for the following reasons: first, the fluorescence was restricted to the stratum lucidum, where mossy fibres exist (Fig. 1) and, in addition, large boutons of 3‐5 μm diameter, which were located en passant along the labelled fibres, could be seen under higher magnification (× 100 objective, Fig.…”

Retrograde Axonal Degeneration (“Dieback”) in the Corticospinal Tract after Transection Injury of the Rat Spinal Cord: A Confocal Microscopy Study

“…Many studies have taken advantage of the combination of DiI and confocal microscopy to examine detailed physiological phenomena such as changes in axon arrangement in the retinofugal pathway of mouse embryos (Chan and Chung, 1999), mossy fiber growth in live hippocampal slices (Yu and Brown, 1994) and even mapping of complex brain circuitry . Furthermore, investigators have used confocal imaging with DiI neuronal tracing to study projections of developing CST axons, and found that more detailed tracing of DiI-labeled axons was achievable and at higher contrast compared with HRP or WGA-HRP labeling in association with light microscopy (Nagashima, 1994).…”