Imaging phluorin-based probes at hippocampal synapses.
Accurate measurement of synaptic vesicle exocytosis and endocytosis is crucial to understanding the molecular basis of synaptic transmission. The fusion of a pH-sensitive green fluorescent protein (pHluorin) to various synaptic vesicle proteins has allowed the study of synaptic vesicle recycling in real time. Two such probes, synaptopHluorin and sypHy, have been imaged at synapses of hippocampal neurons in culture. The combination of these reporters with techniques for molecular interference, such as RNAi allows for the study of molecules involved in synaptic vesicle recycling. Here the authors describe methods for the culture and transfection of hippocampal neurons, imaging of pHluorin-based probes at synapses and analysis of pHluorin signals down to the resolution of individual synaptic vesicles.
Royle
Stephen J
SJ
School of Biomedical Sciences, University of Liverpool, Liverpool, UK.
Granseth
Björn
B
Odermatt
Benjamin
B
Derevier
Aude
A
Lagnado
Leon
L
D000818:Animals; D002478:Cells, Cultured; D004705:Endocytosis; D005089:Exocytosis; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism*; D021621:Imaging, Three-Dimensional / Q000379:methods*; D015335:Molecular Probes / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D038761:Photobleaching; D051381:Rats; D013569:Synapses / Q000378:metabolism*; D014162:Transfection
D015335:Molecular Probes; C485187:PHluorin; D049452:Green Fluorescent Proteins