Internal Dynamics of the Postsynaptic Density

NMDA-type glutamate receptors (NMDARs) shape brain development, synaptic transmission, and disease by acting both as central gatekeepers of synaptic plasticity and as hubs for synaptic protein organization. Strong genetic link between NMDAR subunits and numerous developmental and cognitive disorders places high importance on understanding both the cellular mechanisms that control NMDAR activation and the influence of NMDARs on the synapses where they reside. These receptors are famous as coincidence detectors sensitive to glutamate and postsynaptic voltage, and sensitive to influence at numerous modulatory sites. However, our recent work along with considerable circumstantial evidence from the literature suggests that in fact, activation of these critical receptors is also closely dependent on their subsynaptic positioning and the architecture of the synapse around them. We have found that NMDARs adopt a unique distribution within synapses and are arrayed closely around but offset from just a subset of presynaptic release sites, which modeling predicts will result in high sensitivity to the subsynaptic site of release as well as release probability. Thus, we hypothesize that synapse nanostructure wields strong regulatory control over NMDAR activation. Moreover, our recent work indicates these receptors reciprocally exert direct structural effects on transsynaptic organization. Here, we will dissect this relationship between NMDARs and their synaptic molecular environment. To determine the influence of synaptic nanostructure on receptor activation, we will deploy several information-rich approaches for an unprecedented nanostructure-function investigation of single synapses, including high-throughput optical physiology, post hoc multiplexed nanoimaging, scalable Monte Carlo modeling of these molecularly measured and functionally characterized synapses, and customized machine-learning tools to identify features of synapse protein organization linked to NMDAR signaling. To test directly how the receptors themselves control trans synaptically aligned nanostructure, we will use DNA-PAINT to measure protein organization around and across from NMDARs following knock-in mutagenesis and optical oligomerization, and examine whether these changes help or hinder the induction of plasticity. Finally, recognizing that many aspects of receptor structure remain unknown, particularly in a synaptic context, we here offer a new approach to discern the inner workings of proteins in their native state, and we apply it first to measure the conformational dynamics of NMDARs in synapses. Together, these aims will test multiple new hypotheses about fundamental mechanisms of synapse operation. They will establish a systematic nanostructure-function workflow to link protein abundance and nanostructure to NMDAR-mediated transmission, and generate a new atlas of data-defined synapse subtypes. These aims together offer a deep understanding of one of the most crucial molecules in synaptic transmission, illuminating unexpected cellular mechanisms which control the nature of physiological and pathological plasticity.