Comparison of genetically encoded calcium indicators for monitoring action potentials in mammalian brain by two-photon excitation fluorescence microscopy

Abstract

Imaging calcium transients associated with neuronal activity has yielded important insights into neural physiology. Genetically encoded calcium indicators (GECIs) offer conspicuous potential advantages for this purpose, including exquisite targeting. While the catalogue of available GECIs is steadily growing, many newly developed sensors that appear promising in vitro or in model cells appear to be less useful when expressed in mammalian neurons. We have, therefore, evaluated the performance of GECIs from two of the most promising families of sensors, G-CaMPs [Nat. Biotechnol.2(2), 137-141 (2001)] and GECOs [Science2(2), 1888-1891 (2011)], for monitoring action potentials in rat brain. Specifically, we used two-photon excitation fluorescence microscopy to compare calcium transients detected by G-CaMP3; GCaMP6f; G-CaMP7; Green-GECO1.0, 1.1 and 1.2; Blue-GECO; Red-GECO; Rex-GECO0.9; Rex-GECO1; Carmine-GECO; Orange-GECO; and Yellow-GECO1s. After optimizing excitation wavelengths, we monitored fluorescence signals associated with increasing numbers of action potentials evoked by current injection in CA1 pyramidal neurons in rat organotypic hippocampal slices. Some GECIs, particularly Green-GECO1.2, GCaMP6f, and G-CaMP7, were able to detect single action potentials with high reliability. By virtue of greatest sensitivity and fast kinetics, G-CaMP7 may be the best currently available GECI for monitoring calcium transients in mammalian neurons.

Keywords: G-CaMP; GECO; calcium transients; genetically encoded calcium indicators; organotypic hippocampal slice; two-photon microscopy.

Fig. 1

Simultaneous calcium imaging and electrophysiological recordings. (a) G-CaMP7 expressing CA1 pyramidal neuron; location of line scan is indicated by the white line across the proximal apical dendrite. Increasing fluorescence intensity in this and the following line scan images is represented by colors from black through red and yellow to green, blue and purple. (b) Representative line scans during one action potential and (c) eight action potentials with GECO1.2. The vertical white lines in (b) and (c) and corresponding deflections in (d) and (e) are time stamps, the first indicating the start of current injection, and the second, 50 ms later, serving as an internal time marker. Traces above the line scans show the simultaneously recorded action potentials on the same time scale; these are visible in more detail in panels (f) and (g). (d) and (e) Quantifications of line scans: (d) one action potential and (e) eight action potentials. $X$ axis shows time, $Y$ axis shows $\mathrm{\Delta }F/F$ values. Traces are representations of single line scans. (f) and (g) Simultaneously recorded membrane potentials showing one or eight action potentials.

Fig. 2

(a) Summary of changes in fluorescence intensity corresponding to one to eight action potentials at 100 Hz for all GCaMPs and GECOs tested. (b) Summary of changes in fluorescence intensity corresponding to one to eight action potentials at 100 Hz for all genetically encoded calcium indicators tested, excluding GCaMP6f and G-CaMP7. (c) Summary of changes in fluorescence intensity corresponding to one to eight action potentials at 100 Hz for blue-, orange-, carmine-, red-, and yellow-GECOs. Each data point corresponds to $n=10$ measurements from multiple neurons. Error bars represent standard error of the mean.