Analysis of neurotransmitter release mechanisms by photolysis of caged Ca²⁺ in an autaptic neuron culture system.
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.
Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany.
Andrea
A
Burgalossi
SangYong
S
Jung
Kwun-nok Mimi
KN
Man
Ramya
R
Nair
Wolf J
WJ
Jockusch
Sonja M
SM
Wojcik
Nils
N
Brose
Jeong-Seop
JS
Rhee
D002118:Calcium / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000379:methods*; D003584:Cytological Techniques / Q000379:methods; D004533:Egtazic Acid; D009474:Neurons / Q000378:metabolism*; D018377:Neurotransmitter Agents / Q000378:metabolism*; D010782:Photolysis; D013569:Synapses / Q000378:metabolism*; D013572:Synaptic Vesicles / Q000378:metabolism*; D014466:Ultraviolet Rays
D018377:Neurotransmitter Agents; D004533:Egtazic Acid; D002118:Calcium