Quantitative comparison of genetically encoded Ca indicators in cortical pyramidal cells and cerebellar Purkinje cells

Abstract

Genetically encoded Ca(2+) indicators (GECIs) are promising tools for cell type-specific and chronic recording of neuronal activity. In the mammalian central nervous system, however, GECIs have been tested almost exclusively in cortical and hippocampal pyramidal cells, and the usefulness of recently developed GECIs has not been systematically examined in other cell types. Here we expressed the latest series of GECIs, yellow cameleon (YC) 2.60, YC3.60, YC-Nano15, and GCaMP3, in mouse cortical pyramidal cells as well as cerebellar Purkinje cells using in utero injection of recombinant adenoviral vectors. We characterized the performance of the GECIs by simultaneous two-photon imaging and whole-cell patch-clamp recording in acute brain slices at 33 ± 2°C. The fluorescent responses of GECIs to action potentials (APs) evoked by somatic current injection or to synaptic stimulation were examined using rapid dendritic imaging. In cortical pyramidal cells, YC2.60 showed the largest responses to single APs, but its decay kinetics were slower than YC3.60 and GCaMP3, while GCaMP3 showed the largest responses to 20 APs evoked at 20 Hz. In cerebellar Purkinje cells, only YC2.60 and YC-Nano15 could reliably report single complex spikes (CSs), and neither showed signal saturation over the entire stimulus range tested (1-10 CSs at 10 Hz). The expression and response of YC2.60 in Purkinje cells remained detectable and comparable for at least over 100 days. These results provide useful information for selecting an optimal GECI depending on the experimental requirements: in cortical pyramidal cells, YC2.60 is suitable for detecting sparse firing of APs, whereas GCaMP3 is suitable for detecting burst firing of APs; in cerebellar Purkinje cells, YC2.60 as well as YC-Nano15 is suitable for detecting CSs.

Keywords: acute brain slice; adenovirus; cerebellar Purkinje cell; cortical pyramidal cell; genetically encoded Ca2+ indicators; patch-clamp recording; two-photon imaging.

Figure 1

Adenovirus-mediated expression of GECIs. (A) The domain structure of GECIs. ECFPΔC11, enhanced CFP with C-terminal 11 amino acids deleted; CaM, calmodulin; M13, Ca²⁺/CaM-binding peptide from skeletal muscle myosin light chain kinase; cp173Venus, circularly permuted Venus; cpEGFP, circularly permutated enhanced green fluorescent protein. (B) The structure of recombinant adenovirus vector for expression of GECIs. CAG pro, cytomegalovirus enhancer and β-actin promoter; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element; BGHpA, bovine growth hormone polyadenylation signal. (C) Confocal image of a parasagittal section from an E14:P20 YC2.60 mouse. Each cortical layer (L) position is indicated in white. (D) Confocal image of the neocortex immunostained with anti-CaMKII antibody, a marker for excitatory pyramidal cells. (E) Confocal image of a parasagittal section from an E11:P21 YC2.60 mouse. ML, molecular layer; GCL, granule cell layer. (F) Confocal image of the cerebellum immunostained with anti-IP₃R1 antibody, a marker for Purkinje cells.

Figure 2

Quantitative comparison of GECIs in cortical layer 2/3 pyramidal cells. (A) Maximum intensity projection images of an E14:P19 YC2.60-expressing pyramidal cells (left), and expanded single z-section images within the white boxes (right). A patch pipette is drawn with dotted lines. Line-scan imaging was performed along the proximal apical dendrite as indicated by the red line. (B) Line-scan images of a YC2.60-expressing pyramidal cell in response to 20 action potentials (APs) at 20 Hz evoked during the period indicated. Vertical scale bar, 5 μm. (C) Traces calculated from line-scan images in (B) were averaged across three trials and smoothed by 35 ms moving window. ΔR/R₀, FRET signal change; V_m, membrane potential. The scale bar in V_m inset, 200 ms. (D) ΔR/R₀ (YC) or ΔF/F₀ (GC3) traces in response to 1 (black), 2 (blue), 5 (green), 10 (orange), and 20 (red) APs evoked at 20 Hz. Each trace is the mean across cells (n = 10 cells for YC2.60, 8 for YC3.60, 7 for YC-Nano15, and 6 for GCaMP3). (E) Signal-to-noise ratio (SNR) plotted against the number of APs, calculated for individual pyramidal cells (gray) and the mean across cells (black). For 1 AP, n = 19 for YC2.60, n = 11 for YC3.60, n = 14 for YC-Nano15 and n = 7 for GCaMP3; For 2, 5, 10, and 20 APs, n = 10 for YC2.60, n = 8 for YC3.60, n = 7 for YC-Nano15, and n = 6 for GCaMP3. (F) SNR in response to single APs taken from (E). Each dot represents a value taken from an individual cell, and the bar represents the mean across cells. (G,H) Mean half rise time (G) and mean half decay time (H) of smoothed trial-averaged traces in response to 10 APs at 20 Hz. Error bars show SD. *p < 0.05, **p < 0.01 in Tukey’s post-hoc test following one-way ANOVA.

Figure 3

Quantitative comparison of GECIs in cerebellar Purkinje cells. (A) Maximum intensity projection image (Venus emission) of an E12:P56 YC2.60-expressing Purkinje cell (left), and an expanded single z-section image within the blue box (right). Line-scan imaging was performed along the distal dendrite as indicated by the red line. Rec, an eletrode for patch-clamp recording; stim, an electrode for extracellular stimulation. (B) Smoothed ΔR/R₀ traces of YC2.60 in response to a single complex spike (CS) for individual trials (gray) and the mean across trials (black). (C) ΔR/R₀ (YC) or ΔF/F₀ (GC3) traces in response to 1 (black), 2 (blue), 5 (green), 10 (red) CSs evoked at 10 Hz. Each trace is the mean across cells (n = 8 cells for YC2.60, 7 for YC3.60, 7 for YC-Nano15, and 8 for GCaMP3). (D) SNR plotted against the number of CSs, calculated for individual Purkinje cells (gray) and the mean across cells (black). For 1 CS, n = 17 for YC2.60, n = 11 for YC3.60, n = 12 for YC-Nano15, and n = 8 for GCaMP3; for 2, 5, and 10 CS, n = 8 for YC2.60, n = 7 for YC3.60, n = 7 for YC-Nano15, and n = 8 for GCaMP3. (E) SNR in response to single CSs taken from (D). Each dot represents a value taken from an individual cell, and the bar represents the mean across cells. (F,G) Mean half rise time (F) and mean half decay time (G) of trial-averaged traces in response to 5 CSs at 10 Hz. Error bars show SD. *p < 0.05, **p < 0.01 in Tukey’s post-hoc test following one-way ANOVA.

Figure 4

Stability of YC2.60 expression and responses. (A) Maximum intensity projection image (Venus emission) of an E12:P113 YC2.60-expressing Purkinje cell. (B) ΔR/R₀ traces in response to 1 (black), 2 (blue), 5 (green), 10 (red) CSs evoked at 10 Hz, recorded from YC2.60-expressing Purkinje cells at P113–114. Each trace is the mean across cells (n = 5). (C) SNR plotted against the number of CSs, calculated for individual Purkinje cells (gray) and the mean across cells (black, n = 5).