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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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We thank L. Looger and colleagues for sharing their unpublished acetylcholine sensors that validated some of our results.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.

Author information

Author notes

    • Miao Jing
    •  & Peng Zhang

    These authors contributed equally to this work.

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. Zilkha Neurogenetic Institute, Department of Physiology & Neuroscience, 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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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.

Competing interests

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

Corresponding authors

Correspondence to J Julius Zhu or Yulong Li.

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https://doi.org/10.1038/nbt.4184