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

 描述(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着生物医学研究的一个重要领域，因为普通人群继续享受着更长的健康和寿命。大脑研究面临的一个主要障碍是很难识别活跃在特定行为产生过程中的回路的细胞组成。虽然了解活跃行为中大脑区域之间的所有连接是至关重要的，但也有必要识别在给定体验期间这些回路中哪些元素是活跃的。通过荧光原位杂交(CatFISH)对细胞时间活动的细胞室分析能够确定在离散行为期间活跃的细胞，具有时间特异性；但是，这种技术需要FISH方法，这些方法耗时，涉及阈值决策和成像过程中的荧光检测问题，这些问题可能会影响结果的可靠性，并且可能会影响RNA分子的完整性，从而排除了对细胞转录组的下一代测序。需要的是一种方法，可以绕过FISH方法，直接标记参与感兴趣的特定行为的细胞。这样做的能力将开辟重要的新研究领域，不仅研究与衰老和疾病相关的变化，而且还研究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大鼠在用户指定行为期间激活的回路中的单个细胞。这个系统被设计成模块化的，适应性强，原则上可以用来标记任何大脑区域的任何电活动细胞。我们将在这笔赠款的早期阶段进一步发展这一方法，并将免费与科学界分享由此产生的分子构造。我们还将利用新的激活细胞标记方法来研究老化对电路组成和利用以及激活细胞转录组动力学的作用。赠款的总体目标将是 通过以下具体目标实现。目标1是确定细胞活性标签工具箱(CATT)的分子成分并确定其优先顺序，并将其减少到F344大鼠模型中。目的2将利用CATT来研究与新环境探索和F344大鼠衰老相关的整个大脑回路的变化。我们将结合CATT和Clarity方法研究三个不同年龄-6、12和24个月的电路的细胞组成。目的3将利用CATT来研究F344大鼠海马结构内与衰老相关的转录变化。对于FOCUS，我们将描述目标2中研究的相同年龄的齿状回内CATT标记细胞的转录图谱。我们的总体目标是开发方法来标记在定义的时间段内活跃的细胞，并利用这一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。

 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.