| dc.description.abstracteng | Excitation-secretion coupling at nerve cell synapses is a sub-millisecond process that entails the transduction of an electrical stimulus into synaptic vesicle fusion. Before fusion, synaptic vesicles are physically docked to the presynaptic active zone membrane and functionally primed to become fusion competent. In response to an increase in intracellular calcium concentration after the arrival of an action potential, primed vesicles fuse with the plasma membrane and release their neurotransmitter content into the synaptic cleft. Recent studies combining cryo-fixation methods and three-dimensional electron microscopy analysis proposed that synaptic vesicle docking and priming steps may not reflect independent mechanisms, but rather describe the same molecular process namely full or partial soluble N-ethylmalemide-sensitive factor attachment protein receptor (SNARE) complex formation initiated by members from the UNC-13/Munc13 protein family. However, other studies have challenged the notion of SNARE complex assembly prior to the calcium triggering step in the release process.
In the present study, a combination of organotypic hippocampal slice culture, high-pressure freezing, freeze substitution and electron tomography was used to reinvestigate the role of key synaptic proteins in synaptic vesicle docking in glutamatergic hippocampal spine synapses. This method enables the analysis of synaptic parameters in an in-situ-like setting using lethal mouse mutants that do not survive birth. Loss or reduction of components of the molecular priming machinery, namely Munc13 or CAPS proteins, and of the individual neuronal SNARE proteins Synaptobrevin-2, Syntaxin-1 and synaptosome-associated protein of 25 kDa (SNAP25) caused severe defects in synaptic vesicle membrane-attachment in this experimental setting. Moreover, loss of the calcium (Ca2+)-sensor Synaptotagmin-1, causes a decrease in vesicle numbers in presynaptic terminals in comparison to control synapses and a reduction in membrane-proximal (loosely tethered) and docked vesicles. However, the reduction in the number of membrane-attached synaptic vesicles was milder than it was observed in the absence of the vesicular SNARE ptrotein Synaptobrevin-2, indicating that Synaptotagmin-1 might have a regulatory (e.g. tethering or clamping) but not essential role in synaptic vesicle docking in neurons. Complexin-deficient synapses exhibited no changes in the number of membrane-attached synaptic vesicles, a finding that supports a facilitatory rather than inhibitory role of Complexins prior to synaptic vesicle fusion. These findings indicate that synaptic vesicle membrane-attachment, synaptic vesicle priming and (partial) SNARE complex assembly are respective morphological, functional and molecular manifestations of the same process. | de |