-Abstract - Synapses are the fundamental units of communication in the nervous system and reliable synaptic transmission is central to key nervous system processes such as learning, memory, and sensory adaptation. Moreover, due to dysfunctions at the synapse, many neurological diseases may develop. While electrophysiological studies of synaptic transmission have been around for a long time, only the recent development of optical quantal analysis (OQA) tools has made possible to correlate morphological and structural elements to transmission properties of individual synapses. Studies using OQA have revealed a much larger diversity in synaptic transmission, even among neighboring synapses, than was previously thought. The advent of higher-resolution OQA, such as the one developed in our lab called “QuaSOR”, and super-resolution structural imaging methods opens up an exciting frontier of being able to investigate how neural activity shapes (and is shaped by) synaptic diversity, what are the molecular determinants and mechanisms the set this diversity, and how do these molecular determinants shape synaptic diversity. By using OQA, we've shown that synaptic diversity, as measured by difference in synaptic strength (i.e., probability of action potential evoked transmission; Pr ) is extremely heterogeneous (Pr: 0.01–0.5) within a single neuron synapsing onto a single target cell. This high degree of heterogeneity leads us to the central hypothesis of my thesis which is that synaptic strength is set by a very precise, local distribution of key proteins. To test this hypothesis I will use the model glutamatergic synapse–Drosophila melanogaster larval neuromuscular junction (NMJ)– where I will investigate hundreds of synapses in parallel, in vivo, and address synaptic heterogeneity from both functional and structural perspectives at single synapse resolution (50-100nm). For my thesis I am being trained in synaptic physiology, fly genetics, and advanced super-resolution functional/structural imaging and analysis. In Aim 1 of my thesis, I will establish the degree of functional synaptic heterogeneity in vivo single synapses using QuaSOR. Specifically, I have completed the preliminary experiments investigating the extent of basal synaptic heterogeneity and addressing the relationship between basal strength of synapses (basal Pr) and synaptic adaptation to higher frequencies. Through super-resolution structural imaging experiments proposed for Aim 2, I will investigate some of the key proteins at the synapse and determine whether it's the local quantities, the relative abundance between them (ratios), and/or the nanolocalization within the synapse that shape synaptic diversity. Finally, for Aim 3, through chronic and acute manipulations, I will determine the role of Unc-13a in shaping this diversity. Through the combination of super resolution structural and functional imaging, this proposed work will yield much needed insight into the molecular determinants...