Calcium indicator loading of neurons using single-cell electroporation

  • Thomas NevianEmail author
  • Fritjof Helmchen
Instruments and Techniques


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.


Two-photon microscopy Neocortex Neural network Dendrite Synapse 



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


  1. 1.
    Adelsberger H, Garaschuk O, Konnerth A (2005) Cortical calcium waves in resting newborn mice. Nat Neurosci 8:988–990PubMedCrossRefGoogle Scholar
  2. 2.
    Augustine GJ (1994) Combining patch-clamp and optical methods in brain slices. J Neurosci Methods 54:163–169PubMedCrossRefGoogle Scholar
  3. 3.
    Berridge MJ (1998) Neuronal calcium signaling. Neuron 21:13–26PubMedCrossRefGoogle Scholar
  4. 4.
    Bonnot A, Mentis GZ, Skoch J, O’Donovan MJ (2004) Electroporation loading of calcium sensitive dyes into the central nervous system. J Neurophysiol 93:1793–1808PubMedCrossRefGoogle Scholar
  5. 5.
    Brustein E, Marandi N, Kovalchuk Y, Drapeau P, Konnerth A (2003) “In vivo” monitoring of neuronal network activity in zebrafish by two-photon Ca2+ imaging. Pflügers Arch 446:766–773PubMedCrossRefGoogle Scholar
  6. 6.
    Canatella PJ, Karr JF, Petros JA, Prausnitz MR (2001) Quantitative study of electroporation-mediated molecular uptake and cell viability. Biophys J 80:755–764PubMedGoogle Scholar
  7. 7.
    DeBruin KA, Krassowska W (1999) Modeling electroporation in a single cell. I. Effects of field strength and rest potential. Biophys J 77:1213–1224PubMedGoogle Scholar
  8. 8.
    DeBruin KA, Krassowska W (1999) Modeling electroporation in a single cell. II. Effects of ionic concentrations. Biophys J 77:1225–1233PubMedGoogle Scholar
  9. 9.
    Di Cristo G, Wu C, Chattopadhyaya B, Ango F, Knott G, Welker E, Svoboda K, Huang ZJ (2004) Subcellular domain-restricted GABAergic innervation in primary visual cortex in the absence of sensory and thalamic inputs. Nat Neurosci 7:1184–1186PubMedCrossRefGoogle Scholar
  10. 10.
    Dodt HU, Zieglgänsberger W (1998) Visualization of neuronal form and function in brain slices by infrared videomicroscopy. Histochem J 30:141–152PubMedCrossRefGoogle Scholar
  11. 11.
    Eilers J, Konnerth A (2000) Dye loading with patch pipets. In: Yuste R, Lanni F, Konnerth A (eds) Imaging neurons, a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp 35.31–35.10Google Scholar
  12. 12.
    Gan WB, Grutzendler J, Wong WT, Wong RO, Lichtman JW (2000) Multicolor “DiOlistic” labeling of the nervous system using lipophilic dye combinations. Neuron 27:219–225PubMedCrossRefGoogle Scholar
  13. 13.
    Garaschuk O, Linn J, Eilers J, Konnerth A (2000) Large-scale oscillatory calcium waves in the immature cortex. Nat Neurosci 3:452–459PubMedCrossRefGoogle Scholar
  14. 14.
    Gehl J, Mir LM (1999) Determination of optimal parameters for in vivo gene transfer by electroporation, using a rapid in vivo test for cell permeabilization. Biochem Biophys Res Commun 261:377–380PubMedCrossRefGoogle Scholar
  15. 15.
    Golzio M, Teissie J, Rols MP (2002) Direct visualization at the single-cell level of electrically mediated gene delivery. Proc Natl Acad Sci U S A 99:1292–1297PubMedCrossRefGoogle Scholar
  16. 16.
    Graham LJ, Del Abajo R, Gener T, Fernandez E (2007) A method of combined single-cell electrophysiology and electroporation. J Neurosci Methods 160:69–74PubMedCrossRefGoogle Scholar
  17. 17.
    Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450PubMedGoogle Scholar
  18. 18.
    Haas K, Sin WC, Javaherian A, Li Z, Cline HT (2001) Single-cell electroporation for gene transfer in vivo. Neuron 29:583–591PubMedCrossRefGoogle Scholar
  19. 19.
    Haas K, Jensen K, Sin WC, Foa L, Cline HT (2002) Targeted electroporation in Xenopus tadpoles in vivo-from single cells to the entire brain. Differentiation 70:148–154PubMedCrossRefGoogle Scholar
  20. 20.
    Hasan MT, Friedrich RW, Euler T, Larkum ME, Giese G, Both M, Duebel J, Waters J, Bujard H, Griesbeck O, Tsien RY, Nagai T, Miyawaki A, Denk W (2004) Functional fluorescent Ca2+ indicator proteins in transgenic mice under TET control. PLoS Biol 2:e163PubMedCrossRefGoogle Scholar
  21. 21.
    Helmchen F, Imoto K, Sakmann B (1996) Ca2+ buffering and action potential-evoked Ca2+ signaling in dendrites of pyramidal neurons. Biophys J 70:1069–1081PubMedGoogle Scholar
  22. 22.
    Helmchen F, Svoboda K, Denk W, Tank DW (1999) In vivo dendritic calcium dynamics in deep-layer cortical pyramidal neurons. Nat Neurosci 2:989–996PubMedCrossRefGoogle Scholar
  23. 23.
    Helmchen F, Waters J (2002) Ca2+ imaging in the mammalian brain in vivo. Eur J Pharmacol 447:119–129PubMedCrossRefGoogle Scholar
  24. 24.
    Ho SY, Mittal GS (1996) Electroporation of cell membranes: a review. Crit Rev Biotechnol 16:349–362PubMedCrossRefGoogle Scholar
  25. 25.
    Inoue T, Krumlauf R (2001) An impulse to the brain—using in vivo electroporation. Nat Neurosci 4:1156–1158PubMedCrossRefGoogle Scholar
  26. 26.
    Kaiser KM, Lübke J, Zilberter Y, Sakmann B (2004) Postsynaptic calcium influx at single synaptic contacts between pyramidal neurons and bitufted interneurons in layer 2/3 of rat neocortex is enhanced by backpropagating action potentials. J Neurosci 24:1319–1329PubMedCrossRefGoogle Scholar
  27. 27.
    Kerr JND, Greenberg D, Helmchen F (2005) Imaging input and output of neocortical networks in vivo. Proc Natl Acad Sci U S A 102:14063–14068PubMedCrossRefGoogle Scholar
  28. 28.
    Kettunen P, Demas J, Lohmann C, Kasthuri N, Gong YD, Wong ROL, Gan WB (2002) Imaging calcium dynamics in the nervous system by means of ballistic delivery of indicators. J Neurosci Methods 119:37–43PubMedCrossRefGoogle Scholar
  29. 29.
    Köster HJ, Johnston D (2005) Target cell-dependent normalization of transmitter release at neocortical synapses. Science 308:863–866CrossRefGoogle Scholar
  30. 30.
    Krassowska W, Filev PD (2006) Modeling Electroporation in a Single Cell. Biophys J 77:1213–1224Google Scholar
  31. 31.
    Kreitzer AC, Gee KR, Archer EA, Regehr WG (2000) Monitoring presynaptic calcium dynamics in projection fibers by in vivo loading of a novel calcium indicator. Neuron 27:25–32PubMedCrossRefGoogle Scholar
  32. 32.
    Kuner T, Augustine GJ (2000) A genetically encoded ratiometric indicator for chloride: capturing chloride transients in cultured hippocampal neurons. Neuron 27:447–459PubMedCrossRefGoogle Scholar
  33. 33.
    Lambe EK, Aghajanian GK (2003) Hypocretin (orexin) induces calcium transients in single spines postsynaptic to identified thalamocortical boutons in prefrontal slice. Neuron 40:139–150PubMedCrossRefGoogle Scholar
  34. 34.
    Lin X, Webster P, Li Q, Chen S, Ouyang Y (2003) Optical recordings of Ca2+ signaling activities from identified inner ear cells in cochlear slices and hemicochleae. Brain Res Brain Res Protoc 11:92–100PubMedCrossRefGoogle Scholar
  35. 35.
    Lohmann C, Finski A, Bonhoeffer T (2005) Local calcium transients regulate the spontaneous motility of dendritic filopodia. Nat Neurosci 8:305–312PubMedCrossRefGoogle Scholar
  36. 36.
    Lovell P, Jezzini SH, Moroz LL (2006) Electroporation of neurons and growth cones in Aplysia californica. J Neurosci Methods 151:114–120PubMedCrossRefGoogle Scholar
  37. 37.
    Maravall M, Mainen ZF, Sabatini BL, Svoboda K (2000) Estimating intracellular calcium concentrations and buffering without wavelength ratioing. Biophys J 78:2655–2667PubMedGoogle Scholar
  38. 38.
    Margrie TW, Brecht M, Sakmann B (2002) In vivo, low-resistance, whole-cell recordings from neurons in the anaesthetized and awake mammalian brain. Pflügers Arch 444:491–498PubMedCrossRefGoogle Scholar
  39. 39.
    Margrie TW, Meyer AH, Caputi A, Monyer H, Hasan MT, Schaefer AT, Denk W, Brecht M (2003) Targeted whole-cell recordings in the mammalian brain in vivo. Neuron 39:911–918PubMedCrossRefGoogle Scholar
  40. 40.
    Neumann E, Toensing K, Kakorin S, Budde P, Frey J (1998) Mechanism of electroporative dye uptake by mouse B cells. Biophys J 74:98–108PubMedGoogle Scholar
  41. 41.
    Nevian T, Sakmann B (2004) Single spine Ca2+ signals evoked by coincident EPSPs and backpropagating action potentials in spiny stellate cells of layer 4 in the juvenile rat somatosensory barrel cortex. J Neurosci 24:1689–1699PubMedCrossRefGoogle Scholar
  42. 42.
    Nevian T, Sakmann B (2006) Spine Ca2+ signaling in spike-timing-dependent plasticity. J Neurosci 26:11001–11013PubMedCrossRefGoogle Scholar
  43. 43.
    Nimchinsky EA, Yasuda R, Oertner TG, Svoboda K (2004) The number of glutamate receptors opened by synaptic stimulation in single hippocampal spines. J Neurosci 24:2054–2064PubMedCrossRefGoogle Scholar
  44. 44.
    Nimmerjahn A, Kirchhoff F, Kerr JN, Helmchen F (2004) Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo. Nat Methods 1:31–37PubMedCrossRefGoogle Scholar
  45. 45.
    Nolkrantz K, Farre C, Brederlau A, Karlsson RID, Brennan C, Eriksson PS, Weber SG, Sandberg M, Orwar O (2001) Electroporation of single cells and tissues with an electrolyte-filled capillary. Anal Chem 73:4469–4477PubMedCrossRefGoogle Scholar
  46. 46.
    O’Donovan M, Ho S, Yee W (1994) Calcium imaging of rhythmic network activity in the developing spinal cord of the chick embryo. J Neurosci 14:6354–6369PubMedGoogle Scholar
  47. 47.
    Ohki K, Chung S, Ch’ng YH, Kara P, Reid RC (2005) Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex. Nature 433:597–603PubMedCrossRefGoogle Scholar
  48. 48.
    Olofsson J, Nolkrantz K, Ryttsen F, Lambie BA, Weber SG, Orwar O (2003) Single-cell electroporation. Curr Opin Biotechnol 14:29–34PubMedCrossRefGoogle Scholar
  49. 49.
    Peterlin ZA, Kozloski J, Mao BQ, Tsiola A, Yuste R (2000) Optical probing of neuronal circuits with calcium indicators. Proc Natl Acad Sci U S A 97:3619–3624PubMedCrossRefGoogle Scholar
  50. 50.
    Pologruto TA, Yasuda R, Svoboda K (2004) Monitoring neural activity and [Ca2+] with genetically encoded Ca2+ indicators. J Neurosci 24:9572–9579PubMedCrossRefGoogle Scholar
  51. 51.
    Rae JL, Levis RA (2002) Single-cell electroporation. Pflügers Arch 443:664–670PubMedCrossRefGoogle Scholar
  52. 52.
    Rathenberg J, Nevian T, Witzemann V (2003) High-efficiency transfection of individual neurons using modified electrophysiology techniques. J Neurosci Methods 126:91–98PubMedCrossRefGoogle Scholar
  53. 53.
    Reiff DF, Ihring A, Guerrero G, Isacoff EY, Joesch M, Nakai J, Borst A (2005) In vivo performance of genetically encoded indicators of neural activity in flies. J Neurosci 25:4766–4778PubMedCrossRefGoogle Scholar
  54. 54.
    Roorda RD, Hohl TM, Toledo-Crow R, Miesenbock G (2004) Video-rate nonlinear microscopy of neuronal membrane dynamics with genetically encoded probes. J Neurophysiol 92:609–621PubMedCrossRefGoogle Scholar
  55. 55.
    Ruthazer ES, Akerman CJ, Cline HT (2003) Control of axon branch dynamics by correlated activity in vivo. Science 301:66–70PubMedCrossRefGoogle Scholar
  56. 56.
    Ryttsen F, Farre C, Brennan C, Weber SG, Nolkrantz K, Jardemark K, Chiu DT, Orwar O (2000) Characterization of single-cell electroporation by using patch-clamp and fluorescence microscopy. Biophys J 79:1993–2001PubMedCrossRefGoogle Scholar
  57. 57.
    Sabatini BL, Maravall M, Svoboda K (2001) Ca2+ signaling in dendritic spines. Curr Opin Neurobiol 11:349–356PubMedCrossRefGoogle Scholar
  58. 58.
    Smetters D, Majewska A, Yuste R (1999) Detecting action potentials in neuronal populations with calcium imaging. Methods 18:215–221PubMedCrossRefGoogle Scholar
  59. 59.
    Stosiek C, Garaschuk O, Holthoff K, Konnerth A (2003) In vivo two-photon calcium imaging of neuronal networks. Proc Natl Acad Sci U S A 100:7319–7324PubMedCrossRefGoogle Scholar
  60. 60.
    Stuart GJ, Dodt HU, Sakmann B (1993) Patch-clamp recordings from the soma and dendrites of neurons in brain slices using infrared video microscopy. Pflügers Arch 423:511–518PubMedCrossRefGoogle Scholar
  61. 61.
    Svoboda K, Denk W, Kleinfeld D, Tank DW (1997) In vivo dendritic calcium dynamics in neocortical pyramidal neurons. Nature 385:161–165PubMedCrossRefGoogle Scholar
  62. 62.
    Teruel MN, Meyer T (1997) Electroporation-induced formation of individual calcium entry sites in the cell body and processes of adherent cells. Biophys J 73:1785–1796PubMedGoogle Scholar
  63. 63.
    Teruel MN, Blanpied TA, Shen K, Augustine GJ, Meyer T (1999) A versatile microporation technique for the transfection of cultured CNS neurons. J Neurosci Methods 93:37–48PubMedCrossRefGoogle Scholar
  64. 64.
    Turrigiano GG, Nelson SB (2000) Hebb and homeostasis in neuronal plasticity. Curr Opin Neurobiol 10:358–364PubMedCrossRefGoogle Scholar
  65. 65.
    Umeda T, Ebihara T, Okabe S (2005) Simultaneous observation of stably associated presynaptic varicosities and postsynaptic spines: morphological alterations of CA3-CA1 synapses in hippocampal slice cultures. Mol Cell Neurosci 28:264–274PubMedCrossRefGoogle Scholar
  66. 66.
    Waters J, Larkum M, Sakmann B, Helmchen F (2003) Supralinear Ca2+ influx into dendritic tufts of layer 2/3 neocortical pyramidal neurons in vitro and in vivo. J Neurosci 23:8558–8567PubMedGoogle Scholar
  67. 67.
    Wimmer VC, Nevian T, Kuner T (2004) Targeted in vivo expression of proteins in the calyx of Held. Pflügers Arch 449:319–333PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  1. 1.Abteilung ZellphysiologieMax-Planck-Institut für medizinische ForschungHeidelbergGermany
  2. 2.Institut für PhysiologieUniversität BernBernSwitzerland
  3. 3.Abteilung Neurophysiologie, Institut für HirnforschungUniversität ZürichZürichSwitzerland

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