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.

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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.

Author information

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.

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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