Structural plasticity of spines at giant mossy fiber synapses

The granule cells of the dentate gyrus give rise to thin unmyelinated axons, the mossyfibers. They form giant presynaptic boutons impinging on large complex spines onthe proximal dendritic portions of hilar mossy cells and CA3 pyramidal neurons. Whilethese anatomical characteristics have been known for some time, it remained unclearwhether functional changes at mossy fiber synapses such as long-term potentiation(LTP) are associated with structural changes. Since subtle structural changes mayescape a fine-structural analysis when the tissue is fixed by using aldehydes and isdehydrated in ethanol, rapid high-pressure freezing (HPF) of the tissue was applied.Slice cultures of hippocampus were prepared and incubated in vitro for 2 weeks. Then,chemical LTP (cLTP) was induced by the application of 25mM tetraethylammonium (TEA)for 10min. Whole-cell patch-clamp recordings from CA3 pyramidal neurons revealeda highly significant potentiation of mossy fiber synapses when compared to controlconditions before the application of TEA. Next, the slice cultures were subjected to HPF,cryosubstitution, and embedding in Epon for a fine-structural analysis. When comparedto control tissue, we noticed a significant decrease of synaptic vesicles in mossy fiberboutons and a concomitant increase in the length of the presynaptic membrane. On thepostsynaptic side, we observed the formation of small, finger-like protrusions, emanatingfrom the large complex spines. These short protrusions gave rise to active zones that wereshorter than those normally found on the thorny excrescences. However, the total numberof active zones was significantly increased. Of note, none of these cLTP-induced structuralchanges was observed in slice cultures from Munc13-1 deficient mouse mutants showingseverely impaired vesicle priming and docking. In conclusion, application of HPF allowedus to monitor cLTP-induced structural reorganization of mossy fiber synapses.