CATT: Development and Application of a Neuronal Cell Activity-Tagging Toolbox

CATT：神经元细胞活动标记工具箱的开发与应用

• 批准号: 9105311
• 负责人: CAROL A. BARNES
• 金额: $ 34.55万
• 依托单位: TRANSLATIONAL GENOMICS RESEARCH INST
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-30 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9105311
• 关键词: Affect; Aftercare; Age; Aging; Alzheimer' s Disease; Animals; Appearance; Area; Behavior; Behavior Control; Behavioral Paradigm; Beryllium; Biomedical Research; Brain; Brain Diseases; Brain Mapping; Brain region; Bypass; Cells; Cellular Compartment Analysis; Cellular Structures; Characteristics; Collection; Communities; Detection; Development; Disease; Electroconvulsive Shock; Elements; Environment; Fluorescence; Fluorescent in Situ Hybridization; General Population; Generations; Genes; Genetic Transcription; Goals; Grant; Health; Hippocampal Formation; Image; Inbred F344 Rats; Individual; Investigation; Knowledge; Label; Lasers; Learning; Longevity; Maps; Methodology; Methods; Modeling; Molecular; Neurons; Neurosciences; Nuclear; Pattern; Pentylenetetrazole; Performance; Physiology; Production; Proteins; RNA; Rattus; Reliability of Results; Research Personnel; Role; Seizures; Specific qualifier value; Specificity; Staging; System; Techniques; Tetanus Helper Peptide; Time; Trans-Activators; Transcript; Transgenic Animals; Viral Vector; Work; adeno-associated viral vector; age group; age related; base; brain circuitry; connectome; dentate gyrus; design; driving behavior; experience; granule cell; healthy aging; in vivo; interest; molecular dynamics; mutant; nervous system disorder; neuronal circuitry; next generation; next generation sequencing; normal aging; novel; novel strategies; prevent; promoter; recombinase; therapy development; tool; transcriptome; transcriptome sequencing; treatment group; trend; vector

 DESCRIPTION (provided by applicant): Changes in the brain that are associated with normal aging and age-related disease represent a significant area of biomedical research as the general population continues to enjoy lengthened health and lifespan. One major obstacle confronting the study of the brain has been the difficulty of identifying the cellular composition f circuits active in the production of specific behaviors. While it is critical to understand the oveall wiring between brain regions engaged during active behavior, it is also necessary to identify which elements are active within those circuits during a given experience. Methods such as cellular compartment analysis of temporal activity by fluorescence in situ hybridization (catFISH) are able to identify cells active during discrete behaviors with temporal specificity; however, thi technique requires FISH methods that are time consuming, involve thresholding decisions and fluorescence detection issues during imaging that can impact the reliability of the results, and can affect the integrity of RNA molecules in such a way that precludes next generation sequencing of the cellular transcriptome. What is needed is a method that can bypass FISH methodologies and directly tag cells that participate in a specific behavior of interest. An abilit to do this would open up significant new areas of investigation not only to changes associated with aging and disease but many other areas of neuroscience as well. We propose to develop a viral vector based approach to fluorescently label individual cells within the circuit activated during a user-specified behavior in the rat. This system is designed to be modular, adaptable, and could, in principle, be utilized to label any electrically active cell in any brain region. We ill further develop this approach during the early stages of this grant and will freely share the resulting molecular constructs with the scientific community. We will also utilize the novel activated cell tagging approach to investigate the role of aging on circuit composition and utilization along with activated cell transcriptome dynamics. The overall goal of the grant will be achieved through the following Specific Aims. Aim 1 is to identify and prioritize the molecular components of the cell activity-tagging toolbox (CATT) and reduce it to practice in the F344 rat model. Aim 2 will utilize CATT to investigate the whole brain circuit changes associated with novel environment exploration and aging in the F344 rat. We will investigate the cellular composition of circuits in three different ages - 6, 12, and 24 months - combining CATT with the CLARITY approach. Aim 3 will utilize CATT to investigate the transcriptional changes within the hippocampal formation associated with aging in the F344 rat. For focus, we will characterize the transcriptional profile of CATT-labeled cells within the dentate gyrus from the same ages investigated in Aim 2. Our overall goal is to develop methods to label cells that were active during a defined temporal period and utilize that new approach to investigate the impact of aging on the circuit elements engaged by those behaviors as well as the transcriptional function of those behavior-driven labeled cells.

 描述（由申请人提供）：与正常衰老和年龄相关疾病相关的大脑变化代表了生物医学研究的一个重要领域，因为普通人群的健康和寿命持续延长，这是研究面临的一个主要障碍。大脑一直是识别在产生特定行为时活跃的细胞组成的一个难题，虽然了解活跃行为期间参与的大脑区域之间的整体连接至关重要，但也有必要确定这些元素中哪些元素是活跃的。给定期间的电路通过荧光原位杂交 (catFISH) 进行时间活性的细胞区室分析等方法能够识别具有时间特异性的离散行为期间的细胞活性；然而，这需要耗时的 FISH 方法，涉及阈值技术决策和荧光检测。成像过程中的问题可能会影响结果的可靠性，并可能影响 RNA 分子的完整性，从而妨碍细胞转录组的下一代测序，我们需要一种可以绕过 FISH 方法并直接标记细胞的方法。参加一个做到这一点的能力不仅可以开辟与衰老和疾病相关的变化的重要新研究领域，而且还可以开发一种基于病毒载体的方法来荧光标记单个细胞。该系统设计为模块化、适应性强，原则上可用于标记任何大脑区域的任何电活动细胞。这笔赠款的早期阶段，并将免费分享我们还将利用新的激活细胞标记方法来研究衰老对电路组成和利用以及激活细胞转录组动力学的作用。 通过以下具体目标实现：目标 1 是确定细胞活性标记工具箱 (CATT) 的分子成分并对其进行优先级排序，并将其简化并在 F344 大鼠模型中进行实践；目标 2 将利用 CATT 来研究整个大脑回路的变化。与 F344 大鼠的新环境探索和衰老相关我们将结合 CATT 与 CLARITY 方法研究三个不同年龄（6、12 和 24 个月）的回路的细胞组成。 3 将利用 CATT 研究与 F344 大鼠衰老相关的海马结构内的转录变化。为了重点，我们将表征目标 2 中研究的相同年龄的齿状回内 CATT 标记细胞的转录谱。目标是开发方法来标记在定义的时间段内活跃的细胞，并利用这种新方法来研究衰老对这些行为所涉及的电路元件以及这些行为驱动的标记细胞的转录功能的影响。