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Protein organization and NMDA receptor activation at individual hippocampal synapses.Protein organization and receptor activation coordinate to maintain synaptic efficacy at individual synapses. Therefore, it is important to explore both mechanisms that impact how proteins are arranged and factors that influence receptor activation. Transsynaptic cleft proteins are important for synaptogenesis and synapse function but little is known about how they are organized within the cleft. One such transsynaptic protein is Synaptic Cell Adhesion Molecule 1 (SynCAM 1), which has been shown to be important for synaptogenesis and synapse maintenance but its subsynaptic organization had not been explored. Using a combination of high-resolution imaging approaches, including cryoelectron tomography and super-resolution imaging, it was determined that not only does SynCAM 1 form peri-synaptic puncta, but also that the presence of SynCAM 1 is required for normal protein density distribution within the synaptic cleft. Thus supporting the idea that the synapse is organized into nano-compartments and that SynCAM 1 may be important for this subsynaptic organization. Additionally, NMDA receptor (NMDAR) activation is critical for maintenance and modification of synapse strength. Specifically, NMDAR activation by spontaneous glutamate release has been shown to mediate some forms of synaptic plasticity, as well as synaptic development. Interestingly, there is evidence that within individual synapses each release mode may be segregated such that postsynaptically there are distinct pools of responsive receptors. In order to examine potential regulators of NMDAR activation due to spontaneous glutamate release in cultured hippocampal neurons, I utilized GCaMP6f imaging at single synapses in concert with confocal and super-resolution imaging. Using these single-spine approaches, I found that Ca2+ entry activated by spontaneous release tends to be carried by GluN2B-NMDARs. The amount of NMDAR activation varies greatly both between synapses and within synapses, and is unrelated to spine and synapse size, but does correlate loosely with synapse distance from the soma. Despite the critical role of spontaneous activation of NMDARs in maintaining synaptic function, their activation seems to be controlled by factors other than synapse size or synapse distance from the soma. It is most likely that NMDAR activation by spontaneous release influenced variability in subsynaptic receptor position, release site position, vesicle content, and channel properties.