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Synaptic transmission: communication between


Machinerie d’exocytose

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Two principal kinds of synapses: electrical and chemical Chemical synapses: the predominant means of
communication between neurons
Presynaptic Active Zone

Criteria that define a neurotransmitter:
An early experiment to support the neurotransmitter 1. Must be ppresent at p
y p terminal
hypothesis 2. Must be released by depolarization, Ca++-dependent
3. Specific receptors must be present
Neurotransmitters may be either small molecules or peptides
Neurotransmitter is released in discrete packages, or quanta
Mechanisms and sites of synthesis are different

Smallll molecule
S l l Peptides, or
transmitters are neuropeptides are
synthesized at synthesized in the
terminals, endoplasmic
d l i
packaged into reticulum and
small clear-core transported to the
vesicles (often synapse,
referred to as sometimes they
synaptic vesicles’
vesicles are pprocessed
along the way.
Neuropeptides are
packaged in large

Failure analysis reveals that neurons release many
quanta of neurotransmitter when stimulated
stimulated, that all
contribute to the response

Quantal content: Quantal
Q t l size:
The number of How size of the
quanta released
q individual quanta
by stimulation of
the neuron

From Kristin Harris Lectures.
Quanta correspond to release of individual synaptic

EM images and biochemistry suggest that a MEPP could
be caused by a single vesicle

EM studies revealed correlation between fusion of
vesicles with plasma membrane and size of postsynaptic

From Kristin Harris Lectures.

Stimulation Evoked amplitudes.
4X 1X
i i Mini histogram.



1 mV

Squire Fund. Neurosci.
Calcium influx is necessary for neurotransmitter
CNS synapses and
d quanta.
t release

• A
At synapses withi h only
l a single
i l release
l site,
changing the probability of release (changing
calcium concentration) does not effect the
amplitude of the response (as only zero or one
vesicle is released).
• At synapses with multiple release sites, changing
probability can change the response amplitude as
more transmitter is released.
• At the NMJ a single nerve can elicit a postsynaptic channels
AP given multiquantal release, while at the CNS
p synapses
y p must cooperate,
p , forces a

Calcium influx is sufficient for neurotransmitter release
Synaptic release II

The synaptic vesicle release cycle

1. Tools and Pools

2. Molecular biology and biochemistry of vesicle release:
1. Docking
2 Priming
3. Fusion

3. Recovery and recycling of synaptic vesicles
The synaptic vesicle cycle Synaptic transmission is an adaptatio
of normal vesicle trafficking.

How do we study vesicle dynamics?
y p vesicle release consists of three
Morphological techniques principal steps:
Electron microscopy to obtain static pictures of vesicle distribution; TIRFM (total
internal reflection fluorescence microscopy) to visualize movement of vesicles close
1. Docking
to the membrane
Docked vesicles lie close to plasma membrane (within 30
y g studies nm)
Chromaffin cells
Neuroendocrine cells derived from adrenal medulla with large dense-core vesicles.
Can measure membrane fusion ((capacitance
p measurements), ), or direct release of 1. Priming
catecholamine transmitters using carbon fiber electrodes (amperometry) Primed vesicles can be induced to fuse with the plasma
Neurons membrane by sustained depolarization, high K+, elevated
Measure release of neurotransmitter from a presynaptic cell by quantifying the Ca++, hypertonic
yp sucrose treatment
response of a postsynaptic cell
2. Fusion
Ge et cs
Delete or overexpress proteins in mice, worms, or flies, and analyze phenotype Vesicles fuse with the plasma membrane to release
using the above techniques transmitter. Physiologically this occurs near calcium
channels, but can be induced experimentally over larger area
(see ‘priming’). The ‘active zone’ is the site of physiological
release, and can sometimes be recognized as an electron-
dense structure.
Vesicle release requires many proteins on vesicle and
Neurotransmitter Release plasma membrane

E Neher T Sudhöf P De Camilli

R Jahn

SNAREs: targets of clostridial NTs SNAREs: targets
g of clostridial neurotoxins
C Montecucco

H Niemann
Jahn and Scheller Nature Reviews Molecular Cell Biology 7, 631–643 (2006) | doi:10.1038/nrm2002

JE Rothman D Tareste



d La vésicule s’est arrimée
Syn1A/SNAP-25 a hemifusionné
ou a fusionné
Membrane cible
Les SNAREs n’interagissent
n interagissent Le SNAREpin commence à Le SNAREpin est partiellement
pas encore (d > 8 nm) se former (d ~ 8 nm) assemblé (d < 4 nm)

~ 35 kBT
Role of the linker region
D Bruns

Jahn and Scheller Nature Reviews Molecular Cell Biology 7, 631–643 (2006) | doi:10.1038/nrm2002

A Brunger
The SNARE complex
Vesicles in the reserve pool undergo priming to enter the readily-
releasable pool

At a molecular level, priming corresponds to the assembly of the
SNARE complex

(Sutton et al., Nature 1998)
The SNARE complex

domain, folds
back on itself
“open” syntaxin
doesn’tt fold

Synaptotagmin functions as a calcium sensor,
promoting vesicle fusion
Calcium & exocytosis
Regulation by calcium: through
i ?

off sytt

Syt accelerates membrane fusion in vitro
SNARE & synaptotagmin
Syt acts through SNAREs and lipids

ER Chapman

Annuall Reviews

Regulation by complexin & A complexin-tagmin
complexin tagmin cycle?
T Sudhöf synaptotagmin
y p g C Rosenmund
Synaptic vesicles exist in multiple pools within the nerve terminal

Regulation, regulation (Release stimulated by flash-photolysis of caged calcium)

• Much more is known:
(reserve pool)

Munc 13
Munc-13 Munc 18

N Brose M Verhage

• Much
M h more tto come: B h
Becherer, U
U, R
tti J.J Cell
C ll Tissue
Ti Res
R (2006) 326
Morphologically, vesicles are classified as docked or undocked. Docked vesicles
????? are further subdivided into primed and unprimed pools depending g on whether
they are competent to fuse when cells are treated with high K+, elevated Ca++,
sustained depolarization, or hypertonic sucrose treatment.

In CNS neurons, vesicles are divided into
UNC-18 (or munc-18) is necessary for vesicle docking
Reserve pooll (80
95%) (W i
(Weimer ett al.
l 2003,
2003 Nature
N t N
i 6

Recycling pool (5-20%) 1. unc
18 mutant C. elegans have neurotransmitter release defect

y pool ((0.1-2%; 5-10 synapses
p y p p
per active zone)) 2. unc-18 mutant C. elegans have reduction of docked vesicles

Rizzoli, Betz (2005). Nature Reviews Neuroscience 6:57-69)

A small fraction of vesicles (the recycling pool)
replenishes the RRP upon mild stimulation. Strong
stimulation causes the reserve pool to mobilize and
be released
Unc-18 mutants are defective for calcium-independent release
Unc-18 mutants are defective for evoked and spontaneous release

primed vesicles occasionally fuse in the absence of calcium; a
calcium-independent fusion defect suggests a lack of primed

UNC-18 (munc18) is required for docking:
unc 18 mutants have fewer docked vesicles Summary:

Unc-18 mutants are unable to dock vesicles efficiently.
Impaired docking leads to fewer primed vesicles; fewer
primed vesicles leads to reduced overall neurotransmitter
Synaptic vesicles recycle post-fusion

B Davletov
Endocytosis retrieves synaptic vesicle membrane and
Modern methods to track recycling membrane protein from the plasma
p p membrane followingg fusion
The ATP-ase NSF
disassembles the SNARE
complex, then clathrin-
dependent endocytosis
compensates for exocytosis

P De Camilli