Optical measurement of synaptic glutamate spillover and reuptake by linker optimized glutamate-sensitive fluorescent reporters
Optical measurement of synaptic glutamate spillover and reuptake by linker optimized glutamate-sensitive fluorescent reporters
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DOI：
10.1073/pnas.0712008105
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发表时间：
2008-03-18
影响因子：
11.1
通讯作者：
Tsien, Roger Y.
中科院分区：
文献类型：
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作者：
Hires, Samuel Andrew;Zhu, Yongling;Tsien, Roger Y.
基因编码的谷氨酸浓度传感器是基于蓝色和黄色荧光蛋白之间的FRET包围细菌谷氨酸结合蛋白。由于在生理缓冲液中或在神经元细胞表面上表达时的响应幅度差，这种传感器尚未在神经元中找到定量应用。我们已经改进了我们的谷氨酸传感荧光报告（GluSnFR）的系统优化接头序列和谷氨酸的亲和力。使用SuperGluSnFR，它表现出6.2倍的响应幅度比原来的GluSnFR增加，我们证明了定量光学测量的时间过程中的突触谷氨酸释放，溢出，并在培养的海马神经元与微秒的时间和脊柱大小的空间分辨率的再摄取。在突发发射期间，功能上显著的溢出持续数百毫秒。这些谷氨酸水平似乎足以引发NMDA受体，可能影响树突棘的启动和计算。刺激频率依赖的溢出调制表明非突触神经元通信的机制。
Genetically encoded sensors of glutamate concentration are based on FRET between cyan and yellow fluorescent proteins bracketing a bacterial glutamate-binding protein. Such sensors have yet to find quantitative applications in neurons, because of poor response amplitude in physiological buffers or when expressed on the neuronal cell surface. We have improved our glutamate-sensing fluorescent reporter (GluSnFR) by systematic optimization of linker sequences and glutamate affinities. Using SuperGluSnFR, which exhibits a 6.2-fold increase in response magnitude over the original GluSnFR, we demonstrate quantitative optical measurements of the time course of synaptic glutamate release, spillover, and reuptake in cultured hippocampal neurons with centisecond temporal and spine-sized spatial resolution. During burst firing, functionally significant spillover persists for hundreds of milliseconds. These glutamate levels appear sufficient to prime NMDA receptors, potentially affecting dendritic spike initiation and computation. Stimulation frequency-dependent modulation of spillover suggests a mechanism for nonsynaptic neuronal communication.