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.
We will develop a collection of molecular tools that facilitate the fluorescent labeling of neurons that are active during a user-specified behavioral paradigm in the rat. This significant advance will allow us to specifically dissect out the neuronal circuit changes that occur during healthy aging. A better understanding of age-related differences at this level of detail will dramatically impact our knowledge of how transcription within specific brain networks is impacted by aging, providing the backdrop for understanding age-associated brain diseases like Alzheimer's disease.
