girardeau paul silvestre de ferron benoit. synaptic transmission is limited by the recycling of...
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A READILY RETRIEVABLE POOL OF SYNAPTIC VESICLES
GIRARDEAU Paul
SILVESTRE DE FERRON Benoit
Synaptic transmission is limited by the recycling of synaptic vesicles for many rounds of use
Introduction Synaptic transmission
The main process of vesicle recycling is mediated by clathrin
The formation of clathrin-coated vesicles from the plasma membrane
BUT slow process (sec to mn)
Introduction Synaptic transmission
2 mechanisms identified
Continuous activity Necessity of rapid recycling
Clathrin mediated : Preassembled structures
Pool of preassembled vesicle proteins
At the pre-synaptic surface
Introduction Vesicle recycling
Clathrin-independent : Kiss and run
What are the temporal dynamics of such a « readily retrievable pool » ?
VARIOUS SYNAPTIC PROTEINS AT THE SYNAPTIC VESICULAR MEMBRANE
Synatptotagmin (Syt1) and Synaptobrevin (Syb2) : most numerous vesicular proteins VGAT (vesicular transporter of amino acids in inhibitory neurons)
Labeling:• of Syt1, Syb 2 and VGAT• to test the hypothesis of the pool of preassembled structures • in rat hippocampal neurons
Introduction Vesicle proteins
Methods Exo-endoxytosis of endogenous synaptic vesicle proteins
Synaptotagmin 1 antibody coupled with cypHer (pH sensitive fluorophore) anti-Syt1-cypHer
VGAT antibody coupled with cypHer (pH sensitive fluorophore)
anti-VGAT-cypHer
Normalized fluorescence of anti-Syt1-cypHer
The fluorescence is maximal at pH 5.5(pH of inside synaptic vesicle)
Cypher emits red fluorescence when excited at 640 nm
For studying inhibitory synapses
7,47,37,27,17,06,96,86,76,66,56,46,36,26,16,05,95,85,75,6
5,5
pH
Synaptotagmin 1 antibody coupled with cypHer (pH sensitive fluorophore)
Synaptotagmin 1 Red fluorescence emission
Spontaneous activity
Results Exo-endoxytosis of endogenous synaptic vesicle proteins
5.5
pH
VGAT antibody coupled with cypHer (pH sensitive fluorophore)
VGAT Red fluorescence emission
Results Exo-endoxytosis of endogenous synaptic vesicle proteins
Fluorescence image of hippocampal neurons labeled with anti-VGAT-cypHer
Results Exo-endoxytosis of endogenous synaptic vesicle proteins
Question Exo-endoxytosis of endogenous synaptic vesicle proteins
Are these probes efficiency to report stimulation-dependent exo-endocytosis?
7,4
7,37,27,17,06,96,86,76,66,56,46,36,26,16,05,95,85,75,6
5,5
pH
Synaptotagmin 1 antibody coupled with cypHer (pH sensitive fluorophore)-> Anti-Syt1-cypHer
Synaptotagmin 1
Red fluorescence emission
Elicited APs at 20 Hz(50, 200, 600, 900)
Results Exo-endoxytosis of endogenous synaptic vesicle proteins
7,4
7,37,27,17,06,96,86,76,66,56,46,36,26,16,05,95,85,75,6
5,5
pH
Synaptotagmin 1 antibody coupled with cypHer (pH sensitive fluorophore)-> Anti-Syt1-cypHer
Synaptotagmin 1
Red fluorescence emission
Results Exo-endoxytosis of endogenous synaptic vesicle proteins
Elicited APs at 20 Hz(50, 200, 600, 900)
Results
These probes are efficiency to report stimulation dependent exo-endocytosis
Exo-endoxytosis of endogenous synaptic vesicle proteins
50 APs 100 APs 200 APs
Results Size of the surface pool of synaptic vesicle constituents
200 APs
Previous studies :Endocytic rate after stimulus = Endocytic rate during the stimulus
Fluorescence variations rise linearly with stimulus strength
Question Size of the surface pool of synaptic vesicle constituents
Are endogenous synaptic vesicle proteins already present on the presynaptic membrane?
7,4
7,37,27,17,06,96,86,76,66,56,46,36,26,16,05,95,85,75,6
5,5
pH
Synaptotagmin 1 antibody coupled with cypHer (pH sensitive fluorophore)-> Anti-Syt1-cypHer
Synaptotagmin 1
Red fluorescence emission
Buffer of pH 5,5
Buffer of pH 5,5Proteins already presents
on the presynaptic membrane
Num
ber o
f bou
tons
Results Size of the surface pool of synaptic vesicle constituents
These proteins are able of compensating exocytosis induced by 70 APs
ΔF ~ 50 a.u.
ΔF = size of the surface poolΔF
Methods Labeled antibodies report same recycling kinectics as spH
Dual-color imaging
Exogenous probe
Overexpressed probe
Synaptotagmin 1 antibody coupled with cypHer (pH sensitive fluorophore) anti-Syt1-cypHer
Fluorescence in acidic compartments
Synaptobrevin 2 coupled with pHluorin (pH sensitive GFP) SpH
Fluorescence in neutral compartments
200 APs at 20 Hz
Synaptotagmin 1
Red fluorescence emission
Synaptotagmin 1 antibody coupled with cypHer (pH sensitive fluorophore)-> Anti-Syt1-cypHer
Synaptobrevin 2 coupled with pHluorin (pH sensitive fluorophore)-> SpH
Green fluorescence emission
pH
5,5
7,4
Results Labeled antibodies report same recycling kinectics as spH
Synaptotagmin 1
Red fluorescence emission
Synaptotagmin 1 antibody coupled with cypHer (pH sensitive fluorophore)-> Anti-Syt1-cypHer
pH
5,5
7,4
Synaptobrevin 2 coupled with pHluorin (pH sensitive fluorophore)-> SpH
Green fluorescence emission
Mirror-image signals
200 APs at 20 Hz
Results Labeled antibodies report same recycling kinectics as spH
These probes report exo-endocytosis
Results Labeled antibodies report same recycling kinectics as spH
Same results in inhibitory synapses with VGAT
Question A surface RRetP of synaptic vesicle constituents
Are synaptic vesicle proteins, exo- and endocytosed by the same stimulus,
identical or different?
Vesicle proteins already present on the presynaptic membrane
Methods A surface RRetP of synaptic vesicle constituents
Inactivation of Synaptobrevin 2 fluorescence on the presynaptic membrane TEV cleavage site between Synaptobrevin 2 and pHluorin
Inactivation of Synaptotagmin 1 fluorescence on the vesicles Photobleaching
First experiment
Second experiment
Synaptotagmin 1
Synaptotagmin 1 antibody coupled with cypHer (pH sensitive fluorophore)-> Anti-Syt1-cypHer
pH
5,5
7,4
Synaptobrevin 2 coupled with pHluorin (pH sensitive fluorophore)-> SpH
TEV cleavage site
pH
Tobacco etch virus (TEV) protease
50 APs at 20 Hz
A surface pool of vesicle proteins is
endocytosed
Results A surface RRetP of synaptic vesicle constituents
Synaptotagmin 1
Synaptotagmin 1 antibody coupled with cypHer (pH sensitive fluorophore)-> Anti-Syt1-cypHer
pH
5,5
7,4
Synaptobrevin 2 coupled with pHluorin (pH sensitive fluorophore)-> SpH
pH
Photobleaching
50 APs at 20 Hz
Presorted synaptic vesicle proteins are preferentially endocytosed on exocytosis
Results A surface RRetP of synaptic vesicle constituents
Results A surface RRetP of synaptic vesicle constituents
Endocytosis of Syt1 or VGAT is not perturbed by spH overexression
Without spH overexpression
With spH overexpression
Results A surface RRetP of synaptic vesicle constituents
Readily Retrievable Pool (RRetP)
RRP seems to be counterbalanced by an RRetP
of similar size
Readily Releasable Pool (RRP)
Partial bleaching of the surface pool
Question Spatial organization of the RRetP
How is the functionnal surface pool spatially organized at the presynapse?
Methods Spatial organization of the RRetP
Antibody to Synaptotagmin 1
Secondary antibody
Antibody to Synaptotagmin 1
Secondary antibody
Antibody to RIM (Active Zone)
Secondary antibody
Antibody to Homer1 (Post Synaptic Density)
Secondary antibody
First labeling Second labeling
IsoSTED microscopy4Pi microscopy
Results Spatial organization of the RRetP
H
H
H
R
R
R
Presynaptic PostsynapticR
H
RIM (AZ)
Homer1 (PSD)
Syt1 (?)
?
IsoSTED
Results Spatial organization of the RRetP
H
H
H
R
R
R
?Presynaptic Postsynaptic
R
H
RIM
Homer1
Syt1Axon
d
α
Doghnut-like arrangement around the active zone of the RRetP
Discussion and conclusion
Vesicle proteins already presents on the presynaptic membrane
Presorted synaptic vesicle proteins are preferentially endocytosed on exocytosis
Doghnut-like arrangement around the active zone of the RRetP
RRP seems to be counterbalanced by an RRetP of similar size
Discussion and conclusion
Results A surface RRetP of synaptic vesicle constituents
200 APs
50 APs
Incomplete depletion of the RRetP during the 1st stimulus
Partial replenishment of the RRetP after the 1st stimulus
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