PROJECT SUMMARY/ABSTRACT Memories are stored through long-lasting biological changes in the brain called engrams. An engram is defined as the neural ensemble activated during learning and whose reactivation by the original stimulus results in memory retrieval. Studies have shown that engrams are sparse neuronal ensembles distributed throughout the brain that undergo synaptic modifications and that these ensembles can be manipulated in disease states to alleviate mood and cognitive disorders. Examining the underlying neurobiological mechanisms of engram function will allow for a deeper understanding of how an individual memory is encoded in the brain and how memory processes can be altered in disease states. The methods to visualize and quantify engram populations in the brain rely on immediate early genes (IEGs), which are expressed upon neuronal activation. So far, studies that utilize IEGs to visualize engram populations focus on neuronal ensembles identified by single IEGs, which have been assumed as interchangeable in the field. However, recent evidence has shown that ensembles labeled by different IEGs within an engram are functionally distinct neuronal populations that drive opposing memory-related behaviors. The main goal of this proposal is to identify if ensembles tagged by Arc and c-fos, the most utilized IEGs in memory research, have similar or different roles in memory processes within an engram. I will focus on characterizing the synaptic, anatomical, and functional differences between these ensembles using novel techniques such as a recently generated dual activity-dependent labeling system, electrophysiology, circuit tracing, and simultaneous in vivo calcium imaging and optogenetics. In Aim 1, I will identify and quantify the similarities and differences between Arc+ and c-fos+ ensembles by tagging two engrams in a single mouse. I have recently generated a mouse model in which two engrams can be tagged simultaneously. In Aim 2, I will utilize anatomical viral tracing strategies and electrophysiology to characterize the synaptic excitability and connectivity profile of Arc+ and c-fos+ ensembles. For Aim 3, I will utilize nVoke minimicroscopes developed by Inscopix to perform simultaneous in vivo calcium imaging and optogenetics to identify and quantify how Arc+ and c-fos+ ensembles either similarly or differentially modulate downstream activity. The training plan presented in this F31 application will provide me with technical expertise in electrophysiology and simultaneous optogenetics and in vivo calcium imaging, strengthen my data analysis skills, and help me develop the professional skills necessary to become an independent scientist focused on studying the cellular mechanisms of memory.