techniques for synaptic vesicle recycling 1.electrophysiology 2.imaging 3.electron microscopy fm...
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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: SynaptopHluorinTransgenetic 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 microscopyDefects in synaptic vesicle endocytosis
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Adrenal gland
Chromaffin cells as the model systemChromaffin cells as the model systemfor vesicle cyclingfor 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 systemChromaffin cells as the model systemfor vesicle cyclingfor vesicle cycling
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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
1 nA
1 ms
V = 10 mV
i(t) = (I0-Iss) exp(-t/) + Iss
I0 = V/Rs
Iss = V/(Rs+ RM)
= CM RsRM/(Rs+ RM)
IRM = V/RM
ICM = CM(dV(t)/dt)
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Technique 1: capacitance measurementsSine-wave technique
1 ms
50 mV
90o
IRM
ICM
IRM + ICM
Phase sensitive detector (PSD) splits the current in real and imaginary part and calculates R s, RM and CM
IRM = V/RM ICM = CM(dV(t)/dt)
V(t)=Vosin(2f t)
V(t)=(1/RM) Vosin(2f t)
V(t)= CMVosin(2f t + 90o)
1 F/cm2
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Whole cell capacitance technique
stimulation
Cap
acit
ance
(p
F)
60 fF
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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
Conductance
Capacitance
Patch pipette
Patch pipette
Chromaffin cell
0.1 nS
1 fF
0.5 nS
10 ms
Conductance
Capacitance
Fusion poreconductance
Fusion pore
PM
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Carbon fiber+700 mV
Amperometry detects catecholamine release fromsingle vesicles by oxidization
Amperometrical current
Quantal sizeAmperometry
Chromaffin cellFusion pore
PM
Foot
Spike
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Technique 2: amperometryAmperometry gives information about the release process
Analysis of single spikes
stand-alone foot
kiss-and-runfull 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-Patch amperometry: a method combines amperometry and cell-attached capacitance measurementattached capacitance measurement
Simultaneous detections of fusion and neurotransmitter release of same vesicle.
Carbon fiber
Patch pipette P
atch
pip
ette
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 pAAmperometrical
Im
Re
Gp
1 pAAmperometrical
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