Dynamic Imaging of Synaptic Inhibition

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。本项目的目标是采用clomeleon，一种基因编码的指示蛋白，用于成像大脑中cl依赖性突触抑制。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（FRET）依赖发射的比例与细胞内氯离子（[Cl-]i）的浓度成正比。提出的实验将使用一种遗传策略来靶向小鼠大脑中神经元亚群的克隆克隆表达。这将允许对Cl-的生理功能进行许多新颖的实验分析。在本文提出的第一阶段实验中，荧光成像方法将用于寻找与海马、小脑和皮质组织切片神经元中抑制性突触通路激活相关的静息[Cl-] I的神经元特异性变化和[Cl-] I的变化。在第二阶段，这些实验程序将扩展到体内条件，以成像完整大脑神经网络中突触抑制的时间和空间模式。这项新技术应该提供大脑突触抑制动力学的第一个实验观点，并提供了阐明在正常功能和药物滥用期间大脑活动的许多重要特征的希望。

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.