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元回路的一个基本方面。它位于 学习和记忆，感觉适应，以及许多与疾病相关的过程，如成瘾， 慢性疼痛或再生。在分子水平上，生化机制已经很好地发挥了作用 对此进行了描述，但对这些在神经元内如何在空间和时间上协调却知之甚少 活生生的大脑回路。为了阐明体内可塑性的回路操作，我们在这里建议 开发和验证高灵敏度的红色FRET/FLIM传感器，以便与绿色同时使用 钙传感器，允许对神经元钙活动和生化信号进行成像 个体突触和神经元群体的动力学。我们将重点关注与可塑性相关的 参与突触可塑性和脊柱形态发生的生化事件。FRET/FLIM传感器 将允许准确测量微小变化，即使在大脑存在的情况下也是如此 在行为过程中的运动。在目标1中，我们将开发高灵敏度的红光FRET/FLiM 基于哺乳动物细胞的蛋白质文库和结构导向的大规模传感器 放映。目标2将涉及传感器的体外和体内验证。最后，传感器将是 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。我们计划改进的迭代周期，其中来自生物物理分析的输入 传感器、切片和体内验证，以及来自早期推出用户的反馈 动物模型将被用来创造连续的传感器世代，并且不断增加 性能。结合体内2P FRET/FLIM可塑性和2P荧光成像 钙活动将为研究脑内突触和神经元的可塑性提供有力的新途径。 活着的有机体。

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.