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A genetically encoded fluorescent acetylcholine indicator for in vitro and in vivo studies

Nature Biotechnology volume 36, pages 726–737 (2018)Cite this article

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Abstract

The neurotransmitter acetylcholine (ACh) regulates a diverse array of physiological processes throughout the body. Despite its importance, cholinergic transmission in the majority of tissues and organs remains poorly understood owing primarily to the limitations of available ACh-monitoring techniques. We developed a family of ACh sensors (GACh) based on G-protein-coupled receptors that has the sensitivity, specificity, signal-to-noise ratio, kinetics and photostability suitable for monitoring ACh signals in vitro and in vivo. GACh sensors were validated with transfection, viral and/or transgenic expression in a dozen types of neuronal and non-neuronal cells prepared from multiple animal species. In all preparations, GACh sensors selectively responded to exogenous and/or endogenous ACh with robust fluorescence signals that were captured by epifluorescence, confocal, and/or two-photon microscopy. Moreover, analysis of endogenous ACh release revealed firing-pattern-dependent release and restricted volume transmission, resolving two long-standing questions about central cholinergic transmission. Thus, GACh sensors provide a user-friendly, broadly applicable tool for monitoring cholinergic transmission underlying diverse biological processes.

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Figure 1: Development of GACh sensors.
Figure 2: Characterization of GACh sensors in cultured HEK293T cells and neurons.
Figure 3: GACh2.0 detects rapid ACh application in brain slices.
Figure 4: GACh2.0 reveals firing pattern-dependent restricted volume transmission in MEC.
Figure 5: GACh sensors reveal dynamics of endogenous ACh release in Drosophila.
Figure 6: Attention-engaging visual stimuli evoke ACh release in behaving mice.

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Acknowledgements

We thank L. Looger and colleagues for sharing their unpublished acetylcholine sensors that validated some of our results. We thank Y. Rao for generous sharing of two-photon microscopy. We are also grateful to L. Luo, S. Owen, Y. Rao, and L. Nevin for critical reading of the manuscript. We thank Z. Ye for the help in art designing. This work was supported by the National Basic Research Program of China (973 Program; grant 2015CB856402), The General Program of National Natural Science Foundation of China (project 31671118 and project 31371442), and the Junior Thousand Talents Program of China to Y.L. Additional support comes from NIH grants NS103558 (Y.L. and L.I.Z.), DC008983 (L.I.Z.), MH104227 and MH109475 (Y.Z.), MH109104 and NS022061 (L.W.R.), LH089717 (P.Q.B.), and NS053570, NS091452, NS094980, NS092548, and NS104670 (J.J.Z.). J.J.Z. is the Radboud Professor and Sir Yue-Kong Pao Chair Professor.

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Author notes

  1. Miao Jing and Peng Zhang: These authors contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Membrane Biology, Peking University School of Life Sciences, Beijing, China

    Miao Jing, Jiesi Feng, Jianzhi Zeng, Huoqing Jiang & Yulong Li

  2. PKU-IDG/McGovern Institute for Brain Research, Beijing, China

    Miao Jing, Jiesi Feng, Jianzhi Zeng, Huoqing Jiang & Yulong Li

  3. Peking-Tsinghua Center for Life Sciences, Beijing, China

    Miao Jing, Jiesi Feng, Jianzhi Zeng, Huoqing Jiang, Yan Song & Yulong Li

  4. Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia, USA

    Peng Zhang, Guangfu Wang, Jess C Looby, Nick A Guagliardo, Paula Q Barrett & J Julius Zhu

  5. Center for Life Sciences, School of Life Science and Technology, Harbin Institute of Technology, Harbin, China

    Guangfu Wang

  6. Department of Physiology & Neuroscience, Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, California, USA

    Lukas Mesik & Li I Zhang

  7. Department of Neurobiology and Behavior, Stony Brook University, Stony Brook, New York, USA

    Shaohua Wang, David A Talmage & Lorna W Role

  8. Undergraduate Class of 2019, University of Virginia College of Arts and Sciences, Charlottesville, Virginia, USA

    Jess C Looby

  9. Department of Surgery, University of Virginia School of Medicine, Charlottesville, Virginia, USA

    Linda W Langma

  10. Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, California, USA

    Ju Lu & Yi Zuo

  11. School of Life Sciences, Tsinghua University, Beijing, China

    Minmin Luo

  12. National Institute of Biological Sciences, Beijing, China

    Minmin Luo

  13. School of Medicine, Ningbo University, Ningbo, China

    J Julius Zhu

  14. Donders Institute for Brain, Cognition and Behavior, Radboud University Nijmegen, Nijmegen, the Netherlands

    J Julius Zhu

  15. Department of Physiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    J Julius Zhu

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  1. Miao Jing
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Contributions

J.J.Z. and Y.L. conceived the project. M.J. did GACh screening and optimization as well as its validation in cultured neurons and IPN slices. Y.L., Y.S., Z.J.Z., and H.J. designed and performed the work on transgenic flies. M.J. and J.F. performed experiments related to calcium imaging, GPCR internalization, Tango assay, and FRET measurements. L.M. and L.Z. did in vivo imaging of GACh sensors in mouse visual cortex. M.L. supervised the imaging experiments on MHb-IPN brain slices. P.Z. and G.W. together carried out the other experiments with assistance and advice from S.W., J.C.L., N.A.G., L.W.L., J.L., Y.Z., D.A.T., L.W.R., P.Q.B., and J.J.Z. All authors contributed to data analysis. M.J., P.Z., G.W., J.J.Z., and Y.L. wrote the manuscript with input from other authors.

Corresponding authors

Correspondence to J Julius Zhu or Yulong Li.

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M.J. and Y.L. have filed patent applications whose value might be affected by this publication.

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Jing, M., Zhang, P., Wang, G. et al. A genetically encoded fluorescent acetylcholine indicator for in vitro and in vivo studies. Nat Biotechnol 36, 726–737 (2018). https://doi.org/10.1038/nbt.4184

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