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Techniques for synaptic vesicle recycling 1.Electrophysiology 2.Imaging 3.Electron microscopy FM dyes SynaptopHluorin Quantum dots
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Sankaranarayanan et al, Biophys J 2000 2. Imaging: SynaptopHluorin SynaptopHluorin Miesenbock et al, Nature 1998
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SynaptopHluorin reports synaptic vesicle exocytosis Sankaranarayanan & Ryan, Nat Cell Biol 2000
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2. Imaging: SynaptopHluorin Bafilomycin to separate exo/endocytosis Mani et al, Neuron 2007
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2. Imaging: SynaptopHluorin Transgenetic mice expressing SynaptopHluorin Li et al, PNAS 2005
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2. Imaging: Synaptophysin-pHluorin Granseth et al, Neuron 2006
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Full fusion and kiss-and-run Harata et al, J Neurochem 2006
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Detection of full fusion and kiss-and-run by quantum dots Zhang et al, Science 2009 2. Imaging: quantum dots
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Detection of full fusion and kiss-and-run by quantum dots Zhang et al, Science 2009 2. Imaging: quantum dots
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3. Electron microscopy (1) Docked vesicles (2) Endocytic vesicle biogenesis Rettig & Neher, Science 2002
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Hayashi et al, PNAS 2008 3. Electron microscopy Defects in synaptic vesicle endocytosis
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Adrenal gland Chromaffin cells as the model system for vesicle cycling
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Differences between neurons and chromaffin cells Intracellular vesicle trafficking pathway
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Voets et al, Neuron 2003 Munc-18 is important for vesicle docking
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Adrenal gland Chromaffin cells as the model system for vesicle cycling Techniques: Capacitance measurements Amperometry Big vesicle size (150-450 nm in diameter) Oxidizable catecholamine
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AC and DC current DC - direct current - the polarity of a current source remains the same when the current is DC AC - Alternative current - the polarity of a current source is constantly changing when the current is AC Membrane conductance and capacitance
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Technique 1: capacitance measurements Time domain technique
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Technique 1: capacitance measurements Sine-wave technique 1 F/cm 2
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Whole cell capacitance technique stimulation Capacitance (pF) 60 fF
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Ca 2+ photolysis NP-EGTA
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Whole-cell capacitance technique and Ca 2+ photolysis Rettig & Neher, Science 2002
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Cell-attached capacitance to detect single vesicle fusion Conductance Capacitance Patch pipette Chromaffin cell 0.1 nS 1 fF 0.5 nS 10 ms Conductance Capacitance Fusion pore conductance Fusion pore PM
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Carbon fiber +700 mV Amperometry detects catecholamine release from single vesicles by oxidization Amperometrical current Quantal size Amperometry Chromaffin cell Fusion pore PM Foot Spike
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Technique 2: amperometry Amperometry gives information about the release process Analysis of single spikes stand-alone foot kiss-and-run full fusion
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Different isoform of synaptotagmin controls the choice between full fusion and kiss-and-run Wang et al, Nature 2003
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Patch amperometry: a method combines amperometry and cell- attached capacitance measurement Simultaneous detections of fusion and neurotransmitter release of same vesicle. Carbon fiber Patch pipette Catecholamine release Vesicle capacitance Fusion dynamics Does the fusion-pore size limit neurotransmitter release? Albillos et al., Nature 1997
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500 ms 400 pS 100 pS 1 fF 10 pA Amperometrical Im Re Gp 1 pA Amperometrical 50 pS 0.5 pA Fusion-pore Gp Amperometrical signal The neurotransmitter release is limited by the size of fusion pore Gong et al, Nat Cell Biol 2007
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Neuroendocrine chromaffin cells: 1.Whole-cell capacitance technique 2.Cell-attached capacitance technique 3.Amperometry 4.Patch amperometry
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