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

项目摘要 目标.该提案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相关的化学信使释放（技术开发）。的 第二个目标是应用这些传感器来确定来自不同脑区的星形胶质细胞中的信号异质性 （生物学假说）。 意义和知识差距。星形胶质细胞作为大脑中许多回路和环境的整合者， 它们与其他细胞类型的相互作用在时间和空间上是不同的。严格的先前研究表明，星形胶质细胞是 异质性，因大脑区域和回路而异。异源性可受到与突触相关的转录调控的影响 功能，可塑性，分子传递和蛋白质机器和细胞器的神经胶质传递的基础。星形细胞 异质性也影响这类细胞对损伤和年龄的反应，并可能预测疾病 易损性.星形胶质细胞通常被作为同质群体进行研究，因此需要研究星形胶质细胞的功能。 星形胶质细胞的异质性（例如，信号）来确定星形胶质细胞在发育，对损伤的反应， 以及这种反应如何影响局部回路功能和体内平衡。 解决方案和具体目标。研究胶质传递的一个主要障碍是缺乏具有特异性的测量工具。 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。我们的目标是开发 一个传感器平台，克服了这一障碍，提供直接和快速的测量胶质传输广泛 空间和时间范围。通过一项合作提案，我们建议利用通用和特定的化学品， 基于适配体的电化学（E-AB）传感器的检测能力，具有创新的测量科学， 胶质递质动力学和异质性的测定。通过这种新的测量技术，我们的目标是测试 假设神经胶质递质信号在频率，数量和释放的递质的身份方面有所不同， 电路，以及区域间和区域内的特殊性。我们将1）开发基于适体的电化学（E-AB）传感器， 在3D培养中监测来自细胞群的胶质递质的实时释放。2)开发嵌入式微型E-AB 传感器来监测单个星形胶质细胞释放的胶质递质。3)开发嵌入式纳米级传感器， 从亚细胞区域释放神经胶质递质。4)选择和表征高度特异性的结构转换 核酸适体作为谷氨酸、GABA和D-丝氨酸的结合配偶体，具有nM亲和力，用于目的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.