Identification and Transsynaptic Molecular Context of Docked Synaptic Vesicles by Fluorescence Microscopy

How proteins of the presynaptic active zone (AZ) and postsynaptic density (PSD) assemble into the functional complexes that carry out synaptic function is a major effort in neuroscience. Among the most critical locations within the synapse is the site at which synaptic vesicles (SVs) dock with the presynaptic plasma membrane. The number of docked SVs establishes the readily releasable pool of neurotransmitter that governs the strength and frequency-dependence of neurotransmission, and counting docked SVs remains a central goal in understanding mechanisms of dysfunction in neuropsychiatric disorders. Further, two aspects of the molecular organization around docking sites are critical for the functional impact of individual SVs in synaptic transmission. First, in the AZ, the prevailing model of organization is that proteins essential for SV tethering, Ca2+ channel recruitment, and SV priming all accumulate at docking sites to maximize SV release probability. Second, in the PSD, super-resolution imaging suggests that postsynaptic receptors are preferentially enriched in subregions of the synapse in nanoscale alignment with sites of neurotransmitter release. These models carry strong implications for synaptic plasticity and highlight how structural disorder in synapses may contribute to cognitive dysfunction. However, recent work suggests much more heterogeneity in the functional performance of individual vesicles than predicted by these simple models. For instance, we and others have identified considerable variability of the protein distribution in both the AZ and PSD, suggesting the proteins involved in SV docking may often be separated from those involved in Ca2+ channel localization and that only a subset of SV docking sites accumulate nearby receptors of specific types. Thus, understanding the foundations of synaptic transmission and its regulation requires an efficient means of measuring protein nanoscale organization around docked SVs. While electron microscopy is classically required to identify docked SVs, the ideal assay would enable analysis of the fine-scale organization of numerous pre- and postsynaptic proteins around them. To address this, we introduce and propose to optimize an experimental pipeline to identify docked synaptic vesicles together with their molecular context using a multiplexed super-resolution microscopy approach. The centerpiece technology is RESI, a recent iteration of multiplexed DNA-PAINT that achieves imaging resolution below the size of single proteins using commercialized kit reagents. To establish a compelling and widely useful assay, we will optimize reagents and analysis for discriminating the distance of single SVs from plasma membrane proteins for the identification and quantification of the docked population in both cultured neurons and brain slice, and robustly validate the technique with a number of structural and functional assays. Then, combining the method with analysis based on our published work, we will measure two critical yet unknown aspects of the transcellular molecular context of docked SVs. Together, this work will introduce an optimized new method that we anticipate will be widely impactful for investigating changes to synapse structure during plasticity and disease.