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

使用基于 FRET/FLIM 的优化传感器对大脑活动和可塑性进行多重成像

基本信息

批准号：
10516813
负责人：
Daniel A Dombeck
金额：
$ 115.96万
依托单位：
NORTHWESTERN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-01-01 至 2026-12-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10516813
关键词：
Animal Model Behavior Benchmarking Biochemical Biochemistry Biosensor Brain Brain imaging CREB1 gene Calcium Cell Nucleus Cell Separation Dendrites Development Disease Electric Stimulation Environment Event Feedback Fluorescence Resonance Energy Transfer Generations Genetic Glutamates Goals Hippocampus Hour Image Individual Investigation Ion Channel Label Learning Libraries Link Mammalian Cell Measurement Measures Mediating Memory Methods Molecular Monitor Morphogenesis Morphologic artifacts Movement Mus Natural regeneration Neurites Neuronal Plasticity Neurons Noise Organism Pathway interactions Pattern Performance Photic Stimulation Population Process Property Proteins Public Health Regulation Reporter Research Resolution Sensory Signal Pathway Signal Transduction Slice Spatial Behavior Structure Synapses Synaptic plasticity Time Validation Vertebral column Visual Cortex addiction awake base biophysical analysis calmodulin-dependent protein kinase II chronic pain dark rearing experience experimental study fluorescence imaging fluorophore hemodynamics hippocampal pyramidal neuron improved in vivo injury recovery light scattering millisecond multiplexed imaging neural circuit neuronal circuitry novel novel strategies operation place fields prototype receptor recruit redshift response screening sensor spatiotemporal tool two photon microscopy two-photon way finding

项目摘要

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.

项目概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，例如成瘾、慢性疼痛或再生。在分子水平上生化机制已得到很好的阐明描述过，但人们对它们如何在神经元内的空间和时间上进行协调知之甚少活体大脑的回路。为了阐明体内可塑性的电路运作，我们在这里建议开发并验证可与绿色同时使用的高灵敏度红色 FRET/FLIM 传感器钙传感器，可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态。我们将重点关注与可塑性相关的涉及突触可塑性和脊柱形态发生的生化事件。 FRET/FLIM 传感器即使在大脑存在的情况下，也可以准确测量微小的变化行为过程中的运动。在目标1中，我们将开发高灵敏度的红光FRET/FLIM 使用基于哺乳动物细胞的蛋白质库和结构引导的大规模传感器筛选。目标 2 将涉及传感器的离体和体内验证。最后，传感器将经验证可用于空间导航任务期间突触和神经元可塑性的研究醒着的老鼠。我们计划迭代改进周期，其中生物物理分析的输入传感器的数量、切片和体内验证以及早期推出用户的反馈动物模型将用于创建连续几代传感器，并且数量不断增加表现。结合体内可塑性 2P FRET/FLIM 和 2P 荧光成像钙活性将为研究突触和神经元可塑性提供强大的新方法活的有机体。