Dynamic Imaging of Synaptic Inhibition

虽然有许多方法可以用来对大脑中的兴奋过程进行功能成像，但到目前为止，还没有一种实用的方法来成像大脑中的突触抑制。该项目的目标是采用Clomeleon，一种遗传编码的指示蛋白，来成像大脑中依赖氯的突触抑制。这一指示剂是由氯敏感的黄色荧光蛋白和氯不敏感的青色荧光蛋白融合而成的；这两个荧光团的荧光共振能量转移(FRET)依赖发射的比率与细胞内氯离子浓度([Cl-]i)成正比。拟议中的实验将使用一种遗传策略，将Clomeleon的表达定向到小鼠大脑中的神经元亚群。这应该允许对氯离子的生理功能进行许多新颖的实验分析。在这里提出的第一阶段实验中，将使用荧光成像方法来寻找神经元特异性的静息[Cl-]i变化，以及与海马神经元、小脑和皮质组织切片中抑制性突触通路激活相关的[Cl-]i变化。在第二阶段，这些实验程序将扩展到活体条件下，以成像完整大脑神经网络中突触抑制的时间和空间模式。这项新技术应该会提供大脑中突触抑制动力学的第一个实验视图，并有望阐明正常功能期间和药物滥用导致的大脑活动的许多重要特征。

While many methods are available for functional imaging of excitatory processes in the brain, until now there has been no practical way to image synaptic inhibition in the brain. The goal of this project is to adopt clomeleon, a genetically-encoded indicator protein, for imaging Cl-dependent synaptic inhibition in the brain. This indicator was produced by fusing the chloride-sensitive yellow fluorescent protein with the chloride-insensitive cyan fluorescent protein; the ratio of fluorescence resonance energy transfer (FRET) dependent emission of these two fluorophores varies in proportion to the intracellular concentration of chloride ions ([Cl-]i). The proposed experiments will use a genetic strategy to target expression of clomeleon to subsets of neurons in the mouse brain. This should allow many novel experimental analyses of the physiological functions of Cl-. During the Phase I experiments proposed here, fluorescence imaging methods will be use to look for neuron-specific variations in resting [Cl-]i and changes in [Cl-]i associated with activation of inhibitory synaptic pathways in neurons of hippocampal, cerebellar, and cortical tissue slices. In Phase II, these experimental procedures will be extended to in vivo conditions to image the temporal and spatial patterns of synaptic inhibition in neural networks of the intact brain. This new technology should provide the first experimental views of the dynamics of synaptic inhibition in the brain and offers the promise of elucidating many important features of brain activity during normal function and as a consequence of drug abuse.