Novel Genetic Strategy for Sparse Labeling and Manipulation of Mammalian Neurons

 描述（由申请人提供）：Cajal通过使用高尔基染色剂以揭示神经元整体形态的方式稀疏地和随机地标记细胞，彻底改变了大脑的研究。虽然在过去的15年中，用于果蝇中单个神经元的稀疏和随机标记和操作的遗传工具已被广泛使用，但它们最近才可用于哺乳动物系统，但后者的工具仅限于少数几个系统，其中细胞类型特异性试剂（例如增强剂）可用或涉及繁琐的操作。因此，在本领域中存在开发用于在单细胞水平上分析神经元的稳健试剂的重要需求。事实上，在单个识别的细胞水平上对神经元的分析提供了关于控制神经元形态、连接、生理和可塑性的关键信息。本申请是对BRAIN Initiative RFA-MH-14 - 216的响应。我们提供了概念验证的初步数据的一般方法来标记和操纵单个神经元的小鼠中枢神经系统。使用这种方法（称为MORF），我们创建了一种新的多巴胺D1受体BAC转基因小鼠，可以稀疏和随机标记的D1表达纹状体直接通路介质棘神经元以及海马锥体神经元的子集。标记的神经元显示详细的形态学，包括树突状乔木和突触。我们建议进一步验证和扩展这种技术，使其普遍用于单神经元遗传标记以及哺乳动物大脑中多种神经元细胞类型的遗传操作。此外，我们建议修改MORF，以促进表位标记的突触蛋白从其内源性基因座的图像突触的单个确定的细胞类型。我们将开发和简化成像和计算工具，以获取和登记标准脑图谱中的全脑单神经元形态信息，以便将数据快速传播给研究界。总之，我们提出的计划将开发一种新的遗传导向的单神经元标记工具，该工具在概念上与现有工具不同，并且比现有工具简单得多，沿着简化的成像和映射方法，以便于使用模型提供的丰富的单神经元信息。这里开发的新工具和小鼠资源应该对神经科学和脑疾病相关领域立即有用和有影响力。

 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.