Genetically encoded green fluorescent Ca2+ indicators with improved detectability for neuronal Ca2+ signals - PubMed (original) (raw)

Genetically encoded green fluorescent Ca2+ indicators with improved detectability for neuronal Ca2+ signals

Masamichi Ohkura et al. PLoS One. 2012.

Abstract

Imaging the activities of individual neurons with genetically encoded Ca(2+) indicators (GECIs) is a promising method for understanding neuronal network functions. Here, we report GECIs with improved neuronal Ca(2+) signal detectability, termed G-CaMP6 and G-CaMP8. Compared to a series of existing G-CaMPs, G-CaMP6 showed fairly high sensitivity and rapid kinetics, both of which are suitable properties for detecting subtle and fast neuronal activities. G-CaMP8 showed a greater signal (F(max)/F(min) = 38) than G-CaMP6 and demonstrated kinetics similar to those of G-CaMP6. Both GECIs could detect individual spikes from pyramidal neurons of cultured hippocampal slices or acute cortical slices with 100% detection rates, demonstrating their superior performance to existing GECIs. Because G-CaMP6 showed a higher sensitivity and brighter baseline fluorescence than G-CaMP8 in a cellular environment, we applied G-CaMP6 for Ca(2+) imaging of dendritic spines, the putative postsynaptic sites. By expressing a G-CaMP6-actin fusion protein for the spines in hippocampal CA3 pyramidal neurons and electrically stimulating the granule cells of the dentate gyrus, which innervate CA3 pyramidal neurons, we found that sub-threshold stimulation triggered small Ca(2+) responses in a limited number of spines with a low response rate in active spines, whereas supra-threshold stimulation triggered large fluorescence responses in virtually all of the spines with a 100% activity rate.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Characterization of G-CaMPs in vitro and in HeLa cells.

A, Schematic representation. Mutations are indicated with respect to G-CaMP2. RSET and M13 are tags that encode hexahistidine and a target peptide for Ca2+-bound CaM derived from MLCK, respectively. The amino-acid numbers of EGFP and CaM are indicated in parentheses. B, Dynamic range (F max/F min) and Ca2+ affinity (K d). Error bars, s.d. (n = 3 each). C, Ca2+ titration curve. Curves were fit according to the Hill equation. K d is shown in B. D, Normalized fluorescence and absorbance (inset) spectra of G-CaMP6 and G-CaMP8 in 1 µM Ca2+ or 1 mM EGTA. E, Fluorescence images of HeLa cells expressing G-CaMPs. Bars, 30 µm. F, Time course of the changes (Δ_F_/F) in G-CaMP fluorescence in response to 100 µM ATP. Error bars, s.d. G, Baseline fluorescence and peak responses (Δ_F_/F) to ATP application in HeLa cells.

Figure 2

Figure 2. Characterization of G-CaMPs in cultured hippocampal slices.

A, Expression of G-CaMP6 in hippocampal CA3 pyramidal neurons. Inset: Higher-magnification views are shown in the bottom panels. B, Baseline fluorescence of hippocampal neurons expressing G-CaMP3, G-CaMP6 and G-CaMP8. No significant differences in variance were detected among the three groups (P_>0.05, χ2 = 2.90, Bartlett’s test). Error bars, s.d. (n = 7 each, P_>0.05, Tukey’s test). C, Representative traces of the response (Δ_F/F) to spike trains. The frequency of stimuli was 50 Hz. Right: Magnified views of single spikes. D, Mean responses (Δ_F/F) and SNRs of G-CaMP3 (black), G-CaMP6 (red) and G-CaMP8 (blue). Inset: Magnified views of 1–2 spikes. Error bars, s.e.m. (n = 7 each). E, Rise and decay time constants for the responses to single spikes. Error bars, s.e.m. (n = 7 each; *P<0.05 in Tukey’s post-hoc test following one-way ANOVA). F, Trial-averaged responses of G-CaMP6 to spike trains. Gray, individual traces (n = 10 trials); red, averaged traces. Bars indicate stimulus timing. Inset: Magnified views.

Figure 3

Figure 3. Comparison of G-CaMP responses in acute cortical slices.

A, Confocal image of G-CaMP6-expressing cortical pyramidal cells. The expression of G-CaMP6 was driven by the CAG promoter via in utero plasmid electroporation. Inset: Higher-magnification views are shown in the right panels. B, Representative Δ_F_/F traces in response to 1–4 spikes evoked at 50 Hz. C, Mean responses (Δ_F_/F) of G-CaMP3 and G-CaMP6 to spike trains. Error bars, s.e.m. (G-CaMP3, n = 4 cells; G-CaMP6, n = 5 cells).

Figure 4

Figure 4. Temperature dependence of G-CaMP6 signals.

A, Representative traces of the fluorescence response (Δ_F_/F) of G-CaMP6 to a single spike at 25–28°C and at 37°C. B, Mean responses (Δ_F_/F) of G-CaMP6 to spike trains. Error bars, s.e.m. (n = 6 each). C, Rise and decay time constants of the responses of G-CaMP6 to single spikes. (*P<0.05, paired _t_-test).

Figure 5

Figure 5. Electrophysiological properties of hippocampal neurons expressing G-CaMP6.

A, Left, input resistance. Middle, membrane capacitance. Right, resting potential. Error bars, s.e.m. (n = 6 each). There were no significant differences between the control and G-CaMP6 groups for any of the parameters (_P_>0.05, Student’s _t_-test). B, Left, spontaneous current under the voltage clamp at –70 mV. Middle, amplitude of the excitatory postsynaptic current. Right, frequency of the excitatory postsynaptic current. Error bars, s.e.m. (n = 6 each, _P_>0.05, Student’s _t_-test).

Figure 6

Figure 6. Ca2+ imaging of cholinergic DA motoneurons in freely moving C. elegans.

A, Confocal images of L1 larvae expressing G-CaMP6 (jqEx97) or G-CaMP3 (jqEx216) in the DA motoneurons. In both transgenic strains, DsRed-Express-1 is co-expressed in the DA motoneurons. TL, transmitted-light image. Arrows indicate the DA7 motoneuron analyzed in B. B, Representative spontaneous fluorescence responses (Δ_R_/R) of G-CaMPs from DA7 cholinergic neurons in transgenic worms during locomotion. C, Mean peak responses (Δ_R_/R). Error bars, s.e.m. (n = 10 each from 4 worms, *P = 0.0020, Student’s _t_-test). Movies of the recordings are available as supplementary information (Movies S1 and S2).

Figure 7

Figure 7. Ca2+ Imaging of individual spines in cultured hippocampal slices.

A, Schematic representation of G-CaMP6-actin. B, Schematic drawing of the placement of a stimulation electrode and a patch pipette in a cultured hippocampal slice. C, Z-projection of a representative CA3 pyramidal neuron expressing G-CaMP6-actin. The position of the patch pipette is indicated by dotted lines. Two spines of interest (S1 and S2) in the striatum lucidum are indicated by yellow circles. Inset: High-magnification views of rectangular windows. D, Changes in fluorescence at S1and S2 and membrane potential (V m) upon sub- or supra-threshold electrical stimulation (Stim). E, Mean responses of active spines. Error bars, s.d. (n = 63 and 131 spines for sub- and supra-threshold stimulation, respectively, from 5 slices); *P<0.05, Student’s t_-test. The average Δ_F/F of the soma in response to supra-threshold stimulation was 15±6.5%.

Figure 8

Figure 8. Long-term imaging of Ca2+ activity in spines in a cultured hippocampal pyramidal neuron.

A, Z-projection of a representative CA3 pyramidal neuron expressing G-CaMP6-actin at 8 (upper) and 29 (lower) days in vitro (Div). After 7 days in vitro, the G-CaMP6-actin plasmid was introduced into the neuron via single-cell electroporation. Two spines of interest (S1, S2) are indicated by yellow circles. B, Changes in fluorescence at S1 and S2 upon supra-threshold electrical stimulation (Stim). The average spine Δ_F_/F ratios in response to supra-threshold stimulation were 253±30.5% and 201±46.6% at 8 Div and 29 Div, respectively (n = 25 spines, _P_>0.05, Student’s _t_-test).

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This work was partly supported by the Regional Innovation Cluster Program (City Area Type, Central Saitama Area) and by grants from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) to M.O. (nos. 22500285 and 24111509), T.S. (no. 10J05408), K.G.-A. (no. 22500353) and J.N. (no. 21500379). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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