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

10 9 neurons synapses 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, Fixed at rest Fixed 5ms after stimulation Synaptic vesicles fuse with the plasma membrane

Südhof TC Annu. Rev. Neurosci 27: 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 Held Example 2: chromaffin cell studies of SNAP-25 Viral overexpression techniques: knock-out and rescue

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

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

Technique 1: capacitance measurements Fusion 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 terminal can be patched directly Calyx of Held Example: Wölfel et al, 2003, J. Neurosci. 23: 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 Held Example 2: chromaffin cell studies of SNAP-25 Viral overexpression techniques: knock-out and rescue

Technique 2: amperometry modified from Westerink, 2004, Neurotoxicology 25, mV

Technique 2: amperometry Amperometry 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:

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

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

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

Schneggenburger and Neher, 2000, Nature 406: Calyx of Held Example 1: photorelease of caged-calcium reveals the true calcium-dependence of fast release

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

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

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

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

Koh and Bellen, 2003, Trends in Neurosci. 26, Südhof, 2002, J. Biol. Chem. 277, Synaptotagmins are calcium sensors

Rhee et al., 2005, PNAS 102, 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 Held Example 2: chromaffin cell studies of SNAP-25 Viral overexpression techniques: knock-out and rescue