Investigation of Ca-dependent cellular functions with caged compounds

树突棘作为兴奋性突触连接的优先位点，是多形性的。为了解决树突棘的结构-功能关系，我们使用谷氨酸的双光子释放来允许在单个突触水平上绘制功能性谷氨酸受体。我们对CA1锥体神经元棘的分析表明，AMPA （α-氨基-3-羟基-5-甲基-4-异恶唑ep - ropionic acid）型谷氨酸受体在蘑菇棘中含量丰富（高达150个/棘），但在细棘和丝状足中分布较少。后者可能是沉默突触的结构底物，沉默突触在突触传递的发育和可塑性中起作用。我们的数据表明，功能性AMPA受体的分布与脊柱几何形状密切相关，受体活性在单个脊柱水平上独立调节。用双光子激发成像技术研究了小鼠完整胰岛的胰岛素分泌。胰岛素颗粒的胞吐主要发生在间隙，远离血管。融合孔异常稳定，寿命为1.8秒。在1.4纳米直径的孔打开之前，脂质沿着孔内壁无限制地横向扩散，这支持了这种结构由膜脂组成的观点。当孔隙扩展到12纳米时，颗粒迅速衰减并排出其所含物质。因此，我们的方法在纳米分辨率下揭示了完整组织中的融合孔隙动力学。

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.