Functional analysis of calcium channel-mediated exocytosis in synaptic terminals by FM imaging technique.
OBJECTIVE
Presynaptic voltage-gated Ca(2+) channels mediate rapid Ca(2+) influx into the synaptic terminal which triggers synaptic vesicle exocytosis and neurotransmitter release. The FM 1-43 dye was firstly introduced as a fluorescence probe by Betz and his colleagues in 1992, and has been used to monitor exocytosis, endocytosis and endosomal traffic in a variety of cell types. The present study aims to investigate the feasibility of applying the FM 1-43 dye in the functional analysis of calcium channel-mediated exocytosis in synaptic terminals.


METHODS
The hippocampi were isolated from embryos of pregnant rats, and hippocampal neurons were then transfected with Ds-Red conjugated plasmid. The neurons were then loaded with 8 micromol/L FM 1-43 and 47 mmol/L KCl for 90 s after transfection. After that, 90 mmol/L KCl was employed to induce FM dye destaining, which was recorded by FM imaging system.


RESULTS
The neuron synaptic terminals of rat hippocampus could be effectively stained by the FM 1-43 dye. Besides, the destaining of the labeled neuron terminals was in accordance with the transmitter release, which could be blocked by the application of nifedipine (inhibitor for L-type calcium channel).


CONCLUSIONS
The FM imaging technique is an advanced and effective method for analyzing synaptic vesicle exocytosis and neurotransmitter release, and can be applied in various synaptic functional studies.
Gu
Feng
F
Department of breast pathology, Tianjin Medical University Cancer Institute and Hospital, Key Laboratory of Breast Cancer Prevention and Therapy of Ministry of Education, Key Laboratory of Cancer Prevention and Therapy of Tianjin, Tianjin 300060, China.
Fu
Li
L
Ma
Yong-Jie
YJ
D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D003952:Diagnostic Imaging; D004622:Embryo, Mammalian; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D005260:Female; D006624:Hippocampus / Q000166:cytology; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D009543:Nifedipine / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D011247:Pregnancy; D017729:Presynaptic Terminals / Q000187:drug effects / Q000502:physiology*; D011726:Pyridinium Compounds / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D051381:Rats; D013997:Time Factors; D014162:Transfection / Q000379:methods
D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; C073804:FM1 43; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D011189:Potassium Chloride; D009543:Nifedipine