Protein-based sensors of excitatory synaptic activity

描述(由申请人提供)：这项资助计划为主要的兴奋性神经递质谷氨酸产生基于蛋白质的、可遗传编码的传感器，并原位表达这些融合蛋白，以研究兴奋性突触信号的时空方面。将产生一系列基于神经元谷氨酸转运体(EAAT3或EAAT4)和GFP离子敏感变体融合的结构。传感器结构将分三个阶段进行评估。首先，将在HEK细胞中确定每种信号的特征，以确定谷氨酸是否引起光信号，如果是，则确定pH和氯的敏感性以及这些信号的动力学特性(目标1)。下一步，可行的构建体将被插入适合在大鼠大脑中表达的病毒载体中。将测试构建物响应兴奋性突触活动而产生光信号的能力(目标2)。结合光学和电生理测量，构建将被用来在不同条件下将活跃的突触输入的空间模式映射到神经元(目标3)。这些实验将共同开发和建立一类可遗传编码的兴奋性突触传递指标的实用价值。一旦开发出来，这些指示器有望对神经回路内信息处理的研究产生重要影响，允许对阈值下突触信号进行微创测量。

DESCRIPTION (provided by applicant): This grant proposes to generate protein-based, genetically-encodable sensors for the primary excitatory neurotransmitter glutamate and to express these fusion proteins in situ to study spatiotemporal aspects of excitatory synaptic signaling. A series of constructs based on fusion of a neuronal glutamate transporter (EAAT3 or EAAT4) and ion-sensitive variants of GFP will be generated. Sensor constructs will be evaluated in three stages. First, each will be characterized in HEK cells to establish whether glutamate elicits optical signals, and if so to determine the pH and chloride sensitivity and the kinetic properties of those signals (Aim 1). Next, viable constructs will be inserted into viral vectors suitable for expression in rat brain. The ability of constructs to generate optical signals in response to excitatory synaptic activity will be tested (Aim 2). Combining optical and electrophysiological measurements, constructs will be used to map the spatial pattern of active synaptic inputs to neurons under various conditions (Aim 3). Together these experiments will develop and establish the practical usefulness of a class of genetically-encodable indicators of excitatory synaptic transmission. Once developed, such indicators are expected to have an important impact on the study of information processing within neural circuits, allowing minimally invasive measurement of subthreshold synaptic signals.