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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References
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).
Nicoll, R.A., Kauer, J.A. & Malenka, R.C. The current excitement in long-term potentiation. Neuron 1, 97–103 (1988).
Bliss, T.V.P. & Collingridge, G.L. A synaptic model of memory: long-term potentiation in the hippocampus. Nature 361, 31–39 (1993).
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).
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).
Yang, Y., Wang, X.B., Frerking, M. & Zhou, Q. Spine expansion and stabilization associated with long-term potentiation. J. Neurosci. 28, 5740–5751 (2008).
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).
Engert, F. & Bonhoeffer, T. Dendritic spine changes associated with hippocampal long-term synaptic plasticity. Nature 399, 66–70 (1999).
Maletic-Savatic, M., Malinow, R. & Svoboda, K. Rapid dendritic morphogenesis in CA1 hippocampal dendrites induced by synaptic activity. Science 283, 1923–1927 (1999).
Matus, A. Actin-based plasticity in dendritic spines. Science 290, 754–758 (2000).
Yuste, R. & Bonhoeffer, T. Morphological changes in dendritic spines associated with long-term synaptic plasticity. Annu. Rev. Neurosci. 24, 1071–1089 (2001).
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).
Segal, M. Dendritic spines and long-term plasticity. Nat. Rev. Neurosci. 6, 277–284 (2005).
Hotulainen, P. & Hoogenraad, C.C. Actin in dendritic spines: connecting dynamics to function. J. Cell Biol. 189, 619–629 (2010).
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).
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).
Geinisman, Y. Structural synaptic modifications associated with hippocampal LTP and behavioral learning. Cereb. Cortex 10, 952–962 (2000).
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).
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).
Adrian, M., Dubochet, J., Lepault, J. & McDowall, A.W. Cryo-electron microscopy of viruses. Nature 308, 32–36 (1984).
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).
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).
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).
Zhao, S. et al. Structural plasticity of hippocampal mossy fiber synapses as revealed by high-pressure freezing. J. Comp. Neurol. 520, 2340–2351 (2012).
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).
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).
Lucic, V., Rigort, A. & Baumeister, W. Cryo-electron tomography: The challenge of doing structural biology in situ. J. Cell Biol. 202, 407–419 (2013).
Studer, D., Michel, M. & Muller, M. High pressure freezing comes of age. Scanning Microsc. Suppl. 3, 253–268 (1989).
Studer, D., Graber, W., Al-Amoudi, A. & Eggli, P. A new approach for cryofixation by high-pressure freezing. J. Microsc. 203, 285–294 (2001).
Hohenberg, H., Mannweiler, K. & Muller, M. High-pressure freezing of cell suspensions in cellulose capillary tubes. J. Microsc. 175, 34–43 (1994).
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).
Rostaing, P. et al. Analysis of synaptic ultrastructure without fixative using high-pressure freezing and tomography. Eur. J. Neurosci. 24, 3463–3474 (2006).
Siksou, L. et al. Three-dimensional architecture of presynaptic terminal cytomatrix. J. Neurosci. 27, 6868–6877 (2007).
Siksou, L. et al. A common molecular basis for membrane docking and functional priming of synaptic vesicles. Eur. J. Neurosci. 30, 49–56 (2009).
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).
Frotscher, M., Misgeld, U. & Nitsch, C. Ultrastructure of mossy fiber endings in in vitro hippocampal slices. Exp. Brain Res. 41, 247–255 (1981).
Geiger, J.R.P. et al. Patch-clamp recording in brain slices with improved slicer technology. Pflügers Arch. 443, 491–501 (2002).
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).
Stoppini, L., Buchs, P.A. & Muller, D. A simple method for organotypic cultures of nervous tissue. J. Neurosci. Meth. 37, 173–182 (1991).
Frotscher, M. & Gähwiler, B.H. Synaptic organization of intracellularly stained CA3 pyramidal neurons in slice cultures of rat hippocampus. Neuroscience 24, 541–551 (1988).
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).
Collin, C., Miyaguchi, K. & Segal, M. Dendritic spine density and LTP induction in cultured hippocampal slices. J. Neurophysiol. 77, 1614–1623 (1997).
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).
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).
Deisseroth, K. et al. Next-generation optical technologies for illuminating genetically targeted brain circuits. J. Neurosci. 26, 10380–10386 (2006).
Zhang, F. et al. Multimodal fast optical interrogation of neural circuitry. Nature 446, 633–639 (2007).
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).
Adams, S.R. & Tsien, R.Y. Controlling cell chemistry with caged compounds. Annu. Rev. Physiol. 55, 755–784 (1993).
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).
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).
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).
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).
Hamlyn, L.H. The fine structure of the mossy fibre endings in the hippocampus of the rabbit. J. Anat. 96, 112–120 (1962).
Bamburg, J.R. Proteins of the ADF/cofilin family: essential regulators of actin dynamics. Annu. Rev. Cell Biol. 15, 185–230 (1999).
Arber, S. et al. Regulation of actin dynamics through phosphorylation of cofilin by LIM-kinase. Nature 393, 805–809 (1998).
Yang, N. et al. Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin reorganization. Nature 393, 809–812 (1998).
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).
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).
Racz, B. & Weinberg, R.J. Spatial organization of cofilin in dendritic spines. Neuroscience 138, 447–456 (2006).
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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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.
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D.S. was involved in the development of EM PACT and receives royalties from Leica Microsystems (Vienna, Austria).
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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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DOI: https://doi.org/10.1038/nprot.2014.099
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