Microfluidic patch clamp chips for multi-unit, high-throughput recordings

描述（由申请人提供）：离子通道在神经元和非神经元细胞的生理学、神经系统的信息处理、脑发育以及广泛的神经和非神经疾病（如心血管疾病和不孕症）中发挥关键作用。 直到今天，研究离子通道的黄金标准是膜片钳技术，这是一种费力的技术，需要小心地将移液管的孔径与细胞膜密封（千兆欧姆密封）。 该技术不易于自动化，因此基于移液管的记录的低通量是离子通道靶向化合物的药理学筛选的严重瓶颈。 此外，用移液管同时记录>2-3个细胞是不可能的;因此，对复杂的成熟和发育中的神经网络中的通信的研究限于细胞外记录或细胞内钙活动的光学成像，这两者在提供关于细胞内电活动的详细信息的能力方面都是有限的。 最近有几个研究小组报道了各种设计的膜片钳芯片的成功操作，所有这些都是基于将细胞定位在一个微制造的孔上。 我们最近开发了一种微流体膜片钳芯片，其允许获得千兆欧姆密封，其产量与移液管可实现的产量相当或超过移液管可实现的产量;在内向整流钾离子通道的全细胞记录期间，对单个大鼠嗜碱性白血病细胞的性能进行了评估。 在这里，我们建议延长我们最近的工作，从培养的胚胎皮层切片使用一种新的切片制备，有一个干净的细胞层的切片表面上的记录。 利用多单元膜片钳芯片，我们将研究神经元信号在发育中的皮层网络中的传播。 该项目的成功完成可以揭示神经元通信机制，这些机制是发育（作为正常发育程序的一部分）以及癫痫中活动波传播的基础。 此外，相同的技术将使得能够低成本地筛选离子通道靶向化合物（其构成约25%的药物）对单个离子通道电流的作用（大约50%的与安全性相关的药物从市场撤回是由于对离子通道的不期望的副作用，因此FDA现在建议所有候选药物 通常含有> 10，000种化合物 离子通道在所有已知的脑功能中起关键作用。 使用膜片钳芯片，现在可以监测大量单个解离细胞（但不是脑切片）的离子通道活性。 我们建议开发一种膜片钳芯片设计，用于监测脑切片表面的多个细胞。 我们将使用该设备来研究神经元信号在发育中的皮层网络中的传播。

DESCRIPTION (provided by applicant): Ion channels play key roles in the physiology of neuronal and non-neuronal cells, in information processing in the nervous system, in brain development, and in a broad spectrum of neurological and non-neurological disorders, such as cardiovascular disease and infertility conditions. To this day, the gold standard for studying ion channels is the patch clamp technique, a laborious technique based on carefully sealing the aperture of a pipette against the cell membrane (the gigaohm seal ). The technique is not easily amenable to automation, so the low throughput of pipette-based recordings is a serious bottleneck for pharmacological screening of ion channel-targeting compounds. Furthermore, recording from >2-3 cells simultaneously is not possible with pipettes; as a result, investigations of communication in complex mature and developing neural networks are limited to extracellular recordings or optical imaging of intracellular calcium activity, both of which are limited in their ability to provide detailed information about intracellular electrical activity. Several groups have recently reported the successful operation of various designs of patch clamp chips, all based on positioning the cell against a microfabricated aperture. We have recently developed a microfluidic patch clamp chip that allows for obtaining gigaohm seals with yields comparable to or surpassing those achievable with a pipette; the performance was evaluated on single rat basophilic leukemia cells during whole-cell recordings of the inward- rectifying potassium ion channel. Here we propose to extend our recent work to recordings from cultured embryonic cortical slices using a novel slice preparation that has a clean layer of cells on the surface of the slice. With the multi-unit patch clamp chip we will investigate the propagation of neuronal signals across developing cortical networks. PUBLIC HEALTH RELEVANCE The successful completion of this project could reveal neuronal communication mechanisms that underlie the propagation of activity waves seen in development (as part of the normal developmental program) as well as in epilepsy. Furthermore, the same technology would enable low-cost screening of ion channel-targeting compounds (which constitute ~25% of drugs) for their effects on single ion channel currents (approximately 50% of safety-related withdrawals of drugs from the market are due to undesired side effects on ion channels, so the FDA now recommends that all drug candidates usually pools of >10,000 compounds be patch clamp-tested for their effects on the hERG channel).Ion channels play key roles in all known brain functions. Using patch clamp chips, it is now possible to monitor the ion channel activity of large numbers of single dissociate cells (but not of brain slices). We propose to develop a patch clamp chip design for monitoring multiple cells on the surface of brain slices. We will use the device to investigate the propagation of neuronal signals across developing cortical networks.