1. Techniques for synaptic vesicle recycling 1.Electrophysiology 2.Imaging 3.Electron microscopy FM dyes SynaptopHluorin Quantum dots

2. Sankaranarayanan et al, Biophys J 2000 2. Imaging: SynaptopHluorin SynaptopHluorin Miesenbock et al, Nature 1998

3. SynaptopHluorin reports synaptic vesicle exocytosis Sankaranarayanan & Ryan, Nat Cell Biol 2000

4. 2. Imaging: SynaptopHluorin Bafilomycin to separate exo/endocytosis Mani et al, Neuron 2007

5. 2. Imaging: SynaptopHluorin Transgenetic mice expressing SynaptopHluorin Li et al, PNAS 2005

6. 2. Imaging: Synaptophysin-pHluorin Granseth et al, Neuron 2006

7. Full fusion and kiss-and-run Harata et al, J Neurochem 2006

8. Detection of full fusion and kiss-and-run by quantum dots Zhang et al, Science 2009 2. Imaging: quantum dots

9. Detection of full fusion and kiss-and-run by quantum dots Zhang et al, Science 2009 2. Imaging: quantum dots

10. 3. Electron microscopy (1) Docked vesicles (2) Endocytic vesicle biogenesis Rettig & Neher, Science 2002

11. Hayashi et al, PNAS 2008 3. Electron microscopy Defects in synaptic vesicle endocytosis

12. Adrenal gland Chromaffin cells as the model system for vesicle cycling

13. Differences between neurons and chromaffin cells Intracellular vesicle trafficking pathway

14. Voets et al, Neuron 2003 Munc-18 is important for vesicle docking

15. Adrenal gland Chromaffin cells as the model system for vesicle cycling Techniques: Capacitance measurements Amperometry Big vesicle size (150-450 nm in diameter) Oxidizable catecholamine

16. AC and DC current DC - direct current - the polarity of a current source remains the same when the current is DC AC - Alternative current - the polarity of a current source is constantly changing when the current is AC Membrane conductance and capacitance

17. Technique 1: capacitance measurements Time domain technique

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

19. Whole cell capacitance technique stimulation Capacitance (pF) 60 fF

20. Ca 2+ photolysis NP-EGTA

21. Whole-cell capacitance technique and Ca 2+ photolysis Rettig & Neher, Science 2002

24. Cell-attached capacitance to detect single vesicle fusion Conductance Capacitance Patch pipette Chromaffin cell 0.1 nS 1 fF 0.5 nS 10 ms Conductance Capacitance Fusion pore conductance Fusion pore PM

25. Carbon fiber +700 mV Amperometry detects catecholamine release from single vesicles by oxidization Amperometrical current Quantal size Amperometry Chromaffin cell Fusion pore PM Foot Spike

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

27. Different isoform of synaptotagmin controls the choice between full fusion and kiss-and-run Wang et al, Nature 2003

28. Patch amperometry: a method combines amperometry and cell- attached capacitance measurement Simultaneous detections of fusion and neurotransmitter release of same vesicle. Carbon fiber Patch pipette Catecholamine release Vesicle capacitance Fusion dynamics Does the fusion-pore size limit neurotransmitter release? Albillos et al., Nature 1997

29. 500 ms 400 pS 100 pS 1 fF 10 pA Amperometrical Im Re Gp 1 pA Amperometrical 50 pS 0.5 pA Fusion-pore Gp Amperometrical signal The neurotransmitter release is limited by the size of fusion pore Gong et al, Nat Cell Biol 2007

30. Neuroendocrine chromaffin cells: 1.Whole-cell capacitance technique 2.Cell-attached capacitance technique 3.Amperometry 4.Patch amperometry