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3-D characterisation of mossy fiber synapses by cryomethods

English title 3-D characterisation of mossy fiber synapses by cryomethods
Applicant Studer Daniel
Number 105822
Funding scheme Project funding (Div. I-III)
Research institution Institut für Anatomie Medizinische Fakultät Universität Bern
Institution of higher education University of Berne - BE
Main discipline Neurophysiology and Brain Research
Start/End 01.10.2004 - 30.09.2007
Approved amount 191'409.00
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Keywords (6)

electron microscopy; tomography; cryofixation; high pressure freezing; freeze-substitution; cryosectioning

Lay Summary (English)

Lead
Lay summary
The objective of biological transmission electron microscopy (TEM) is toobserve biological structures in a state as close as possible to theliving organism and if possible in three dimensions (electron tomography[ET]). The investigation of biological samples close to their native statecan be achieved successfully by cryomethods. The most straight on way isVCO: vitrifiying (solidification by cooling as rapid that water does notcrystallise) small specimens, cutting them into cryo-sections andobserving them directly on a cryo stage in the TEM at best conceivableresolution. Alternatively freeze-substitution and embedding is used (thisapproach leads to improved structural preservation compared withconventional chemical fixation). Particularly for immuno-cytochemistrythis is a good and practicable way.
Since 1989 we are working on the improvement of cryomethods and we arestill enthusiastic to further develop them. In order to elaborateimportant ultrastructural information we will focus on the analysis ofmossy fiber synapses in the hippocampus of new born rats in order toclarify their true structure and dimensions. We intend to investigatefresh brain tissue and brain slices in culture in close collaboration withM. Frotscher (Anatomy, University Freiburg i. B., Germany). Samplessubjected to VCO or freeze-substitution and immunolabeling will beinvestigated in our lab in Bern and in the lab of J. Dubochet (LAU,Université de Lausanne). In the lab of A. Verkleij and A. Koster(Cellbiology, University of Utrecht, NL) we will have the possibility toapply electron tomography techniques to our sections. We hope to get abetter three dimensional description of the mossy fiber synapseultrastructure.
The importance of the outlined project will be above all a betterunderstanding of the true structure and dimensions of mossy fibersynapses. These findings may be the starting point for detailedphysiological experiments and in particular for realistic simulations ofglutamatergic neurotransmission at a defined central synapse. Thescientific community will profit from a once again improved powerful toolto look at relatively large biological samples close to their nativestate. Medicine, too, will profit from VCO in diagnostics and pathology,because the method is very fast (one to few hours from sample collectionto micrographs). Diatome (Nidau, Switzerland) a worldwide leading companyin the production and selling of diamond knives for ultramicrotomy,depends partly on the technical developments realized in our lab, tomaintain their position in the market. And last but not least cryomethodshave become more and more popular in the ultrastructural analysis, asdocumented by an increasing number of publications. Our proposalestablishes synergies with the recently started program of the Europeancommunity: “New Electron Microscopy Approaches for Studying ProteinComplexes and Cellular Supramolecular Architecture”(http://europa.eu.int/comm/research/fp6/p1/firstcallresult_en.html). Twoof the fourteen engaged laboratories (Dubochet and Verkleij) willcollaborate with us.
Direct link to Lay Summary Last update: 21.02.2013

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Associated projects

Number Title Start Funding scheme
118394 Characterisation of the three dimensional nanostructure of nerve tissue and cell cultures using cryomethods 01.10.2007 Project funding (Div. I-III)
59296 Vitrifying, cutting, observing - the dream method for electron microscopy 01.10.2000 Project funding (Div. I-III)

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