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
109 neurons1013 synapses1015 synaptic vesicles
The quantal hypothesis
Bernard KatzHeuser and Reese
The fusion of one synaptic vesicle corresponds to one spontaneous electrical event
Heuser and Reese, 1981, J. Cell Biol. 88, 564-580
Fixed atrest
Fixed 5msafterstimulation
Synaptic vesicles fuse with the plasma membrane
Südhof TC. 2004. Annu. Rev. Neurosci 27:509-554
Synaptic vesicles engage in a cycle of exo- and endocytosis
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
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
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
Technique 1: capacitance measurementsFusion of large secretory vesicles
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
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
Technique 2: amperometry
modified from Westerink, 2004, Neurotoxicology 25, 461-470
+650 mV
Technique 2: amperometryAmperometry gives information about the release process
Analysis of single spikes
‚stand-alone foot‘
‚kiss-and-run‘
full fusion
Technique 2: amperometry combined with capacitance measurements (patch-amperometry)
Albillos et al., 1997, Nature 389: 509-512.
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
Technique 3: calcium uncagingThe distribution of vesicles and calcium channels
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
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
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
Getting to the molecular questionsWhich proteins are doing what?
Munc18-1
AT Brunger, 2001
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
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
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
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
Koh and Bellen, 2003, Trends in Neurosci. 26, 413-422
Südhof, 2002, J. Biol. Chem. 277, 7629-7632.
Synaptotagmins are calcium sensors
Rhee et al., 2005,PNAS 102, 18664-9
Example 3: synaptotagmin 1 is the fast calcium sensor for synaptic release
• 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
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