Tran, O.; Bertelli, S.; Hughes, H.; Lozo, L.; Plested, A.; Torok, K.

The first genetically encoded single fluorescent protein-based glutamate sensor, iGluSnFR opened a new era of imaging neuronal activity at the level of circuits as well as at synapses. With slow off-kinetics, iGluSnFR could only resolve low frequency glutamate release. A binding site variant of iGluSnFR, iGluu with significantly faster off-kinetics, enabled the resolution of high (100 Hz) frequency glutamate release in hippocampal slices at individual CA3-CA1 synapses. Moreover, iGluu revealed impairment of glutamate retrieval in HD mice models pointing to defective glutamate transport. Recently, the iGluSnFR3 generation (SF-Venus-iGluSnFR. v857) has been developed with increased dynamic range and rapid on-kinetics, making it attractive for in vivo imaging. However, similarly to iGluSnFR, glutamate off-kinetics of iGluSnFR3 were predicted to limit it to resolving low frequency release only. We undertook to improve the kinetic properties of iGluSnFR3 for resolving high (100 Hz) frequency glutamate release. We generated and characterized iGlu3fast, an ultrafast decay variant of iGluSnFR3. For iGlu3Fast we obtained a decay rate constant of 340 s-1, ~5-fold faster than iGluSnFR3 at 71 s-1, at 20oC. Furthermore, iGlu3Fast superseded iGluSnFR3 with a 42-fold glutamate-induced fluorescence increase compared to 26 for iGluSnFR3. Thus, with rapid off-kinetics comparable to that of iGluu as well as significantly increased fluorescence dynamic range and preserved rapid on-kinetics, iGlu3fast represents an excellent novel sensor for imaging high frequency glutamate release at all levels of organisation. Two novel variants with superslow off-kinetics are also reported, expanding the range of applications in neurobiology.