CAREER: Aberrant Rewiring of Neurons after Injury - Intracellular Interactions in Vivo

大脑中的神经元与其他类型的细胞相互作用。其中一种是小胶质细胞，它是星形细胞，用它们的臂状分支来观察神经元之间的连接。理解这些相互作用很重要，因为神经连接的变化是学习和记忆形成的基础。主要研究者假设小胶质细胞可能调节神经连接的这种变化。该项目将研究小胶质细胞是否直接参与断开神经元，以及这些行为是否会因损伤而中断。作为对损伤的反应，小胶质细胞失去了它们的分支，首席研究员假设这阻止了它们正确调节神经连接。这项工作将使用一种特殊设计的显微镜，对活大脑中的细胞进行成像。为了使细胞可见，小胶质细胞将被设计成在显微镜下看起来是绿色的，并且，使用该项目开发的策略，每个神经元将被标记以独特的颜色。通过这种方法，可以可视化小胶质细胞在正常条件下和损伤后如何与神经元相互作用。分析这样的图像是复杂的，需要接受过高级数学训练的神经生物学家。该项目将开发软件和教学工具，使未受过工程师培训的科学家能够理解图像分析背后的数学。首席研究员将创建一个基于调查的模块，关于这些想法称为“超越图像”，这将介绍高中学生图像分析和数字处理。该模块将通过康奈尔合作推广学校的4-H STEM网站等网站提供给各地的教师。由于神经分支小且密集，因此所提出的神经连接的小胶质细胞调制的成像具有挑战性。一项技术创新将是使用一种病毒株的组合来感染神经元，每种病毒株都会产生不同颜色的荧光蛋白。每个神经元将被标记有自己独特的颜色代码，使其分支能够区分。使用病毒的一个优点是，标记细胞的类型和位置都可以改变，以优化颜色编码。与其等待神经元偶然断开连接，激光将被用来精确地切断神经元的分支。延时成像将捕捉小胶质细胞和神经元对现在无用的切断的分支的反应。预测是，小胶质细胞将通过吞噬和移除失效连接的过程伸出手来。然而，随着损伤，小胶质细胞可能错误地允许异常连接逗留或可能不适当地损害正常连接。

Neurons in the brain interact with other cell types. One type, microglia, are star-shaped cells that survey the connections between neurons with their arm-like branches. It is important to understand these interactions because changes in neural connections underlie learning and memory formation. The Principal Investigator hypothesizes that microglia might regulate such changes in neural connectivity. This project will investigate whether microglia are directly involved in disconnecting neurons, and whether these actions are disrupted by injury. In response to injury, microglia lose their branches, and the Principal Investigator hypothesizes that this prevents them from properly regulating neural connections. The work will use a specially-designed microscope that images cells within the living brain. To make the cells visible, the microglia will be engineered to look green under the microscope, and, using a strategy developed in this project, each neuron will be labeled with a unique color. With this approach, it will be possible to visualize how microglia interact with neurons under normal conditions and after injury. Analyzing such images is complicated and requires neurobiologists who are trained to use high-level mathematics. This project will develop software and teaching tools to make the mathematics behind image analysis understandable to scientists who were not trained as engineers. The Principal Investigator will create an inquiry-based module about these ideas called "Beyond the Image," which will introduce high school students to image analysis and digital processing. The module will be made available to teachers everywhere through websites such as the Cornell Cooperative Extension School's 4-H STEM site. Because neural branches are small and densely packed, the proposed imaging of microglial modulation of neural connections is challenging. A technical innovation will be to infect neurons using a combination of virus strains that each generate a different color of fluorescent protein. Each neuron will be labeled with its own unique color code, enabling its branches to be distinguished. An advantage of using viruses is that both the type and location of labeled cells can be varied to optimize color-coding. Rather than wait for neurons to disconnect by chance, a laser will be used to precisely cut a branch off of a neuron. Time-lapse imaging will capture the reactions of microglia and neurons to the now useless, severed branch. The prediction is that microglia will reach out with processes to engulf and remove defunct connections. However, with injury, the microglia might erroneously allow aberrant connections to linger or might inappropriately damage normal connections.