PROJECT SUMMARY / ABSTRACT Memories are thought to be stored through lasting physical and chemical changes in the brain. The biological substrate of a memory is known as an engram or memory trace. An engram is the neural ensemble activated during learning and whose reactivation by the original stimulus results in memory retrieval. Modern technological advances have allowed scientists to visualize and manipulate engrams, showing that they exist in various brain regions and their content can be altered. These strategies have taken advantage of immediate early genes (IEGs) such as Arc, which are expressed upon neuronal activation. Transgenic and viral tools, such as the ArcCreERT2 mice and the Robust Activity Marking (RAM) system, have been engineered to label cells in an activity-dependent manner. Although these tools have furthered our understanding of how a single engram is represented in the brain, they have not been combined to label multiple engrams. Thus, this research plan focuses on identifying multiple engrams in a single mouse and characterizing the synaptic mechanisms underlying multiple engram storage. To achieve these goals, I have developed and characterized a novel viral strategy that allows for the labeling of previously activated Arc+ ensembles. I have combined this viral strategy with the ArcCreERT2 x enhanced yellow fluorescent protein (EYFP) mice to ultimately label multiple Arc+ ensembles and visualize multiple engrams in a single mouse. Using this novel combinatorial transgenic/viral strategy, I have shown that the engrams of exposures to a single environment are reactivated to a greater extent those representing exposures to different environments. Moreover, the synaptic mechanisms that contribute to the storage of multiple memories in the hippocampus is poorly understood. To this end, during the F99 phase, I propose to use ex vivo electrophysiology to identify synaptic signatures (e.g., excitability, connectivity) of co-labeled Arc+ ensembles. A greater understanding of the synaptic properties of the ensembles that are recruited to multiple engrams will contribute to our overall understanding of how memories are co-stored in the brain. For the K00 phase, I will investigate the population-level dynamics of hippocampal ensembles during memory processes using large-scale recording techniques and computational models. These studies will illuminate our understanding of the computations performed by the hippocampus that support memory processes and will allow us to generate more precise hypotheses for how these computations might go awry in memory-related disorders, such as Alzheimer’s Disease (AD). The completion of this research proposal will provide me with training in electrophysiology, strengthen my technical and professional skills, and facilitate my transition to a postdoctoral position in a laboratory that studies population dynamics of neural ensembles and computational methods.