PARENT R35 ABSTRACT Direct examination of presynaptic processes has historically been limited by the resolution constraints of conventional light microscopy. As a result, much of what we know about vesicle movement, fusion, and recycling relies on inferences from indirect electrophysiological and/or biochemical assays, or from electron micrographs that reflect a single instant of a dynamic system. The long-term goal of my research program is to understand the fundamental mechanisms of synaptic transmission at central synapses, including details of spatiotemporal dynamics under normal conditions, and what disruptions lead to disease states. Current projects in the lab address two central knowledge gaps. First, we directly probe and track dynamic presynaptic processes in living tissue by applying our own novel, nanoscale resolution imaging technology. Using this approach, we will, for the first time, visualize these processes at the level of single synaptic vesicles within identified synapses. We have already made significant contributions using this approach, including the discovery that synaptic vesicle dynamics are active, not passive, and are controlled by actin cytoskeleton and myosin motors. The second major knowledge gap we address is the contribution of presynaptic deficits to pathophysiology of Fragile X syndrome (FXS). FXS is the most common known cause of heritable intellectual disability and autism. Our recent findings have triggered a necessary shift in the field towards considering the contributions of presynaptic mechanisms in addition to postsynaptic mechanisms, thus creating an entirely new array of diagnostic and therapeutic possibilities. Continuing work in this area will focus on linking presynaptic defects with abnormalities at the circuit level and the implications of these abnormalities for behavior and cognition. Sustained funding through this R35 mechanism will support a multipronged approach to these important neurobiological questions that will maximize the potential for synergy and translational impact.