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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References
Nicolelis, M.A. & Ribeiro, S. Multielectrode recordings: the next steps. Curr. Opin. Neurobiol. 12, 602–606 (2002).
Tsien, R.Y. Monitoring cell calcium. in Calcium as a Cellular Regulator (Carafoli, E. & Klee, C., eds.) 28–54 (Oxford University Press, New York, 1999).
Miyawaki, A. et al. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882–887 (1997).
Tank, D.W., Sugimori, M., Connor, J.A. & Llinas, R.R. Spatially resolved calcium dynamics of mammalian Purkinje cells in cerebellar slice. Science 242, 773–777 (1988).
Stosiek, C., Garaschuk, O., Holthoff, K. & Konnerth, A. In vivo two-photon calcium imaging of neuronal networks. Proc. Natl. Acad. Sci. USA 100, 7319–7324 (2003).
Kerr, J.N., Greenberg, D. & Helmchen, F. Imaging input and output of neocortical networks in vivo. Proc. Natl. Acad. Sci. USA 102, 14063–14068 (2005).
Ohki, K., Chung, S., Ch'ng, Y.H., Kara, P. & Reid, R.C. Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex. Nature 433, 597–603 (2005).
Kerr, J.N. et al. Spatial organization of neuronal population responses in layer 2/3 of rat barrel cortex. J. Neurosci. 27, 13316–13328 (2007).
Ohki, K. & Reid, R.C. Specificity and randomness in the visual cortex. Curr. Opin. Neurobiol. 17, 401–407 (2007).
Palmer, A.E. & Tsien, R.Y. Measuring calcium signaling using genetically targetable fluorescent indicators. Nat. Protocols 1, 1057–1065 (2006).
Palmer, A.E. et al. Ca2+ indicators based on computationally redesigned calmodulin-peptide pairs. Chem. Biol. 13, 521–530 (2006).
Kerr, R. et al. Optical imaging of calcium transients in neurons and pharyngeal muscle of C. elegans. Neuron 26, 583–594 (2000).
Wang, J.W., Wong, A.M., Flores, J., Vosshall, L.B. & Axel, R. Two-photon calcium imaging reveals an odor-evoked map of activity in the fly brain. Cell 112, 271–282 (2003).
Higashijima, S., Masino, M.A., Mandel, G. & Fetcho, J.R. Imaging neuronal activity during zebrafish behavior with a genetically encoded calcium indicator. J. Neurophysiol. 90, 3986–3997 (2003).
Hasan, M.T. et al. Functional fluorescent Ca2+ indicator proteins in transgenic mice under TET control. PLoS Biol. 2, e163 (2004).
Heim, N. et al. Improved calcium imaging in transgenic mice expressing a troponin C-based biosensor. Nat. Methods 4, 127–129 (2007).
Klein, R.L. et al. Dose and promoter effects of adeno-associated viral vector for green fluorescent protein expression in the rat brain. Exp. Neurol. 176, 66–74 (2002).
Schnepp, B.C., Jensen, R.L., Chen, C.L., Johnson, P.R. & Clark, K.R. Characterization of adeno-associated virus genomes isolated from human tissues. J. Virol. 79, 14793–14803 (2005).
Girod, A. et al. Genetic capsid modifications allow efficient re-targeting of adeno-associated virus type 2. Nat. Med. 5, 1052–1056; erratum 5, 1438 (1999).
Shevtsova, Z., Malik, J.M., Michel, U., Bahr, M. & Kugler, S. Promoters and serotypes: targeting of adeno-associated virus vectors for gene transfer in the rat central nervous system in vitro and in vivo. Exp. Physiol. 90, 53–59 (2005).
Zhu, P. et al. Silencing and un-silencing of tetracycline-controlled genes in neurons. PLoS ONE 2, e533 (2007).
Wirth, D. et al. Road to precision: recombinase-based targeting technologies for genome engineering. Curr. Opin. Biotechnol. 18, 411–419 (2007).
Denk, W., Strickler, J.H. & Webb, W.W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).
Hahn, T.T., Sakmann, B. & Mehta, M.R. Phase-locking of hippocampal interneurons' membrane potential to neocortical up-down states. Nat. Neurosci. 9, 1359–1361 (2006).
Margrie, T.W. et al. Targeted whole-cell recordings in the mammalian brain in vivo. Neuron 39, 911–918 (2003).
Margrie, T.W., Brecht, M. & Sakmann, B. In vivo, low-resistance, whole-cell recordings from neurons in the anaesthetized and awake mammalian brain. Pflugers Arch. 444, 491–498 (2002).
Lee, A.K., Manns, I.D., Sakmann, B. & Brecht, M. Whole-cell recordings in freely moving rats. Neuron 51, 399–407 (2006).
de Kock, C.P., Bruno, R.M., Spors, H. & Sakmann, B. Layer- and cell-type-specific suprathreshold stimulus representation in rat primary somatosensory cortex. J. Physiol. (Lond.) 581, 139–154 (2007).
Greenberg, D.S., Houweling, A.R. & Kerr, J.N. Population imaging of ongoing neuronal activity in the visual cortex of awake rats. Nat. Neurosci. 11, 749–751 (2008).
Theer, P., Hasan, M.T. & Denk, W. Two-photon imaging to a depth of 1000 microm in living brains by use of a Ti:Al2O3 regenerative amplifier. Opt. Lett. 28, 1022–1024 (2003).
Nakai, J., Ohkura, M. & Imoto, K. A high signal-to-noise Ca2+ probe composed of a single green fluorescent protein. Nat. Biotechnol. 19, 137–141 (2001).
Wickersham, I.R. et al. Monosynaptic restriction of transsynaptic tracing from single, genetically targeted neurons. Neuron 53, 639–647 (2007).
During, M.J. Adeno-associated virus as a gene delivery system. Adv. Drug Deliv. Rev. 27, 83–94 (1997).
Kugler, S., Hahnewald, R., Garrido, M. & Reiss, J. Long-term rescue of a lethal inherited disease by adeno-associated virus-mediated gene transfer in a mouse model of molybdenum-cofactor deficiency. Am. J. Hum. Genet. 80, 291–297 (2007).
Stoppini, L., Buchs, P.A. & Muller, D. A simple method for organotypic cultures of nervous tissue. J. Neurosci. Methods 37, 173–182 (1991).
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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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.
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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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DOI: https://doi.org/10.1038/nmeth.1242
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