1. Jakob B. Sørensen Research group leader “Molecular mechanism of exocytosis” Max-Planck-Institut für biophysikalische Chemie Am Fassberg 11 37077 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

2. 10 9 neurons 10 13 synapses 10 15 synaptic vesicles

3. The quantal hypothesis Bernard KatzHeuser and Reese The fusion of one synaptic vesicle corresponds to one spontaneous electrical event

4. Heuser and Reese, 1981, J. Cell Biol. 88, 564-580 Fixed at rest Fixed 5ms after stimulation Synaptic vesicles fuse with the plasma membrane

5. Südhof TC. 2004. Annu. Rev. Neurosci 27:509-554 Synaptic vesicles engage in a cycle of exo- and endocytosis

6. 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 Held Example 2: chromaffin cell studies of SNAP-25 Viral overexpression techniques: knock-out and rescue

7. Technique 1: capacitance measurements Time domain technique 1  F/cm 2 I

8. Technique 1: capacitance measurements Sine-wave technique 1  F/cm 2

9. Technique 1: capacitance measurements Fusion of large secretory vesicles

10. 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 terminal can be patched directly Calyx of Held Example: Wölfel et al, 2003, J. Neurosci. 23:7059-7063. Capacitance changes report on both exocytosis and endocytosis

11. 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 Held Example 2: chromaffin cell studies of SNAP-25 Viral overexpression techniques: knock-out and rescue

12. Technique 2: amperometry modified from Westerink, 2004, Neurotoxicology 25, 461-470 +650 mV

13. Technique 2: amperometry Amperometry gives information about the release process Analysis of single spikes ‚stand-alone foot‘ ‚kiss-and-run‘ full fusion

14. Technique 2: amperometry combined with capacitance measurements (patch-amperometry) Albillos et al., 1997, Nature 389: 509-512.

15. 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 using fast cyclic voltammetry

16. Technique 3: calcium uncaging The distribution of vesicles and calcium channels

17. Nitrophenyl-EGTA K D = 80 nM Break-down products K D ~ 2 mM Technique 3: calcium uncaging Ca 2+ -uncaging results in a homogeneous calcium concentration

18. 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

19. Data:Experimental setup: NP-EGTA Fura-2/Furaptra Technique 1-3: capacitance measurements, amperometry and calcium uncaging

20. Getting to the molecular questions Which proteins are doing what? Munc18-1 AT Brunger, 2001

21. Example 2: knock-out of SNAP-25 abolishes secretion - overexpression rescues secretion eGFPSNAP-25 Snap-25 Sørensen J.B., Nagy G. et al. 2003, Cell 114, 75-86. SNAP-25 knock-out

22. 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

23. 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 Held Example 2: chromaffin cell studies of SNAP-25 Viral overexpression techniques: knock-out and rescue

24. Technique 5: Autaptic Microisland Culture of Hippocampal Neurons Postsynaptic current AP Synaptic plasticityYesNo Hippocamp. autaptic Chromaffin cells Molecular manipulation Knock-out miceYesYes OverexpressionYesYes Direct Presynaptic measurementsNoYes Distinction of vesicle pools(No)Yes

25. Koh and Bellen, 2003, Trends in Neurosci. 26, 413-422 Südhof, 2002, J. Biol. Chem. 277, 7629-7632. Synaptotagmins are calcium sensors

26. Rhee et al., 2005, PNAS 102, 18664-9 Example 3: synaptotagmin 1 is the fast calcium sensor for synaptic release

27. 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

28. 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 Held Example 2: chromaffin cell studies of SNAP-25 Viral overexpression techniques: knock-out and rescue