Analysis of neurotransmitter release mechanisms by photolysis of caged Ca²⁺ in an autaptic neuron culture system
2012 | journal article; research paper. A publication with affiliation to the University of Göttingen.
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Analysis of neurotransmitter release mechanisms by photolysis of caged Ca²⁺ in an autaptic neuron culture system
Burgalossi, A.; Jung, S. ; Man, K. Man, Kwun-nok Mimi ; Nair, R.; Jockusch, W. J; Wojcik, S. M & Brose, N. et al. (2012)
Nature Protocols, 7(7) pp. 1351-1365. DOI: https://doi.org/10.1038/nprot.2012.074
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- Authors
- Burgalossi, Andrea; Jung, SangYong ; Man, Kwun-nok Mimi ; Nair, Ramya; Jockusch, Wolf J; Wojcik, Sonja M ; Brose, Nils ; Rhee, Jeong-Seop
- Abstract
- Neurotransmitter release is triggered by membrane depolarization, Ca²⁺ influx and Ca²⁺ sensing by the release machinery, causing synaptic vesicle (SV) fusion with the plasma membrane. Interlinked is a complex membrane cycle in which vesicles are tethered to the release site, primed, fused and recycled. As many of these processes are Ca²⁺ dependent and simultaneously occurring, it is difficult to dissect them experimentally. This problem can be partially circumvented by controlling synaptic Ca²⁺ concentrations via UV photolysis of caged Ca²⁺. We developed a culture protocol for Ca²⁺ uncaging in small synapses on the basis of the generation of small glia cell islands with single neurons on top, which are sufficiently small to be covered with a UV-light flash. Neurons are loaded with the photolabile Ca²⁺-chelator nitrophenyl-EGTA and Ca²⁺ indicators, and a UV flash is used to trigger Ca²⁺-uncaging and SV fusion. The protocol takes three weeks to complete and provides unprecedented insights into the mechanisms of transmitter release.
- Issue Date
- 2012
- Journal
- Nature Protocols
- ISSN
- 1754-2189
- Language
- English