Techniques for synaptic vesicle recycling

1. Electrophysiology

2. Imaging

3. Electron microscopy

• FM dyes• SynaptopHluorin• Quantum dots

Sankaranarayanan et al, Biophys J 2000

2. 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: SynaptopHluorinTransgenetic 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 2009

2. Imaging: quantum dots

Detection of full fusion and kiss-and-run by quantum dots

Zhang et al, Science 2009

2. Imaging: quantum dots

3. Electron microscopy

(1) Docked vesicles(2) Endocytic vesicle biogenesis

Rettig & Neher, Science 2002

Hayashi et al, PNAS 2008

3. Electron microscopyDefects in synaptic vesicle endocytosis

Adrenal gland

Chromaffin cells as the model systemChromaffin cells as the model systemfor vesicle cyclingfor 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 systemChromaffin cells as the model systemfor vesicle cyclingfor vesicle cycling

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

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)

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

Whole cell capacitance technique

stimulation

Cap

acit

ance

(p

F)

60 fF

Ca2+ photolysis

NP-EGTA

Whole-cell capacitance technique and Ca2+ photolysis

Rettig & Neher, Science 2002

Cell-attached capacitance to detect single vesicle fusion

Conductance

Capacitance

Patch pipette

Patch pipette

Chromaffin cell

0.1 nS

1 fF

0.5 nS

10 ms

Conductance

Capacitance

Fusion poreconductance

Fusion pore

PM

Carbon fiber+700 mV

Amperometry detects catecholamine release fromsingle vesicles by oxidization

Amperometrical current

Quantal sizeAmperometry

Chromaffin cellFusion pore

PM

Foot

Spike

Technique 2: amperometryAmperometry gives information about the release process

Analysis of single spikes

stand-alone foot

kiss-and-runfull 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-Patch amperometry: a method combines amperometry and cell-attached capacitance measurementattached capacitance measurement

Simultaneous detections of fusion and neurotransmitter release of same vesicle.

Carbon fiber

Patch pipette P

atch

pip

ette

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 pAAmperometrical

Im

Re

Gp

1 pAAmperometrical

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