Dynamic imaging of synaptic inhibition in the brain

描述（由申请人提供）：虽然有许多方法可用于大脑兴奋性过程的功能成像，但到目前为止，还没有实用的方法来成像大脑中的突触抑制。这个项目的目标是使用Clomeleon，一种基因编码的指示蛋白，来成像大脑中氯依赖的突触抑制。该指示剂是由氯化物敏感的黄色荧光蛋白与氯化物不敏感的青色荧光蛋白融合而成；这两种荧光团的荧光共振能量转移依赖发射的比例与细胞内氯离子（[Cl]i）的浓度成正比。本实验旨在优化Clomeleon技术，以成像活脑组织中抑制回路的时空动态。首先，我们将使用诱变来改变Clomeleon的氯离子结合特性。这将增强Clomeleon在突触抑制相关范围内报告[Cl]i变化的能力。其次，我们将使用两种遗传策略将Clomeleon的表达靶向到小鼠大脑中的神经元亚群。第三，我们将使用荧光成像方法来测量与表达clomeleon的小鼠小脑、杏仁核和上丘神经元中抑制性突触通路激活相关的[Cl]i的变化。虽然这些实验程序将在体外使用，但希望我们对Clomeleon技术的改进最终将允许对完整大脑神经网络中突触抑制的时间和空间模式进行成像。这项新技术将提供大脑突触抑制动力学的第一个空间解析视图，并为阐明在正常功能、精神和神经疾病以及药物滥用期间大脑活动的许多重要特征提供了希望。

DESCRIPTION (provided by applicant): 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 use Clomeleon, a genetically-encoded indicator protein, to image chloride-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 dependent emission of these two fluorophores varies in proportion to the intracellular concentration of chloride ions ([Cl]i). The proposed experiments are designed to optimize the Clomeleon technique for imaging the spatial and temporal dynamics of inhibitory circuits in living brain tissue. First, we will use mutagenesis to change the chloride binding properties of Clomeleon. This will enhance the ability of Clomeleon to report changes in [Cl]i in the range relevant for synaptic inhibition. Second, we will use two genetic strategies to target expression of Clomeleon to subsets of neurons in the mouse brain. Third, we will use fluorescence imaging methods to measure changes in [Cl]i associated with activation of inhibitory synaptic pathways in neurons of slices of the cerebellum, amygdala, and superior colliculus from these Clomeleon-expressing mice. While these experimental procedures will be used in vitro, it is hoped that our improvements in Clomeleon technology eventually will allow imaging of the temporal and spatial patterns of synaptic inhibition in neural networks of the intact brain. This new technology should provide the first spatially-resolved 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, during psychiatric and neurological disorders, and as a consequence of drug abuse.