Regulation of the release probability (Pr) of single synaptic vesicles (SVs) from active zones (AZs) determines the presynaptic contribution to synaptic strength. Dynamic regulation of synaptic strength is thought to underlie learning and memory, so fundamental knowledge of the molecular mechanisms governing Pr is critical to understanding these vital neuronal processes. The Drosophila neuromuscular junction (NMJ) is a widely-used model synapse for studying Pr due to its accessibility and genetic toolkit. In this system, two glutamatergic motor neuron classes (termed 1b and 1s) with distinct excitability and synaptic properties each form hundreds of AZs onto individual body wall muscles. At each AZ, structural proteins position SVs near voltage-gated calcium channels. Calcium influx is detected by calcium-sensing proteins on SV membranes, triggering SV release. However, calcium is also quickly buffered once inside the cell, making the distance between SVs and calcium channels extremely important for determining Pr. Recent studies have shown that Pr is heterogeneous across AZs formed by 1b and 1s and can vary as much as 50-fold between neighboring AZs. However, the sources of this heterogeneity have not been fully identified. Accumulating evidence suggests that structural AZ proteins play a large role in regulating Pr by regulating position of SVs relative to calcium channels. Super-resolution stimulated emission depletion (STED) imaging reveals that AZ structural proteins vary widely in their nanoscale morphology across individual AZs, suggesting that heterogeneity in AZ morphology could explain heterogeneity in Pr across AZs. Additionally, recent data indicates that average AZ morphology and Pr are significantly different between AZs formed by 1b and 1s, suggesting that a difference in gene expression between these neurons is likely to control cell-wide AZ morphology. Single-cell RNA sequencing indicates that Toll-6 is one of the most differentially expressed genes between 1b and 1s neurons, and preliminary data indicates that it is capable of affecting AZ morphology and electrophysiology making it a likely candidate underlying difference in AZ properties between these neuronal classes. To determine how AZ morphology correlates with Pr across 1b and 1s neurons, a novel genetically encoded fluorescent release sensor and STED microscopy will be used on Drosophila NMJs to compare nanoscale AZ morphology and Pr at single AZs. An exogenous calcium buffer will be used to investigate importance of morphology in determining nanodomain distance coupling between SVs and calcium channels. To determine how Toll-6 affects cell-wide AZ morphology and Pr, Toll-6 expression levels in 1b and 1s neurons will be individually genetically manipulated. This project will be carried out in the lab of Dr. Troy Littleton in the Picower Institute for Learning and Memory (PILM) at the Massachusetts Institute of Technology (MIT). All necessary equipment is available through...