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

追踪和操纵斑马鱼神经元回路的基因编码方法的开发和验证 - 多样性补充

• 批准号: 10731536
• 负责人: CARLOS LOIS
• 金额: $ 17.72万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10731536
• 关键词: Address; Affect; African American; African American population; Alzheimer' s Disease; Anatomy; Animal Model; Animals; Bachelor' s Degree; Behavior; Behavioral; Behavioral Assay; Biological Assay; Biological Models; Brain; Brain Diseases; Brain Mapping; Brain imaging; Communities; Complex; Data; Decision Making; Development; Development Plans; Disease; Doctor of Philosophy; Drosophila genus; Education; Electroporation; Embryo; Ensure; Feedback; Fellowship; Fishes; Flow Cytometry; Genes; Genetic; Goals; Health; Image; Jasminum; Journals; Mentors; Methods; Modality; Modeling; Modernization; Modification; Molecular; Names; Neurons; Neurosciences; Optics; Oral; Population; Postdoctoral Fellow; Process; Property; Publishing; Research; Schizophrenia; Scientific Advances and Accomplishments; Scientist; Sleep; Statistical Methods; Synapses; System; Techniques; Technology; Training; Transgenic Organisms; United States National Institutes of Health; Validation; Vertebrates; Visual; Visualization; Writing; Zebrafish; analytical method; anatomical tracing; autism spectrum disorder; brain research; career; career development; experience; experimental study; genetic manipulation; graduate student; member; motor learning; neuronal circuitry; neuropsychiatric disorder; new technology; parent grant; parent project; postsynaptic neurons; pre-doctoral; single-cell RNA sequencing; skills; success; symposium; synergism; tool

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. 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. 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, 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 is constrained by a lack of methods to identify and perturb synaptically connected neurons. In preliminary studies, we developed a TRACT system that can identify anterograde monosynaptic connections between neurons in the zebrafish brain. In the parent grant, 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 synaptically connected neurons. This diversity supplement application describes an experimental and conceptual career development plan for a graduate student whose experimental goals are to (1) determine whether TRACT can function in all neurons in the brain, (2) determine whether transient expression or electroporation can be used to avoid the need to generate transgenic fish and thus expedite the use of TRACT for diverse neuronal populations, and (3) use flow cytometry and single cell RNA sequencing to identify synaptically connected neurons in a comprehensive and high-throughput manner. This experimental plan directly relates to the parent grant by further developing the TRACT system in zebrafish in experiments that are separate from, yet synergize with, the experiments described in the parent grant. Together, the parent grant and diversity supplement have the potential to establish a powerful new technology for mapping brain circuits that will increase the usefulness of zebrafish as a model system to study vertebrate neuronal circuit function, 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.

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。当前可用 这些方法具有局限性，使得可视化这些大脑接线图具有挑战性。此外还有 迫切需要一种方法，不仅可以揭示大脑的连接，而且可以从基因上 修改电路中连接的神经元的功能特性。我们最近开发了一个基因系统 命名为TRACT，并使用果蝇证明它具有这两种特征。然而，许多复杂的 大脑功能无法在果蝇中进行检查，了解它们需要使用脊椎动物 动物斑马鱼已成为研究复杂脑过程的有用脊椎动物模型， 相对简单但保守的脊椎动物大脑，光学透明度，对大规模行为的适应性， 分析，在仅仅5天的发育后出现复杂的行为，以及越来越多的遗传学特征。 这些工具允许观察和操纵行为动物的神经回路。然而， 由于缺乏识别和干扰突触连接神经元的方法，斑马鱼的研究受到限制。在 在初步研究中，我们开发了一个TRACT系统，可以识别顺行单突触连接 神经元之间的联系。在家长补助金中，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基因操纵突触连接的神经元。这种多样性补充 应用程序描述了一个实验性和概念性的职业发展计划的研究生， 实验目标是（1）确定TRACT是否可以在大脑中的所有神经元中发挥作用，（2）确定 瞬时表达或电穿孔是否可用于避免产生转基因鱼的需要， 从而加快了TRACT在不同神经元群体中的应用，以及（3）使用流式细胞术和单细胞 RNA测序以全面和高通量的方式鉴定突触连接的神经元。 该实验计划通过进一步开发斑马鱼中的TRACT系统直接关系到父母补助金 在实验是独立的，但协同作用，实验中描述的母基金。 父母补助金和多样性补助金一起有可能建立一种强大的新技术 用于绘制大脑回路，这将增加斑马鱼作为研究脊椎动物模型系统的有用性 神经元回路功能，揭示神经元回路的一般原则，这些原则是特定行为的基础， 模拟复杂的大脑疾病，如自闭症、阿尔茨海默病和精神分裂症。