Dynamic imaging of synaptic inhibition in the brain

大脑突触抑制的动态成像

• 批准号: 7020736
• 负责人: GEORGE J. AUGUSTINE
• 金额: $ 33.84万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7020736
• 关键词: amygdala; bioimaging /biomedical imaging; brain imaging /visualization /scanning; cerebellar Purkinje cell; cerebellum; chloride ion; fluorescence resonance energy transfer; fluorescent dye /probe; genetically modified animals; ionophores; laboratory mouse; superior colliculus; synapses

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.

描述（由申请人提供）：虽然许多方法可用于脑中兴奋性过程的功能成像，但直到现在还没有对脑中突触抑制进行成像的实用方法。该项目的目标是使用Clomeleon，一种遗传编码的指示蛋白，来成像大脑中的氯依赖性突触抑制。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依赖性发射的比率与氯离子（[Cl]i）的细胞内浓度成比例地变化。拟议的实验旨在优化Clomeleon技术成像的空间和时间动态的抑制电路在活的脑组织。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在与突触抑制相关的范围内报告[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这些克隆龙表达小鼠的小脑、杏仁核和上级丘切片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。这项新技术应该提供第一个空间分辨的意见，在大脑中的突触抑制的动态，并提供了阐明许多重要功能的大脑活动在正常的功能，精神和神经系统疾病，并作为药物滥用的结果的承诺。