Advertisement

Two-Photon Imaging of Dendritic Calcium Dynamics In Vivo

  • Lucy M. PalmerEmail author
Protocol
Part of the Neuromethods book series (NM, volume 148)

Abstract

Developments in microscopes and indicators over the past decade have paved the way for recording neural dynamics in vivo. Previously inaccessible small neuronal processes, such as dendrites and axons, can now be probed in vivo using two-photon microscopy, revealing their important role in information processing during behaviour. To perform such experiments, various tools, techniques and considerations are required. In this chapter, the procedures for recording dendritic calcium dynamics in vivo are detailed in a step-by-step manner. The various sources of calcium contributing to the recorded transients are discussed, and details are given on how to identify them from the characteristics of the recorded fluorescence transients. These procedural details and considerations regarding two-photon calcium imaging of dendritic activity are put in context of the importance of such recordings for understanding neural function during behaviour.

Keywords

Two-photon Dendrites In vivo Behaviour Procedures Calcium imaging Awake 

References

  1. 1.
    Deiters O (1865) Untersuchungen über Gehirn und Rückenmark des Menschen und der Säugethiere. Vieweg, BraunschweigCrossRefGoogle Scholar
  2. 2.
    Denk W, Strickler JH, Webb WW (1990) Two-photon laser scanning fluorescence microscopy. Science 248:73–76PubMedCrossRefGoogle Scholar
  3. 3.
    Berridge MJ (1998) Neuronal calcium signaling. Neuron 21:13–26PubMedCrossRefGoogle Scholar
  4. 4.
    Chen TW, Wardill TJ, Sun Y, Pulver SR, Renninger SL, Baohan A, Schreiter ER, Kerr RA, Orger MB, Jayaraman V, Looger LL, Svoboda K, Kim DS (2013) Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499:295–300PubMedPubMedCentralCrossRefGoogle Scholar
  5. 5.
    Helmchen F, Imoto K, Sakmann B (1996) Ca2+ buffering and action potential-evoked Ca2+ signaling in dendrites of pyramidal neurons. Biophys J 70:1069–1081PubMedPubMedCentralCrossRefGoogle Scholar
  6. 6.
    Markram H, Lubke J, Frotscher M, Sakmann B (1997) Regulation of synaptic efficacy by coincidence of postsynaptic APs and EPSPs. Science 275:213–215PubMedCrossRefGoogle Scholar
  7. 7.
    Letzkus JJ, Kampa BM, Stuart GJ (2006) Learning rules for spike timing-dependent plasticity depend on dendritic synapse location. J Neurosci 26:10420–10429PubMedPubMedCentralCrossRefGoogle Scholar
  8. 8.
    Stuart G, Schiller J, Sakmann B (1997) Action potential initiation and propagation in rat neocortical pyramidal neurons. J Physiol 505:617–632PubMedPubMedCentralCrossRefGoogle Scholar
  9. 9.
    Clark BA, Monsivais P, Branco T, London M, Häusser M (2005) The site of action potential initiation in cerebellar Purkinje neurons. Nat Neurosci 8:137–139PubMedCrossRefGoogle Scholar
  10. 10.
    Palmer LM, Stuart GJ (2006) Site of action potential initiation in layer 5 pyramidal neurons. J Neurosci 26:1854–1863PubMedPubMedCentralCrossRefGoogle Scholar
  11. 11.
    Destexhe A, Lang EJ, Pare D (1998) Somato-dendritic interactions underlying action potential generation in neocortical pyramidal cells in vivo. In: Bower J (ed) Computational neuroscience: trends in research. Plenum Press, New York, pp 167–172CrossRefGoogle Scholar
  12. 12.
    Kole MH, Stuart GJ (2012) Signal processing in the axon initial segment. Neuron 73:235–247PubMedCrossRefPubMedCentralGoogle Scholar
  13. 13.
    Stuart GJ, Sakmann B (1994) Active propagation of somatic action potentials into neocortical pyramidal cell dendrites. Nature 367:69–72PubMedCrossRefPubMedCentralGoogle Scholar
  14. 14.
    Spruston N, Schiller Y, Stuart G, Sakmann B (1995) Activity-dependent action potential invasion and calcium influx into hippocampal CA1 dendrites. Science 268:297–300PubMedCrossRefPubMedCentralGoogle Scholar
  15. 15.
    Häusser M, Stuart G, Racca C, Sakmann B (1995) Axonal initiation and active dendritic propagation of action potentials in substantia nigra neurons. Neuron 15:637–647PubMedCrossRefPubMedCentralGoogle Scholar
  16. 16.
    Larkum ME, Rioult MG, Lüscher HR (1996) Propagation of action potentials in the dendrites of neurons from rat spinal cord slice cultures. J Neurophysiol 75:154–170PubMedCrossRefPubMedCentralGoogle Scholar
  17. 17.
    Bischofberger J, Jonas P (1997) Action potential propagation into the presynaptic dendrites of rat mitral cells. J Physiol 504:359–365PubMedPubMedCentralCrossRefGoogle Scholar
  18. 18.
    Williams SR, Stuart GJ (2000) Action potential backpropagation and somato-dendritic distribution of ion channels in thalamocortical neurons. J Neurosci 20:1307–1317PubMedPubMedCentralCrossRefGoogle Scholar
  19. 19.
    Lemon N, Turner RW (2000) Conditional spike backpropagation generates burst discharge in a sensory neuron. J Neurophysiol 84:1519–1530PubMedCrossRefPubMedCentralGoogle Scholar
  20. 20.
    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 23:8558–8567PubMedPubMedCentralCrossRefGoogle Scholar
  21. 21.
    Larkum ME, Watanabe S, Lasser-Ross N, Rhodes P, Ross WN (2008) Dendritic properties of turtle pyramidal neurons. J Neurophysiol 99:683–694PubMedCrossRefPubMedCentralGoogle Scholar
  22. 22.
    Bathellier B, Margrie TW, Larkum ME (2009) Properties of piriform cortex pyramidal cell dendrites: implications for olfactory circuit design. J Neurosci 29:12641–12652PubMedPubMedCentralCrossRefGoogle Scholar
  23. 23.
    Ledergerber D, Larkum ME (2010) Properties of layer 6 pyramidal neuron apical dendrites. J Neurosci 30:13031–13044PubMedPubMedCentralCrossRefGoogle Scholar
  24. 24.
    Casale AE, McCormick DA (2011) Active action potential propagation but not initiation in thalamic interneuron dendrites. J Neurosci 31:18289–18302PubMedPubMedCentralCrossRefGoogle Scholar
  25. 25.
    Feldman DE, Brecht M (2005) Map plasticity in somatosensory cortex. Science 310:810–815PubMedCrossRefPubMedCentralGoogle Scholar
  26. 26.
    Egger V, Feldmeyer D, Sakmann B (1999) Coincidence detection and changes of synaptic efficacy in spiny stellate neurons in rat barrel cortex. Nat Neurosci 2:1098–1105PubMedCrossRefPubMedCentralGoogle Scholar
  27. 27.
    Feldman DE (2000) Timing-based LTP and LTD at vertical inputs to layer II/III pyramidal cells in rat barrel cortex. Neuron 27:45–56PubMedCrossRefPubMedCentralGoogle Scholar
  28. 28.
    Sjostrom PJ, Turrigiano GG, Nelson SB (2001) Rate, timing, and cooperativity jointly determine cortical synaptic plasticity. Neuron 32:1149–1164PubMedCrossRefPubMedCentralGoogle Scholar
  29. 29.
    Holmgren CD, Zilberter Y (2001) Coincident spiking activity induces long-term changes in inhibition of neocortical pyramidal cells. J Neurosci 21:8270–8277PubMedPubMedCentralCrossRefGoogle Scholar
  30. 30.
    Froemke RC, Dan Y (2002) Spike-timing-dependent synaptic modification induced by natural spike trains. Nature 416:433–438PubMedCrossRefGoogle Scholar
  31. 31.
    Stuart GJ, Häusser M (2001) Dendritic coincidence detection of EPSPs and action potentials. Nat Neurosci 4:63–71PubMedCrossRefGoogle Scholar
  32. 32.
    Svoboda K, Denk W, Kleinfeld D, Tank DW (1997) In vivo dendritic calcium dynamics in neocortical pyramidal neurons. Nature 385:161–165PubMedCrossRefGoogle Scholar
  33. 33.
    Hill DN, Varga Z, Jia H, Sakmann B, Konnerth A (2013) Multibranch activity in basal and tuft dendrites during firing of layer 5 cortical neurons in vivo. Proc Natl Acad Sci U S A 110:13618–13623PubMedPubMedCentralCrossRefGoogle Scholar
  34. 34.
    Palmer LM, Shai AS, Reeve JE, Anderson HL, Paulsen O, Larkum ME (2014) NMDA spikes enhance action potential generation during sensory input. Nat Neurosci 17:383–390PubMedCrossRefPubMedCentralGoogle Scholar
  35. 35.
    Larkum ME, Zhu JJ, Sakmann B (1999) A new cellular mechanism for coupling inputs arriving at different cortical layers. Nature 398:338–341PubMedCrossRefPubMedCentralGoogle Scholar
  36. 36.
    Schiller J, Schiller Y, Stuart G, Sakmann B (1997) Calcium action potentials restricted to distal apical dendrites of rat neocortical pyramidal neurons. J Physiol 505:605–616PubMedPubMedCentralCrossRefGoogle Scholar
  37. 37.
    Perez-Garci E, Larkum ME, Nevian T (2013) Inhibition of dendritic Ca2+ spikes by GABAB receptors in cortical pyramidal neurons is mediated by a direct Gi/o-βγ-subunit interaction with Cav1 channels. J Physiol 591:1599–1612PubMedCrossRefPubMedCentralGoogle Scholar
  38. 38.
    Larkum ME, Zhu JJ (2002) Signaling of layer 1 and whisker-evoked Ca2+ and Na+ action potentials in distal and terminal dendrites of rat neocortical pyramidal neurons in vitro and in vivo. J Neurosci 22:6991–7005PubMedPubMedCentralCrossRefGoogle Scholar
  39. 39.
    Larkum ME, Nevian T, Sandler M, Polsky A, Schiller J (2009) Synaptic integration in tuft dendrites of layer 5 pyramidal neurons: a new unifying principle. Science 325:756–760PubMedCrossRefPubMedCentralGoogle Scholar
  40. 40.
    Harnett MT, Xu NL, Magee JC, Williams SR (2013) Potassium channels control the interaction between active dendritic integration compartments in layer 5 cortical pyramidal neurons. Neuron 79:516–529PubMedCrossRefGoogle Scholar
  41. 41.
    Harnett MT, Magee JC, Williams SR (2015) Distribution and function of HCN channels in the apical dendritic tuft of neocortical pyramidal neurons. J Neurosci 35:1024–1037PubMedPubMedCentralCrossRefGoogle Scholar
  42. 42.
    Xu NL, Harnett MT, Williams SR, Huber D, O'Connor DH, Svoboda K, Magee JC (2012) Nonlinear dendritic integration of sensory and motor input during an active sensing task. Nature 492:247–251PubMedCrossRefGoogle Scholar
  43. 43.
    Takahashi N, Oertner TG, Hegemann P, Larkum ME (2016) Active cortical dendrites modulate perception. Science 354:1587–1590PubMedCrossRefGoogle Scholar
  44. 44.
    Gambino F, Pages S, Kehayas V, Baptista D, Tatti R, Carleton A, Holtmaat A (2014) Sensory-evoked LTP driven by dendritic plateau potentials in vivo. Nature 515:116–119PubMedCrossRefGoogle Scholar
  45. 45.
    Cichon J, Gan WB (2015) Branch-specific dendritic Ca2+ spikes cause persistent synaptic plasticity. Nature 520:180–185PubMedPubMedCentralCrossRefGoogle Scholar
  46. 46.
    Major G, Polsky A, Denk W, Schiller J, Tank DW (2008) Spatiotemporally graded NMDA spike/plateau potentials in basal dendrites of neocortical pyramidal neurons. J Neurophysiol 99:2584–2601PubMedCrossRefGoogle Scholar
  47. 47.
    Polsky A, Mel BW, Schiller J (2004) Computational subunits in thin dendrites of pyramidal cells. Nat Neurosci 7:621–627PubMedCrossRefGoogle Scholar
  48. 48.
    Nevian T, Larkum ME, Polsky A, Schiller J (2007) Properties of basal dendrites of layer 5 pyramidal neurons: a direct patch-clamp recording study. Nat Neurosci 10:206–214PubMedCrossRefGoogle Scholar
  49. 49.
    Branco T, Häusser M (2011) Synaptic integration gradients in single cortical pyramidal cell dendrites. Neuron 69:885–892PubMedPubMedCentralCrossRefGoogle Scholar
  50. 50.
    Lavzin M, Rapoport S, Polsky A, Garion L, Schiller J (2012) Nonlinear dendritic processing determines angular tuning of barrel cortex neurons in vivo. Nature 490:397–401PubMedCrossRefPubMedCentralGoogle Scholar
  51. 51.
    Mao T, O’Connor DH, Scheuss V, Nakai J, Svoboda K (2008) Characterization and subcellular targeting of GCaMP-type genetically-encoded calcium indicators. PLoS One 3:e1796PubMedPubMedCentralCrossRefGoogle Scholar
  52. 52.
    Bollmann JH, Engert F (2009) Subcellular topography of visually driven dendritic activity in the vertebrate visual system. Neuron 61:895–905PubMedPubMedCentralCrossRefGoogle Scholar
  53. 53.
    Takahashi N, Kitamura K, Matsuo N, Mayford M, Kano M, Matsuki N, Ikegaya Y (2012) Locally synchronized synaptic inputs. Science 335:353–356PubMedCrossRefPubMedCentralGoogle Scholar
  54. 54.
    Winnubst J, Cheyne JE, Niculescu D, Lohmann C (2015) Spontaneous activity drives local synaptic plasticity in vivo. Neuron 87:399–410PubMedCrossRefPubMedCentralGoogle Scholar
  55. 55.
    Wilson DE, Whitney DE, Scholl B, Fitzpatrick D (2016) Orientation selectivity and the functional clustering of synaptic inputs in primary visual cortex. Nat Neurosci 19:1003–1009PubMedPubMedCentralCrossRefGoogle Scholar
  56. 56.
    Jia H, Rochefort NL, Chen X, Konnerth A (2010) Dendritic organization of sensory input to cortical neurons in vivo. Nature 464:1307–1312PubMedCrossRefPubMedCentralGoogle Scholar
  57. 57.
    Chen X, Leischner U, Rochefort NL, Nelken I, Konnerth A (2011) Functional mapping of single spines in cortical neurons in vivo. Nature 475:501–505PubMedCrossRefPubMedCentralGoogle Scholar
  58. 58.
    Koch C, Zador A (1993) The function of dendritic spines: devices subserving biochemical rather than electrical compartmentalization. J Neurosci 13:413–422PubMedPubMedCentralCrossRefGoogle Scholar
  59. 59.
    Yuste R, Denk W (1995) Dendritic spines as basic functional units of neuronal integration. Nature 375:682–684PubMedCrossRefPubMedCentralGoogle Scholar
  60. 60.
    Palmer LM, Stuart GJ (2009) Membrane potential changes in dendritic spines during action potentials and synaptic input. J Neurosci 29:6897–6903PubMedPubMedCentralCrossRefGoogle Scholar
  61. 61.
    Harnett MT, Makara JK, Spruston N, Kath WL, Magee JC (2012) Synaptic amplification by dendritic spines enhances input cooperativity. Nature 491:599–602PubMedPubMedCentralCrossRefGoogle Scholar
  62. 62.
    Shimomura O, Johnson FH, Saiga Y (1962) Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol 59:223–239PubMedCrossRefPubMedCentralGoogle Scholar
  63. 63.
    Ashley CC, Ridgway EB (1968) Simultaneous recording of membrane potential, calcium transient and tension in single muscle fibers. Nature 219:1168–1169PubMedCrossRefPubMedCentralGoogle Scholar
  64. 64.
    Tsien RY (1980) New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures. Biochemistry 19:2396–2404PubMedCrossRefPubMedCentralGoogle Scholar
  65. 65.
    Varga Z, Jia H, Sakmann B, Konnerth A (2011) Dendritic coding of multiple sensory inputs in single cortical neurons in vivo. Proc Natl Acad Sci U S A 108:15420–15425PubMedPubMedCentralCrossRefGoogle Scholar
  66. 66.
    Chen X, Rochefort NL, Sakmann B, Konnerth A (2013) Reactivation of the same synapses during spontaneous up states and sensory stimuli. Cell Rep 4:31–39PubMedCrossRefPubMedCentralGoogle Scholar
  67. 67.
    Palmer LM, Schulz JM, Murphy SC, Ledergerber D, Murayama M, Larkum ME (2012) The cellular basis of GABAB-mediated interhemispheric inhibition. Science 335:989–993PubMedCrossRefPubMedCentralGoogle Scholar
  68. 68.
    Grienberger C, Chen X, Konnerth A (2014) NMDA receptor-dependent multidendrite Ca2+ spikes required for hippocampal burst firing in vivo. Neuron 81:1274–1281PubMedCrossRefPubMedCentralGoogle Scholar
  69. 69.
    Lu R, Sun W, Liang Y, Kerlin A, Bierfeld J, Seelig JD, Wilson DE, Scholl B, Mohar B, Tanimoto M, Koyama M, Fitzpatrick D, Orger MB, Ji N (2017) Video-rate volumetric functional imaging of the brain at synaptic resolution. Nat Neurosci 20:620–628PubMedPubMedCentralCrossRefGoogle Scholar
  70. 70.
    Looger LL, Griesbeck O (2012) Genetically encoded neural activity indicators. Curr Opin Neurobiol 22:18–23PubMedCrossRefGoogle Scholar
  71. 71.
    Mittmann W, Wallace DJ, Czubayko U, Herb JT, Schaefer AT, Looger LL, Denk W, Kerr JN (2011) Two-photon calcium imaging of evoked activity from L5 somatosensory neurons in vivo. Nat Neurosci 14:1089–1093PubMedCrossRefGoogle Scholar
  72. 72.
    Gentet LJ, Kremer Y, Taniguchi H, Huang ZJ, Staiger JF, Petersen CC (2012) Unique functional properties of somatostatin-expressing GABAergic neurons in mouse barrel cortex. Nat Neurosci 15:607–612PubMedCrossRefGoogle Scholar
  73. 73.
    Sheffield ME, Dombeck DA (2014) Calcium transient prevalence across the dendritic arbour predicts place field properties. Nature 517:200–204PubMedPubMedCentralCrossRefGoogle Scholar
  74. 74.
    Sachidhanandam S, Sreenivasan V, Kyriakatos A, Kremer Y, Petersen CC (2013) Membrane potential correlates of sensory perception in mouse barrel cortex. Nat Neurosci 16:1671–1677PubMedCrossRefGoogle Scholar
  75. 75.
    Micallef AH, Takahashi N, Larkum ME, Palmer LM (2017) A reward-based behavioral platform to measure neural activity during head-fixed behavior. Front Cell Neurosci 11:156PubMedPubMedCentralCrossRefGoogle Scholar
  76. 76.
    Dana H, Mohar B, Sun Y, Narayan S, Gordus A, Hasseman JP, Tsegaye G, Holt GT, Hu A, Walpita D, Patel R, Macklin JJ, Bargmann CI, Ahrens MB, Schreiter ER, Jayaraman V, Looger LL, Svoboda K, Kim DS (2016) Sensitive red protein calcium indicators for imaging neural activity. Elife 5:e12727.  https://doi.org/10.7554/eLife.12727 PubMedPubMedCentralCrossRefGoogle Scholar
  77. 77.
    Collot M, Wilms CD, Bentkhayet A, Marcaggi P, Couchman K, Charpak S, Dieudonne S, Hausser M, Feltz A, Mallet JM (2015) CaRuby-Nano: a novel high affinity calcium probe for dual color imaging. Elife 4:05808.  https://doi.org/10.7554/eLife.05808 CrossRefGoogle Scholar
  78. 78.
    Reddy GD, Saggau P (2005) Fast three-dimensional laser scanning scheme using acousto-optic deflectors. J Biomed Opt 10:064038.  https://doi.org/10.1117/1.2141504 PubMedCrossRefPubMedCentralGoogle Scholar
  79. 79.
    Reddy GD, Kelleher K, Fink R, Saggau P (2008) Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity. Nat Neurosci 11:713–720PubMedCentralCrossRefGoogle Scholar
  80. 80.
    Göbel W, Helmchen F (2007) New angles on neuronal dendrites in vivo. J Neurophysiol 98:3770–3779PubMedCrossRefPubMedCentralGoogle Scholar
  81. 81.
    Göbel W, Helmchen F (2007) In vivo calcium imaging of neural network function. Physiology (Bethesda) 22:358–365Google Scholar
  82. 82.
    Katona G, Kaszas A, Turi GF, Hajos N, Tamas G, Vizi ES, Rozsa B (2011) Roller Coaster Scanning reveals spontaneous triggering of dendritic spikes in CA1 interneurons. Proc Natl Acad Sci U S A 108:2148–2153PubMedPubMedCentralCrossRefGoogle Scholar
  83. 83.
    Katona G, Szalay G, Maak P, Kaszas A, Veress M, Hillier D, Chiovini B, Vizi ES, Roska B, Rozsa B (2012) Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes. Nat Methods 9:201–208PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Florey Institute of Neuroscience and Mental Health, University of MelbourneParkvilleAustralia

Personalised recommendations