Synaptic transmission is maintained by a delicate, subsynaptic molecular architecture, and even mild alterations in synapse structure drive functional changes during experience-dependent plasticity and pathological disorder1,2. Key to this architecture is how the distribution of presynaptic vesicle fusion sites corresponds to the position of receptors in the postsynaptic density. However, despite long recognition that this spatial relationship modulates synaptic strength3, it has not been precisely described, due in part to the limited resolution of light microscopy. Using localization microscopy, we report here that key proteins mediating vesicle priming and fusion are mutually co-enriched within nanometer-scaled subregions of the presynaptic active zone. Through development of a new method to map vesicle fusion positions within single synapses, we found that action potential evoked fusion was guided by this protein gradient and occurred preferentially in confined areas with higher local density of RIM within the active zones. These presynaptic RIM nanoclusters closely aligned with concentrated postsynaptic receptors and scaffolding proteins4–6, suggesting a transsynaptic molecular “nanocolumn.” Thus, we propose that the nanoarchitecture of the active zone directs action potential evoked vesicle fusion to occur preferentially at sites directly opposing postsynaptic receptor-scaffold ensembles. Remarkably, NMDA receptor activation triggered distinct phases of plasticity in which postsynaptic reorganization was followed by transsynaptic nanoscale realignment. This architecture thus suggests a simple organizational principle of CNS synapses to maintain and modulate synaptic efficiency.

SUMMARY Scaffolding molecules at the postsynaptic membrane form the foundation of excitatory synaptic transmission by establishing the architecture of the postsynaptic density (PSD), but the small size of the synapse has precluded measurement of PSD organization in live cells. We measured the internal structure of the PSD in live neurons at approximately 25 nm resolution using photoactivated localization microscopy (PALM). We found that four major PSD scaffold molecules are each organized in distinctive nanodomains ~80 nm in diameter, intrasynaptic protein ensembles that undergo striking changes over time. Further, the dense subdomains of PSD-95 were preferentially enriched in AMPA receptors more than NMDA receptors. Chronic suppression of activity triggered changes in PSD interior architecture that may help amplify synaptic plasticity. The observed clustered architecture of the PSD controlled the amplitude and variance of simulated postsynaptic currents, suggesting several ways in which PSD interior organization may regulate the strength and plasticity of neurotransmission.

Endocytosis is a fundamental mechanism by which neurons control intercellular signaling, nutrient uptake, and synaptic transmission. This process is carried out by the assembly of clathrin coats and the budding of clathrin-coated vesicles from the neuronal plasma membrane. Here, we demonstrate that in young neurons, clathrin assembly and disassembly occur rapidly, locally, and repeatedly at "hot spots" throughout dendrites and at the tips of dendritic filopodia. In contrast, clathrin coats in mature dendrites reside in stable, long-lasting zones at sites of endocytosis, where clathrin undergoes continuous exchange with local cytosolic pools. In dendritic spines, endocytic zones lie lateral to the postsynaptic density (PSD) where they develop and persist independent of synaptic activity, akin to the PSD itself. These results reveal the presence of a novel specialization dedicated to endocytosis near the postsynaptic membrane.

Formation of mature excitatory synapses requires the assembly and delivery of NMDA receptors to the neuronal plasma membrane. A key step in the trafficking of NMDA receptors to synapses is the exit of newly assembled receptors from the endoplasmic reticulum (ER). Here we report the identification of an RXR-type ER retention/retrieval motif in the C-terminal tail of the NMDA receptor subunit NR1 that regulates receptor surface expression in heterologous cells and in neurons. In addition, we show that PKC phosphorylation and an alternatively spliced consensus type I PDZ-binding domain suppress ER retention. These results demonstrate a novel quality control function for alternatively spliced C-terminal domains of NR1 and implicate both phosphorylation and potential PDZ-mediated interactions in the trafficking of NMDA receptors through early stages of the secretory pathway.