Development and Validation of a Genetically Encoded Method to Trace and Manipulate Neuronal Circuits in Zebrafish

项目摘要 确定神经元在大脑中是如何相互连接的是实现这一目标的重要而必要的一步。 了解大脑活动如何引起行为，以及疾病如何干扰大脑活动。不幸的是， 当前可用的方法具有限制，使得可视化这些脑布线图具有挑战性。在 此外，迫切需要一种方法，不仅可以揭示大脑的连通性， 也可以通过基因改变连接在电路中的神经元的功能特性。我们最近开发了一个 该基因系统命名为TRACT，并显示使用果蝇，它拥有这两个特点。但是，在这方面， 许多复杂的大脑功能无法在果蝇中进行研究，了解它们需要使用 脊椎动物。近年来，斑马鱼已成为一种有用的脊椎动物模型进行研究 复杂的大脑过程，由于其相对简单但保守的脊椎动物大脑，光学透明度， 胚胎和幼虫发育阶段，对大规模行为测定的适应性， 在仅仅5天的发育后就有了复杂的行为，还有一套越来越多的遗传工具， 和操纵动物的神经回路。然而，斑马鱼对神经科学的有用性 研究受到缺乏识别和扰动突触连接的神经元的方法的限制。在 在初步研究中，我们开发了一种TRACT系统，可用于识别顺行单突触 斑马鱼大脑中神经元之间的连接。在这里，我们建议进一步发展TRACT作为一种工具， 通过开发额外的解剖追踪模式，并通过建立 使用TRACT对突触连接的神经元进行遗传操纵和功能表征。这些 这些能力将增加斑马鱼作为研究脊椎动物神经元回路的模型系统的有用性 功能，既揭示了神经元回路的一般原则，这些神经元回路是特定行为的基础， 复杂的大脑疾病，如自闭症，阿尔茨海默病和精神分裂症。

PROJECT SUMMARY Identifying how neurons are connected to each other in the brain is an important and necessary step towards understanding how brain activity gives rise to behavior, and how it is perturbed by disease. Unfortunately, currently available methods have limitations that make it challenging to visualize these brain wiring diagrams. In addition, there is an urgent need for a method that will make it possible not only to unveil brain connectivity, but also to genetically modify the functional properties of neurons connected in a circuit. We recently developed a genetic system named TRACT and showed using Drosophila that it possesses both of these features. However, many complex brain functions cannot be examined in Drosophila, and understanding them will require the use of vertebrate animals. In recent years the zebrafish has emerged as a useful vertebrate animal model to study complex brain processes due to its relatively simple yet conserved vertebrate brain, optical transparency during embryonic and larval stages of development, amenability to large-scale behavioral assays, the emergence of complex behaviors after only 5 days of development, and a growing suite of genetic tools that allow observation and manipulation of neuronal circuits in behaving animals. However, the usefulness of zebrafish for neuroscience research is constrained by a lack of methods to identify and perturb synaptically connected neurons. In preliminary studies, we developed a TRACT system that can be used to identify anterograde monosynaptic connections between neurons in the zebrafish brain. Here we propose to further develop TRACT as a tool for transneuronal tracing in zebrafish by developing additional anatomical tracing modalities, and by establishing the use of TRACT to genetically manipulate and functionally characterize synaptically connected neurons. These capabilities will increase the usefulness of zebrafish as a model system to study vertebrate neuronal circuit function, both to reveal general principles of neuronal circuits that underlie specific behaviors, and to model complex brain disorders such as autism, Alzheimer’s disease and schizophrenia.