Faculty Bibliography

The mossy fiber (MF)-CA3 synapse in the hippocampus is unique in the CNS because of its wide dynamic range of transmitter release during short- and long-term plasticity. The presynaptic mechanisms underlying the fidelity of transmission were investigated for the MF-CA3 synapses. The relative size of readily releasable pool (RRP) of vesicles was estimated by counting the number of docked vesicles at an active zone (AZ) on the transmission electron microscopy (TEM) image. The size of the releasable pool and the exo-endocytosis kinetics were directly measured from individual large MF boutons in hippocampal slices of transgenic mice that selectively express synaptopHluorin (SpH), a pH-sensitive GFP fused to the lumenal aspect of one of the vesicular membrane proteins, VAMP-2, in these boutons. Here we found (1) there are distinct two vesicle pools, the resting pool which is resistant to exocytosis, and the releasable pool, (2) the initially docked vesicles are easily depleted and the RRP is maintained by refilling from the reserve subpopulation of releasable pool ("reserve" releasable pool), and (3) the contribution of rapid reuse of recycled vesicles is relatively small. Therefore, the fidelity of transmission is suggested to be ensured by the rapid refilling rate of RRP.

We generated six transgenic mouse lines in which synaptopHluorin (SpH), one of green fluorescent protein-based sensors of vesicular exocytosis, was expressed under the control of neuron-specific Thy-1.2 promoter. In situ hybridization study revealed that SpH mRNA was expressed in a broad spectrum of brain regions in four of them, whereas in others it was expressed in the specific regions of the hippocampus. In one particular line, SpH immunoreactivity was specifically observed in the mossy fiber presynaptic terminals of both hippocampus and cerebellar cortex. The fluorescence intensity of these presynaptic terminals was somewhat decreased by acidic buffer superfusion and greatly increased by vesicular neutralization of pH, indicating that the SpH molecules are mainly distributed in the synaptic vesicles. The exocytosis-dependent fluorescence increment was measured upon activation of a single presynaptic terminal. These transgenic lines are expected to facilitate morphological and physiological studies of presynaptic terminals in a variety of regions of the brain.