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Flash-and-Freeze Electron Microscopy: Coupling Optogenetics with High-Pressure Freezing

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Nanoscale Imaging of Synapses

Part of the book series: Neuromethods ((NM,volume 84))

Abstract

A complete understanding of neuronal functions requires observation of the synapse with high spatial and temporal resolution. Recently, we developed a method to combine optogenetics with electron microscopy to capture dynamic changes in synaptic morphology during neurotransmission. First we generated transgenic C. elegans animals expressing channelrhodopsin in specific neurons. We stimulated these neurons in intact animals using a home-built light stimulation device and modified specimen holders for the Leica EM Pact2 high-pressure freezer. Samples were subsequently frozen 20 ms after the light stimulus. We demonstrated that synaptic vesicle fusion intermediates could be captured using this technique. This method can be readily applied to other light-activatable molecules, such as caged compounds, light-switchable ligands, and photoactivatable proteins, to study dynamic changes in cells.

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References

  1. Eccles SJC (1964) The physiology of synapses. Springer, Berlin

    Book  Google Scholar 

  2. Heuser JE, Reese TS, Dennis MJ, Jan Y, Jan L, Evans L (1979) Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release. J Cell Biol 81(2):275–300

    Article  CAS  PubMed  Google Scholar 

  3. Smith JE, Reese TS (1980) Use of aldehyde fixatives to determine the rate of synaptic transmitter release. J Exp Biol 89:19–29

    CAS  PubMed  Google Scholar 

  4. Rosenmund C, Stevens CF (1997) The rate of aldehyde fixation of the exocytotic machinery in cultured hippocampal synapses. J Neurosci Methods 76(1):1–5

    Article  PubMed  Google Scholar 

  5. Heuser JE, Reese TS (1981) Structural changes after transmitter release at the frog neuromuscular junction. J Cell Biol 88(3):564–580

    Article  CAS  PubMed  Google Scholar 

  6. Torri-Tarelli F, Grohovaz F, Fesce R, Ceccarelli B (1985) Temporal coincidence between synaptic vesicle fusion and quantal secretion of acetylcholine. J Cell Biol 101(4):1386–1399

    Article  CAS  PubMed  Google Scholar 

  7. Kachar B, Christakis NA, Reese TS, Lane NJ (1986) The intramembrane structure of septate junctions based on direct freezing. J Cell Sci 80:13–28

    CAS  PubMed  Google Scholar 

  8. Arsenault AL, Ottensmeyer FP, Heath IB (1988) An electron microscopic and spectroscopic study of murine epiphyseal cartilage: analysis of fine structure and matrix vesicles preserved by slam freezing and freeze substitution. J Ultrastruct Mol Struct Res 98(1):32–47

    Article  CAS  PubMed  Google Scholar 

  9. Neill PJ, Smith JH, Doughty BL, Kemp M (1988) The ultrastructure of the Schistosoma mansoni egg. Am J Trop Med Hyg 39(1):52–65

    CAS  PubMed  Google Scholar 

  10. Moor H (1987) Theory and practice of high pressure freezing. Cryotechniques in Biological Electron Microscopy. Springer, Berlin, pp 175–191

    Book  Google Scholar 

  11. Moor H, Riehle U (1968) Snap-freezing under high pressure: a new fixation technique for freeze-etching. Proc Fourth Eur Reg Conf Electron Microsc 2:33–34

    Google Scholar 

  12. Dubochet J (2007) The physics of rapid cooling and its implications for cryoimmobilization of cells. Cell Electron Microsc 79:7–21

    Article  CAS  Google Scholar 

  13. Rasmussen DH, Macaulay MN, MacKenzie AP (1975) Supercooling and nucleation of ice in single cells. Cryobiology 12(4):328–339

    Article  CAS  PubMed  Google Scholar 

  14. Yizhar O, Fenno LE, Davidson TJ, Mogri M, Deisseroth K (2011) Optogenetics in neural systems. Neuron 71(1):9–34

    Article  CAS  PubMed  Google Scholar 

  15. Sineshchekov OA, Jung K-H, Spudich JL (2002) Two rhodopsins mediate phototaxis to low- and high-intensity light in Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A 99(13):8689–8694

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Nagel G, Ollig D, Fuhrmann M, Kateriya S, Musti AM, Bamberg E, Hegemann P (2002) Channelrhodopsin-1: a light-gated proton channel in green algae. Science 296(5577):2395–2398

    Article  CAS  PubMed  Google Scholar 

  17. Nagel G, Szellas T, Huhn W, Kateriya S, Adeishvili N, Berthold P, Ollig D, Hegemann P, Bamberg E (2003) Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc Natl Acad Sci U S A 100(24):13940–13945

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  18. Govorunova EG, Spudich EN, Lane CE, Sineshchekov OA, Spudich JL (2011) New channelrhodopsin with a red-shifted spectrum and rapid kinetics from Mesostigma viride. MBio 2(3):e00115–00111

    Article  PubMed Central  PubMed  Google Scholar 

  19. Gunaydin LA, Yizhar O, Berndt A, Sohal VS, Deisseroth K, Hegemann P (2010) Ultrafast optogenetic control. Nat Neurosci 13(3):387–392

    Article  CAS  PubMed  Google Scholar 

  20. Lin JY (2012) Optogenetic excitation of neurons with channelrhodopsins: light instrumentation, expression systems, and channelrhodopsin variants. Prog Brain Res 196:29–47

    Article  CAS  PubMed  Google Scholar 

  21. Lin JY, Lin MZ, Steinbach P, Tsien RY (2009) Characterization of engineered channelrhodopsin variants with improved properties and kinetics. Biophys J 96(5):1803–1814

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  22. Boyden ES, Zhang F, Bamberg E, Nagel G, Deisseroth K (2005) Millisecond-timescale, genetically targeted optical control of neural activity. Nat Neurosci 8(9):1263–1268

    Article  CAS  PubMed  Google Scholar 

  23. Douglass AD, Kraves S, Deisseroth K, Schier AF, Engert F (2008) Escape behavior elicited by single, channelrhodopsin-2-evoked spikes in zebrafish somatosensory neurons. Curr Biol 18(15):1133–1137

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  24. Leifer AM, Fang-Yen C, Gershow M, Alkema MJ, Samuel ADT (2011) Optogenetic manipulation of neural activity in freely moving Caenorhabditis elegans. Nat Methods 8(2):147–152

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  25. Liu Q, Hollopeter G, Jorgensen EM (2009) Graded synaptic transmission at the Caenorhabditis elegans neuromuscular junction. Proc Natl Acad Sci 106(26):10823–10828

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  26. Liewald JF, Brauner M, Stephens GJ, Bouhours M, Schultheis C, Zhen M, Gottschalk A (2008) Optogenetic analysis of synaptic function. Nat Methods 5(10):895–902

    Article  CAS  PubMed  Google Scholar 

  27. Nagel G, Brauner M, Liewald JF, Adeishvili N, Bamberg E, Gottschalk A (2005) Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses. Curr Biol 15(24):2279–2284

    Article  CAS  PubMed  Google Scholar 

  28. Watanabe S, Rost BR, Camacho-Perez M, Davis MW, Söhl-Kielczynski B, Rosenmund C, Jorgesen EM. Ultrafast endocytosis at mouse hippocampal synapses. Nature (advance online publication)

    Google Scholar 

  29. Watanabe S, Liu Q, Davis MW, Hollopeter G., Thomas N, Jorgensen NB, Jorgensen EM (2013) Ultrafast endocytosis at Caenorhabditis elegans neuromuscular junctions. Elife 2, e00723

    Google Scholar 

  30. Milburn T, Matsubara N, Billington AP, Udgaonkar JB, Walker JW, Carpenter BK, Webb WW, Marque J, Denk W, McCray JA (1989) Synthesis, photochemistry, and biological activity of a caged photolabile acetylcholine receptor ligand. Biochemistry 28(1):49–55

    Article  CAS  PubMed  Google Scholar 

  31. Walker JW, McCray JA, Hess GP (1986) Photolabile protecting groups for an acetylcholine receptor ligand. Synthesis and photochemistry of a new class of o-nitrobenzyl derivatives and their effects on receptor function. Biochemistry 25(7):1799–1805

    Article  CAS  PubMed  Google Scholar 

  32. Wieboldt R, Gee KR, Niu L, Ramesh D, Carpenter BK, Hess GP (1994) Photolabile precursors of glutamate: synthesis, photochemical properties, and activation of glutamate receptors on a microsecond time scale. Proc Natl Acad Sci U S A 91(19):8752–8756

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  33. Banghart M, Borges K, Isacoff E, Trauner D, Kramer RH (2004) Light-activated ion channels for remote control of neuronal firing. Nat Neurosci 7(12):1381–1386

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  34. Bartels E, Wassermann NH, Erlanger BF (1971) Photochromic activators of the acetylcholine receptor. Proc Natl Acad Sci U S A 68(8):1820–1823

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  35. Adams SR, Tsien RY (1993) Controlling cell chemistry with caged compounds. Annu Rev Physiol 55(1):755–784

    Article  CAS  PubMed  Google Scholar 

  36. Kennedy MJ, Hughes RM, Peteya LA, Schwartz JW, Ehlers MD, Tucker CL (2010) Rapid blue-light-mediated induction of protein interactions in living cells. Nat Methods 7(12):973–975

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  37. Levskaya A, Weiner OD, Lim WA, Voigt CA (2009) Spatiotemporal control of cell signalling using a light-switchable protein interaction. Nature 461(7266):997–1001

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  38. Yazawa M, Sadaghiani AM, Hsueh B, Dolmetsch RE (2009) Induction of protein-protein interactions in live cells using light. Nat Biotechnol 27(10):941–945

    Article  CAS  PubMed  Google Scholar 

  39. McDonald KL, Morphew M, Verkade P, Müller-Reichert T (2007) Recent advances in high-pressure freezing: equipment- and specimen-loading methods. Methods Mol Biol 369:143–173

    Article  CAS  PubMed  Google Scholar 

  40. Müller-Reichert T, Mäntler J, Srayko M, O’Toole E (2008) Electron microscopy of the early Caenorhabditis elegans embryo. J Microsc 230(2):297–307

    Article  PubMed  Google Scholar 

  41. Verkade P (2008) Moving EM: the Rapid Transfer System as a new tool for correlative light and electron microscopy and high throughput for high-pressure freezing. J Microsc 230(Pt 2):317–328

    Article  CAS  PubMed  Google Scholar 

  42. Miller TM, Heuser JE (1984) Endocytosis of synaptic vesicle membrane at the frog neuromuscular junction. J Cell Biol 98(2):685–698

    Article  CAS  PubMed  Google Scholar 

  43. McDonald K, Schwarz H, Müller-Reichert T, Webb R, Buser C, Morphew M (2010) “Tips and tricks” for high-pressure freezing of model systems. Methods Cell Biol 96:671–693

    Article  PubMed  Google Scholar 

  44. Mueller-Reichert T (2010) Electron microscopy of model systems, vol 96, 1st edn. Academic, New York

    Google Scholar 

  45. McDonald K (2007) Cryopreparation methods for electron microscopy of selected model systems. Methods Cell Biol 79:23–56

    Article  CAS  PubMed  Google Scholar 

  46. McDonald KL, Webb RI (2011) Freeze substitution in 3 hours or less. J Microsc 243(3):227–233

    Article  CAS  PubMed  Google Scholar 

  47. Rostaing P, Weimer RM, Jorgensen EM, Triller A, Bessereau J-L (2004) Preservation of immunoreactivity and fine structure of adult C. elegans tissues using high-pressure freezing. J Histochem Cytochem 52(1):1–12

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We would like to thank the Grass Foundation and the Marine Biological Laboratory at Woods Hole for providing us space and equipment required for performing the freezing experiments. Qiang Liu performed freezing experiments using the optical fiber. We would like to thank Roger Y. Tsien for providing us a construct for ChIEF and Eddie Hujber for critical reading of the manuscript. The research was supported by the US National Institutes of Health (NS034307) and the Marine Biological Laboratory (The Dart Scholars Program and the Grass Fellowship). EMJ is an investigator of the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Department of Biology and Howard Hughes Medical Institute, University of Utah, Salt Lake City, UT, USA

    Shigeki Watanabe, M. Wayne Davis & Erik M. Jorgensen

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  1. Shigeki Watanabe
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  2. M. Wayne Davis
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  3. Erik M. Jorgensen
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Editors and Affiliations

  1. Interdisciplinary Institute for Neuroscience, University of Bordeaux, Bordeaux, France

    U. Valentin Nägerl

  2. Institute of Biology at Ecole Normale Supérieure, Ecole Normale Supérieure, Paris, France

    Antoine Triller

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Watanabe, S., Davis, M.W., Jorgensen, E.M. (2014). Flash-and-Freeze Electron Microscopy: Coupling Optogenetics with High-Pressure Freezing. In: Nägerl, U., Triller, A. (eds) Nanoscale Imaging of Synapses. Neuromethods, vol 84. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4614-9179-8_3

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  • DOI: https://doi.org/10.1007/978-1-4614-9179-8_3

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4614-9178-1

  • Online ISBN: 978-1-4614-9179-8

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