Simultaneous monitoring of three key neuronal functions in primary neuronal cultures.
The coupling of Ca(2+) influx to synaptic vesicle (SV) recycling in nerve terminals is essential for neurotransmitter release and thus neuronal communication. Both of these parameters have been monitored using fluorescent reporter dyes such as fura-2 and FM1-43 in single central nerve terminals. However, their simultaneous monitoring has been hampered by the proximity of their fluorescence spectra, resulting in significant contamination of their signals by bleedthrough. We have developed an assay that simultaneously monitors both SV recycling and changes in intracellular free Ca(2+) ([Ca(2+)](i)) in cultured neurons using the reporter dyes FM4-64 and fura-2AM. By monitoring both fura-2 and FM4-64 emission in the far red range, we were able to visualize functionally independent readouts of both SV recycling and [Ca(2+)](i) independent of fluorescence bleedthrough. We were also able to incorporate an assay of cell viability without any fluorescence bleedthrough from either fura-2 or FM4-64 signals, using the dye SYTOX Green. We propose that this assay of three key neuronal functions could be simply translated into a high content screening format for studies investigating small molecule inhibitors of these processes.
Evans
Gareth John Owen
GJ
Centre for Integrative Physiology, Hugh Robson Building, George Square, University of Edinburgh, Edinburgh EH8 9XD, UK.
Cousin
Michael Alan
MA
D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000502:physiology; D001681:Biological Assay / Q000379:methods*; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology*; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D005089:Exocytosis / Q000502:physiology; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives; D008856:Microscopy, Fluorescence / Q000379:methods; D009474:Neurons / Q000166:cytology / Q000502:physiology*; D009482:Neurophysiology / Q000379:methods*; D009930:Organic Chemicals; D017729:Presynaptic Terminals / Q000378:metabolism*; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D051381:Rats; D017207:Rats, Sprague-Dawley; D009435:Synaptic Transmission / Q000502:physiology; D013572:Synaptic Vesicles / Q000378:metabolism*; D046509:Tissue Culture Techniques / Q000379:methods
C092350:FM 4-64; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; C402795:SYTOX Green; C049925:fura-2-am; D002118:Calcium; D016257:Fura-2