Multiplex Imaging of Brain Activity and Plasticity with Optimized FRET/FLIM-based Sensors

项目摘要 可塑性是所有物种神经回路的一个基本方面。它位于 学习和记忆，感觉适应，以及许多疾病相关的过程，如成瘾， 慢性疼痛或再生。在分子水平上， 描述，但很少有人知道这些是如何协调在空间和时间内神经元 活体大脑的回路为了阐明可塑性在体内的回路运作，我们在这里提出， 开发和验证高灵敏度的红色FRET/FLIM传感器，可与绿色传感器同时使用 钙传感器，允许神经元钙活动和生化信号的成像 单个突触和神经元群体的动力学。我们将专注于塑料连接 参与突触可塑性和脊柱形态发生的生化事件。FRET/FLIM 将允许精确测量微小的变化，即使在大脑存在的情况下， 行为过程中的动作。在目标1中，我们将开发高灵敏度的红光发射FRET/FLIM 使用基于哺乳动物细胞的蛋白质文库和结构引导的大规模 筛选目标2将涉及离体和体内传感器的验证。最后，传感器将 经验证可用于空间导航任务中突触和神经元可塑性的研究， 清醒的老鼠我们计划进行迭代周期的改进，其中生物物理分析的输入 的传感器，切片和体内验证以及早期推出用户的反馈， 动物模型将被用于创建连续的传感器世代， 性能结合2 P FRET/FLIM的体内可塑性和2 P荧光成像 钙活性将为研究突触和神经元的可塑性提供一种强有力的新方法， 活的有机体。

Project Summary Plasticity is a fundamental aspect of neuronal circuits across all species. It is at the base of learning and memory, sensory adaption, and many disease-related processes such as addiction, chronic pain or regeneration. On the molecular level biochemical mechanisms have been well described, but little is known on how these are coordinated in space and time within neuronal circuits of living brains. To elucidate the circuit operation of plasticity in vivo we here propose to develop and validate highly sensitive, red FRET/FLIM sensors for simultaneous use with green calcium sensors, allowing imaging of neuronal calcium activity and biochemical signaling dynamics in individual synapses and neuronal populations. We will focus on plasticity-linked biochemical events involved in synaptic plasticity and spine morphogenesis. FRET/FLIM sensors will allow for the accurate measurement of small changes, even in the presence of brain movement during behavior. In aim 1, we will develop highly sensitive red-emitting FRET/FLIM sensors using mammalian cell based protein libraries and structurally-guided large scale screening. Aim 2 will involve validation of sensors ex vivo and in vivo. Finally, sensors will be validated for use in studies on synaptic and neuronal plasticity during spatial navigation tasks in awake mice. We plan for iterative cycles of improvements in which input from biophysical analysis of sensors, validation in slices and in vivo and feed-back from early roll-out users with different animal models will be used to create successive sensor generations with ever increasing performance. Combined in vivo 2P FRET/FLIM of plasticity and 2P fluorescence imaging of calcium activity will provide a powerful new approach to study synaptic and neuronal plasticity in living organisms.