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. 2012 Jun 21;7(7):1351-65.
doi: 10.1038/nprot.2012.074.

Analysis of Neurotransmitter Release Mechanisms by Photolysis of Caged Ca²⁺ in an Autaptic Neuron Culture System

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Analysis of Neurotransmitter Release Mechanisms by Photolysis of Caged Ca²⁺ in an Autaptic Neuron Culture System

Andrea Burgalossi et al. Nat Protoc. .

Abstract

Neurotransmitter release is triggered by membrane depolarization, Ca(2+) influx and Ca(2+) 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 Ca(2+) dependent and simultaneously occurring, it is difficult to dissect them experimentally. This problem can be partially circumvented by controlling synaptic Ca(2+) concentrations via UV photolysis of caged Ca(2+). We developed a culture protocol for Ca(2+) 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 Ca(2+)-chelator nitrophenyl-EGTA and Ca(2+) indicators, and a UV flash is used to trigger Ca(2+)-uncaging and SV fusion. The protocol takes three weeks to complete and provides unprecedented insights into the mechanisms of transmitter release.

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