Investigation of Ca-dependent cellular functions with caged compounds

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Dendritic spines serve as preferential sites of excitatory synaptic connections and are pleomorphic. To address the structure- function relationship of the dendritic spines, we used two-photon uncaging of glutamate to allow mapping of functional glutamate receptors at the level of the single synapse. Our analyses of the spines of CA1 pyramidal neurons reveal that AMPA (α-amino-3-hydroxyl-5-methyl-4-isoxazolep ropionic acid)-type glutamate receptors are abundant (up to 150/spine) in mushroom spines but sparsely distributed in thin spines and filopodia. The latter may be serving as the structural substrates of the silent synapses that have been proposed to play roles in development and plasticity of synaptic transmission. Our data indicate that distribution of functional AMPA receptors is tightly correlated with spine geometry and that receptor activity is independently regulated at the level of single spines.Insulin secretion from intact mouse pancreatic islets was investigated with two-photon excitation imaging. Insulin granule exocytosis occurred mainly toward the interstitial space, away from blood vessels. The fusion pore was unusually stable with a lifetime of 1.8 seconds. Opening of the 1.4-nanometerdiameter pore was preceded by unrestricted lateral diffusion of lipids along the inner wall of the pore, supporting the idea that this structure is composed of membrane lipids. When the pore dilated to 12 nanometers, the granules rapidly attened and discharged their contents. Thus, our methodology reveals fusion pore dynamics in intact tissues at nanometer resolution.