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Spontaneous network activity visualized by ultrasensitive Ca2+ indicators, yellow Cameleon-Nano

Abstract

We report ultrasensitive Ca2+ indicators, yellow cameleon-Nano (YC-Nano), developed by engineering the Ca2+-sensing domain of a genetically encoded Ca2+ indicator, YC2.60 or YC3.60. Their high Ca2+ affinities (Kd = 15–140 nM) and large signal change (1,450%) enabled detection of subtle Ca2+ transients associated with intercellular signaling dynamics and neuronal activity, even in 100,000-cell networks. These indicators will be useful for studying information processing in living multicellular networks.

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Figure 1: Design and properties of YC-Nano Ca2+ indicators.
Figure 2: Comparison of peak amplitude and time constant between YC3.60 and YC-Nano15 expressed in layer 2/3 pyramidal neurons in response to trains of action potentials.
Figure 3: Spontaneous motor activities in living zebrafish embryos.

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Acknowledgements

We thank A. Nagasaki and Y. Kuramoto for instruction and assistance with the experiment using Dictyostelium cell, T. Kotani and S. Higashijima for assistance and instruction on fish embryo imaging, and T. Shimogori for instruction on in utero injection. This work was partly supported by a Grant-in-Aid for Young Scientists (A) of the Japan Society for the Promotion of Science and Scientific Research on Advanced Medical Technology of the Ministry of Labor, Health and Welfare of Japan to T.N. and a grant from Precursory Research for Embryonic Science and Technology of the Japan Science and Technology Agency to T.N. and K.H.

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Authors

Contributions

K.H. and T.N. invented YC-Nano variants. Y.Y., M.H., A.M., T. Michikawa and K.M. established the method of the expression of Ca2+ indicators in neurons using adenoviral vectors; K.H. and T. Matsuda performed experiments other than electrophysiology and Ca2+ imaging in brain slices. K.H. and T. Matsu-ura performed stopped-flow spectrometry. Y.Y. performed electrophysiology and Ca2+ imaging in brain slices; K.H., K.K., Y.Y., T. Michikawa and T.N. analyzed data. K.H., Y.Y., T. Michikawa and T.N. wrote the manuscript. T.N. supervised the study.

Corresponding author

Correspondence to Takeharu Nagai.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Tables 1–2 and Supplementary Notes 1–5 (PDF 1898 kb)

Supplementary Video 1

Comparative visualization of Ca2+ dynamics in early aggregating stage of Dictyostelium cells visualized by YC-Nano15 and YC2.60. (MOV 908 kb)

Supplementary Video 2

Ca2+ dynamics in aggregating Dictyostelium cells visualized by YC-Nano15. (MOV 1606 kb)

Supplementary Video 3

Ca2+ dynamics in aggregating Dictyostelium cells visualized by YC2.60. (MOV 224 kb)

Supplementary Video 4

Twitching behavior of zebrafish embryo. (MOV 196 kb)

Supplementary Video 5

Spontaneous motor activities in living zebrafish embryos visualized by YC-Nano50. (MOV 2568 kb)

Supplementary Video 6

Spontaneous motor activities in living zebrafish embryos visualized by YC3.60. (MOV 1520 kb)

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Horikawa, K., Yamada, Y., Matsuda, T. et al. Spontaneous network activity visualized by ultrasensitive Ca2+ indicators, yellow Cameleon-Nano. Nat Methods 7, 729–732 (2010). https://doi.org/10.1038/nmeth.1488

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