title: 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.
authors.affiliation: 1] Institute of Anatomy, University of Bern, Bern, Switzerland. [2].
authors.forename: Daniel
authors.initials: D
authors.lastname: Studer
authors.affiliation: 1] Institute for Structural Neurobiology, Center for Molecular Neurobiology Hamburg (ZMNH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany. [2].
authors.forename: Shanting
authors.initials: S
authors.lastname: Zhao
authors.affiliation: Institute for Structural Neurobiology, Center for Molecular Neurobiology Hamburg (ZMNH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Xuejun
authors.initials: X
authors.lastname: Chai
authors.affiliation: IST Austria (Institute of Science and Technology Austria), Klosterneuburg, Austria.
authors.forename: Peter
authors.initials: P
authors.lastname: Jonas
authors.affiliation: Institute of Anatomy, University of Bern, Bern, Switzerland.
authors.forename: Werner
authors.initials: W
authors.lastname: Graber
authors.affiliation: Institute of Anatomy and Cell Biology, University of Freiburg, Freiburg, Germany.
authors.forename: Sigrun
authors.initials: S
authors.lastname: Nestel
authors.affiliation: Institute for Structural Neurobiology, Center for Molecular Neurobiology Hamburg (ZMNH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Frotscher
mesh_terms: D001921:Brain / Q000166:cytology*; D020285:Cryoelectron Microscopy / Q000379:methods*; D009474:Neurons / Q000648:ultrastructure*; D011312:Pressure; D013569:Synapses / Q000502:physiology* / Q000648:ultrastructure*