Chronic Ca2+ imaging of cortical neurons with long-term expression of GCaMP-X

  1. Jinli Geng
  2. Yingjun Tang
  3. Zhen Yu
  4. Yunming Gao
  5. Wenxiang Li
  6. Yitong Lu
  7. Bo Wang
  8. Huiming Zhou
  9. Ping Li
  10. Nan Liu
  11. Ping Wang
  12. Yubo Fan
  13. Yaxiong Yang  Is a corresponding author
  14. Zengcai Guo  Is a corresponding author
  15. Xiaodong Liu  Is a corresponding author
  1. Beihang University, China
  2. Tsinghua University, China
  3. Yunnan University, China
  4. Zhejiang University, China

Abstract

Dynamic Ca2+ signals reflect acute changes in membrane excitability (e.g. responses to stimuli), and also mediate intracellular signaling cascades that normally take longer time to manifest (e.g., regulations of transcription). In both cases, chronic Ca2+ imaging has been often desired, but largely hindered by unexpected cytotoxicity intrinsic to GCaMP, a popular series of genetically-encoded Ca2+ indicators. Here, we demonstrate the performance of GCaMP-X in chronic Ca2+ imaging with long-term probe expression in cortical neurons, which has been designed to eliminate the unwanted interactions between conventional GCaMP indicators and endogenous (apo)calmodulin-binding proteins. By expressing in live adult mice at high levels over an extended time frame, GCaMP-X indicators showed less damage and improved performance in two-photon imaging of acute Ca2+ responses to whisker deflection or spontaneous Ca2+ fluctuations. Chronic Ca2+ imaging data (³1 month) were acquired from cultured cortical neurons expressing GCaMP-X, unveiling that spontaneous/local Ca2+ transients would progressively develop into autonomous/global Ca2+ oscillations. Besides the morphological indices of neurite length and soma size, the major metrics of oscillatory Ca2+, including rate, amplitude and synchrony were also examined along with the multiple stages (from neonatal to mature) during neural development. Dysregulations of both neuritogenesis and Ca2+ oscillations were observed typically in 2-3 weeks, which were exacerbated by stronger or prolonged expression of GCaMP. In comparison, neurons expressing GCaMP-X exhibited significantly less damage. By varying the timepoints of virus infection or drug induction, GCaMP-X outperformed GCaMP similarly in cultured mature neurons. These data altogether highlight the unique importance of oscillatory Ca2+ to morphology and health of neurons, presumably underlying the differential performance between GCaMP-X and GCaMP. In summary, GCaMP-X provides a viable option for Ca2+ imaging applications involving long-time and/or high-level expression of Ca2+ probes.

Data availability

The plasmids of pEGFP-N1-GCaMP7b-XC (178361) and pEGFP-N1-GCaMP7b-XN (178362) are available on Addgene. Source data for WB and Co-IP are organized as four ZIP files. The data in details associated with the main figures have been deposited to Dryad (https://doi.org/10.5061/dryad.zw3r22893). Other data and information are available from the corresponding author upon reasonable request.

The following data sets were generated

Article and author information

Author details

  1. Jinli Geng

    Advanced Innovation Center for Biomedical Engineering, Beihang University, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  2. Yingjun Tang

    Tsinghua-Peking Joint Center for Life Sciences, Tsinghua University, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  3. Zhen Yu

    Advanced Innovation Center for Biomedical Engineering, Beihang University, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  4. Yunming Gao

    Advanced Innovation Center for Biomedical Engineering, Beihang University, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  5. Wenxiang Li

    Advanced Innovation Center for Biomedical Engineering, Beihang University, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  6. Yitong Lu

    Advanced Innovation Center for Biomedical Engineering, Beihang University, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  7. Bo Wang

    Advanced Innovation Center for Biomedical Engineering, Beihang University, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  8. Huiming Zhou

    Tsinghua-Peking Joint Center for Life Sciences, Tsinghua University, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  9. Ping Li

    Advanced Innovation Center for Biomedical Engineering, Beihang University, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  10. Nan Liu

    Center for Life Sciences, Yunnan University, Kunming, China
    Competing interests
    The authors declare that no competing interests exist.
  11. Ping Wang

    Laboratory for Biomedical Engineering of Ministry of Education, Zhejiang University, Hangzhou, China
    Competing interests
    The authors declare that no competing interests exist.
  12. Yubo Fan

    Advanced Innovation Center for Biomedical Engineering, Beihang University, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  13. Yaxiong Yang

    Advanced Innovation Center for Biomedical Engineering, Beihang University, Beijing, China
    For correspondence
    yangyaxiong@buaa.edu.cn
    Competing interests
    The authors declare that no competing interests exist.
  14. Zengcai Guo

    Tsinghua-Peking Joint Center for Life Sciences, Tsinghua University, Beijing, China
    For correspondence
    guozengcai@tsinghua.edu.cn
    Competing interests
    The authors declare that no competing interests exist.
  15. Xiaodong Liu

    Advanced Innovation Center for Biomedical Engineering, Beihang University, Beijing, China
    For correspondence
    liu-lab@buaa.edu.cn
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3171-9611

Funding

National Natural Science Foundation of China (81971728)

  • Xiaodong Liu

Natural Science Foundation of Beijing Municipality (7191006)

  • Xiaodong Liu

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: Procedures involving animals have been approved by local institutional ethical committees (IACUC in Tsinghua University and Beihang University),

Copyright

© 2022, Geng et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Jinli Geng
  2. Yingjun Tang
  3. Zhen Yu
  4. Yunming Gao
  5. Wenxiang Li
  6. Yitong Lu
  7. Bo Wang
  8. Huiming Zhou
  9. Ping Li
  10. Nan Liu
  11. Ping Wang
  12. Yubo Fan
  13. Yaxiong Yang
  14. Zengcai Guo
  15. Xiaodong Liu
(2022)
Chronic Ca2+ imaging of cortical neurons with long-term expression of GCaMP-X
eLife 11:e76691.
https://doi.org/10.7554/eLife.76691

Share this article

https://doi.org/10.7554/eLife.76691

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