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.

Grant Number: 1R21MH141559-01
NIH Institute/Center: NIH
Principal Investigator: Thomas Blanpied