Techniques for synaptic vesicle recycling

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Techniques for synaptic vesicle recycling
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2. Imaging SynaptopHluorin
SynaptopHluorin
Miesenbock et al, Nature 1998
Sankaranarayanan et al, Biophys J 2000
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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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2. Imaging quantum dots
Detection of full fusion and kiss-and-run by
quantum dots
Zhang et al, Science 2009
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2. Imaging quantum dots
Detection of full fusion and kiss-and-run by
quantum dots
Zhang et al, Science 2009
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3. Electron microscopy

1. Docked vesicles
2. Endocytic vesicle biogenesis

Rettig Neher, Science 2002
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3. Electron microscopy
Defects in synaptic vesicle endocytosis
Hayashi et al, PNAS 2008
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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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Munc-18 is important for vesicle docking
Voets et al, Neuron 2003
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Chromaffin cells as the model system for vesicle
cycling
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Membrane conductance and capacitance
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Technique 1 capacitance measurements Time
domain technique
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Technique 1 capacitance measurementsSine-wave
technique
1 ?F/cm2
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Whole cell capacitance technique
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Ca2 photolysis
NP-EGTA
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Whole-cell capacitance technique and Ca2
photolysis
Rettig Neher, Science 2002
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Cell-attached capacitance to detect single
vesicle fusion
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Amperometry detects catecholamine release
from single vesicles by oxidization
Amperometry
Amperometrical current
Chromaffin cell
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Technique 2 amperometryAmperometry gives
information about the release process
Analysis of single spikes
kiss-and-run
full fusion
stand-alone foot
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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.
Albillos et al., Nature 1997
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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
• Whole-cell capacitance technique
• Cell-attached capacitance technique
• Amperometry
• Patch amperometry