Glutamatergic signaling via NMDA receptors (NMDARs) is critical in synaptic plasticity, excitotoxicity, and numerous degenerative and cognitive disorders. Each NMDAR comprises four subunits: two obligatory GluN1 subunits and two GluN2A-D subunits. Notably, NMDARs with different subunit compositions display different channel kinetics and protein interactions, supporting the widely held notion that subunit composition dictates NMDAR function. The subsynaptic organization of NMDARs influences the probability of their activation during synaptic transmission, and extrasynaptic receptors are thought to play roles in cell death, plasticity and gene expression. However, despite evidence for subunit-specific NMDAR nanoclustering, to date there is almost no information about the nanoscale distribution of specific NMDAR subunits within synapses or with respect to sites of vesicular release. Thus, to elucidate the organization of synaptic and extrasynaptic NMDARs with respect to key postsynaptic molecules and presynaptic release sites, in the first aim of my project, I will use a combination of CRISPR-mediated gene tagging and nanobody labeling with DNA-Exchange PAINT super-resolution imaging. These measures will provide a comprehensive map of major NMDAR subunits and provide insight into their roles in neurons. Next, though many NMDARs contain GluN1 and two identical GluN2 subunits, in many brain regions, NMDARs commonly are “triheteromeric” receptors containing both GluN2A and GluN2B with GluN1. However, despite their deduced importance, there are no methods to unambiguously distinguish triheteromeric NMDARs from other subtypes in neurons, and thus their distribution and subcellular trafficking remain mysterious. To overcome this, I have designed a method for direct visualization of triheteromeric NMDARs using bimolecular fluorescent complementation (BiFC). I tagged GluN2A and GluN2B with two parts of a modified fluorescent protein that complement to produce fluorescence only when an NMDAR is assembled containing both the GluN2A and GluN2B subunits (split-tagged NMDARs). In the second aim of my proposal, following a series of validation experiments, I will use these split-tagged NMDARs to address two straightforward but longstanding questions surrounding NMDARs. First, though GluN2A and GluN2B traffic with different dynamics and target subsynaptic regions in part through differing interactions with scaffolding molecules, it is unknown which subunit dominates synaptic integration of triheteromeric receptors. Using my new probes, I will provide the first direct measure of triheteromeric synaptic enrichment. Second, the rate of NMDAR turnover in synapses is critical for determining synaptic strength and plasticity, yet the exchange rate and mobile fraction of triheteromeric receptors have not previously been possible to measure. Using FRAP, I will time determine if triheteromeric NMDARs display unique turnover compared to other NMDARs. Completion of this work...