Micro-and nano-mechanical and chemical guidance of neurons

描述（由申请人提供）：pi提议开发新的微纳米制造系统，可用于监测物理和化学指导线索对细胞行为的同时和竞争效应。特别是，这项工作将集中于理解不同类型的信号如何“竞争”以引起神经元的轴突延伸。许多团体研究了不同因素单独或组合的影响，但据我们所知，没有人研究过单个因素和因素组合之间的竞争。这一信息将揭示外部信号对极化事件的影响以及参与发育和神经再生的轴突引导机制，并可能为帮助神经修复的治疗系统提供理想的特性。为了测试不同的信号如何促进神经突的延伸，将使用微纳米制造技术来创建独特的几何图形，这些图形可以同时独立地向神经元提供物理或机械线索（例如，凹槽，支柱）和化学线索（例如，生长因子，粘附配体）。特别是，沟槽底物与固定神经生长因子和netrin-1之间的竞争，以及这些刺激的组合，将研究极化（轴突起始）和轴突伸长和转向响应。将制作三种不同的装置几何形状：(1)“平行”几何形状，用于测试极化和细胞体对两种刺激之间竞争作出的决定；(2)“十字形”图案，用于测试极化和细胞体对三种或更多刺激之间竞争作出的决定；(3)“分支”图案，用于测试轴突引导和生长锥对两种或更多刺激作出的决定。大鼠胚胎海马神经元将被精确地定位在设备上，使它们有相同的概率遇到每个单独的线索。这将允许细胞在相互竞争的因素之间做出“决定”，要么定义一个轴突，要么将一个现有的轴突延伸到想要的信号。图像分析和荧光显微镜将用于测量神经突伸长，并监测轴突和树突标记物对每个线索的反应。这三种不同的装置将作为具体目标1的一部分进行制作和优化，信号竞争对大鼠海马神经元极化和轴突转向的影响将分别在具体目标2和3中进行评估。

DESCRIPTION (provided by applicant): The PIs propose to develop novel micro- and nano-fabricated systems that can be used to monitor the simultaneous and competing effects of physical and chemical guidance cues on cell behavior. In particular, this work will focus on understanding how different types of signals "compete" to give rise to axonal extension from neurons. Many groups have studied the impact of different factors individually or in combination, but no one, to our knowledge, has looked at competition between individual factors and combinations of factors. This information will shed insight into the effect of external signals on polarization events and axonal guidance mechanisms involved in development and nerve regeneration, and could potentially provide insight into the desirable characteristics for therapeutic systems to aid nerve repair. To test how different signals contribute to neurite extension, micro- and nano-fabrication techniques will be used to create unique patterned geometries that simultaneously, and yet independently, present the neuron with physical or mechanical cues (e.g., grooves, pillars) and chemical cues (e.g., growth factors, adhesive ligands). In particular, the competition between grooved substrates and immobilized nerve growth factor and netrin-1, and combinations of these stimuli, will be studied for both polarization (axon initiation) and axon elongation and steering responses. Three different device geometries will be fabricated: (1) a "parallel" geometry to test polarization and decisions made at the cell body in response to competition between two stimuli, (2) a "cross-shaped" pattern to test polarization and decisions made at the cell body in response to competition between three or more stimuli, and (3) a "branched" pattern to test axon guidance and decisions made at the growth cone in response to two or more stimuli. Rat embryonic hippocampal neurons will be precisely micropositioned on the devices such that they have equal probability of encountering each separate cue. This will allow the cells to make "decisions" between competing factors by either defining an axon or extending an existing axon toward a desired signal. Image analysis and fluorescence microscopy will be used to measure neurite elongation and monitor the presence of axonal and dendritic markers in response to each cue. The three different proposed devices will be fabricated and optimized as part of Specific Aim 1 and the effects of signal competition on polarization and axon steering in rat hippocampal neurons will be assessed in Specific Aims 2 and 3, respectively.