CAREER: Understanding calcium communication in neurons with nanomagnetic forces and engineered network patterns

细胞是生命系统的基本生物单元，钙离子是调节细胞通讯和功能的重要信号。在中枢神经系统内，生理水平的钙在神经活动和跨神经元网络的信号传输中起重要作用。相反，高度升高的钙离子水平与功能失调的通信相关，并可能导致神经元网络的退化。最近，已经证明磁性纳米颗粒可以用于机械地打开钙通道，允许钙离子通过细胞膜进入神经元。这种有意的、力介导的钙离子流入如何与神经元内的结构相互作用，以及它如何在神经元网络内传播，对于理解力介导的脑细胞通讯和退行性疾病非常重要。该奖项将资助一项研究计划，该计划将纳米材料与神经生物学系统联系起来，旨在捕获和量化实验生长的受控神经网络中的钙信号传输。参与该项目的研究生和本科生都将受益于结合纳米工具和神经工程的增强型研究基础设施。这些纳米工具也将提供给更广泛的研究团体，他们对分析变性或干细胞发育过程中通信模式的变化感兴趣。最后，该项目将涉及创建一个跨学科的生物微机电系统（BioMEMS）课程，以促进蒙大拿州的STEM劳动力，并为对将工程数据转化为视觉艺术特别感兴趣的学生举办科学交流夏季研讨会。通过纳米磁力的刺激是一种在磁场内操作磁性纳米颗粒以在相关物体上施加机械力的工具。在生物学背景下，该项目认为表达机械敏感离子通道和受体的脑细胞是纳米磁刺激的目标。最近，在体外神经网络的膜上施加纳米磁力已被证明会引发钙离子的流入。然而，力刺激与其他神经生理事件的关系仍然未知。特别是，亚细胞生物力学与创新的神经元细胞筛选平台的整合尚未与纳米磁力刺激结合使用。这种知识差距目前限制了纳米磁刺激更广泛的实验适用性。因此，该项目通过揭示神经元与神经元之间的通信如何受到力介导的细胞内钙信号变化的影响来解决纳米磁力刺激的当前局限性。拟议的工作将系统地分析这种力介导的钙离子流入神经元网络的速度有多快，以及神经元网络的结构如何影响诱发信号的空间传输。钙荧光测定法将与电生理学结合使用，以评估啮齿动物原代神经元亚细胞钙刺激的时空方面。此外，该项目开发了高度并行化的细胞纳米操作阵列和评估工具，具有低分辨率活细胞钙成像，以绘制跨神经元培养物的钙信号传播。这项提议的研究结果将为将来使用机械刺激作为精确的神经生理学工具铺平道路。基于这些知识，该项目将开发设计指南，为未来的神经疗法使用磁性纳米粒子介导的力stimulation.This奖项反映了NSF的法定使命，并已被认为是值得通过评估使用基金会的智力价值和更广泛的影响审查标准的支持。

Calcium ions are essential signals to regulate communication and function in cells, which are the basic biological units of living systems. Within the central nervous system, physiological levels of calcium play an important role in neural activity and in transporting signals across neuronal networks. In contrast, highly elevated levels of calcium ions are associated with dysfunctional communication and can lead to the degeneration of the neuronal network. Recently, it has been demonstrated that magnetic nanoparticles can be used to mechanically open calcium channels allowing calcium ions to enter neurons through the cell membrane. How this intentional, force-mediated influx of calcium ions interacts with structures inside neurons and how it spreads within a neuronal network is highly important for understanding force-mediated brain cell communication and degenerative diseases. This award will fund a research program that links nanomaterials to neurobiological systems and aims to capture and to quantify calcium signal transport in experimentally grown, controlled neuronal networks. Both graduate and undergraduate students involved in the project will benefit from an enhanced research infrastructure combining nanotools and neuroengineering. These nanotools will also become available to a broader research community interested in analyzing changes in communication patterns during degeneration or stem cell development. Finally, the project will involve the creation of an interdisciplinary bio micro-electro-mechanical system (BioMEMS) course to advance the STEM workforce in Montana, and science communication summer workshops for students specifically interested in transforming engineering data into visual arts. Stimulation through nanomagnetic forces is a tool that operates magnetic nanoparticles within magnetic fields to impose a mechanical force on associated objects. Within a biological context, this project considers brain cells that express mechanically sensitive ion channels and receptors as the target for nanomagnetic stimulation. Applying nanomagnetic forces to the membrane of neural networks in vitro has been shown to trigger an influx of calcium ions, recently. How the force stimulation relates to other neurophysiological events, however, remains unknown. In particular, the integration of subcellular biomechanics with innovative neuronal cell screening platforms has not been used in combination with nanomagnetic force stimulation, previously. This knowledge gap currently limits the broader experimental applicability of nanomagnetic stimulation. Hence, this project addresses current limitations of nanomagnetic force stimulation by unraveling how neuron-to-neuron communication is impacted by force-mediated intracellular changes in calcium signaling. The proposed work will systematically analyze how fast this force-mediated calcium influx in neuronal networks occurs, and how the architecture of a neuronal network impacts the spatial transport of evoked signals. Calcium fluorometry will be employed in combination with electrophysiology to assess the spatiotemporal aspects of subcellular calcium stimulation in rodent primary neurons. Furthermore, this project develops highly parallelized arrays of cell nano-manipulation and assessment tools with low-resolution live-cell calcium imaging to map calcium signal propagation across neuronal cultures. Research results from this proposal will pave the way to future use of mechanical stimulation as a precise neurophysiological tool. Based on this knowledge, the project will develop design guidelines for future neuro-therapeutics using magnetic nanoparticle-mediated force stimulation.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.