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: 8798137
• 负责人: CAROL A. BARNES
• 金额: $ 32.82万
• 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/8798137
• 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; Gene Expression Profile; 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; RNA Sequences; 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; dentate gyrus; design; driving behavior; experience; granule cell; healthy aging; in vivo; interest; molecular dynamics; mutant; nervous system disorder; next generation; next generation sequencing; normal aging; novel; novel strategies; prevent; promoter; public health relevance; recombinase; therapy development; tool; 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，因为普通人群继续享受延长的健康和寿命。大脑研究面临的一个主要障碍是，很难确定在产生特定行为时活跃的回路的细胞组成。虽然了解活跃行为期间大脑区域之间的神经连接至关重要，但也有必要确定在特定体验期间这些回路中哪些元素是活跃的。诸如通过荧光原位杂交（catFISH）进行的时间活动的细胞区室分析的方法能够鉴定在具有时间特异性的离散行为期间活跃的细胞;然而，该技术需要耗时的FISH方法，涉及成像期间的阈值决定和荧光检测问题，这会影响结果的可靠性，并且可以影响RNA分子的完整性，从而妨碍细胞转录组的下一代测序。所需要的是一种可以绕过FISH方法并直接标记参与感兴趣的特定行为的细胞的方法。这样做的能力将开辟重要的新研究领域，不仅是与衰老和疾病有关的变化，而且还有神经科学的许多其他领域。我们建议开发一种基于病毒载体的方法来荧光标记在大鼠中用户指定行为期间激活的回路内的单个细胞。该系统被设计成模块化的，可适应的，并且原则上可以用于标记任何大脑区域中的任何电活性细胞。我们将在该资助的早期阶段进一步发展这种方法，并将与科学界自由分享由此产生的分子构建体。我们还将利用新的活化细胞标记方法来研究老化对电路组成和利用沿着活化细胞转录组动态的作用。赠款的总体目标将是 通过以下具体目标实现。目的1是识别和优先考虑细胞活性标记工具箱（CATT）的分子组分，并将其应用于F344大鼠模型。目的二利用CATT技术研究F344大鼠探索新环境和衰老过程中全脑回路的变化。我们将研究在三个不同的年龄- 6，12和24个月-结合CATT与重复性方法的电路的细胞组成。目的3利用CATT技术研究F344大鼠海马结构内转录水平的变化。为了聚焦，我们将描述目标2中研究的相同年龄的齿状回内CATT标记细胞的转录谱。我们的总体目标是开发方法来标记在特定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的影响以及这些行为驱动的标记细胞的转录功能。