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


Author(s): Studer, Daniel; Zhao, Shanting; Chai, Xuejun; Jonas, Peter; Graber, Werner A; Nestel, Sigrun; Frotscher, Michael
Article Title: Capture of activity-induced ultrastructural changes at synapses by high-pressure freezing of brain tissue
Affiliation IST Austria
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 â ̂1/450 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.
Journal Title: Nature Protocols
Volume: 9
Issue 6
ISSN: 1750-2799
Publisher: Nature Publishing Group  
Date Published: 2014-06-01
Start Page: 1480
End Page: 1495
DOI: 10.1038/nprot.2014.099
Open access: no