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

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.