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, 15 (11), 936-939

Stability, Affinity, and Chromatic Variants of the Glutamate Sensor iGluSnFR


Stability, Affinity, and Chromatic Variants of the Glutamate Sensor iGluSnFR

Jonathan S Marvin et al. Nat Methods.

Erratum in


Single-wavelength fluorescent reporters allow visualization of specific neurotransmitters with high spatial and temporal resolution. We report variants of intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) that are functionally brighter; detect submicromolar to millimolar amounts of glutamate; and have blue, cyan, green, or yellow emission profiles. These variants could be imaged in vivo in cases where original iGluSnFR was too dim, resolved glutamate transients in dendritic spines and axonal boutons, and allowed imaging at kilohertz rates.


Fig. 1.
Fig. 1.. SF-iGluSnFR.A184S detects orientation selective glutamate transients in ferret visual cortex.
a) Two-photon standard-deviation projection of SF-iGluSnFR.A184S expressed in ferret visual cortex (190 μm depth, scale bar 100 μm). b) Fluorescence changes (ΔF/F) to randomly oriented drifting gratings (dashed lines) shown for both a soma (ROI 1) and nearby neuropil (ROI 2) in an individual trial. c) Trial-averaged stimulus-evoked responses (shown for ROIs 1 and 2) reveal robust orientation tuning. Neuropil peak amplitudes were greater (~30–40% ΔF/F) than that for soma ROIs (~5–10% ΔF/F). Responses shown as the mean (black) and standard error (grey) over 10 presentations of each grating direction and a blank (grey screen) period. d) Two-photon standard-deviation projection of an isolated dendritic segment with active spines revealed with SF-iGluSnFR.A184S (scale bar 5 μm). e) Fluorescence (ΔF/F) during visual stimulation for individual dendritic spines (grey) and nearby dendritic segments (cyan). f) Same as in (c) for an individual dendritic spine (grey) and nearby dendrite. Spines exhibit selective and robust responses to drifting gratings. Each response is shown as the mean (black) and standard error (grey) over 8 presentations of each grating direction and a blank (grey screen) period. Dendritic spine responses can have differential tuning from nearby dendritic segments (cyan). (g) Peak responses are plotted as a function of stimulus direction, showing robust selectivity of spines and larger responses than nearby dendritic segments. Images were collected at 30 Hz. Data points are average of 8 experiments, with error bars showing SEM. Measurements for a-c were repeated for 47 ROI pairs across 3 fields of view with similar results. Measurements for d-f were repeated from a total of 76 spine ROIs from 3 dendrites across 3 fields of view with similar results. See also Supp. Video 1.
Fig. 2.
Fig. 2.. 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.
Fig. 3.
Fig. 3.. Utility of SF-Venus-iGluSnFR in cultured neurons and in vivo.
(a-c) High-speed (1016 Hz frame rate) two-photon imaging of a cultured neuron expressing SF-Venus-iGluSnFR.A184V. a) RuBi-glutamate was uncaged for 10 msec. at each of two 5-micron spots (arrowheads) on two adjacent dendrites (Supp. Video 2). Color saturation denotes the glutamate transient amplitude; grey tones indicate no evoked response. Color hue denotes response timing (see scale bars). The yellow line indicates the axis of the kymograph shown in (c). Representative example of 4 trials. b) Recorded traces at the centers of the uncaging locations shown in (a). c) Top: Kymograph showing the response amplitude over time along the dendrite surrounding uncaging location 1. Traces are approximate maximum-likelihood solutions for a spatially multiplexed recording acquired with a high-power 1030 nm fiber laser. These recordings show a single trial without averaging. Bottom: simulation (Methods) of diffusion of glutamate at the surface of a planar coverslip surrounded by 3D solution, following uncaging in a 5 μm spot with a 0.8 NA beam at time 0. Intensity scale is log-transformed. (d-f) Recording of visually-evoked spine transients in isolated SF-Venus-iGluSnFR.A184S labeled dendrites in mouse visual cortex, imaged at 1030 nm excitation. See also Supp. Video 3. d) Motion-aligned average image. e) Mean responses (top) and tuning curves (bottom) of ROIs indicated in d, for 20 trials of each of the 8 oriented moving grating stimuli. Dark lines/markers denote mean, shaded regions/error bars denote SEM. Black bar denotes the stimulus period. f) pixel intensity of ROI 1 for eight consecutive stimulus presentations of different directions, illustrating detectable responses in single trials. Colored bars denote stimuli as in (e). The spine response is selective to the stimulus orientation. (d-f) representative example of 7 mice.

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