Brainbow: Novel tools for studying the development of neuronal circuits

描述(申请人提供)：人脑的组织依赖于数千亿个神经元之间的精确连接。这些连接中的绝大多数是在神经系统发育期间建立的，此时神经元找到并连接到适当的靶点。到目前为止，对神经元回路发育和成熟的详细研究一直受到技术限制的阻碍，这些限制阻碍了许多相互作用的神经元的清晰可视化。然而，新的“脑弓”转基因小鼠使用随机表达的多个荧光蛋白，以允许清楚地识别复杂神经元回路中的多个单独成分。这些新工具通过允许人们提取以前无法获取的关于特定神经回路的信息，极大地提高了人们研究神经元连接及其发展的能力。使用共聚焦成像方法，可以通过直接可视化整个神经系统中许多相互作用的神经元的轴突和树突来构建清晰的电路图。然而，现有的图像分析方法不足以处理大数据集，需要在现有技术中取得新的进步。这项研究计划旨在创建和利用计算机算法来自动化大型神经元数据集的重建，并生成新的脑弓小鼠品系以改进分析。然后，这些新的工具将被用来彻底调查年轻小脑中神经元电路是如何发展的，以及在一种名为共济失调的发育性小脑疾病的小鼠模型中，小脑电路是如何异常形成的。这些研究将有助于更好地了解哺乳动物大脑中神经元电路的发展和功能。公共卫生相关性这项工作的目标是了解发育中的大脑是如何形成电路的。当神经发育出错时，人类会出现大量的疾病；对大脑回路的更好理解可以导致对疾病的适当治疗。这项研究将开发新的工具，帮助我们了解导致大脑内正常和异常连接的发育机制。

DESCRIPTION (provided by applicant): The organization of the human brain relies upon precise connectivity among hundreds of billions of neurons. The vast majority of these connections are established during nervous system development, when neurons find and connect to appropriate targets. To date, detailed studies of neuronal circuit development and maturation have been hampered due to technical limitations that prevent the clear visualization of many interacting neurons. New "Brainbow" transgenic mice, however, use random expression of multiple fluorescent proteins to allow for the clear identification of multiple individual components within complex neuronal circuits. These new tools greatly enhance one's ability to study neuronal connectivity and its development by allowing one to extract previously inaccessible information about specific neural circuits. Using confocal imaging methods, clear circuit diagrams can be constructed by direct visualization of the axons and dendrites of many interacting neurons throughout the nervous system. However, existing methods of image analysis are not sufficient for large Brainbow datasets, requiring new advancements in current techniques. This research proposal aims to create and utilize computer algorithms for automating the reconstruction of large neuronal datasets, and to generate new Brainbow mouse lines for improved analysis. These new tools will then be used to conduct a thorough investigation into how neuronal circuitry develops in the young cerebellum, and how cerebellar circuitry may form abnormally in an ataxic mouse model of the developmental cerebellar disease known as ataxia telangectasia. These studies will help to provide a better understanding of how neuronal circuits develop and function in the mammalian brain. PUBLIC HEALTH RELEVANCE The goal of this work is to understand how circuits form in the developing brain. There are a great deal of human disorders that arise when neurological development goes awry; a better understanding of brain circuitry can lead to appropriate treatments for disease. This research will develop new tools that will help us to understand the developmental mechanisms that lead to normal and abnormal connectivity within the brain.