Micro-and nano-mechanical and chemical guidance of neurons

描述(由申请人提供)：PI建议开发新的微米和纳米制造系统，可以用来监测物理和化学指导信号对细胞行为的同时和竞争影响。特别是，这项工作将侧重于了解不同类型的信号如何“竞争”引起神经元的轴突延伸。许多小组研究了不同因素单独或组合在一起的影响，但据我们所知，没有人研究过个别因素之间的竞争和因素的组合。这些信息将深入了解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，并可能为治疗系统提供帮助神经修复的理想特征。为了测试不同的信号如何影响神经突起的延伸，微米和纳米制造技术将被用来创建独特的图案化几何图形，这些几何图形同时又独立地向神经元提供物理或机械线索(例如沟槽、柱子)和化学线索(例如生长因子、粘附性配体)。特别是，将研究凹槽底物和固定的神经生长因子和netrin-1之间的竞争，以及这些刺激的组合，以同时研究极化(轴突启动)和轴突延长和转向反应。将制造三种不同的器件几何形状：(1)用于测试极化和在细胞体上做出的决定以响应两个刺激之间的竞争的“平行”几何形状；(2)用于测试极化和在细胞体上响应于三个或更多刺激之间的竞争而作出的决定的“十字形”图案；以及(3)用于测试轴突引导和在生长锥处对两个或更多刺激作出的决定的“分支”模式。大鼠胚胎海马神经元将被精确地定位在设备上，以便它们有相同的机会遇到每一个单独的提示。这将允许细胞通过定义轴突或向所需信号延伸现有轴突，在相互竞争的因素之间做出“决定”。图像分析和荧光显微镜将被用来测量轴突伸长，并监测轴突和树突标记的存在，以响应每一条线索。这三种不同的设备将作为特定目标1的一部分进行制造和优化，并将分别在特定目标2和3中评估信号竞争对大鼠海马神经元极化和轴突引导的影响。

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.