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

钙离子是调节细胞通讯和功能的重要信号，细胞是生命系统的基本生物单位。在中枢神经系统内，生理水平的钙在神经活动和通过神经网络传输信号方面发挥着重要作用。相比之下，钙离子水平的高度升高与通讯功能障碍有关，并可能导致神经元网络的退化。最近，有研究表明，磁性纳米颗粒可以机械地打开钙离子通道，使钙离子通过细胞膜进入神经元。这种有意识的、力介导的钙离子内流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中传播，对于理解力介导的脑细胞通讯和退行性疾病非常重要。该奖项将资助一项将纳米材料与神经生物系统联系起来的研究计划，旨在捕获并量化实验培养的受控神经元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研究基础设施。这些纳米工具也将提供给更广泛的研究社区，他们对分析退化或干细胞发育过程中通讯模式的变化感兴趣。最后，该项目将包括开设一个跨学科的生物微电子机械系统(BioMEMS)课程，以促进蒙大拿州STEM劳动力的发展，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暑期讲习班。通过纳米磁力的刺激是一种在磁场中操作磁性纳米颗粒以对相关物体施加机械力的工具。在生物学背景下，该项目将表达机械敏感离子通道和受体的脑细胞视为纳米磁刺激的目标。最近，在体外对神经网络的细胞膜施加纳米磁力已被证明会触发钙离子的内流。然而，力刺激与其他神经生理事件的关系尚不清楚。特别是，将亚细胞生物力学与创新的神经元细胞筛选平台相结合，以前还没有与纳米磁力刺激结合使用。这一知识缺口目前限制了纳米磁刺激更广泛的实验适用性。因此，这个项目解决了目前纳米磁力刺激的局限性，通过揭示力介导的细胞内钙信号变化是如何影响神经元之间的通信的。这项拟议的工作将系统地分析这种力介导的钙离子内流在神经元网络中发生的速度，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相结合来评估啮齿动物初级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和评估工具阵列，使用低分辨率活细胞钙成像来绘制跨神经元培养的钙信号传播图。这项提议的研究结果将为未来使用机械刺激作为一种精确的神经生理工具铺平道路。基于这些知识，该项目将为未来使用磁性纳米颗粒介导力刺激的神经疗法制定设计指南。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.