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

Yoshiyuki Yamada

doi:10.3389/fncel.2011.00018

Abstract

Genetically encoded Ca2+ 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.

Highlights

Understanding the spatio-temporal patterns of neuronal activity underlying brain function is one of the fundamental goals in neuroscience research, and requires techniques for the large-scale recording in living animals
These results show that in utero injection of recombinant adenoviral vectors carrying Genetically encoded Ca2+ indicators (GECIs) could lead to their specific expression in cortical pyramidal cells as well as cerebellar Purkinje cells, successfully reproducing our previous results
Half rise time values (Figure 2G) of YC2.60 (185 ± 55 ms, n = 10) and yellow cameleon (YC)-Nano15 (159 ± 28 ms, n = 7) were significantly smaller than those of YC3.60 (214 ± 28 ms, n = 8) and GCaMP3 (288 ± 18 ms, n = 6), whereas half decay time values (Figure 2H) of YC2.60 (2.31 ± 0.95 s, n = 10) and YC-Nano15 (2.38 ± 0.92 s, n = 7) were significantly larger than those of YC3.60 (0.40 ± 0.06 s, n = 8) and GCaMP3 (0.22 ± 0.06 s, n = 6). These results show that in cortical pyramidal cells, YC2.60 would be suitable for reliable detection of sparse firing of action potentials (APs), while GCaMP3 would be suitable for detecting large number of APs due to its large signal-to-noise ratio (SNR) and fast signal decay

Summary

Introduction

Understanding the spatio-temporal patterns of neuronal activity underlying brain function is one of the fundamental goals in neuroscience research, and requires techniques for the large-scale recording in living animals. The repertoire of in vivo multi-cell recording techniques has been enriched by the recent establishment of in vivo Ca2+ imaging, a combination of multi-photon imaging and bolus loading of synthetic Ca2+ dyes (Stosiek et al, 2003). It has contributed to unveiling the functional micro-architecture of many brain regions (Ohki et al, 2005, 2006; Kerr et al, 2005, 2007; Sullivan et al, 2005; Rothschild et al, 2010; Smith and Häusser, 2010), which was difficult to achieve using classical electrode-based techniques. The lack of cell type specificity, the unrepeatability, and the short-lived nature (typically less than 1 day) of imaging using synthetic Ca2+ dyes has remained an obstacle for further applications. Encoded Ca2+ indicators (GECIs; for review, Miyawaki, 2005; Mank and Griesbeck, 2008), which are Ca2+-

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