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

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

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

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

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

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

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

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

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

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

Hayashi et al, PNAS Electron microscopy Defects in synaptic vesicle endocytosis

Adrenal gland Chromaffin cells as the model system for vesicle cycling

Differences between neurons and chromaffin cells Intracellular vesicle trafficking pathway

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

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

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

Technique 1: capacitance measurements Time domain technique

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

Whole cell capacitance technique stimulation Capacitance (pF) 60 fF

Ca 2+ photolysis NP-EGTA

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

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

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

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

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

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

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

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