Single-spike detection in vitro and in vivo with a genetic Ca2+ sensor

Abstract

Measurement of population activity with single-action-potential, single-neuron resolution is pivotal for understanding information representation and processing in the brain and how the brain's responses are altered by experience. Genetically encoded indicators of neuronal activity allow long-term, cell type–specific expression. Fluorescent Ca2+ indicator proteins (FCIPs), a main class of reporters of neural activity, initially suffered, in particular, from an inability to report single action potentials in vivo. Although suboptimal Ca2+-binding dynamics and Ca2+-induced fluorescence changes in FCIPs are important factors, low levels of expression also seem to play a role. Here we report that delivering D3cpv, an improved fluorescent resonance energy transfer–based FCIP, using a recombinant adeno-associated virus results in expression sufficient to detect the Ca2+ transients that accompany single action potentials. In upper-layer cortical neurons, we were able to detect transients associated with single action potentials firing at rates of <1 Hz, with high reliability, from in vivo recordings in living mice.

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Figure 1: D3cpv expression in neurons.
Figure 2: Responses to synaptic stimulation in organotypic slices.
Figure 3: Whole-cell recordings in organotypic slices.
Figure 4: Responses in mouse somatosensory cortex to sensory stimulation.
Figure 5: Simultaneous electrical and optical recording of action potentials in vivo.

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Acknowledgements

We thank A. Karpova and W. Mittmann for reading the manuscript and for helpful suggestions, D.S. Greenberg for modifying the automatic detection algorithm, A. Migala for rat hippocampal organotypic slices, I. Wunderlich for help with the virus preparation, J.A. Kleinschmidt (German Cancer Center, Heidelberg) for providing helper plasmids and E. Heil for art work. This work was supported by the Max Planck Society, Collaborative Research Grant (SFB636/A4), the Volkswagen Foundation (AZ: I/80 704) and the Schloessmann Foundation.

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Authors

Contributions

A.E.P. and R.Y.T. provided D3cpv. Experiments and data analysis: S.A., M.T.H. and W.D. (cell-attached and whole-cell experiments in rat hippocampal slices); D.J.W. and J.N.D.K. (targeted in vivo patch recordings); S.M.z.A.B., W.D. and M.T.H. (rat hippocampal slices and in vivo activity responses); R.S. and M.T.H. (construct design); Y.Y. and M.T.H. (molecular biology); S.K., Y.Y. and M.T.H. (virus production); Y.Y., R.S. and M.T.H. (expression analyses); and M.B. and M.T.H. (in vivo injections). M.T.H. led the project; W.D. and M.T.H. wrote the paper.

Corresponding author

Correspondence to Mazahir T Hasan.

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Supplementary Figures 1 and 2, Supplementary Methods (PDF 1103 kb)

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Wallace, D., zum Alten Borgloh, S., Astori, S. et al. Single-spike detection in vitro and in vivo with a genetic Ca2+ sensor. Nat Methods 5, 797–804 (2008). https://doi.org/10.1038/nmeth.1242

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