title: Analysis of synaptic ultrastructure without fixative using high-pressure freezing and tomography.
abstract: Electron microscopy allows the analysis of synaptic ultrastructure and its modifications during learning or in pathological conditions. However, conventional electron microscopy uses aldehyde fixatives that alter the morphology of the synapse by changing osmolarity and collapsing its molecular components. We have used high-pressure freezing (HPF) to capture within a few milliseconds structural features without aldehyde fixative, and thus to provide a snapshot of living synapses. CA1 hippocampal area slices from P21 rats were frozen at -173 degrees C under high pressure to reduce crystal formation, and synapses on dendritic spines were analysed after cryosubstitution and embedding. Synaptic terminals were larger than after aldehyde fixation, and synaptic vesicles in these terminals were less densely packed. Small filaments linked the vesicles in subgroups. The postsynaptic densities (PSDs) exhibited filamentous projections extending into the spine cytoplasm. Tomographic analysis showed that these projections were connected with the spine cytoskeletal meshwork. Using immunocytochemistry, we found as expected GluR1 at the synaptic cleft and CaMKII in the PSD. Actin immunoreactivity (IR) labelled the cytoskeletal meshwork beneath the filamentous projections, but was very scarce within the PSD itself. ProSAP2/Shank3, cortactin and Ena/VASP-IRs were concentrated on the cytoplasmic face of the PSD, at the level of the PSD projections. Synaptic ultrastructure after HPF was different from that observed after aldehyde fixative. The boutons were larger, and filamentous components were preserved. Particularly, filamentous projections were observed linking the PSD to the actin cytoskeleton. Thus, synaptic ultrastructure can be analysed under more realistic conditions following HPF.
authors.affiliation: INSERM U789, Ecole Normale Supérieure, 46 rue d'Ulm, 75005 Paris, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Rostaing
authors.forename: Eleonore
authors.initials: E
authors.lastname: Real
authors.forename: Léa
authors.initials: L
authors.lastname: Siksou
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Lechaire
authors.forename: Thomas
authors.initials: T
authors.lastname: Boudier
authors.forename: Tobias M
authors.initials: TM
authors.lastname: Boeckers
authors.forename: Frank
authors.initials: F
authors.lastname: Gertler
authors.forename: Eckart D
authors.initials: ED
authors.lastname: Gundelfinger
authors.forename: Antoine
authors.initials: A
authors.lastname: Triller
authors.forename: Serge
authors.initials: S
authors.lastname: Marty
mesh_terms: D000447:Aldehydes / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D051356:Cortactin / Q000378:metabolism; D015925:Cryopreservation / Q000379:methods*; D005615:Freezing; D006624:Hippocampus / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission / Q000379:methods; D016253:Microscopy, Immunoelectron / Q000379:methods; D051381:Rats; D017470:Receptors, Glutamate / Q000378:metabolism; D013569:Synapses / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D016707:Tissue Fixation / Q000379:methods; D014054:Tomography
chemical_list: D000447:Aldehydes; D051356:Cortactin; D017470:Receptors, Glutamate; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases