Analysis of neurotransmitter release mechanisms by photolysis of caged Ca2+ in an autaptic neuron culture system

A Burgalossi, SY Jung, KM Man, R Nair, WJ Jockusch… - Nature protocols, 2012 - nature.com
A Burgalossi, SY Jung, KM Man, R Nair, WJ Jockusch, SM Wojcik, N Brose, JS Rhee
Nature protocols, 2012nature.com
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 …
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.
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