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Capture of activity-induced ultrastructural changes at synapses by high-pressure freezing of brain tissue

Abstract

Electron microscopy (EM) allows for the simultaneous visualization of all tissue components at high resolution. However, the extent to which conventional aldehyde fixation and ethanol dehydration of the tissue alter the fine structure of cells and organelles, thereby preventing detection of subtle structural changes induced by an experiment, has remained an issue. Attempts have been made to rapidly freeze tissue to preserve native ultrastructure. Shock-freezing of living tissue under high pressure (high-pressure freezing, HPF) followed by cryosubstitution of the tissue water avoids aldehyde fixation and dehydration in ethanol; the tissue water is immobilized in 50 ms, and a close-to-native fine structure of cells, organelles and molecules is preserved. Here we describe a protocol for HPF that is useful to monitor ultrastructural changes associated with functional changes at synapses in the brain but can be applied to many other tissues as well. The procedure requires a high-pressure freezer and takes a minimum of 7 d but can be paused at several points.

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Figure 1: Equipment for HPF.
Figure 2: Punching and placement of slice culture onto specimen carrier.
Figure 3: Hippocampal CA3 region in a slice culture after HPF.
Figure 4: Fine structure of microbiopsy material and of acute slice preparations following HPF.
Figure 5: Post-embedding immunogold labeling of thin sections from slice cultures for p-cofilin.
Figure 6: Application of HPF to the study of structural synaptic plasticity.

References

  1. Bliss, T.V.P. & Lomo, T. Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path. J. Physiol. 232, 331–356 (1973).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  2. Nicoll, R.A., Kauer, J.A. & Malenka, R.C. The current excitement in long-term potentiation. Neuron 1, 97–103 (1988).

    CAS  PubMed  Article  Google Scholar 

  3. Bliss, T.V.P. & Collingridge, G.L. A synaptic model of memory: long-term potentiation in the hippocampus. Nature 361, 31–39 (1993).

    CAS  PubMed  Article  Google Scholar 

  4. Whitlock, J.R., Heynen, A.J., Shuler, M.G. & Bear, M.F. Learning induces long-term potentiation in the hippocampus. Science 313, 1093–1097 (2006).

    CAS  PubMed  Article  Google Scholar 

  5. Bliss, T., Collingridge, G. & Morris, R. Synaptic plasticity in the hippocampus. in The Hippocampus Book (eds. Andersen, P., Morris, R., Amaral, D., Bliss, T. & O'Keefe, J.) 343–474 (Oxford University Press, 2007).

  6. Yang, Y., Wang, X.B., Frerking, M. & Zhou, Q. Spine expansion and stabilization associated with long-term potentiation. J. Neurosci. 28, 5740–5751 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  7. Hosokawa, T., Rusakov, D.A., Bliss, T.V.P. & Fine, A. Repeated confocal imaging of individual dendritic spines in the living hippocampal slice: evidence for changes in length and orientation associated with chemically induced LTP. J. Neurosci. 15, 5560–5573 (1995).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  8. Engert, F. & Bonhoeffer, T. Dendritic spine changes associated with hippocampal long-term synaptic plasticity. Nature 399, 66–70 (1999).

    CAS  PubMed  Article  Google Scholar 

  9. Maletic-Savatic, M., Malinow, R. & Svoboda, K. Rapid dendritic morphogenesis in CA1 hippocampal dendrites induced by synaptic activity. Science 283, 1923–1927 (1999).

    CAS  PubMed  Article  Google Scholar 

  10. Matus, A. Actin-based plasticity in dendritic spines. Science 290, 754–758 (2000).

    CAS  PubMed  Article  Google Scholar 

  11. Yuste, R. & Bonhoeffer, T. Morphological changes in dendritic spines associated with long-term synaptic plasticity. Annu. Rev. Neurosci. 24, 1071–1089 (2001).

    CAS  PubMed  Article  Google Scholar 

  12. Matsuzaki, M., Honkura, N., Ellis-Davies, G.C. & Kasai, H. Structural basis of long-term potentiation in single dendritic spines. Nature 429, 761–766 (2004).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. Segal, M. Dendritic spines and long-term plasticity. Nat. Rev. Neurosci. 6, 277–284 (2005).

    CAS  PubMed  Article  Google Scholar 

  14. Hotulainen, P. & Hoogenraad, C.C. Actin in dendritic spines: connecting dynamics to function. J. Cell Biol. 189, 619–629 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  15. Buchs, P.A. & Muller, D. Induction of long-term potentiation is associated with major ultrastructural changes of activated synapses. Proc. Natl. Acad. Sci. USA 93, 8040–8045 (1996).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  16. Toni, N., Buchs, P.A., Nikonenko, I., Bron, C.R. & Muller, D. LTP promotes formation of multiple spine synapses between a single axon terminal and a dendrite. Nature 402, 421–425 (1999).

    CAS  PubMed  Article  Google Scholar 

  17. Geinisman, Y. Structural synaptic modifications associated with hippocampal LTP and behavioral learning. Cereb. Cortex 10, 952–962 (2000).

    CAS  PubMed  Article  Google Scholar 

  18. Harris, K.M., Fiala, J.C. & Ostroff, L. Structural changes at dendritic spine synapses during long-term potentiation. Philos. Trans. Roy. Soc. B. 358, 745–748 (2003).

    Article  Google Scholar 

  19. Heuser, J.E. et al. Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release. J. Cell Biol. 81, 275–300 (1979).

    CAS  PubMed  Article  Google Scholar 

  20. Adrian, M., Dubochet, J., Lepault, J. & McDowall, A.W. Cryo-electron microscopy of viruses. Nature 308, 32–36 (1984).

    CAS  PubMed  Article  Google Scholar 

  21. Moor, H., Bellin, G., Sandri, C. & Akert, K. The influence of high pressure freezing on mammalian nerve tissue. Cell Tissue Res. 209, 201–216 (1980).

    CAS  PubMed  Article  Google Scholar 

  22. Sartori, N., Richter, K. & Dubochet, J. Vitrification depth can be increased more than 10-fold by high-pressure freezing. J. Microsc. 172, 55–61 (1993).

    CAS  Article  Google Scholar 

  23. Studer, D., Michel, M., Wohlwend, M., Hunziker, E.B. & Buschmann, M.D. Vitrification of articular cartilage by high-pressure freezing. J. Microsc. 179, 321–332 (1995).

    CAS  PubMed  Article  Google Scholar 

  24. Zhao, S. et al. Structural plasticity of hippocampal mossy fiber synapses as revealed by high-pressure freezing. J. Comp. Neurol. 520, 2340–2351 (2012).

    PubMed  Article  Google Scholar 

  25. Studer, D., Humbel, B.M. & Chiquet, M. Electron microscopy of high pressure frozen samples: bridging the gap between cellular ultrastructure and atomic resolution. Histochem. Cell Biol. 130, 877–889 (2008).

    CAS  PubMed  Article  Google Scholar 

  26. Zuber, B., Nikonenko, I., Klauser, P., Muller, D. & Dubochet, J. The mammalian central nervous synaptic cleft contains a high density of periodically organized complexes. Proc. Natl. Acad. Sci. USA 102, 19192–19197 (2005).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  27. Lucic, V., Rigort, A. & Baumeister, W. Cryo-electron tomography: The challenge of doing structural biology in situ. J. Cell Biol. 202, 407–419 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  28. Studer, D., Michel, M. & Muller, M. High pressure freezing comes of age. Scanning Microsc. Suppl. 3, 253–268 (1989).

    CAS  PubMed  Google Scholar 

  29. Studer, D., Graber, W., Al-Amoudi, A. & Eggli, P. A new approach for cryofixation by high-pressure freezing. J. Microsc. 203, 285–294 (2001).

    CAS  PubMed  Article  Google Scholar 

  30. Hohenberg, H., Mannweiler, K. & Muller, M. High-pressure freezing of cell suspensions in cellulose capillary tubes. J. Microsc. 175, 34–43 (1994).

    CAS  PubMed  Article  Google Scholar 

  31. Sumakovic, M. et al. UNC-108/RAB-2 and its effector RIC-19 are involved in dense core vesicle maturation in Caenorhabditis elegans. J. Cell Biol. 186, 897–914 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. Rostaing, P. et al. Analysis of synaptic ultrastructure without fixative using high-pressure freezing and tomography. Eur. J. Neurosci. 24, 3463–3474 (2006).

    PubMed  Article  Google Scholar 

  33. Siksou, L. et al. Three-dimensional architecture of presynaptic terminal cytomatrix. J. Neurosci. 27, 6868–6877 (2007).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  34. Siksou, L. et al. A common molecular basis for membrane docking and functional priming of synaptic vesicles. Eur. J. Neurosci. 30, 49–56 (2009).

    PubMed  Article  Google Scholar 

  35. Vanhecke, D. et al. A rapid microbiopsy system to improve the preservation of biological samples prior to high-pressure freezing. J. Microsc. 212, 3–12 (2003).

    CAS  PubMed  Article  Google Scholar 

  36. Frotscher, M., Misgeld, U. & Nitsch, C. Ultrastructure of mossy fiber endings in in vitro hippocampal slices. Exp. Brain Res. 41, 247–255 (1981).

    CAS  PubMed  Google Scholar 

  37. Geiger, J.R.P. et al. Patch-clamp recording in brain slices with improved slicer technology. Pflügers Arch. 443, 491–501 (2002).

    CAS  PubMed  Article  Google Scholar 

  38. Gähwiler, B.H., Capogna, M., Debanne, D., McKinney, R.A. & Thompson, S.M. Organotypic slice cultures: a technique has come of age. Trends Neurosci. 20, 471–477 (1997).

    PubMed  Article  Google Scholar 

  39. Stoppini, L., Buchs, P.A. & Muller, D. A simple method for organotypic cultures of nervous tissue. J. Neurosci. Meth. 37, 173–182 (1991).

    CAS  Article  Google Scholar 

  40. Frotscher, M. & Gähwiler, B.H. Synaptic organization of intracellularly stained CA3 pyramidal neurons in slice cultures of rat hippocampus. Neuroscience 24, 541–551 (1988).

    CAS  PubMed  Article  Google Scholar 

  41. Bonhoeffer, T., Staiger, V. & Aertsen, A. Synaptic plasticity in rat hippocampal slice cultures: local 'Hebbian' conjunction of pre- and postsynaptic stimulation leads to distributed synaptic enhancement. Proc. Natl. Acad. Sci. USA 86, 8113–8117 (1989).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  42. Collin, C., Miyaguchi, K. & Segal, M. Dendritic spine density and LTP induction in cultured hippocampal slices. J. Neurophysiol. 77, 1614–1623 (1997).

    CAS  PubMed  Article  Google Scholar 

  43. Leutgeb, J.K., Frey, J.U. & Behnisch, T. LTP in cultured hippocampal-entorhinal cortex slices from young adult (P25-30) rats. J. Neurosci. Methods 130, 19–32 (2003).

    PubMed  Article  Google Scholar 

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

    CAS  PubMed  Article  Google Scholar 

  45. Deisseroth, K. et al. Next-generation optical technologies for illuminating genetically targeted brain circuits. J. Neurosci. 26, 10380–10386 (2006).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  46. Zhang, F. et al. Multimodal fast optical interrogation of neural circuitry. Nature 446, 633–639 (2007).

    CAS  PubMed  Article  Google Scholar 

  47. del Rio, J.A. & Soriano, E. Regenerating cortical connections in a dish: the entorhino-hippocampal organotypic slice co-culture as tool for pharmacological screening of molecules promoting axon regeneration. Nat. Protoc. 5, 217–226 (2010).

    CAS  PubMed  Article  Google Scholar 

  48. Adams, S.R. & Tsien, R.Y. Controlling cell chemistry with caged compounds. Annu. Rev. Physiol. 55, 755–784 (1993).

    CAS  PubMed  Article  Google Scholar 

  49. Bischofberger, J., Engel, D., Li, L., Geiger, J.R.P. & Jonas, P. Patch-clamp recording from mossy fiber terminals in hippocampal slices. Nat. Protoc. 1, 2075–2081 (2006).

    CAS  PubMed  Article  Google Scholar 

  50. Augustin, I., Rosenmund, C., Sudhof, T.C. & Brose, N. Munc13-1 is essential for fusion competence of glutamatergic synaptic vesicles. Nature 400, 457–461 (1999).

    CAS  PubMed  Article  Google Scholar 

  51. Reynolds, E.S. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208–212 (1963).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  52. Blackstad, T.W. & Kjaerheim, A. Special axodendritic synapses in the hippocampal cortex: electron and light microscopic studies on the layer of mossy fibers. J. Comp. Neurol. 117, 113–159 (1961).

    Article  Google Scholar 

  53. Hamlyn, L.H. The fine structure of the mossy fibre endings in the hippocampus of the rabbit. J. Anat. 96, 112–120 (1962).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Bamburg, J.R. Proteins of the ADF/cofilin family: essential regulators of actin dynamics. Annu. Rev. Cell Biol. 15, 185–230 (1999).

    CAS  Article  Google Scholar 

  55. Arber, S. et al. Regulation of actin dynamics through phosphorylation of cofilin by LIM-kinase. Nature 393, 805–809 (1998).

    CAS  PubMed  Article  Google Scholar 

  56. Yang, N. et al. Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin reorganization. Nature 393, 809–812 (1998).

    CAS  PubMed  Article  Google Scholar 

  57. Chen, L.Y., Rex, C.S., Casale, M.S., Gall, C.M. & Lynch, G. Changes in synaptic morphology accompany actin signaling during LTP. J. Neurosci. 27, 5363–5372 (2007).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  58. Rex, C.S. et al. Different Rho GTPase-dependent signaling pathways initiate sequential steps in the consolidation of long-term potentiation. J. Cell Biol. 186, 85–97 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  59. Racz, B. & Weinberg, R.J. Spatial organization of cofilin in dendritic spines. Neuroscience 138, 447–456 (2006).

    CAS  PubMed  Article  Google Scholar 

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Acknowledgements

We thank K. Miller for her help with Supplementary Video 1 and N. Brose (Max Planck Institute for Experimental Medicine) for providing the Munc13-1 mutants. This study was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 780 and FR 620/12-1 to M.F.) and the Swiss National Foundation (grant no. 3100AO_118394 to D.S.). M.F. is Senior Research Professor for Neuroscience of the Hertie Foundation.

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Authors

Contributions

D.S., S.Z., W.G., P.J., S.N. and M.F. carried out the various experiments together that formed the basis of the present protocol. X.C. did the genotyping (Munc13-1 mutants and wild-type littermates), and S.N. performed the immunogold labeling experiments. S.Z. produced Supplementary Video 1. D.S., S.Z. and M.F. developed the protocol and wrote the paper.

Corresponding author

Correspondence to Michael Frotscher.

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Competing interests

D.S. was involved in the development of EM PACT and receives royalties from Leica Microsystems (Vienna, Austria).

Supplementary information

Supplementary Methods

Immunogold labelling for p-coffin. (PDF 84 kb)

Procedure of high-pressure freezing.

We show starting the HPF machine (EM PACT2) and manipulations carried out in steps 25 – 49 of the PROCEDURE to high-pressure freeze tissue samples. (MOV 57198 kb)

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Studer, D., Zhao, S., Chai, X. et al. Capture of activity-induced ultrastructural changes at synapses by high-pressure freezing of brain tissue. Nat Protoc 9, 1480–1495 (2014). https://doi.org/10.1038/nprot.2014.099

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