High-speed imaging of glutamate release with genetically encoded sensors

Abstract

This Protocol describes the design, in vitro characterisation and imaging applications of iGluSnFR-based genetically-encoded glutamate indicators (GEGIs) in tissue culture of rat hippocampus TWEET A new protocol for the design, characterisation and high-speed imaging applications of genetically-encoded glutamate indicators (GEGIs). COVER TEASER High-speed imaging of glutamate release Up to three primary research articles where the protocol has been used and/or developed. 1. Helassa, N. et al. Ultrafast glutamate sensors resolve high-frequency release at Schaffer collateral synapses.

A Practical Guide to Using CV Analysis for Determining the Locus of Synaptic Plasticity

“…However, it appears that the locus of plasticity expression can be reliably and easily estimated with CV analysis-using alternative methods such as analysis of NMDA:AMPA ratio, paired-pulse ratio, or quantal analysis in parallel with CV analysis generally give rise to the same interpretation (Reid and Clements, 1999;. Modern and more direct methods based on optical activation or readout are especially attractive alternatives (Jensen et al, 2017(Jensen et al, , 2019Durst et al, 2019;Ellis-Davies, 2019;MacDougall and Fine, 2019;Mitchell et al, 2019;Padamsey et al, 2019), since they in many cases are virtually free of assumptions. Still, all methods come with their own advantages and caveats, e.g., analysis of pairedpulse ratio may erroneously suggest presynaptic expression for NMDA-only silent synapses that undergo postsynaptic expression (Poncer and Malinow, 2001), glutamate uncaging can necessarily only explore postsynaptic expression, and dyes used with optical methods may distort plasticity mechanisms by buffering calcium (MacDougall and Fine, 2019).…”

“…For further information regarding these techniques, we invite the reader to the review by Glasgow et al (2019) in this research topic. Furthermore, modern techniques enable more direct measurements of locus of expression, e.g., using 2-photon glutamate uncaging (Ellis-Davies, 2019; Mitchell et al, 2019), optical glutamate sensors (Jensen et al, 2017(Jensen et al, , 2019Durst et al, 2019), or optical quantal analysis (Oertner et al, 2002;Emptage et al, 2003;MacDougall and Fine, 2019;Padamsey et al, 2019). These more advanced methods may however require expensive specialized equipment.…”

Freeze-frame imaging of synaptic activity using SynTagMA

“…by employing genetically encoded sensors of vesicular pH (Ariel and Ryan, 2010;Miesenbock et al, 1998). In intact brain tissue, however, monitoring more than one synapse with high temporal resolution becomes very challenging (Dürst et al, 2019;Iacaruso et al, 2017). To create a more permanent tag of active synapses, Ca 2+ precipitation in active spines was used to create an electron-dense label that can be detected by electron microscopy (Toni et al, 1999).…”

Increased and synchronous recruitment of release sites underlies hippocampal mossy fiber presynaptic potentiation

“…Glutamate release from single hMFBs was visualized using the genetically encoded ultrafast glutamate sensor iGlu u (Helassa et al, 2018) that has been used for high-speed glutamate imaging before (Dürst et al, 2019;Dvorzhak et al, 2019;Dvorzhak and Grantyn, 2020).…”