jakob b. sørensen research group leader “molecular mechanism of exocytosis” max-planck-institut...
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Jakob B. SørensenResearch group leader “Molecular mechanism of exocytosis”
Max-Planck-Institut für biophysikalische ChemieAm Fassberg 1137077 Göttingen
Vesicle membrane fusion mediating fast signal transmission - molecular aspects
The Graduate School of Neuroscience,Faculty of Health Sciences, University of Copenhagen
Ph.D. course: Molecular Neurobiology
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109 neurons1013 synapses1015 synaptic vesicles
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The quantal hypothesis
Bernard KatzHeuser and Reese
The fusion of one synaptic vesicle corresponds to one spontaneous electrical event
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Heuser and Reese, 1981, J. Cell Biol. 88, 564-580
Fixed atrest
Fixed 5msafterstimulation
Synaptic vesicles fuse with the plasma membrane
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Südhof TC. 2004. Annu. Rev. Neurosci 27:509-554
Synaptic vesicles engage in a cycle of exo- and endocytosis
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How can we measure the fusion of secretory/synaptic vesicles in real time?
Detection of added membrane: membrane capacitance
Detection of released neurotransmitter: amperometry
Detection using the postsynaptic cell: autaptic hippo-campal neurons
Detection using fluorescent tracers: next talk by Jürgen Klingauf
Example 3: neuronal studies of synaptotagmin 1
Stimulation method: calcium uncaging
Example 1: calyx of HeldExample 2: chromaffin cell studies of SNAP-25
Viral overexpression techniques: knock-out and rescue
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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)
1 F/cm2
I
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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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Technique 1: capacitance measurementsFusion of large secretory vesicles
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Technique 1: capacitance measurements Limitations
In most neurons, the release of synaptic vesicles occur at the end of a long axon, which does not allow electrical measurements.
However, some synapses are so large that the presynaptic terminalcan be patched directly
Calyx of HeldExample:
Wölfel et al, 2003, J. Neurosci. 23:7059-7063.
Capacitance changes report on both exocytosis and endocytosis
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How can we measure the fusion of secretory/synaptic vesicles in real time?
Detection of added membrane: membrane capacitance
Detection of released neurotransmitter: amperometry
Detection using the postsynaptic cell: autaptic hippo-campal neurons
Detection using fluorescent tracers: next talk by Jürgen Klingauf
Example 3: neuronal studies of synaptotagmin 1
Stimulation method: calcium uncaging
Example 1: calyx of HeldExample 2: chromaffin cell studies of SNAP-25
Viral overexpression techniques: knock-out and rescue
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Technique 2: amperometry
modified from Westerink, 2004, Neurotoxicology 25, 461-470
+650 mV
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Technique 2: amperometryAmperometry gives information about the release process
Analysis of single spikes
‚stand-alone foot‘
‚kiss-and-run‘
full fusion
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Technique 2: amperometry combined with capacitance measurements (patch-amperometry)
Albillos et al., 1997, Nature 389: 509-512.
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Technique 2: Amperometry Limitations
No access to the release site in synapses
Only a few neurotransmitters/hormones (adrenaline, noradrenaline, dopamine, serotonine, histamine) can be oxidized
Other methods: detection of neurotransmitter type usingfast cyclic voltammetry
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Technique 3: calcium uncagingThe distribution of vesicles and calcium channels
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Nitrophenyl-EGTAKD = 80 nM
Break-down productsKD ~ 2 mM
p(Ca)
ICa
Ca -DMN photoproducts + Ca2+
Flash
Technique 3: calcium uncagingCa2+-uncaging results in a homogeneous
calcium concentration
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Schneggenburger and Neher, 2000, Nature 406: 889-893
Calyx of Held
Example 1: photorelease of caged-calcium reveals the true calcium-dependence of fast release
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Data:Experimental setup:
NP-EGTAFura-2/Furaptra
30
20
10
0
543210Time (s)
8.0
7.8
7.6
7.4
7.2
200
100
0
1.21.00.80.60.4Time (s)
Fast burst
Slow burst
= 18 ms
= 132 ms
Preflash calcium
Flash
Technique 1-3: capacitance measurements, amperometry and calcium uncaging
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Getting to the molecular questionsWhich proteins are doing what?
Munc18-1
AT Brunger, 2001
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Example 2: knock-out of SNAP-25 abolishes secretion - overexpression rescues secretion
3020100
[Ca2+
] (
M)
80
60
40
20
0
I Am
p (p
A)
543210Time (s)
300
200
100
0
Cm
(fF
)
Control (+/+; +/-)Knock-out (-/-)
300
200
100
0
Cm
(fF
)
60
40
20
0
I Am
p (
pA
)
543210Time (s)
403020100
[Ca2+
] (
M)
Control (+/+;+/-)Knock-out (-/-) overexpressing SNAP-25A
eGFPSNAP-25
Snap-25
Sørensen J.B., Nagy G. et al. 2003, Cell 114, 75-86.
SNAP-25 knock-out
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Technique 4: Viral overexpression‘knock-out and rescue’
• Semliki Forest virus: RNA virus, very high expression level, lethal
Adenovirus 5: DNA virus, moderate expression level, fast onset
Lentivirus: retrovirus (HIV-1), moderate expression level, slower onset
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How can we measure the fusion of secretory/synaptic vesicles in real time?
Detection of added membrane: membrane capacitance
Detection of released neurotransmitter: amperometry
Detection using the postsynaptic cell: autaptic hippo-campal neurons
Detection using fluorescent tracers: next talk by Jürgen Klingauf
Example 3: neuronal studies of synaptotagmin 1
Stimulation method: calcium uncaging
Example 1: calyx of HeldExample 2: chromaffin cell studies of SNAP-25
Viral overexpression techniques: knock-out and rescue
![Page 24: Jakob B. Sørensen Research group leader “Molecular mechanism of exocytosis” Max-Planck-Institut für biophysikalische Chemie Am Fassberg 11 37077 Göttingen](https://reader036.vdocument.in/reader036/viewer/2022062304/56649d4b5503460f94a2806a/html5/thumbnails/24.jpg)
Technique 5:Autaptic Microisland Culture of Hippocampal Neurons
1 nA5 ms
Postsynaptic current
AP
Synaptic plasticity Yes No
Hippocamp.autaptic
Chromaffincells
Molecular manipulationKnock-out mice Yes YesOverexpression Yes Yes
Direct Presynaptic measurements No Yes
Distinction of vesicle pools (No) Yes
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Koh and Bellen, 2003, Trends in Neurosci. 26, 413-422
Südhof, 2002, J. Biol. Chem. 277, 7629-7632.
Synaptotagmins are calcium sensors
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Rhee et al., 2005,PNAS 102, 18664-9
Example 3: synaptotagmin 1 is the fast calcium sensor for synaptic release
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• Presynaptic
- number of synapses/active zones
- action potential waveform
- modulation of Ca-currents
- Ca++ buffers
- loading of synaptic vesicles
- Fusion of vesicles
• Synaptic
- morphology of synaptic cleft
• Postsynaptic
- desensitization of receptors
- number and clustering of receptors
- block by Polycations
Limitations of using postsynaptic neurons for the detection of neurotransmitter release
Factors that could modify measured postsynaptic currents
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How can we measure the fusion of secretory/synaptic vesicles in real time?
Detection of added membrane: membrane capacitance
Detection of released neurotransmitter: amperometry
Detection using the postsynaptic cell: autaptic hippo-campal neurons
Detection using fluorescent tracers: next talk by Jürgen Klingauf
Example 3: neuronal studies of synaptotagmin 1
Stimulation method: calcium uncaging
Example 1: calyx of HeldExample 2: chromaffin cell studies of SNAP-25
Viral overexpression techniques: knock-out and rescue
![Page 29: Jakob B. Sørensen Research group leader “Molecular mechanism of exocytosis” Max-Planck-Institut für biophysikalische Chemie Am Fassberg 11 37077 Göttingen](https://reader036.vdocument.in/reader036/viewer/2022062304/56649d4b5503460f94a2806a/html5/thumbnails/29.jpg)
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