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Two-Photon Imaging of Neuronal Network Dynamics in Neocortex

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Optical Imaging of Neocortical Dynamics

Part of the book series: Neuromethods ((NM,volume 85))

Abstract

Optical investigation of fast neuronal network dynamics in the intact neocortex—using appropriate activity-dependent indicators—requires single-cell resolution at large imaging depths and sufficient acquisition speed. These requirements are met by two-photon laser scanning microscopy, which has become one of the key methods for functional measurements of neuronal population activity in vivo, primarily in combination with calcium indicators. In this chapter we focus on various advanced two-photon imaging techniques that were recently developed to improve scanning speed, to enable 3D sampling from large numbers of neurons, or to extend imaging towards measurements in freely behaving animals. In general, sampling speed and population size trade off against each other. Currently, about 1,000 neurons can be measured with good signal-to-noise ratio at 1–10 Hz or a few tens of neurons can be optically recorded at 1 kHz. Measurements of local network activity have been used to either characterize spatial distributions of functional properties, such as orientation tuning in visual cortex, or reveal neuronal activation patterns on a fast time scale. We illustrate these new opportunities with examples from in vivo two-photon calcium imaging in mouse visual cortex. The chapter concludes with a discussion of the advantages and limitations of the various techniques and of future perspectives. The direct observation of neuronal network dynamics in living animals no doubt will help to elucidate principles of operation of neocortical microcircuits.

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References

  1. Helmchen F, Denk W (2005) Deep tissue two-photon microscopy. Nat Methods 2:932–940

    Article  CAS  PubMed  Google Scholar 

  2. Garaschuk O, Milos RI, Grienberger C, Marandi N, Adelsberger H, Konnerth A (2006) Optical monitoring of brain function in vivo: from neurons to networks. Pflugers Arch 453:385–396

    Article  CAS  PubMed  Google Scholar 

  3. Grewe BF, Helmchen F (2009) Optical probing of neuronal ensemble activity. Curr Opin Neurobiol 19:520–529

    Article  CAS  PubMed  Google Scholar 

  4. Grienberger C, Konnerth A (2012) Imaging calcium in neurons. Neuron 73:862–885

    Article  CAS  PubMed  Google Scholar 

  5. Tsien RY (1981) A non-disruptive technique for loading calcium buffers and indicators into cells. Nature 290:527–528

    Article  CAS  PubMed  Google Scholar 

  6. Stosiek C, Garaschuk O, Holthoff K, Konnerth A (2003) In vivo two-photon calcium imaging of neuronal networks. Proc Natl Acad Sci USA 100:7319–7324

    Article  CAS  PubMed  Google Scholar 

  7. Garaschuk O, Milos RI, Konnerth A (2006) Targeted bulk-loading of fluorescent indicators for two-photon brain imaging in vivo. Nat Protoc 1:380–386

    Article  CAS  PubMed  Google Scholar 

  8. Kerr JND, Greenberg D, Helmchen F (2005) Imaging input and output of neocortical networks in vivo. Proc Natl Acad Sci USA 102:14063–14068

    Article  CAS  PubMed  Google Scholar 

  9. Sato TR, Gray NW, Mainen ZF, Svoboda K (2007) The functional microarchitecture of the mouse barrel cortex. PLoS Biol 5:e189

    Article  PubMed Central  PubMed  Google Scholar 

  10. Grewe BF, Langer D, Kasper H, Kampa BM, Helmchen F (2010) High-speed in vivo calcium imaging reveals neuronal network activity with near-millisecond precision. Nat Methods 7:399–405

    Article  CAS  PubMed  Google Scholar 

  11. 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 433:597–603

    Article  CAS  PubMed  Google Scholar 

  12. Ohki K, Chung S, Kara P, Hübener M, Bonhoeffer T, Reid RC (2006) Highly ordered arrangement of single neurons in orientation pinwheels. Nature 442:925–928

    Article  CAS  PubMed  Google Scholar 

  13. Greenberg DS, Houweling AR, Kerr JN (2008) Population imaging of ongoing neuronal activity in the visual cortex of awake rats. Nat Neurosci 11:749–751

    Article  CAS  PubMed  Google Scholar 

  14. Mrsic-Flogel TD, Hofer SB, Ohki K, Reid RC, Bonhoeffer T, Hübener M (2007) Homeostatic regulation of eye-specific responses in visual cortex during ocular dominance plasticity. Neuron 54:961–972

    Article  CAS  PubMed  Google Scholar 

  15. Li Y, Van Hooser SD, Mazurek M, White LE, Fitzpatrick D (2008) Experience with moving visual stimuli drives the early development of cortical direction selectivity. Nature 456:952–956

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Kara P, Boyd JD (2009) A micro-architecture for binocular disparity and ocular dominance in visual cortex. Nature 458:627–631

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  17. Rochefort NL, Garaschuk O, Milos RI, Narushima M, Marandi N, Pichler B, Kovalchuk Y, Konnerth A (2009) Sparsification of neuronal activity in the visual cortex at eye-opening. Proc Natl Acad Sci USA 106:15049–15054

    Article  CAS  PubMed  Google Scholar 

  18. Ko H, Hofer SB, Pichler B, Buchanan KA, Sjöström PJ, Mrsic-Flogel TD (2011) Functional specificity of local synaptic connections in neocortical networks. Nature 473:87–91

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  19. 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 9:201–208

    Article  CAS  PubMed  Google Scholar 

  20. Roth MM, Helmchen F, Kampa BM (2012) Distinct functional properties of primary and posteromedial visual area of mouse neocortex. J Neurosci 32(28):9716–9726

    Article  CAS  PubMed  Google Scholar 

  21. Bandyopadhyay S, Shamma SA, Kanold PO (2010) Dichotomy of functional organization in the mouse auditory cortex. Nat Neurosci 13:361–368

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  22. Rothschild G, Nelken I, Mizrahi A (2010) Functional organization and population dynamics in the mouse primary auditory cortex. Nat Neurosci 13:353–360

    Article  CAS  PubMed  Google Scholar 

  23. Bathellier B, Ushakova L, Rumpel S (2012) Discrete neocortical dynamics predict behavioral categorization of sounds. Neuron 76:435–449

    Article  CAS  PubMed  Google Scholar 

  24. Kerr JN, de Kock CP, Greenberg DS, Bruno RM, Sakmann B, Helmchen F (2007) Spatial organization of neuronal population responses in layer 2/3 of rat barrel cortex. J Neurosci 27:13316–13328

    Article  CAS  PubMed  Google Scholar 

  25. Winship IR, Murphy TH (2008) In vivo calcium imaging reveals functional rewiring of single somatosensory neurons after stroke. J Neurosci 28:6592–6606

    Article  CAS  PubMed  Google Scholar 

  26. Dombeck DA, Graziano MS, Tank DW (2009) Functional clustering of neurons in motor cortex determined by cellular resolution imaging in awake behaving mice. J Neurosci 29:13751–13760

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  27. Helmchen F, Imoto K, Sakmann B (1996) Ca2+ buffering and action potential-evoked Ca2+ signaling in dendrites of pyramidal neurons. Biophys J 70:1069–1081

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  28. Yaksi E, Friedrich RW (2006) Reconstruction of firing rate changes across neuronal populations by temporally deconvolved Ca2+ imaging. Nat Methods 3:377–383

    Article  CAS  PubMed  Google Scholar 

  29. Vogelstein JT, Watson BO, Packer AM, Yuste R, Jedynak B, Paninski L (2009) Spike inference from calcium imaging using sequential Monte Carlo methods. Biophys J 97:636–655

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  30. Dombeck DA, Khabbaz AN, Collman F, Adelman TL, Tank DW (2007) Imaging large-scale neural activity with cellular resolution in awake, mobile mice. Neuron 56:43–57

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  31. Huber D, Gutnisky DA, Peron S, O’Connor DH, Wiegert JS, Tian L, Oertner TG, Looger LL, Svoboda K (2012) Multiple dynamic representations in the motor cortex during sensorimotor learning. Nature 484:473–478

    Article  CAS  PubMed  Google Scholar 

  32. Helmchen F, Fee MS, Tank DW, Denk W (2001) A miniature head-mounted two-photon microscope. High-resolution brain imaging in freely moving animals. Neuron 31:903–912

    Article  CAS  PubMed  Google Scholar 

  33. Sawinski J, Wallace DJ, Greenberg DS, Grossmann S, Denk W, Kerr JN (2009) Visually evoked activity in cortical cells imaged in freely moving animals. Proc Natl Acad Sci USA 106:19557–19562

    Article  CAS  PubMed  Google Scholar 

  34. Bonin V, Histed MH, Yurgenson S, Reid RC (2011) Local diversity and fine-scale organization of receptive fields in mouse visual cortex. J Neurosci 31:18506–18521

    Article  CAS  PubMed  Google Scholar 

  35. Keller GB, Bonhoeffer T, Hübener M (2012) Sensorimotor mismatch signals in primary visual cortex of the behaving mouse. Neuron 74:809–815

    Article  CAS  PubMed  Google Scholar 

  36. Andermann ML, Kerlin AM, Reid RC (2010) Chronic cellular imaging of mouse visual cortex during operant behavior and passive viewing. Front Cell Neurosci 4:3

    PubMed Central  PubMed  Google Scholar 

  37. Göbel W, Helmchen F (2007) New angles on neuronal dendrites in vivo. J Neurophysiol 98:3770–3779

    Article  PubMed  Google Scholar 

  38. Lillis KP, Eng A, White JA, Mertz J (2008) Two-photon imaging of spatially extended neuronal network dynamics with high temporal resolution. J Neurosci Methods 172:178–184

    Article  PubMed Central  PubMed  Google Scholar 

  39. Wilson NR, Runyan CA, Wang FL, Sur M (2012) Division and subtraction by distinct cortical inhibitory networks in vivo. Nature 488:343–348

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  40. Göbel W, Kampa BM, Helmchen F (2007) Imaging cellular network dynamics in three dimensions using fast 3D laser scanning. Nat Methods 4:73–79

    Article  PubMed  Google Scholar 

  41. 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 USA 108:2148–2153

    Article  PubMed  Google Scholar 

  42. Grewe BF, Voigt FF, van’t Hoff M, Helmchen F (2011) Fast two-layer two-photon imaging of neuronal cell populations using an electrically tunable lens. Biomed Opt Express 2:2035–2046

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  43. Kirkby PA, Srinivas Nadella KM, Silver RA (2010) A compact Acousto-Optic Lens for 2D and 3D femtosecond based 2-photon microscopy. Opt Express 18:13721–13745

    Article  PubMed Central  PubMed  Google Scholar 

  44. Reddy GD, Kelleher K, Fink R, Saggau P (2008) Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity. Nat Neurosci 11:713–720

    Article  PubMed Central  Google Scholar 

  45. Zeng S, Lv X, Zhan C, Chen WR, Xiong W, Jacques SL, Luo Q (2006) Simultaneous compensation for spatial and temporal dispersion of acousto-optical deflectors for two-dimensional scanning with a single prism. Opt Lett 31:1091–1093

    Article  PubMed  Google Scholar 

  46. Kampa BM, Göbel W, Helmchen F (2011) Measuring neuronal population activity using 3D laser scanning. Cold Spring Harb Protoc 2011:1340–1349

    Article  PubMed  Google Scholar 

  47. Kampa BM, Roth MM, Göbel W, Helmchen F (2011) Representation of visual scenes by local neuronal populations in layer 2/3 of mouse visual cortex. Front Neural Circuits 5:18

    Article  PubMed  Google Scholar 

  48. Botcherby E, Smith C, Booth M, Juskaitis R, Wilson T (2010) Arbitrary-scan imaging for two-photon microscopy. Proc SPIE 7569:756917–756918

    Article  Google Scholar 

  49. Botcherby EJ, Smith CW, Kohl MM, Debarre D, Booth MJ, Juskaitis R, Paulsen O, Wilson T (2012) Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates. Proc Natl Acad Sci USA 109:2919–2924

    Article  CAS  PubMed  Google Scholar 

  50. Ranganathan GN, Koester HJ (2010) Optical recording of neuronal spiking activity from unbiased populations of neurons with high spike detection efficiency and high temporal precision. J Neurophysiol 104:1812–1824

    Article  PubMed  Google Scholar 

  51. Kurtz R, Fricke M, Kalb J, Tinnefeld P, Sauer M (2006) Application of multiline two-photon microscopy to functional in vivo imaging. J Neurosci Methods 151:276–286

    Article  PubMed  Google Scholar 

  52. Niesner R, Andresen V, Neumann J, Spiecker H, Gunzer M (2007) The power of single and multibeam two-photon microscopy for high-resolution and high-speed deep tissue and intravital imaging. Biophys J 93:2519–2529

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  53. Nikolenko V, Watson BO, Araya R, Woodruff A, Peterka DS, Yuste R (2008) SLM microscopy: scanless two-photon imaging and photostimulation with spatial light modulators. Front Neural Circuits 2:5

    Article  PubMed Central  PubMed  Google Scholar 

  54. Cheng A, Gonçalves J, Golshani P, Arisaka K, Portera-Cailliau C (2011) Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing. Nat Methods 8:139–142

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  55. Harvey CD, Coen P, Tank DW (2012) Choice-specific sequences in parietal cortex during a virtual-navigation decision task. Nature 484:62–68

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  56. Helmchen F (2002) Miniaturization of fluorescence microscopes using fibre optics. Exp Physiol 87:737–745

    Article  PubMed  Google Scholar 

  57. Flusberg BA, Cocker ED, Piyawattanametha W, Jung JC, Cheung ELM, Schnitzer MJ (2005) Fiber-optic fluorescence imaging. Nat Methods 2:941–950

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  58. Lee CM, Engelbrecht CJ, Soper TD, Helmchen F, Seibel EJ (2010) Scanning fiber endoscopy with highly flexible, 1 mm catheterscopes for wide-field, full-color imaging. J Biophotonics 3:385–407

    Article  PubMed Central  PubMed  Google Scholar 

  59. Engelbrecht CJ, Johnston RS, Seibel EJ, Helmchen F (2008) Ultra-compact fiber-optic two-photon microscope for functional fluorescence imaging in vivo. Opt Express 16:5556–5564

    Article  CAS  PubMed  Google Scholar 

  60. Piyawattanametha W, Cocker ED, Burns LD, Barretto RP, Jung JC, Ra H, Solgaard O, Schnitzer MJ (2009) In vivo brain imaging using a portable 2.9 g two-photon microscope based on a microelectromechanical systems scanning mirror. Opt Lett 34:2309–2311

    Article  PubMed Central  PubMed  Google Scholar 

  61. Flusberg BA, Jung JC, Cocker ED, Anderson EP, Schnitzer MJ (2005) In vivo brain imaging using a portable 3.9 gram two-photon fluorescence microendoscope. Opt Lett 30:2272–2274

    Article  PubMed  Google Scholar 

  62. Flusberg BA, Nimmerjahn A, Cocker ED, Mukamel EA, Barretto RP, Ko TH, Burns LD, Jung JC, Schnitzer MJ (2008) High-speed, miniaturized fluorescence microscopy in freely moving mice. Nat Methods 5:935–938

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  63. Ghosh KK, Burns LD, Cocker ED, Nimmerjahn A, Ziv Y, Gamal AE, Schnitzer MJ (2011) Miniaturized integration of a fluorescence microscope. Nat Methods 8:871–878

    Article  CAS  PubMed  Google Scholar 

  64. Nimmerjahn A, Kirchhoff F, Kerr JN, Helmchen F (2004) Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo. Nat Methods 1:31–37

    Article  CAS  PubMed  Google Scholar 

  65. Buzsaki G (2004) Large-scale recording of neuronal ensembles. Nat Neurosci 7:446–451

    Article  CAS  PubMed  Google Scholar 

  66. Kerlin AM, Andermann ML, Berezovskii VK, Reid RC (2010) Broadly tuned response properties of diverse inhibitory neuron subtypes in mouse visual cortex. Neuron 67:858–871

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  67. Langer D, Helmchen F (2012) Post hoc immunostaining of GABAergic neuronal subtypes following in vivo two-photon calcium imaging in mouse neocortex. Pflugers Arch 463:339–354

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  68. Runyan CA, Schummers J, Van Wart A, Kuhlman SJ, Wilson NR, Huang ZJ, Sur M (2010) Response features of parvalbumin-expressing interneurons suggest precise roles for subtypes of inhibition in visual cortex. Neuron 67(5):847–57

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  69. Bock DD, Lee WC, Kerlin AM, Andermann ML, Hood G, Wetzel AW, Yurgenson S, Soucy ER, Kim HS, Reid RC (2011) Network anatomy and in vivo physiology of visual cortical neurons. Nature 471:177–182

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  70. Lütcke H, Helmchen F (2011) Two-photon imaging and analysis of neural network dynamics. Rep Prog Phys 74(8):086602

    Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Biological Sciences, Department of Applied Physics, Stanford University, James H. Clark Center for Biomedical Engineering and Sciences, Stanford, CA, USA

    Benjamin F. Grewe

  2. Brain Research Institute, University of Zurich, Zurich, Switzerland

    Fritjof Helmchen & Björn M. Kampa

Authors
  1. Benjamin F. Grewe
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  2. Fritjof Helmchen
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  3. Björn M. Kampa
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Editors and Affiliations

  1. University of Zurich, Switzerland

    Bruno Weber

  2. University of Zurich, Switzerland

    Fritjof Helmchen

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Grewe, B.F., Helmchen, F., Kampa, B.M. (2014). Two-Photon Imaging of Neuronal Network Dynamics in Neocortex. In: Weber, B., Helmchen, F. (eds) Optical Imaging of Neocortical Dynamics. Neuromethods, vol 85. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-785-3_9

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  • DOI: https://doi.org/10.1007/978-1-62703-785-3_9

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-784-6

  • Online ISBN: 978-1-62703-785-3

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