Developing Electrochemical Sensors to Enable Quantitative Measure of Gliotransmitter Release from Astrocytes

项目总结 目标。这项建议的主要目标是开发化学特定的电化学传感器，以提供 快速和直接测量生理上相关的化学信使释放(技术开发)。这个 第二个目标是应用这些传感器来确定不同脑区星形胶质细胞的信号异质性。 (生物学假说)。 意义和知识鸿沟。星形胶质细胞在大脑中的许多回路和环境中扮演着积分者的角色 它们如何与其他类型的细胞相互作用，在时间和空间上可能会有所不同。严格的先前研究表明，星形胶质细胞 异质性，因脑区和脑回路而异。与突触相关的转录调控可以影响异质性 功能，可塑性，分子传递和蛋白质机械和细胞器，神经胶质传递不足。星形胶质细胞 异质性还影响这类细胞对侮辱和年龄的反应，并可能预测疾病 脆弱性。星形胶质细胞通常被作为一个同质群体来研究，因此有必要研究功能 星形胶质细胞的异质性(例如，信号)，以确定星形胶质细胞在发育、对损伤的反应、 和神经退行性疾病以及这种反应如何影响局部电路功能和动态平衡。 解决方案和具体目标。研究神经胶质传递的一个主要障碍是缺乏拥有 结合时空和化学特异性来研究来自星形胶质细胞的动态分子信号。我们的目标是发展 传感器平台克服了这一障碍，通过提供对广泛的神经胶质传输的直接和快速测量 空间和时间范围。通过一项合作提案，我们建议利用通用的和特定的化学品 电化学、适配子(E-AB)传感器的检测能力与创新的测量科学 神经胶质递质动力学和异质性的测定。利用这项新的测量技术，我们的目标是测试 假设神经胶质递质信号根据释放的递质的频率、数量和身份而不同 巡回、区域间和区域内的专一性。我们将1)开发基于适体(E-AB)的电化学传感器 监测3D培养中细胞群体中胶质递质的实时释放。2)开发凹陷的微尺度E-AB 监测单个星形胶质细胞释放神经胶质递质的传感器。3)开发凹陷的纳米级传感器以监测 亚细胞区的神经胶质递质的释放。4)选择和表征高度特定的结构转换 核酸适体作为谷氨酸、GABA和D-丝氨酸的结合伙伴，具有NM亲和力，用于AIMS 1-3。

PROJECT SUMMARY Objectives. The primary objective of this proposal is to develop chemically specific electrochemical sensors to provide rapid and direct measurements of physiologically relevant chemical messenger release (technology development). The secondary objective is to apply these sensors to determine signaling heterogeneity in astrocytes from different brain regions (biological hypothesis). Significance and Knowledge Gap. Astrocytes act as integrators across many circuits and environments in the brain and how they interact with other cell types can vary in time and space. Rigorous prior research demonstrates that astrocytes are heterogenous, varying by brain region and circuit. Heterogeneity can be affected by transcriptional control related to synapse function, plasticity, molecular transmission and protein machinery and organelles that underly gliotransmission. Astrocyte heterogeneity also affects how this class of cells respond to insults and age and can potentially be predictive of disease vulnerability. Astrocytes have typically been studied as a homogenous population, thus there is a need to study functional heterogeneity of astrocytes (e.g., signaling) to determine functional roles of astrocytes in development, response to injuries, and neurodegenerative disease and how this response influences local circuit function and homeostasis. Solution and Specific Aims. A major barrier to studying gliotransmission is the lack of measurement tools that possess the combined spatiotemporal and chemical specificity to study dynamic molecular signaling from astrocytes. We aim to develop a sensor platform that overcomes this barrier by providing direct and rapid measurement of gliotransmission over broad spatial and temporal ranges. Through a collaborative proposal we propose to leverage the universal and specific chemical detection abilities of electrochemical, aptamer-based (E-AB) sensors with innovative measurement science for rapid determination of gliotransmitter dynamics and heterogeneity. With this new measurement technology, we aim to test the hypothesis that gliotransmitter signaling varies in terms of the frequency, amount, and identity of transmitters released with circuit-, and inter- and intraregional specificity. We will 1) Develop electrochemical, aptamer-based (E-AB) sensors to monitor real-time release of gliotransmitters from a cell population in 3D culture. 2) Develop recessed, microscale E-AB sensors to monitor the release of gliotransmitters from single astrocytes. 3) Develop recessed, nanoscale sensors to monitor the release of gliotransmitters from sub-cellular regions. 4) Select and characterize highly specific structure-switching nucleic acid aptamers as binding partners for glutamate, GABA, and D-serine with nM affinity for use in aims 1-3.