Stability, affinity and chromatic variants of the glutamate sensor iGluSnFR

Jonathan S. Marvin; Benjamin Scholl; Daniel E. Wilson; Kaspar Podgorski; Abbas Kazemipour; Johannes Alexander Müller; Susanne Schoch; Francisco José Urra Quiroz; Nelson Rebola; Huan Bao; Justin P. Little; Ariana N. Tkachuk; Edward Cai; Adam W. Hantman; Samuel S.-H. Wang; Victor J. DePiero; Bart G. Borghuis; Edwin R. Chapman; Dirk Dietrich; David A. DiGregorio; David Fitzpatrick; Loren L. Looger

doi:10.1038/s41592-018-0171-3

Fig. 2.

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SF-iGluSnFR.S72A permits resolution of multiple glutamate release events in cultured neurons and in single cerebellar granule cell boutons, acting as a proxy for high-frequency activity.

a-c) Cultured mouse hippocampal neurons. a) Averaged traces of SF-iGluSnFR.S72A (blue, n=4), SF-iGluSnFR.A184V (red, n=4) and iGluSnFR.A184V (black, n=5) response to paired electrical stimuli (100 msec interval). Light traces represent individual trials throughout the figure; dark traces show average. Asterisks indicate times of stimulation. Insets show averaged response trials in which the first and second stimulus each trigger quantal release events. b) Ratio of the amplitudes of the second over the first fluorescent responses to two consecutive quantal glutamate release events. The ratio is slightly larger than unity most likely due to the fact that slight cross-talk from neighboring release sites adds to the second response. Dots denote individual experiments, horizontal and vertical lines indicate mean and standard deviation, respectively. c) The faster off-rate of S72A allows observation of short-term plasticity during 20 Hz trains of synaptic activity. The conversion of facilitation of vesicle release to depression with increased extracellular calcium is most clearly reported by SF-iGluSnFR.S72A (blue, similar results were obtained in 5 independent experiments) when compared to SF-iGluSnFR.A184V (red, 5 experiments) and iGluSnFR.A184V (black, 4 experiments). Traces have been scaled to maximal response for clarity. d-g) Cerebellar granule cell boutons. d) Two-photon fluorescence image of granule cells (arrows) and its axonal boutons (circle) expressing SF-iGluSnFR.A184V (GL, granular layer; ML, molecular layer) in an acute brain slice. e) Single trials (red) and mean traces (blue, average of 3 trials; black, average of 10 trials) of SF-iGluSnFR.A184V responses to 20 Hz extracellular stimulation. Similar results in 11 boutons. f) Normalized averaged fluorescence traces from single boutons expressing GCaMP6f (GC6f; black, 2 mM [Ca²⁺]_e), SF-iGluSnFR.A184V (A184V; red, 1.5 mM [Ca²⁺]_e) and SF-iGluSnFR.S72A (S72A;blue, 1.5 mM [Ca²⁺]_e) in response to single APs. Results repeated in n=10 boutons expressing GC6f; n=12, A184V; n=7, S72A. g) Summary plot of signal-to-noise ratio (SNR; mean ± SD; n boutons=7, GC6f; n=12, A184V; n=7, S72A; ** P=0.0033, ## P=0.0096). Multiple comparisons were performed with the Kruskal-Wallis test and Dunn’s multiple comparisons test. h) Population-averaged response at same calcium concentration to 20 Hz stimulation normalized to the peak of the first response (right; n boutons=5, GC6f, 1.5 mM [Ca²⁺]_e; n=17, A184V; n=6, S72A) and population-averaged response at same calcium concentration to 100 Hz normalized to the maximum amplitude (middle; n boutons=9, GC6f 1.5 mM [Ca²⁺]_e; n=9, A184V; n=9, S72A) or to the peak of the first response (right; n boutons=9, A184V; n=9, S72A). Black arrows indicate 60 μsec extracellular voltage pulse times.