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Genetically-encoded optical sensors to study purinergic signaling

用于研究嘌呤能信号传导的基因编码光学传感器

基本信息

批准号：
8995713
负责人：
Mathew Tantama
金额：
$ 22.96万
依托单位：
PURDUE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-02-01 至 2018-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8995713
关键词：
Adenosine Affinity Astrocytes Axon Behavior Binding Biosensor Blood flow Brain Brain Pathology Calcium Cell surface Cells Communication Communities Detection Electrophysiology (science)Engineering Epilepsy Extracellular Space Feedback G-Protein-Coupled Receptors Glutamates Goals Health Image Immunity In Vitro Kinetics Lead Life Locales Measures Mediating Membrane Methods Microglia Nervous system structure Neurodegenerative Disorders Neuroglia Neurons Neurotransmitters Nucleotidases Optics P2X-receptor Pain Photons Physiological Physiology Presynaptic Terminals Process Property Protein Engineering Protocols documentation Purinoceptor Resolution Role Signal Transduction Slice Stroke Synapses Synaptic Transmission Synaptic plasticity System Testing Time Tissues Vertebral column Vesicle Work age related analytical tool animal tissue autocrine body system brain cell cell type extracellular in vivo live cell microscopy meetings multi-photon neuroregulation optical sensor paracrine receptor response sensor tool transmission process

项目摘要

DESCRIPTION (provided by applicant): Extracellular ATP mediates purinergic signaling throughout the nervous system, and purinergic signaling mechanisms have been associated with multiple brain pathologies including stroke, aging-related neurodegenerative diseases, and epilepsy. Therefore, methods to accurately and precisely detect extracellular ATP are essential to the study of its physiological role. To this end, our broad goal is to develop a set of quantitative optical tools to image and study the signaling dynamics of extracellular ATP in real-time at central synapses. In particular, evidence in recent decades suggests that astrocytes modulate synaptic transmission and plasticity through activity-dependent release of gliotransmitters that include extracellular ATP. Spillover of neurotransmitters during synaptic activity can activate G-protein coupled receptors on perisynaptic astrocytes. It is proposed that this activation of astrocyte receptors elicits a calcium response that can lead to release of ATP as a gliotransmitter. Subsequently, the released ATP or its metabolite adenosine can bind to and activate neuronal purinergic receptors, causing either homosynaptic or heterosynaptic neuromodulation. Thus, extracellular ATP released from astrocytes might act as a feedback signal to modulate synaptic efficacy and network behavior. However, there is an unmet need for new analytical tools to measure extracellular ATP, and the limitations of current detection methods have impeded the resolution of important questions regarding the mechanisms and physiological relevance of ATP as a gliotransmitter. To meet this need, the central hypothesis of this proposal is that genetically-encoded fluorescent biosensors can be engineered to sense extracellular ATP with sensor properties suitable for studying physiologically relevant purinergic signaling. We will test our hypothesis in two working aims: Aim 1 is to engineer cell surface-tethered biosensors to quantitatively image ATP release and clearance; Aim 2 is to use these new biosensors to measure activity- dependent ATP release and clearance in primary cultures of astrocytes and neurons as well as in brain slices. Upon completion of these aims, we will be able to provide both new optical tools for measuring extracellular ATP and imaging protocols that are of broad use to the purinergic signaling community.

描述（由申请人提供）：细胞外ATP介导整个神经系统的嘌呤能信号传导，嘌呤能信号传导机制与多种脑病理学相关，包括中风、衰老相关神经退行性疾病和癫痫。因此，准确和精确地检测细胞外ATP的方法对于研究其生理作用至关重要。为此，我们的主要目标是开发一套定量光学工具，以实时成像和研究细胞外ATP在中枢突触的信号动力学。特别是，近几十年来的证据表明，星形胶质细胞调节突触传递和可塑性，通过活性依赖性释放的胶质递质，包括细胞外ATP。在突触活动期间神经递质的溢出可以激活突触周星形胶质细胞上的G蛋白偶联受体。有人提出，星形胶质细胞受体的这种激活引起钙反应，可导致释放ATP作为胶质递质。随后，释放的ATP或其代谢物腺苷可以结合并激活神经元嘌呤能受体，引起同突触或异突触神经调节。因此，从星形胶质细胞释放的细胞外ATP可能作为反馈信号调节突触效能和网络行为。然而，有一个未满足的需要，新的分析工具来测量细胞外ATP，目前的检测方法的局限性，阻碍了解决重要问题的机制和生理相关性的ATP作为神经胶质递质。为了满足这一需求，该提案的中心假设是，基因编码的荧光生物传感器可以被工程化以感测细胞外ATP，其传感器特性适合于研究生理相关的嘌呤能信号传导。我们将在两个工作目标中测试我们的假设：目标1是设计细胞表面束缚的生物传感器以定量成像ATP释放和清除;目标2是使用这些新的生物传感器来测量星形胶质细胞和神经元以及脑切片的原代培养物中的活性依赖性ATP释放和清除。完成这些目标后，我们将能够提供新的光学工具，用于测量细胞外ATP和成像协议，广泛使用的嘌呤能信号社区。