Brain circuit mapping using light inducible recombinase systems

项目摘要 哺乳动物的大脑由大量错综复杂的神经元组成， 分子、解剖和生理特征。为了理解这些独立建筑的作用 大脑的块，这将是至关重要的，以开发时空精确的工具，将允许神经元 亚型特异性单细胞水平分析。在这个项目中，我们建议开发分子工具， 允许高通量的单细胞基因组修饰，并应用它们以在功能上和 在哺乳动物脑内的细胞类型特异性回路的形态学特征。为了实现这一目标，我们 一种新的方法将是使用光来触发位点特异性DNA修饰酶。位点特异性DNA 重组酶已经是非常有用的工具，用于解剖的功能和遗传组成部分， 神经系统，因为它们能够精确地修改细胞内的单个基因。但他们 缺乏以高时空精度进行调节以精确靶向单个细胞的能力。 我们修改了其中两种酶-Cre和Dre-通过将它们与基于真菌的光结合， 诱导蛋白质，使得在光诱导时可以触发酶的活性。 我们建议进一步将相同的策略应用于Flp重组酶，以富集Flp重组酶。 交叉方法。然后，我们将使用这些重组酶来产生Cre依赖性小鼠系 这样我们就可以将光诱导重组限制在单独或联合的神经元亚型中， 与现有的Cre驱动器线组合。使用这些线，我们将执行光诱导聚焦 重组，以便进行荧光标记的稀疏细胞类型特异性基因组重组。 报告员在轴突和树突细节中完全重建单个神经元亚型。此外我们 将联合收割机结合这种方法与双光子激发进行单细胞狂犬病追踪实验。 我们的研究结果将为详细分析神经元类型特异性功能和 解剖电路，将有可能链接遗传身份，形态，连接和 功能

Project Summary Mammalian brain is composed of vast numbers of intricately interconnected neurons with various molecular, anatomical and physiological identities. To understand the roles of these individual building blocks of the brain, it will be critical to develop spatio-temporally precise tools that will allow neuronal subtype specific single cell level analysis. In this project, we propose to develop molecular tools that will allow high throughput single cell genomic modifications and apply them in order to functionally and morphologically characterize cell type specific circuits within the mammalian brain. To achieve this, our approach will be to use light to trigger site-specific DNA modification enzymes. The site-specific DNA recombinases have been extremely useful tools for dissecting the functional and genetic components of the nervous system due to their ability to precisely modify individual genes within cells. However, they lack the ability to be regulated with high spatiotemporal accuracies to precisely target individual cells. We modified two of these enzymes -Cre and Dre- by combining them with a fungal-based light inducible protein in such a way that upon light induction the activity of the enzymes can be triggered. We propose to further apply the same strategy to the Flp recombinase in order to enrich the intersectional approach. We will then use these recombinases to generate Cre dependent mouse lines so that we can restrict the light inducible recombination to further subtypes of neurons either alone or in combination with existing Cre driver lines. Using these lines we will perform light induced focal recombination in order to perform sparse cell type specific genomic recombination of fluorescent reporters to fully reconstruct individual neuron subtypes in axonal and dendritic detail. Furthermore we will combine this approach with 2-photon excitation to perform single cell rabies tracing experiments. Our results will set the foundation for extremely detailed analysis of neuron type specific functional and anatomical circuits that will make it possible to link genetic identity, morphology, connectivity and function.