techniques for synaptic vesicle recycling 1.electrophysiology 2.imaging 3.electron microscopy fm...
TRANSCRIPT
Techniques for synaptic vesicle recycling
1. Electrophysiology
2. Imaging
3. Electron microscopy
• FM dyes• SynaptopHluorin• Quantum dots
Sankaranarayanan et al, Biophys J 2000
2. Imaging: SynaptopHluorin
SynaptopHluorin
Miesenbock et al, Nature 1998
SynaptopHluorin reports synaptic vesicle exocytosis
Sankaranarayanan & Ryan, Nat Cell Biol 2000
2. Imaging: SynaptopHluorin
Bafilomycin to separate exo/endocytosis
Mani et al, Neuron 2007
2. Imaging: SynaptopHluorinTransgenetic mice expressing SynaptopHluorin
Li et al, PNAS 2005
2. Imaging: Synaptophysin-pHluorin
Granseth et al, Neuron 2006
Full fusion and kiss-and-run
Harata et al, J Neurochem 2006
Detection of full fusion and kiss-and-run by quantum dots
Zhang et al, Science 2009
2. Imaging: quantum dots
Detection of full fusion and kiss-and-run by quantum dots
Zhang et al, Science 2009
2. Imaging: quantum dots
3. Electron microscopy
(1) Docked vesicles(2) Endocytic vesicle biogenesis
Rettig & Neher, Science 2002
Hayashi et al, PNAS 2008
3. Electron microscopyDefects in synaptic vesicle endocytosis
Adrenal gland
Chromaffin cells as the model systemChromaffin cells as the model systemfor vesicle cyclingfor vesicle cycling
Differences between neurons and chromaffin cells
Intracellular vesicle trafficking pathway
Voets et al, Neuron 2003
Munc-18 is important for vesicle docking
Adrenal gland
Chromaffin cells as the model systemChromaffin cells as the model systemfor vesicle cyclingfor vesicle cycling
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
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)
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
Whole cell capacitance technique
stimulation
Cap
acit
ance
(p
F)
60 fF
Ca2+ photolysis
NP-EGTA
Whole-cell capacitance technique and Ca2+ photolysis
Rettig & Neher, Science 2002
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
Carbon fiber+700 mV
Amperometry detects catecholamine release fromsingle vesicles by oxidization
Amperometrical current
Quantal sizeAmperometry
Chromaffin cellFusion pore
PM
Foot
Spike
Technique 2: amperometryAmperometry gives information about the release process
Analysis of single spikes
stand-alone foot
kiss-and-runfull fusion
Different isoform of synaptotagmin controls the choice between full fusion and kiss-and-run
Wang et al, Nature 2003
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
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
Neuroendocrine chromaffin cells:
1.Whole-cell capacitance technique
2.Cell-attached capacitance technique
3.Amperometry
4.Patch amperometry