Abstract
Information transfer at synapses occurs when vesicles fuse with the plasma membrane to release neurotransmitters, which then bind to receptors at the postsynaptic membrane. The process of neurotransmitter release varies dramatically between different synapses, but little is known about how this heterogeneity emerges. The development of super-resolution microscopy has revealed that synaptic proteins are precisely organised within and between the two parts of the synapse and that this precise spatiotemporal organisation fine-tunes neurotransmission. However, it remains unclear if variability in release probability could be attributed to the nanoscale organisation of one or several proteins of the release machinery. To begin to address this question, we have developed a pipeline for correlative functional and super-resolution microscopy, taking advantage of recent technological advancements enabling multicolour imaging. Here we demonstrate the combination of live imaging of SypHy-RGECO, a unique dual reporter that simultaneously measures presynaptic calcium influx and neurotransmitter release, with post hoc immunolabelling and multicolour single molecule localisation microscopy, to investigate the structure-function relationship at individual presynaptic boutons.
Highlights
Received: 07 December 2021 Accepted: 12 January 2022Published: 15 February 2022Citation: Jackson RE, Compans B and Burrone J (2022) Correlative Live-Cell and Super-Resolution Imaging to Link Presynaptic Molecular OrganisationWith Function
In this study we have described the use of a correlative approach combining functional imaging and multicolour 3D-dSTORM to investigate presynaptic structure and function at individual synapses
In synapses with a single subsynaptic domains (SSDs), the density of the protein cluster was increased in comparison to the individual densities of the multiple SSDs, suggesting different strategies can be employed to arrange the same number of total proteins
Summary
Received: 07 December 2021 Accepted: 12 January 2022Published: 15 February 2022With Function. Synapses are remarkably heterogenous subcellular compartments, formed of a presynaptic terminal and a postsynaptic element that vary in structure, function and molecular composition. Calcium influx through voltage gated calcium channels (VGCCs) triggers the fusion of a neurotransmitter-filled synaptic vesicle (SV) with the plasma membrane at the active zone (AZ), a specialised site containing the necessary machinery for vesicle tethering, docking and release (Südhof, 2012). Individual synapses display a high degree of heterogeneity in all stages of this process, differing in their expression of calcium channel subtypes (Luebke et al, 1993; Wheeler et al, 1994; Reid et al, 1997; Gasparini et al, 2001), the level of calcium influx (Ermolyuk et al, 2012) and the sensitivity of release to calcium (Ermolyuk et al, 2012; Jackson and Burrone, 2016; Rebola et al, 2019). Neurotransmitter release is a probabilistic process, with SV
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