An improved genetically encoded red fluorescent Ca2+ indicator for detecting optically evoked action potentials

Abstract

Genetically encoded Ca(2+) indicators (GECIs) are powerful tools to image activities of defined cell populations. Here, we developed an improved red fluorescent GECI, termed R-CaMP1.07, by mutagenizing R-GECO1. In HeLa cell assays, R-CaMP1.07 exhibited a 1.5-2-fold greater fluorescence response compared to R-GECO1. In hippocampal pyramidal neurons, R-CaMP1.07 detected Ca(2+) transients triggered by single action potentials (APs) with a probability of 95% and a signal-to-noise ratio >7 at a frame rate of 50 Hz. The amplitudes of Ca(2+) transients linearly correlated with the number of APs. The expression of R-CaMP1.07 did not significantly alter the electrophysiological properties or synaptic activity patterns. The co-expression of R-CaMP1.07 and channelrhodpsin-2 (ChR2), a photosensitive cation channel, in pyramidal neurons demonstrated that R-CaMP1.07 was applicable for the monitoring of Ca(2+) transients in response to optically evoked APs, because the excitation light for R-CaMP1.07 hardly activated ChR2. These technical advancements provide a novel strategy for monitoring and manipulating neuronal activity with single cell resolution.

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

A, Schematic structures of R-CaMPs. Mutations are indicated with respect to R-GECO1. RSET and M13 are a tag that encodes hexahistidine and a target peptide for a Ca²⁺-bound CaM derived from MLCK, respectively. The amino-acid numbers of mApple and CaM are indicated in parentheses. B, Ca²⁺ affinity (K _d) and dynamic range (F _max/F _min). Error bars, s.d. (n = 3 each). C, Ca²⁺ titration curve. Curves were fit according to the Hill equation. The K _d is shown in B. Error bars, s.d. (n = 3 each). D, Normalized fluorescence and absorbance (inset) spectra of R-CaMP1.07 in 1 µM Ca²⁺ or 1 mM EGTA. E, Fluorescence images of HeLa cells expressing red fluorescent GECIs. F, Mean ΔF/F responses to the application of 100 µM ATP in HeLa cells. Error bars, s.d. (n = 163 cells for R-GECO1, 182 cells for R-CaMP1.01 and 166 cells for R-CaMP1.07). G, Baseline fluorescence and peak responses (ΔF/F) to the application of 100 µM ATP in HeLa cells. Error bars, s.d.

Figure 2. Comparison of the performance of R-GECO1 and R-CaMP1.07 in hippocampal pyramidal cells.

A, A confocal image showing a CA3 pyramidal cell expressing R-CaMP1.07 in a cultured slice. B, Mean fluorescent intensities of neurons expressing R-GECO1 and R-CaMP1.07. Error bars, s.e.m. (n = 6 cells each). P>0.05, Student’s t-test. C, Representative ΔF/F traces in response to trains of 1–6 APs delivered at 50 Hz for a pyramidal cell expressing R-GECO1 (left) and R-CaMP1.07 (right). The same traces for 1 AP are magnified in the right panel. D, Mean ΔF/F response and SNR plotted against the number of APs in R-GECO1 (black) and R-CaMP1.07 (red). Insets are magnified views of 1–2 APs. Error bars, s.e.m. (n = 5 cells each). E, The mean rise and decay time constant calculated from Ca²⁺ transients evoked by single APs. Error bars, s.e.m. (n = 5 cells each). P>0.05, Student’s t-test.

Figure 3. Electrophysiological properties of hippocampal neurons expressing R-CaMP1.07.

A, Average input resistance (left), membrane capacitance (middle), or resting potential (right) did not significantly differ between control and R-CaMP1.07 groups. Error bars, s.e.m. (n = 7 cells each). P>0.05, Student’s t-test. B, Representative spontaneous EPSCs recorded from a control and R-CaMP1.07-expressing cell at a holding potential of −70 mV (left). Cumulative probability of amplitudes of EPSCs is shown in the middle panel (Control, n = 11062 events; R-CaMP1.07, n = 10265 events). P>0.05, Kolmogorov-Smirnov test. Frequency of EPSCs is shown in the right panel. Error bars, s.e.m. (n = 7 cells each). P>0.05, Student’s t-test.

Figure 4. Simultaneous monitoring and manipulation of neuronal activity by co-expression of R-CaMP1.07 and ChR2.

A, A projection image of a CA3 pyramidal cell expressing R-CaMP1.07 and ChR2. The cell was whole-cell recorded to measure the number of APs. B, Imaging of ChR2-triggered-APs by changes in the R-CaMP1.07 fluorescence (top). APs were evoked by a pulse of 470-nm light with a duration of 300 to 3000 ms (blue region indicates time of photostimulation). Putative Ca²⁺ increases during the photostimulation are represented by broken lines (Bottom). The number of APs was recorded using the current-clamp mode. C, ΔF/F amplitude of the R-CaMP1.07 responses as a function of the number of APs induced by photostimulation. The peak ΔF/F amplitudes were calculated from the first frames after termination of the photostimulation. Individual data are plotted as black dots and their averages are shown in red. Error bars, s.e.m. (n = 4 cells).