Microfluidic System for Monitoring Gliotransmitter Release

星形胶质细胞调节突触传递的确切机制尚不清楚。它也不是 明确这些细胞的功能障碍，包括那些由滥用药物引起的功能障碍，可能会导致大脑紊乱。 这项提议的目标是开发一种方法，使星形胶质细胞在 微流控平台可通过以下方式实时测量细胞内钙离子浓度([钙]i) 荧光探针与神经活性物质的分泌同时对各种刺激作出反应。 在我们强大的初步数据的指导下，我们将通过以下方式实现这项建议的目标 具体目标：1)同时监测D-丝氨酸和谷氨酸的分泌，同时进行[Ca~(2+)]i成像 星形胶质细胞培养，以及2)星形胶质细胞培养释放三磷酸腺苷的定量。在第一个目标中，我们将制定一种 用于高时间分辨率监测星形胶质细胞伯胺释放的微流控系统 同时进行细胞内[Ca~(2+)]i显像。在第二个目标中，我们将整合一种酶分析 三磷酸腺苷可以监测星形胶质细胞的所有主要神经胶质递质。这项研究 在本申请中提出的是创新的，因为它将利用广泛的方法来测量 星形胶质细胞释放的多种因子与同步的[钙]i成像相结合，提供了高度的 神经胶质传递的动态视图。这一系统的结果将是重要的，因为它将提供第一个 一种能够以自动方式同时调查所有主要胶质递质释放的工具 对多种兴奋剂的反应。最终，该设备将为未来的研究提供一种新的工具 星形胶质细胞生物学及其在神经保护行为中的作用，三方突触传递， 以及滥用药物的影响。

The precise mechanisms through which astrocytes modulate synaptic transmission are not clear. It is also not clear how dysfunction in these cells, including those caused by drugs of abuse, can lead to brain disorders. The objective of this proposal is to develop a method that will allow the culture of astrocytes within a microfluidic platform to enable real-time measurements of intracellular Ca2+ concentration ([Ca2+]i) via fluorescent probes simultaneously with secretion of neuroactive substances in response to various stimuli. Guided by our strong preliminary data, we will obtain the objective of this proposal by pursuing the following specific aims: 1) monitoring D-serine and glutamate secretion simultaneous with [Ca2+]i imaging within astrocyte cultures, and 2) quantitation of ATP release from astrocyte cultures. In the first aim, we will develop a microfluidic system to monitor, with high time resolution, the release of primary amines from astrocytes simultaneous with intracellular [Ca2+]i imaging. In the second aim, we will integrate an enzymatic assay for adenosine triphosphate to allow monitoring of all the major gliotransmitters from astrocytes. The research proposed in this application is innovative because it will utilize a broad approach to the measurement of multiple factors released from astrocytes combined with simultaneous [Ca2+]i imaging, providing a highly dynamic view of gliotransmission. The results of this system will be significant because it will provide the first tool capable of investigating release of all major gliotransmitters simultaneously in an automated fashion in response to a number of stimulants. Ultimately, this device will produce a novel tool for future studies regarding astrocyte biology and the roles these cells have in neuroprotective behavior, tripartite synaptic transmission, and the effects of drugs of abuse.