Novel Genetic Strategy for Sparse Labeling and Manipulation of Mammalian Neurons

 描述(申请人提供)：Cajal通过使用高尔基染色来稀疏和随机地标记细胞，揭示了神经元的整体形态，从而使大脑研究发生了革命性的变化。虽然在过去的15年里，用于稀疏和随机标记和操纵果蝇单个神经元的遗传工具已经被广泛使用，但它们最近才可用于哺乳动物系统，但后一种工具仅限于少数细胞类型特异性试剂(例如增强子)可用的系统或涉及繁琐的操作。因此，在单细胞水平上开发用于神经元分析的强健试剂是该领域的重要需要。事实上，在单个确定的细胞水平上对神经元的分析提供了关于神经元形态、连通性、生理学和可塑性控制的关键信息。此应用程序是对Brain Initiative RFA-MH-14-216的响应。我们为标记和操纵小鼠中枢神经系统中的单个神经元的一般方法提供了概念验证的初步数据。利用这种方法(称为Morf)，我们创造了一种新型的多巴胺D1受体BAC转基因小鼠，它可以稀疏和随机地标记纹状体直接通路中的棘神经元以及海马锥体神经元的一部分。标记的神经元显示了详细的形态，包括树突枝和突触。我们建议进一步验证和扩展这项技术，使其在哺乳动物大脑中的单个神经元遗传标记以及对多种神经细胞类型的遗传操作中都具有普遍用途。此外，我们建议修改Morf，以便于从其内源基因座对突触蛋白进行表位标记，以成像单一已识别细胞类型的突触。我们将开发和简化成像和计算工具，以获取全脑单个神经元的形态信息，并将其登记在标准的脑图谱中，以便将数据快速传播给研究界。总之，我们提议的计划将开发一种新的遗传导向的单神经元标记工具，该工具在概念上不同于现有的工具，并且大大简化了现有的标记工具，以及简化的成像和映射方法，以促进使用由模型提供的丰富的单神经元信息。这里开发的新工具和鼠标资源应该立即对神经科学和脑疾病相关领域有用和有影响。

 DESCRIPTION (provided by applicant): Cajal revolutionized the study of the brain through the use of the Golgi stain to label cells sparsely and stochastically in a fashion that revealed a neuron's morphology in its entirety. Although genetic tools for sparse and stochastic labeling and manipulation of single neurons in Drosophila have been used extensively over the past 15 years, they have only recently become available for mammalian systems, but the latter tools are limited to only a few systems for which cell-type specific reagents (e.g. enhancers) are available or otherwise involve cumbersome manipulations. Thus, there is an important need in the field to develop robust reagents for analysis of neurons at the level of single cells. Indeed, analysis of neurons at the single identified cellular level provides critical information on the control of neuronal morphology, connectivity, physiology and plasticity. This application is in response to BRAIN Initiative RFA-MH-14-216. We provide proof-of-concept preliminary data for a general method to label and manipulate single neurons in the mouse central nervous system. Using this method (called MORF), we created a novel dopamine D1 receptor BAC transgenic mouse that can sparsely and stochastically label a subset of D1-expressing striatal direct pathway medium spiny neurons as well as hippocampal pyramidal neurons. The labeled neurons reveal detailed morphology including dendritic arbors and synapses. We propose to further validate and expand this technique to be of general use for both single-neuron genetic labeling as well as genetic manipulation for multiple neuronal cell types in the mammalian brain. In addition, we propose to modify MORF to facilitate epitope tagging of synaptic proteins from their endogenous loci to image synapses of single identified cell types. We will develop and streamline imaging and computational tools to acquire and register brain-wide single neuron morphological information in a standard brain atlas for rapid dissemination of data to the research community. In summary, our proposed plan will develop a novel genetically-directed single neuron labeling tool that is conceptually different and drastically simpler than those currently available, along with streamlined imaging and mapping methods to facilitate the use of rich single neuron information provided by the models. The novel tools and mouse resources developed here should be immediately useful and impactful for neuroscience and brain disease-related fields.