PROJECT SUMMARY/ABSTRACT The discrimination of highly similar episodes is termed behavioral pattern separation. This episodic memory process is altered by stress and decreased in humans with and rodent models for a range of brain disorders, including post-traumatic stress disorder (PTSD). Behavioral pattern separation is also susceptible to “load”; it is harder to discriminate episodes that are very similar (high load) vs. different (low load). Defining the underlying circuitry in memory stages and load-sensitivity is a key step to a future where poor behavioral pattern separation might be treated via circuit-based manipulations. The focus of this application is the role of the lateral entorhinal cortex (LEC) in behavioral pattern separation. The anatomical connections of the LEC suggest it is central to this stress-sensitive process. The LEC is innervated by polymodal-, emotion, and stress-linked brain regions. The LEC innervates downstream hippocampal regions critical for behavioral pattern separation, including the dentate gyrus (DG). In fact LEC layer IIa stellate fan cells (LECIIa fan cells) send glutamate directly to two key DG cells, DG granule cells and adult-generated neurons, which are both critical for “high load” pattern separation and are very sensitive to stress. Excellent human imaging and rodent lesion and neural recording studies also suggest the LEC has a role in behavioral pattern separation. However, the LEC’s causal role in orchestrating behavioral pattern separation and its memory stages is untested. The lack of data on LEC’s role is striking given that the LEC is vulnerable to stress, aging, and disease. A link between LEC and the poor pattern separation seen in age and disease — including in stress-induced cognitive disorders like PTSD — remains correlative. Direct evidence of the LEC’s role in behavioral pattern separation is paramount to clear understanding of cortical-hippocampal circuitry and its function in nonpathological and pathological states. In this revised R01 application, we propose three aims to provide fundamental understanding of how LECIIa fan cells are involved in behavioral pattern separation, during what memory stage and which memory load, and how the LEC-DG circuit activity could be manipulated to overcome stress-induced disruption of pattern separation. Aim 1. Test if the encoding and consolidation of behavioral pattern separation rely on the activity of LECIIa fan cell terminals in the DG. Aim 2. Test if behavioral pattern separation performance/retrieval is modulated by the activity of the LEC fan cell-DG circuit. Aim 3. Test if repeated stress disrupts behavioral pattern separation performance/retrieval in a way that can be reversed by LEC-DG circuit stimulation. The data from these Aims will fill major knowledge gaps in the existing models of the neural circuitry that supports behavioral pattern separation. They will provide essential behavioral and mechanistic insight to understand poor pattern s...