jakob b. sørensen research group leader “molecular mechanism of exocytosis” max-planck-institut...

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