Alignment of synaptic release to receptors

Abstract

Healthy synapse function relies on precise structural alignment between the presynaptic active zone (AZ) and the postsynaptic density (PSD). A central question is how the distribution of presynaptic vesicle fusion sites corresponds to the position of receptors in the postsynaptic density. The question is important because receptor activation at glutamatergic synapses is limited by both biophysical properties of the receptors themselves and their distance from vesicle release sites. Thus, release events can fail to activate all synaptic receptors. Recently, our lab and others found that PSD scaffolding proteins cluster receptors into nanometer-scale subregions. This organization is expected to increase the impact of fusion site organization: simulations show that EPSC amplitude at synapses with clustered receptor distributions is greatest when vesicle fusion is aligned with postsynaptic clusters. However, it has not been previously possible to precisely localize where within the AZ vesicles fuse, or how that relates to distributions of postsynaptic receptors. The significance of this issue is emphasized by the growing list of diseases in which cognitive and behavioral deficits appear to stem from disruption of synapse function caused by mutations of genes that encode synaptic proteins. Using localization microscopy, Dr. Ai-hui Tang in the lab previously found that, like PSD scaffold proteins, key proteins mediating vesicle priming and fusion (particularly RIM1/2) are mutually co-enriched within nanometer-scaled subregions of the AZ. Here, I investigated the functional consequence of that organization by directly mapping vesicle fusion sites. I developed a novel technique to localize single-vesicle fusion with high spatial resolution, which I call "pHluorin uncovering sites of exocytosis" or pHuse. Using this approach, I mapped the pattern of evoked or spontaneous vesicle fusion at individual AZs of cultured hippocampal neurons. Spontaneous release of neurotransmitter was previously thought to be biological noise but has recently been linked to physiological functions distinct from those of evoked release. My data using pHuse indicate that evoked and spontaneous fusion in fact occur over different subregions of the terminal. Furthermore, I found that action potential evoked fusion occurred preferentially in confined areas with higher local density of RIM1 proteins within AZs. In collaboration with Dr. Tang, I found that these high local density RIM areas closely aligned with concentrated postsynaptic receptors and scaffolding proteins1-3, defining 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.