Chronic Ca2+ imaging of cortical neurons with long-term expression of GCaMP-X
- Beihang University, China
- Tsinghua University, China
- Yunnan University, China
- 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.
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Chronic Ca2+ imaging of cortical neurons with long-term expression of GCaMP-XDryad Digital Repository, doi:10.5061/dryad.zw3r22893.
Article and author information
Author details
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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