Protein-based sensors of excitatory synaptic activity

描述（由申请人提供）：该资助计划为主要兴奋性神经递质谷氨酸生成基于蛋白质的、遗传可编码的传感器，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构建体。传感器结构将分三个阶段进行评估。首先，将在HEK细胞中对每种细胞进行表征，以确定谷氨酸是否引发光信号，如果是，则确定pH和氯化物敏感性以及这些信号的动力学性质（Aim 1）。接下来，将有活力的构建体插入到适合在大鼠脑中表达的病毒载体中。构建体产生光信号以响应兴奋性突触活动的能力将被测试（目的2）。结合光学和电生理测量，构建将用于绘制各种条件下神经元活跃突触输入的空间模式（目的3）。这些实验将共同发展和建立一类兴奋性突触传递的遗传可编码指标的实际用途。一旦开发出来，这些指标有望对神经回路内信息处理的研究产生重要影响，使阈下突触信号的微创测量成为可能。

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.