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Calcium indicator loading of neurons using single-cell electroporation

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Abstract

Studies of subcellular Ca2+ signaling rely on methods for labeling cells with fluorescent Ca2+ indicator dyes. In this study, we demonstrate the use of single-cell electroporation for Ca2+ indicator loading of individual neurons and small neuronal networks in rat neocortex in vitro and in vivo. Brief voltage pulses were delivered through glass pipettes positioned close to target cells. This approach resulted in reliable and rapid (within seconds) loading of somata and subsequent complete labeling of dendritic and axonal arborizations. By using simultaneous whole-cell recordings in brain slices, we directly addressed the effect of electroporation on neurons. Cell viability was high (about 85%) with recovery from the membrane permeabilization occurring within a minute. Electrical properties of recovered cells were indistinguishable before and after electroporation. In addition, Ca2+ transients with normal appearance could be evoked in dendrites, spines, and axonal boutons of electroporated cells. Using negative-stains of somata, targeted single-cell electroporation was equally applicable in vivo. We conclude that electroporation is a simple approach that permits Ca2+ indicator loading of multiple cells with low background staining within a short amount of time, which makes it especially well suited for functional imaging of subcellular Ca2+ dynamics in small neuronal networks.

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Acknowledgements

We would like to thank B. Sakmann for his support and M. Kaiser, R. Rödel, and K. Schmidt for their excellent technical assistance.

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  1. Thomas Nevian

    Present address: Institut für Physiologie, Universität Bern, Bühlplatz 5, 3012, Bern, Switzerland

  2. Fritjof Helmchen

    Present address: Abteilung Neurophysiologie, Institut für Hirnforschung, Universität Zürich, Winterthurerstr. 190, 8057, Zürich, Switzerland

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  1. Abteilung Zellphysiologie, Max-Planck-Institut für medizinische Forschung, Jahnstr. 29, 69120, Heidelberg, Germany

    Thomas Nevian & Fritjof Helmchen

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  1. Thomas Nevian
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Correspondence to Thomas Nevian.

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Nevian, T., Helmchen, F. Calcium indicator loading of neurons using single-cell electroporation. Pflugers Arch - Eur J Physiol 454, 675–688 (2007). https://doi.org/10.1007/s00424-007-0234-2

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