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，并且是多形性的。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以允许在单个突触水平上映射功能性谷氨酸受体。对CA 1区锥体神经元棘的分析表明，AMPA（α-amino-3-hydroxy-5-methyl-4-isoxazolep ropionic acid）型谷氨酸受体在蘑菇棘中含量丰富（达150个/棘），而在细棘和丝状伪足中分布稀疏。后者可能是沉默突触的结构基质，沉默突触被认为在突触传递的发育和可塑性中发挥作用。我们的数据表明，AMPA受体的功能分布与脊柱的几何形状密切相关，受体活性是独立调节的单spines.Insulin分泌完整的小鼠胰岛的双光子激发成像研究。胰岛素颗粒胞吐主要发生在远离血管的间质空间。融合孔异常稳定，寿命为1.8秒。在1.4纳米直径的孔的开放之前，脂质沿着孔的内壁不受限制地侧向扩散，支持这种结构由膜脂质组成的想法。当孔扩张到12纳米时，颗粒迅速变细并排出其内容物。因此，我们的方法揭示了纳米分辨率的完整组织中的融合孔动力学。

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.