Novel fluorescent sensors for imaging neuromodulation

用于神经调节成像的新型荧光传感器

基本信息

批准号：
10201786
负责人：
Yang DAN
金额：
$ 100.29万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-15 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10201786
关键词：
Acetylcholine Acute Affinity Agonist Amino Acid Sequence Animal Behavior Animals Area Argipressin Axon Behavior Behavioral Behavioral Paradigm Binding Binding Sites Brain Cell physiology Chemicals Cholecystokinin Cognition Communities Corpus striatum structure Coupled Data Dimensions Disease Dopamine Eating Electrophysiology (science)Endocannabinoids Engineering Feedback Fiber Fluorescence G-Protein-Coupled Receptors Goals Health Human Hypothalamic structure Image In Vitro Kinetics Lead Learning Libraries Ligand Binding Ligands Lipids Mammalian Cell Measures Membrane Membrane Proteins Memory Methods Microdialysis Midbrain structure Mission Molecular Conformation Monitor Moods Mus Nervous system structure Neuromodulator Neurons Neuropeptides Oxytocin Pathologic Peptides Performance Photometry Physiological Play Preparation Process Public Health Receptor Activation Research Resolution Role Signal Transduction Signaling Molecule Site Sleep Sleep Wake Cycle Slice Social Behavior Specificity Technology Therapeutic United States National Institutes of Health Universities Validation Vasopressins Viral Yang addiction base cell type detection of nutrient enhanced green fluorescent protein experience experimental study imaging modality in vivo in vivo evaluation in vivo imaging in vivo monitoring lens millisecond monoamine motor learning nanomolar nervous system disorder neural circuit neuroregulation non-invasive monitor novel pain sensation preservation prototype real time monitoring receptor relating to nervous system response screening sensor sleep regulation tool transmission process two-photon

项目摘要

SUMMARY Animal behaviors are orchestrated by the sophisticated nervous system, which is dynamically regulated by neuromodulators including lipids and neuropeptides. Endocannabinoids (eCBs) are neurolipids exist broadly in the brain and regulate learning and memory, addiction, pain sensation, and food intake. Among neuropeptides, cholecystokinin (CCK) is involved in nutrient sensing, food intake, and sleep regulation, and oxytocin (OXT) and vasopressin (AVP) play important roles in various aspects of social behaviors. However, how and when lipid and neuropeptide transmission occur in the brain are largely unclear. Existing methods (e.g. microdialysis) that measures brain chemical content suffer from low temporal and spatial resolution. Additionally, since neurolipid and neuropeptide releases often require repetitive neuronal firing and can occur at both axonal and dendritic sites, activity of the neuromodulator- releasing neurons cannot reliably predict where and when neurolipids and neuropeptides are released. Here we propose to develop a set of new tools for long-term monitoring of neurolipids and neuropeptides. Our strategy taps into their natural receptors, human G protein-coupled receptors (GPCRs), which are coupled to GFP. In the presence of neurolipids or neuropeptides, these GPCR Activation-Based (GRAB) sensors transform ligand binding-induced conformational changes into rapid fluorescent signals. We aim to develop and optimize neurolipid and neuropeptide GRAB sensors with >500% fluorescence change (dF/F) and 10- nanomolar affinity in vitro and validate these novel tools in brain slices ex vivo and mouse behavioral paradigms in vivo. In Aim 1, we will develop GRAB sensors for endocannabinoids, CCK, vasopressin, and OXT by systematically varying key sites involved in ligand binding, conformational change, etc. In Aim 2, we will validate the performance of these sensors in brain slice following long-term expression using viral tools. In Aim 3, we will use three different imaging methods (fiber photometry, epifluorescence and 2-photon imaging coupled with GRIN lens) in different behavioral paradigms to test in vivo performance of the novel GRAB sensors in mice. Feedback from experiments in Aims 2-3 will guide iterative optimization in Aim 1. Successful completion of our proposal will yield a suite of powerful tools and technical approaches, which will greatly facilitate studies of neurolipids and neuropeptides under both physiological and pathological conditions, helping reveal disease mechanisms, providing therapeutic guidance, and eventually benefiting human health.

概括动物行为由复杂的神经系统精心策划，即由包括脂质和神经肽在内的神经调节剂动态调节。内源性大麻素（ECB）是神经脂质的大脑中广泛存在，调节学习和记忆，成瘾，疼痛感和食物摄入量。在神经肽中，胆囊动蛋白（CCK）参与营养感应，食物摄入和睡眠调节，催产素（OXT）和加压素（AVP）在社会行为的各个方面发挥重要作用。但是，如何以及何时脂质和大脑中发生神经肽的传播在很大程度上不清楚。现有方法（例如微透析）测量脑化学含量的时间较低和空间含量解决。此外，由于神经脂肪和神经肽释放通常需要重复神经元发射，并且可以在轴突和树突部位发生，神经调节剂的活性释放神经元无法可靠地预测神经脂质和何时神经肽和神经肽是发行。在这里，我们建议开发一组新工具，用于长期监测神经脂质和神经肽。我们的策略点击了他们的天然受体，人类G蛋白耦合受体（GPCR），与GFP耦合。在存在神经脂质或神经肽的情况下这些基于GPCR激活（GRAB）传感器会改变配体结合诱导的构象变化为快速荧光信号。我们旨在开发和优化神经脂质和神经肽的捕获传感器> 500％荧光变化（DF/F）和10-- 体外纳摩尔亲和力，并在脑切片中验证这些新工具在体内和小鼠体内行为范例。在AIM 1中，我们将开发用于内源性大麻素的传感器， CCK，加压素和oxt通过系统地变化的配体结合的关键位点，构象变化等。在AIM 2中，我们将验证这些传感器在大脑中的性能使用病毒工具长期表达后切片。在AIM 3中，我们将使用三种不同的成像方法（纤维光度法，落荧光和2光子成像与笑镜头）在不同行为范式中测试新型Grab传感器的体内性能老鼠。 AIMS 2-3中实验的反馈将指导AIM 1中的迭代优化。成功完成我们的建议将产生一套强大的工具和技术方法，这将极大地促进对神经脂质和神经肽的研究生理和病理状况，有助于揭示疾病机制，提供治疗指导，最终使人类健康受益。