Neurons have the remarkable ability to process and respond to complex stimuli such as physical exercise and changes in an organism?s external environment. The value of exercise for brain health cannot be underestimated as its effects impact mood, learning and memory as well as prevention and rehabilitation and recovery from neurological illness. However, experimental effort largely has been focused on the effects of sustained exercise over periods of weeks or months (1-3), which can involve direct effects on the CNS as well as indirect effects through alterations in multiple organ systems. Likewise, most attention in exercise-induced hippocampal plasticity has been directed at newborn granule cells (1, 3-7), but plasticity occurs in the far more numerous mature granule cells as well (8). Aside from its sustained benefits, acute exercise has also been linked to short term increases in learning and memory (9, 10) that are likely mediated by the hippocampus (11-13). How this occurs at the molecular level is not clear. Thus we decided to examine how a single episode of exercise affects neural activity and impacts brain function. We developed a novel approach for in vivo analysis of dentate granule cells activated by a single episode of voluntary exercise. Our approach, akin to an impulse function in engineering terms, allowed us to examine exercise-induced synaptic and molecular changes over a period of days post-exercise. Mature dentate granule cells, activated by voluntary exercise during a two-hour window, were permanently marked using Fos-TRAP mice (14, 15), in which the immediate early gene promoter linked to a fluorescent reporter, permanently marks activated granule cells. The single episode of exercise resulted in selective increases in synaptic function and dendritic spine density in the outer molecular layer of the dentate gyrus, the lamina receiving contextual information from entorhinal cortex. The top upregulated gene in RNAseq of exercised-activated cells was Mtss1L, a previously understudied gene coding for an I-BAR-domain protein. As BAR domains sense and induce membrane curvature, we hypothesize that Mtss1L is an early effector of dendritic spine and synapse formation following stimuli such as exercise. Our preliminary data lead to a number of interesting questions that will be addressed in this proposal. Namely: 1. Where is Mtss1L localized and why are the effects on synapses limited to a specific lamina in the dentate gyrus?; What are the effects of other I-BAR family members as several are expressed at synapses but only Mtss1L is activity-dependent?; and 3. Do exercise-induced synaptic changes prime specific synapses for learning and memory by salient stimuli? Our approach provides the cellular- and temporal-specificity to link physiologically- and clinically-relevant stimuli in vivo (exercise) to individual synapses and expression of specific genes contributing to structural plasticity in the hippocampus.