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、神经系统的信息处理、大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。时至今日，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，其基础是将移液器的孔小心地密封在细胞膜上（千兆赫密封）。该技术不容易实现自动化，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，用移液器同时记录> - 2-3个细胞是不可能的；因此，对复杂成熟和发育中的神经网络通信的研究仅限于细胞外记录或细胞内钙活性的光学成像，这两种方法在提供细胞内电活动的详细信息方面都受到限制。几个小组最近报道了各种膜片钳芯片设计的成功操作，所有这些设计都是基于将细胞定位在微加工孔径上。我们最近开发了一种微流体膜片钳芯片，可以获得与移液管相当或超过可实现的产量的千兆赫密封件；在对单个大鼠嗜碱性白血病细胞进行向内整流钾离子通道全细胞记录时，对其性能进行了评价。在这里，我们建议将我们最近的工作扩展到从培养的胚胎皮层切片中记录，使用一种新的切片制备方法，在切片表面有一层干净的细胞。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在发育（作为正常发育计划的一部分）和癫痫中看到的活动波传播基础的神经元通信机制。此外，同样的技术将使低成本筛选离子通道靶向化合物（约占药物的25%）对单个离子通道电流的影响成为可能（大约50%的与安全相关的药物从市场上撤回是由于对离子通道的不良副作用，因此FDA现在建议所有候选药物（通常为100万到10000种化合物）都要进行膜片钳测试，以确定它们对hERG通道的影响）。离子通道在所有已知的大脑功能中起着关键作用。使用膜片钳芯片，现在有可能监测大量单个游离细胞的离子通道活动（但不包括脑切片）。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.