Dynamic Imaging of Synaptic Inhibition

突触抑制的动态成像

• 批准号: 6535592
• 负责人: GEORGE J. AUGUSTINE
• 金额: $ 30.8万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-10 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6535592
• 关键词: brain cell; brain imaging /visualization /scanning; brain mapping; cerebellum; cerebral cortex; chloride ion; confocal scanning microscopy; developmental neurobiology; fluorescence resonance energy transfer; fluorescent dye /probe; gene expression; genetically modified animals; genome; hippocampus; ionophores; laboratory mouse; nerve /myelin protein; neural information processing; neurons; reagent /indicator; synapses; synaptogenesis; transposon /insertion element; voltage /patch clamp

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.

虽然许多方法可用于脑中兴奋过程的功能成像，但直到现在还没有实用的方法来成像脑中的突触抑制。该项目的目标是采用clomeleon，一种遗传编码的指示蛋白，用于成像脑中Cl依赖性突触抑制。该指示剂是通过将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产生的;这两种荧光团的荧光共振能量转移（FRET）依赖性发射的比率与细胞内氯离子浓度（[Cl-]i）成比例地变化。拟议的实验将使用遗传策略将克隆蛋白的表达靶向小鼠大脑中的神经元亚群。这将允许许多新的实验分析的生理功能的Cl-。在这里提出的第一阶段实验中，荧光成像方法将被用来寻找神经元特异性的变化，在休息[Cl-]i和[Cl-]i的变化与海马，小脑和皮质组织切片的神经元中的抑制性突触通路的激活。在第二阶段，这些实验程序将扩展到在体内条件下成像的时间和空间模式的突触抑制在神经网络的完整的大脑。这项新技术应该提供了第一个实验的观点突触抑制的动力学在大脑中，并提供了承诺，阐明许多重要的功能，在正常的大脑活动，并作为药物滥用的后果。