Abstract
Neurons in the nervous system can change their functional properties over time. At present, there are no techniques that allow reliable monitoring of changes within identified neurons over repeated experimental sessions. We increased the signal strength of troponin C–based calcium biosensors in the low-calcium regime by mutagenesis and domain rearrangement within the troponin C calcium binding moiety to generate the indicator TN-XXL. Using in vivo two-photon ratiometric imaging, we show that TN-XXL exhibits enhanced fluorescence changes in neurons of flies and mice. TN-XXL could be used to obtain tuning curves of orientation-selective neurons in mouse visual cortex measured repeatedly over days and weeks. Thus, the genetically encoded calcium indicator TN-XXL allows repeated imaging of response properties from individual, identified neurons in vivo, which will be crucial for gaining new insights into cellular mechanisms of plasticity, regeneration and disease.
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Acknowledgements
We would like to thank A. Moritz for technical assistance. This work was supported by the Max Planck Society, DFG priority program grant SP1172 to O.G., the Wellcome Trust (S.B.H., T.D.M.-F.) and a 'Bsik' grant from SenterNovem (The Netherlands) to C.L.
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M.M. performed all molecular biology and spectroscopy to engineer TN-XXL; S.D. and V.S. performed electrophysiology in hippocampal slices; T.H., D.F.R. and A.B. imaged and evaluated TN-XXL in Drosophila; C.L. performed in utero electroporation; A.F.S., S.B.H., T.D.M.-F., T.B. and M.H. performed and evaluated experiments in mouse visual cortex; O.G., M.H., D.F.R., M.M. and A.F.S. wrote the manuscript; all authors contributed to editing the manuscript.
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M.M. and O.G. are listed as inventors in an international PCT patent application assigned to the Max Planck Society, which includes as claims the calcium biosensor described in this manuscript.
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Mank, M., Santos, A., Direnberger, S. et al. A genetically encoded calcium indicator for chronic in vivo two-photon imaging. Nat Methods 5, 805–811 (2008). https://doi.org/10.1038/nmeth.1243
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DOI: https://doi.org/10.1038/nmeth.1243
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