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

描述(申请人提供)：离子通道在神经细胞和非神经细胞的生理学、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中发挥关键作用，如心血管疾病和不孕症。直到今天，研究离子通道的金标准是膜片钳技术，这是一种基于小心地将吸管的孔密封在细胞膜上的费力的技术(千兆欧密封)。这项技术不易实现自动化，因此基于吸管的记录的低通量是离子通道靶向化合物药理筛选的严重瓶颈。此外，使用吸管不可能同时记录&gt；2-3细胞；因此，对复杂、成熟和发展中的神经网络中的通信的研究仅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。几个小组最近报告了各种设计的膜片钳芯片的成功操作，这些芯片都是基于将细胞对准微型制造的孔来定位的。我们最近开发了一种微流控膜片钳芯片，它允许获得千兆欧密封，其产量与移液管相当或超过；在单个大鼠嗜碱性白血病细胞上对内向整流钾离子通道进行全细胞记录期间，对其性能进行了评估。在这里，我们建议将我们最近的工作扩展到使用一种新的切片制备方法从培养的胚胎皮质切片中进行记录，这种切片制备方法在切片表面具有一层干净的细胞层。利用多单位膜片钳芯片，我们将研究神经元信号在发育中的皮层网络中的传播。公共卫生相关性该项目的成功完成可能揭示神经通讯机制，这些机制是发育中(作为正常发育计划的一部分)以及癫痫中所看到的活动波传播的基础。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(大约50%的安全相关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是&gt；10,000种化合物池进行膜片钳测试其对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，现在可以监测大量单个分离细胞(但不是脑片)的离子通道活动。我们建议开发一种用于监测脑片表面多个细胞的膜片钳芯片设计。我们将使用该设备来研究神经元信号在发育中的皮质网络中的传播。

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.