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.
Key WordsEndocytosis exocytosis hippocampal synapses imaging neurons synaptic vesicle synaptopHluorin sypHy
Superecliptic pHluorin and monomeric RFP cDNAs were kind gifts from Drs James Rothman and Roger Tsien. This work was supported by the Medical Research Council (MRC), the Swedish Research Council and the Human Frontiers Science Program (HFSP).
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