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Nature Reviews Neuroscience | AOP, published online 18 July 2007; doi:10.1038/nrn2191
The sequence of events that underlie
quantal transmission at central
glutamatergic synapses
John E. Lisman*, Sridhar Raghavachari‡ and Richard W. Tsien§
Abstract | The properties of synaptic transmission were first elucidated at the neuromuscular
junction. More recent work has examined transmission at synapses within the brain. Here we
review the remarkable progress in understanding the biophysical and molecular basis of the
sequential steps in this process. These steps include the elevation of Ca2+ in microdomains of
the presynaptic terminal, the diffusion of transmitter through the fusion pore into the synaptic
cleft and the activation of postsynaptic receptors. The results give insight into the factors that
control the precision of quantal transmission and provide a framework for understanding
synaptic plasticity.
*Brandeis University,
Department of Biology, MS
008, 415 South Street,
Waltham, Massachusetts
02454-9110, USA. ‡Duke
University, Department of
Neurobiology, Box 3209,
Duke University Medical
Center, Durham, North
Carolina 27710, USA.
§
Stanford University School of
Medicine, Department of
Molecular & Cellular
Physiology, Beckman Center,
Room B100, 279 Campus
Drive, Stanford, California
94305-5345, USA.
Correspondence to J.E.L.
e-mail: lisman@brandeis.edu
doi:10.1038/nrn2191
Published online 18 July 2007
The basic properties of synaptic transmission were
elucidated in pioneering work at the neuromuscular
junction (NMJ). Studies showed that the arrival of the
action potential at the presynaptic terminal activates
voltage-dependent Ca2+ channels and that the resulting
elevation of intracellular Ca2+ stimulates transmitter
release1. Furthermore, it was shown that the amplitude
of the postsynaptic response varies by an integral multiple of a stereotyped quanta. Later anatomical work2,3
provided evidence that synaptic vesicles are the basis of
quantal transmission. Ca2+ stimulates the formation
of a fusion pore between such vesicles and the presynaptic membrane. The neurotransmitter contained in the
vesicle passes through this pore into the synaptic cleft,
where it activates postsynaptic receptors. Computer
simulations showed that the properties of this diffusion,
together with the properties of the postsynaptic receptors, could satisfactorily account for the kinetics of the
quantal response4. The basic steps in the overall process
of transmission are illustrated in FIG.1.
Chemical transmission at mammalian central synapses proved more difficult to study because of the
synapses’ small size. However, methodological advances
have now made it possible to study at least some central
synapses with great precision. Much of this work has
utilized two giant synapses, the Calyx of Held5 and the
mossy fibre synapses in the hippocampal CA3 (Cornu
Ammonis) region6. At these structures, it is possible to
make simultaneous intracellular recordings from the
pre- and postsynaptic sites. Such recordings (FIG. 2a)
show that the synaptic delay between the arrival of an
NATURE REVIEWS | NEUROSCIENCE
action potential at the presynaptic terminal and the onset
of the excitatory postsynaptic current (EPSC) is ~0.6 ms
(~1 ms to EPSC midpoint). In this Review we discuss
the sequence of molecular events that occur during this
period, and how they determine the properties of the
quantal response. Our focus will be on synapses that
use the neurotransmitter glutamate, the most common
excitatory transmitter in the CNS. We aim to convey how
biophysical and molecular methods have provided a deep
understanding of synaptic transmission, an understanding that will be vital for discovering the modifications of
transmission that underlie learning and memory.
Properties of Ca2+ elevation that trigger release
Pioneering work on the squid giant synapse showed how
action potentials stimulate the release of synaptic vesicles by triggering the opening of voltage-dependent Ca2+
channels in the presynaptic terminal7,8. Recent work has
investigated this issue at vertebrate central glutamatergic
synapses6; the results at mossy fibre synapses are shown
in FIG.2a. In the first stage of a two-stage process, Ca2+
channels (primarily P/Q- and N-type9–13) open rapidly
(0.1 ms) during the peak of the action potential (see
BOX 1 for a description of Ca2+ channels). However, the
Ca2+ current is small because the voltage is so positive
that the electrochemical driving force is low. The second
stage occurs during the descending phase of the action
potential; the lowered voltage augments the driving force
and thereby strongly increases the Ca2+ influx. This twostage process accounts for a substantial part (0.4 ms) of
the synaptic delay at giant synapses (FIG. 2a).
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Depolarization
opens Ca2+
channels
Action potential in
bouton generated
by opening of
Na+ channels
+
+
+
Presynaptic
bouton
+
+
+ +
+
+
+
+
+
+
+
+
+
+
+
+
++
+ Ca2+
+
++++
+
+
+
+
+
++
+
+
+
++
Ca2+ elevation
occurs in
microdomain
Ca2+ binds to
synaptotagmin
causing opening
of fusion pore
Glutamate passes
through fusion
pore and diffuses
in the cleft
Ca2+
+
+
Dendritic
spine
+ + +
+
+
+
+
+
+
+
+
+
+
+
Opening of
AMPA channel
generates EPSC
Figure 1 | Steps in the process of chemical synaptic transmission. These steps occur in both vertebrates and invertebrates,
at the neuromuscular junction and central synapses. Cartoons based on a drawing by J. A. Ernst and A. Brunger.
Synapse
The term synapse can be used
either in a structural sense or to
describe an entire connection.
According to the structural
definition, a synapse consists
of a single presynaptic active
zone and postsynaptic density,
together with the specialized
membranes and cleft
in-between. Synapse diameter
is between 0.2 and 1 micron.
At most dendritic spines, there
is a single such synapse. Giant
synaptic connections have
many structural synapses; the
mossy fibre boutons in CA3
have over 10, whereas the
Calyx of Held has
approximately 50.
Quanta
The elementary building block
of the EPSC, which contains an
integral number of these
events. Quantal size is derived
from the distance between the
peaks in the amplitude
histogram of the EPSC, and
equals the amplitude of the
mEPSC at synapses where
these events are uniquantal.
Fusion pore
Provides the passage from the
interior of the vesicle into
the synaptic cleft, through
which neurotransmitter
diffuses.
Excitatory postsynaptic
current
(EPSC). The inward
postsynaptic membrane
current evoked by a single
presynaptic action potential.
It is measured using the
voltage-clamp technique.
Intriguingly, there are indications that the smaller
synapses of the cerebellum may have found a onestage solution. At these synapses, substantial Ca 2+
entry occurs at the peak of the action potential14. This
is perhaps because fast K+ channels prevent the peak
of the action potential from becoming too positive,
and thereby retain the Ca2+ driving force15. For such
a solution to work, Ca2+ channels must open rapidly,
and there is at least one example to show that this can
occur16.
Properties of the Ca2+ signal in the microdomain. As
Ca2+ enters the presynaptic terminal, it can be effective immediately because only the concentration near
the Ca2+ channels themselves has to rise, rather than
that of the bulk cytoplasm. This is because the Ca2+
sensors that trigger vesicle release are strategically
located within a ‘microdomain’ near the Ca2+ channels.
The first support for this concept came from work on
the giant synapse of the squid17,18, and indicated that the
Ca2+ concentration could rise to 200 μM in the microdomain. An important advance came with the development of structural methods19–21 that localized the Ca2+
channels. At the NMJ, it was determined that vesicles
are positioned within 20 nm of the Ca2+ channels — so
close that the vesicle actually hinders diffusion of Ca2+
away from the channel. Strongly constrained simulations indicate that vesicle release is due to Ca2+ entry
through just one or two nearby (within ~10–20nm)
Ca2+ channels22.
Recent work has analysed microdomain properties at
central glutamatergic synapses23,24 and revealed a different picture. In particular, work in the laboratories of Bert
Sakmann and Erwin Neher provided evidence about
what occurs in the microdomains at the Calyx of Held.
The release of synaptic vesicles was monitored during
defined Ca2+ changes produced by uncaging methods.
Once the relationship between Ca2+ levels and release
rate was determined, it was used in reverse to predict
the Ca2+ changes that trigger the release produced by
598 | ADVANCE ONLINE PUBLICATION
an action potential. It was concluded that Ca2+ in the
microdomain rises to 25 μM25 (or 10 μM using a different method26). The duration of this elevation is about
300 μs (FIG. 2b). In complementary experiments, the
artificial production of such brief Ca2+ signals triggered
release that satisfactorily mimicked those produced by
an action potential27.
Although much is now known about the structure of
central synapses (FIG. 3), the distance between the Ca2+
channels and the vesicles has not been directly measured. The fact that Ca2+ channels can bind directly to
vesicle proteins suggests that some close associations
occur28–30. However, the influence of exogenous Ca2+
buffer on release is greater than would be expected if
Ca2+ has to move only tens of nanometers before activating release. This suggests that the microdomain at
central synapses is considerably larger than at the NMJ.
Models that account for the buffer effects suggest that
approximately ten Ca2+ channels influence release within
a microdomain of ~200 nm31, a dimension only slightly
smaller than the diameter of central synapses (FIG. 3).
Both modelling32 and experimental results33 further
suggest that the Ca2+ channels are clustered, resulting in
Ca2+ gradients within the microdomain. A consequence
of these gradients is that different vesicles will have
different release probabilities.
Importantly, the results of the studies on the Calyx of
Held described above were obtained in young animals.
A recent study34 found that the size of the microdomain
is developmentally regulated. As animals mature, the
effect of slow Ca2+ buffers on release diminishes, indicating a tighter spatial coupling between Ca2+ channels
and sites of vesicle release, as has been found at the
NMJ and squid giant synapse. Further progress in this
field will probably require the localization of the Ca2+
channels at central synapses, and the elucidation of
what a release site actually is. Specialized ‘triangular’
particles can be seen in the active zone34–37 (FIG. 3a), but
the hypothesis that they organize vesicles for release
remains to be tested. Recent work with tomographic
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© 2007 Nature Publishing Group
500 pA
EPSC
1 nA
20
150
10
0
0
0
Synaptic delay 0.5 ms
1
2
3
d
12
[Glu] (mM)
Ca2+ current
300
Local [Ca]i (μM)
100 mV
c
30
Release rate
Local [Ca]i
Response (pA)
b
Action potential
Release rate
(vesicles per ms)
a
6
mEPSC data
Simulation
0
4
0.0
Time (ms)
0.3
Time (ms)
0.6
Open receptors
Bound receptors
Glutamate in
hotspot
2
40
30
20
1
10
0
Number of receptors
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0
0
0.1
0.2
0.3
0.4
0.5
Time (ms)
Figure 2 | Timing of steps in transmission. a | Simultaneous recording of the membrane potential and currents from a
mossy fibre presynaptic terminal and a CA3 hippocampal postsynaptic cell allows the determination of synaptic delay. The
presynaptic Ca2+ current was determined by blocking all other conductances and imposing an action potential waveform
by voltage clamp (at physiological temperature). After subtraction of the capacitative current, what remains is the Ca2+
current. b | The Ca2+ elevation and the vesicle release rate as a function of time after an action potential at the Calyx of Held
(at room temperature). c | A mEPSC waveform recorded from a CA1 hippocampal dendrite is compared with a simulated
(average) waveform based on a model of glutamate diffusion in the cleft and a kinetic model of the AMPA channel. d | Postrelease steps in the generation of the mEPSC. The glutamate concentration in the cleft (averaged over the hotspot of AMPA
channel activation) rises rapidly after opening of the fusion pore. The number of receptors with at least one glutamate
bound follows the glutamate elevation with little delay, but channels open with a substantial further delay due to the time
required for conformational transitions. Simulations as in REF. 77. AMPA, α-amino-3-hydroxy-5-methylisoxazole-4-propionic
acid; CA, Cornu Ammonis; EPSC, excitatory postsynaptic current; mEPSC, miniature excitatory postsynaptic current. Part a
reproduced with permission from REF. 6 © (2000) Cell Press. Part b reproduced with permission from Nature REF. 25 ©
(2000) Macmillan Publishers Ltd. Part c reproduced with permission from REF. 77 © (2004) American Physiological Society.
Electrochemical driving
force
The difference between the
Nernst equilibrium potential
for an ion and the membrane
voltage. The current through
a channel is the product of the
channel conductance and the
electrochemical driving force.
Uncaging methods
The standard form of a
compound is modified by
chemical adducts, which can
be cleaved by bright light to
produce the standard form.
One application of this
technology allows rapid lightinduced Ca2+ elevation.
Release site
A site in the active zone where
the vesicle is released. It
remains unclear whether there
is a structural basis for such
sites, or whether release can
occur anywhere in the active
zone provided that the
appropriate vesicle priming has
occurred.
analysis of hippocampal synapses frozen under high
pressure is beginning to provide data relevant to this
question. As shown in FIG. 3c , large synapses have
several clusters of active zone material. These clusters
contain the triangular particles mentioned above.
Importantly, docked vesicles occur only within these
clusters. Moreover, unidentified, specialized filaments
(FIG. 3b) seem to guide vesicles to triangular particles.
The clarification of the function of these structures
will refine our understanding of what happens in the
microdomain. However, regardless of what these findings show, the importance of the microdomain is clear:
within it, Ca2+ channel opening leads to rapid (~50 μs)
Ca2+ elevation32; after the channels close, diffusion out
of the microdomain quickly reduces the Ca2+ signal31,
thereby ensuring that there is no further activation of
the Ca2+ sensor.
The Ca2+ sensor and fusion pore
Once Ca2+ elevation occurs in the microdomain, it
triggers the release of vesicles with a delay of about
300 μs25,38 (FIG. 2b) (the delay will be shorter if Ca2+ levels
are higher). The first reaction that occurs during this
period is the binding of Ca2+ to the sensors that trigger
vesicle release. Early work suggested that the sensor
becomes activated only after the binding of many
(four to five) Ca2+ ions39, something which has been
confirmed using direct methods25,26,40. Importantly,
during an action potential, only a fraction of the sensors become saturated, and this helps to account for
why only a fraction (less than one-fifth) of the readily
releasable pool of vesicles is released25. The time that
it takes to load the sensors can be calculated to be
approximately 200–300 μs (assuming that binding
takes place near the diffusion limit (108 M–1 s–1) and
that the Ca2+ concentration equals 30 μM). This leaves
NATURE REVIEWS | NEUROSCIENCE
only a short time (~100 μs) for the complex to actually
produce a vesicle release25.
The fact that the sensors are both unsaturated and
nonlinear detectors makes the release highly sensitive
to the Ca2+ concentration. This has important consequences for the modulation of transmission and shortterm synaptic plasticity. During such changes, the release
produced by the action potential can be enhanced
by residual Ca 2+ from a previous action potential,
or by modulation of the voltage-dependent channels that shape the Ca2+ entry produced by an action
potential24,41–43.
The period of elevated release rate. Once Ca2+ fills the
sites on the Ca2+ sensor to form an active complex,
vesicle release can occur. Actual release occurs probabilistically until the concentration of the complex falls44,45.
At the Calyx of Held, this period of elevated release
lasts for approximately 300 μs; because the vesicle
is released probablistically during this period, there is
a jitter in the time of actual vesicle release (FIG. 2b).
This jitter explains why the rising edge of the multiquantal EPSC is slower than that of the quantal event
(FIG. 2a,c). The jitter compromises the temporal precision of the synapse, and one would expect it to be
minimized. Consistent with this expectation, the release
of Ca2+ from synaptotagmin (the probable Ca2+ sensor
for fast vesicle release) is rapid46, making the lifetime of
the active complex only slightly longer than the period
of Ca2+ elevation. In some cells, the time-dependence of
the release rate shows a long tail, producing what is
termed ‘delayed release’. Although the basis of this effect
is not completely known, a tail of Ca2+ elevation appears
to be important. This elevation is due to the effect of
endogenous Ca2+ buffers that have been loaded with Ca2+
during the action potential47.
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Box 1 | Voltage-dependent Ca2+ channels that control transmitter release
Voltage-dependent Ca2+ channels are often classified according to their biophysical and
pharmacological properties133–135. The channels that support mammalian
neurotransmission (N-, P/Q- and R-type), among other functions, are different from
those that dominate excitation – contraction or excitation –transcription coupling
(L-type) or pacemaker activity (T-type and L-type). The presynaptic Ca2+ channels are
exemplified by N-type channels, first discovered in recordings from the cell bodies of
sensory neurons136 and later found to support neurotransmitter release from
sympathetic neurons137 and cholinergic nerve terminals in the ciliary ganglion138,139.
P/Q- and R-type channels were discovered in the early 1990s13,140–142, and also contribute
in varying degrees to neurotransmission at various CNS synapses143-145, including the
Calyx of Held146. P/Q-, N- and R-type channels are composed of pore-forming subunits
known as CaV2.1, CaV2.2 and CaV2.3, respectively147; each is the specific target of a
blocking toxin (ω-AgaIVA, ω-CTx-GVIA and SNX-482)148. Auxiliary subunits (α2-δ and β)
contribute to proper cellular trafficking and the function of the pore-forming α1
subunits134,149.
At most CNS synapses, neurosecretion is supported by more than one type of channel,
but some synapses may be heavily dominated by a single channel type (for example,
certain inhibitory neurons rely almost entirely on N-type). At some synapses, N-type
channels develop early, but are gradually replaced by P/Q-type channels150.
Why are there different kinds of presynaptic Ca2+ channel? Almost certainly, they are
not a result of evolutionary pressure for different biophysical properties. Indeed, basic
features such as extreme Ca2+ selectivity, high open channel flux rate and steeply
voltage-dependent channel gating are similar for P/Q-, N- and R-type currents. In all
cases, selectivity and permeation are conferred by a pore that is continually occupied by
at least one Ca2+ ion, which prevents similarly sized and more abundant Na+ ions from
rushing through151,152. Despite overall similarities, there are substantial differences in
channel regulation, and this is likely to be the main reason for channel diversity. For
example, P/Q- and N-type channels appear to differ in their susceptibility to modulation
by G proteins in at least some systems153,154. The structural motifs in α1 subunits support
various forms of regulation, for example, by G proteins and by protein kinases (see
figure). Along with the release machinery itself, the presynaptic Ca2+ channels constitute
a key convergence point for the regulation of neurosecretion. Several neurological
diseases arise from genetic modifications of P/Q-, but not N-type, channels155; however,
N-type channel-mediated neurotransmission is the target of therapeutic agents used to
treat intractable pain156. CaM, calmodulin; CaMKII, Calmodulin kinase II; CBD,
calmodulin binding domain; nCaBP, neuronal calcium-binding protein; PKC, protein
kinase C; SNAREs, Soluble N-ethylmaleimide-sensitive factor attachment protein
receptors; Gβγ, βγ subunit of G protein.
I
II
III
IV
+
1 2 3 45
+
+
1 23 45
+
Outside
+
1 2345
+
6
+
1 23 4 5
+
6
6
6
Inside
C terminus
N terminus
P
CaMKII
CBD
PKC
Synaptotagmin as the Ca2+ sensor. There is now substantial evidence that the Ca2+ sensor for rapid vesicle release
is synaptotagmin 1 (REF. 48); knockout of this protein
in mice abolishes rapid transmission49. Other studies
have shown that the C2B calcium binding domain of
synaptotagmin I acts as the Ca2+ sensor for synchronized,
rapid release of vesicles, while suppressing the slower,
asynchronous component of vesicle release50,51.
As mentioned above, the Ca2+ sensor acts as if it is
cooperatively activated by the binding of four to five Ca2+
ions. The basis of this cooperativity remains unclear.
The C2B domain on synaptotagmin has five Ca2+ binding sites, suggesting that cooperative properties within
synaptotagmin itself may determine the cooperativity of
release. Consistent with this, mutations in synaptotagmin that affect Ca2+ binding reduce this cooperativity52.
However, important aspects of the release process are
not understood. Vesicles contain fifteen synaptotagmin
molecules53, but it is unclear how they work together to
produce release. Also uncertain is how the cooperativity
is influenced by other molecules54 and the interaction of
multiple SNARE complexes55,56.
Opening of the fusion pore by the activated sensor.
Progress has been made in understanding the mechanism
by which the Ca2+–synaptotagmin complex stimulates
vesicle release. The vesicle is held at the synapse by the
SNARE complex48 (FIG. 4a), some components of which
(v-SNAREs) bind to the vesicle membrane, while others
(t-SNAREs) bind to the plasma membrane. Recent structural and membrane labelling studies indicate that the
vesicle may be primed for release by hemifusion36,57,58, a
process in which the proximal leaflets of the plasma and
vesicle membranes fuse (allowing transfer of membrane
markers), without fusion of the distal leaflets (FIG. 4a).
Fusion of the distal leaflets is thought to occur when
the Ca2+–synaptotagmin complex displaces complexin
from the SNARE complex. Complexin may function to
clamp the hemifused state so that its release allows full
fusion and the formation of the fusion pore59–62 (FIG. 4a).
It has generally been thought that the fusion pore is
lipid lined, but there are now some indications that it
is protein-lined63. According to the latter view, synaptobrevin (a v-SNARE) and syntaxin (a t-SNARE) form a
type of ion channel (FIG. 4a) that opens to form the fusion
pore under the influence of the Ca2+–synaptotagmin
complex64. Consistent with the electrostatic interactions
that are characteristic of a standard ion channel, current
through the fusion pore can be bidirectionally modified
by changing the charge on what is thought to be the pore
wall formed by syntaxin65. The controversy over whether
the pore is lipid- or protein-lined only relates to the early
stages of the process; in both scenarios a protein channel
would no longer be involved if the process proceeded to
complete fusion (FIG. 4a).
SNAREs
nCaBP’s/CaM
Gβγ
Figure courtesy of W. A. Catterall
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Conductance and modes of the fusion pore. The diameter of the fusion pore may seem like a technical detail,
but it has surprisingly important consequences. Indeed,
pore size may even have a role in the synaptic plasticity
implicated in learning and memory (see BOX 2 ).
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Synaptic
cleft
a
Active
zone
b
c
Presynaptic
bouton
Synaptic
vesicle
Docked
vesicle
d
Hotspot
Triangular
particle
Dendritic spine
250nm
Postsynaptic
density
0.5 μm
Figure 3 | Synapse structure and the localization of activated AMPA channels. a | An electron micrograph of the
hippocampal CA1 region, showing the presynaptic bouton, synaptic vesicles, active zone, synaptic cleft, postsynaptic
density and spine. This is the first image of synapses in a slice preparation made by rapid freezing, a method that avoids the
artefacts of fixation. The diameter of the active zone defines the synapse diameter. The presynaptic active zone has dense
material that is coextensive with the postsynaptic density, but is more difficult to visualize. The picture shows a dense
particle (triangular) in the active zone, which may be involved in organizing vesicle release. b | A tomographic
reconstruction of the active zone and filaments (pink) leading to the triangular particle attached to the presynaptic
membrane (white). These filaments may organize the process of bringing vesicles (yellow) to the membrane, where they
become docked (blue). c | A tomographic reconstruction of the entire active zone showing the modular organization of
dense active zone material (yellow) and docked vesicles (blue). d | Postsynaptic densities of hippocampal CA1 synapses
reconstructed from serial sections of electron micrographs. The three shown are representative of the different sizes found.
The red disc shows the size of the hotspot where most of the mEPSC is generated near a site of vesicle release77. This region
is only a small fraction of the synapse area. The vesicle is assumed to be released at the centre of the red disc. Part a kindly
provided by S. Marty, using methods from REF. 178. Parts b,c reproduced with permission from REF. 179 © (2007) Society for
Neuroscience. Postsynaptic densities in part d downloaded from Synapse Web by K. M. Harris. CA, Cornu Ammonis.
Impedance method
A rapidly oscillating voltage is
applied by voltage-clamp. The
in-phase component of the
resulting current is termed
the ‘real’ component and can
be related to patch
conductance. The out-of-phase
component is called the
imaginary component and can
be related to patch capacitance.
The advantage of this method is
that both conductance and
capacitance can be measured
simultaneously.
Monte-Carlo simulation
A simulation that involves
keeping track of the position
and state of each molecule. At
each short time step the
computer calculates the new
position or state of each
molecule according to the
probability of each change.
Measurements of pore properties using impedance
methods were first made in non-neuronal cells66, and
recently this method67 was used with patch recordings
from the presynaptic membrane at the Calyx of Held
(the secretory face was exposed by sucking away the
postsynaptic cell). Using this method, the properties
of each released vesicle could be determined from the
change in patch capacitance and conductance (FIG. 4b).
Changes in patch conductance provide information
about the diameter of the fusion pore. Several important
conclusions were drawn. First, the initial opening may
involve either a small or large fusion pore. Second, some
openings are followed by closure, a process termed ‘kissand-run’ which had been previously suggested by various methods68–75. In this mode the vesicle can be reused,
whereas in the ‘full fusion’ mode, the vesicle membrane
is added to the plasma membrane and a vesicle is subsequently reformed by endocytosis at extrasynaptic sites.
An important finding was that kiss-and-run can occur
even after large fusion pore formation, whereas the formation of small fusion pores is not necessarily followed
by kiss-and-run. These results indicate that fusion pore
size and the mode of vesicle recovery are not linked in an
obligatory way. Although the existence of kiss-and-run is
generally accepted, initially in endocrine cells and more
recently in nerve terminals, the fraction of release events
that use this mode is still uncertain. The functional significance of fusion pore size is discussed at the end of
the next section.
NATURE REVIEWS | NEUROSCIENCE
Glutamate diffusion and AMPA channel activation
When the fusion pore opens at glutamatergic synapses,
glutamate (at an initial concentration within the vesicle
of 200 mM76) diffuses through the pore and dilutes
as it spreads throughout the cleft. There it encounters
AMPA (α-amino-3-hydroxy-5-methylisoxazole-4propionic acid) channels at a density of ~1,000 per
μm2 (REFS 77,78). Recent measurements indicate that
diffusion in the cleft is only somewhat lower than
the free value 79. Monte-Carlo computer simulations
can be used to describe the diffusion of glutamate
through the pore and in the cleft. If the properties of
glutamate diffusion and the properties of the AMPA
channel (as described by a kinetic model) are correctly
understood, it should be possible to incorporate these
properties into computer simulations and predict the
amplitude and kinetics of miniature EPSCs (mEPSCs).
These small spontaneous events generally arise from
the spontaneous release of single vesicles (see below
for a qualification).
The development of a kinetic model of the AMPA
channel (see BOX 3) has been based on the measurement of how channels in excised outside-out patches
respond to ultra-fast, controlled applications of glutamate. Experiments of this kind revealed that micromolar levels of glutamate can bind to single subunits of
the channel (there are four glutamate-binding subunits;
see BOX 3) and cause the channel to adopt a long-lived
desensitized state without opening. By contrast, high
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a
Plasma membrane
Hemifusion
Vesicle
Docked vesicle
Vesicle
SNARE
complex
Neurotransmitter
Synaptobrevin II
(v-SNARE)
SNAP-25
50 aF
Im
Vesicle membrane
Syntaxin (t-SNARE)
b
10 pS
2s
Re
Cytoplasm
50 aF
Im
Plasma membrane
Extracellular
space
Extracellular
space
Plasma membrane
c
Lipid-lined fusion pore
EPSC (pA)
Protein-lined fusion pore
5 pS
Re
0
–6
1 nm; 50 pS
2 nm; 200 pS
4 nm; 800 pS
8 nm; 3,200 pS
–12
0
1
2
3
4
Time (ms)
Figure 4 | Vesicle fusion and fusion pores. a | Two potential mechanisms of formation of the fusion pore between
the synaptic vesicle (upper membrane; pink) and the plasma membrane (lower membrane; green). In the top left panel the
vesicle is in the ‘docked’ state in which it is held near the plasma membrane by the SNARE complex. In the top right panel,
the vesicle and plasma membrane have their distal leaflets in a hemifused state that is primed for release. During the
release process, a protein-lined pore (lower left panel) is formed by two of the SNARE proteins, syntaxin and
synaptobrevin. This step may be reversible, or may be followed by a transition to a lipid-lined pore (bottom right panel).
An alternative model is that fusion pore opening always involves the formation of a lipid-lined pore. b | Kiss-and-run
events and full-fusion events observed by capacitance measurements at the presynaptic secretory face of the Calyx of
Held. Real and imaginary components give information about the fusion pore conductance and membrane capacitance
(in attoFarads) respectively. Top traces: the first event on the left-hand side is an increase in capacitance followed shortly
thereafter by an equal decrease, indicating a kiss-and-run event. No conductance change is detectable (large fusion pores
having conductance greater than 288 pS cannot be detected for technical reasons). The second event is longer and thus
indicates full fusion. Bottom traces: kiss-and-run event in which a conductance change was detectable, indicating a small
fusion pore; the average pore conductance of such events is 66 pS. c | The simulated average mEPSC depends on the size
of the fusion pore (diameters and conductance are given). For the smallest pore, the resulting current would be too small
to detect given the presence of noise, creating a ‘whispering’ form of silent synapse. Im, imaginary component of
impedance; mEPSC, miniature excitatory postsynaptic current; Re, real component of impedance; SNAP, soluble
N-ethylmaleimide-sensitive fusion protein attachment protein; SNARE, SNAP receptor. Part a modified with permission
from REF. 63 © (2006) Annual Reviews. Part b modified with permission from Nature REF. 67 © (2006) Macmillan Publishers
Ltd. Calculations in part c are from the model described in REF. 77.
Kinetic model
The kinetic model of an ion
channel specifies the rate
constants for the binding and
unbinding of neurotransmitter
to sites on the channel, and the
rate of conformational
transitions between various
states that control channel
opening and closing, as well as
transitions to a desensitized
state.
levels (near mM) of glutamate induce rapid opening
of the channel followed by desensitization. The results
can be understood as a race between opening and
desensitization; if only one glutamate binds, desensitization wins. The more glutamate that binds, the higher
the probability of opening before desensitization
and the higher the single channel conductance. BOX 3
gives further information about the kinetics of opening
and desensitization and shows a recently derived structural model of the AMPA channel that provides insight
into the mechanism of activation and desensitization.
An important requirement for comparing predicted
to actual mEPSCs is to have accurately measured
mEPSCs. This is generally not simple to achieve
because mEPSCs are generated in dendrites, and
special procedures are required to record such events
602 | ADVANCE ONLINE PUBLICATION
without electrotonic distortion 80,81. In the study by
Smith et al.80, sucrose was applied to a small dendritic
region and the resulting mEPSCs were recorded
intradendritically from that region (measurements
were taken in slices of the hippocampal CA1 region).
This procedure minimized electrotonic distortion and
revealed that the rise-time (0.1 ms) of the mEPSC is
faster than previously suspected.
Figure 2c shows the mEPSC recorded by this method,
as well as the predictions of a Monte-Carlo computer
simulation. It is clear that the amplitude and rise-time
are adequately accounted for. Given that there were
no freely adjustable parameters in the simulations, the
agreement of theory and experiment argues that the main
determinants of the mEPSC amplitude and rise-time are
reasonably well understood.
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Box 2 | Experimental support for the importance of the mode of transmitter release
Evidence for whispering synapses: partially silent synapses
• At the Calyx of Held, a small fraction of release events have a low conductance fusion pore. These could give rise to the
small fraction of mEPSCs that have small amplitudes and slow rise-times67.
• EPSCs at the fly neuromuscular junction show a slow hump on the decay phase. The hump might be the result of
prolonged release of transmitter from the vesicle due to a small fusion pore. Consistent with this, the hump is selectively
reduced by a low affinity antagonist, indicating a low glutamate concentration75.
• Inhibiting desensitization with cyclothiazide dramatically increases the frequency of mEPSCs without affecting
the amount of glutamate released157 or the NMDAR-mediated EPSC (but see also REF. 158). One explanation is that the
fusion pore conductance for many spontaneous release events is so low that AMPA channels preferentially desensitize
without opening; if desensitization is blocked, the channels will open, accounting for the enhanced frequency of
mEPSCs. In a variant of this hypothesis, synapses are modular and can be ‘partially silent’; some modules are AMPAsilent because they both release glutamate slowly and lack AMPA channels, whereas others are AMPA-functional
because they release glutamate rapidly and contain AMPA channels. In cyclothiazide, the slowly released glutamate in
silent modules can activate AMPA channels in nearby functional modules77,129.
• At single synapses in culture, release was stimulated by an electrode that was placed near the terminal. Remarkably,
some events produced only NMDA responses, whereas others had both AMPA and NMDA components159. These results
can be explained by the heterogeneity in the speed of glutamate release from different vesicles at the same synapse160.
• Acting through G protein βγ subunits, serotonin promotes kiss-and-run release, as determined by a fluorescent marker
of vesicular turnover. This change is accompanied by a reduction in quantal size, suggesting that presynaptic inhibition
can occur through the modulation of vesicle fusion properties161.
Enhancement of fusion pore conductance may contribute to LTP
• A low affinity antagonist results in less blockage of the EPSC after LTP induction than before LTP157. This indicates a
higher concentration of glutamate in the synaptic cleft after LTP. The number of vesicles released, as judged by NMDARmediated EPSCs, was not increased. It was concluded that the increase in glutamate concentration in the cleft was the
result of LTP increasing the fusion pore diameter. In a variant of this hypothesis, LTP both adds AMPA channels and
increases the fusion pore diameter in silent modules, making them functional129.
LTD-induced reduction in fusion pore conductance may contribute to reduced transmission
• After LTD induction, there is an increase in the number of synaptic boutons that take up the fluorescent membrane
marker FM1-43 into vesicles but do not release it during synaptic stimulation162. Because FM1-43 is lipophilic, it leaves a
vesicle much more slowly than it enters. The reduced release after LTD induction may occur because the release mode
has switched to kiss-and-run, thus allowing little time for the dye to exit the vesicle. AMPA, α-amino-3-hydroxy-5methylisoxazole-4-propionic acid; EPSC, excitatory postsynaptic current; LTD, long-term depression; LTP, long-term
potentiation; mEPSC, miniature EPSC; NMDA, N-methyl-d-aspartate; NMDAR, NMDA receptor.
mEPSC
(miniature EPSC). An mEPSC is
a spontaneously occurring
synaptic event caused by
spontaneous vesicle release. It
is generally measured after
blocking action potentials with
tetrodotoxin to insure that
there is no release due to
spontaneous action potentials.
The amplitude of mEPSCs is
determined by AMPA channel
density and is taken as a
measure of postsynaptic
processes in traditional quantal
analysis. However, mEPSC
amplitude can also be affected
by vesicle glutamate
concentration, multi-vesicular
release and glutamate release
mode.
Outside-out patch
A variant of the patch-clamp
technique, in which a patch of
plasma membrane covers the
tip of the electrode. The
outside of the membrane is
exposed to bathing solution.
Simulations of this kind can provide insight into
the timing of events that occur between the opening of the fusion pore and the generation of AMPA
channel current (FIG. 2d): peak glutamate elevation in
the cleft occurs with a latency of ~80 μs; binding of
glutamate to the channel occurs in ~10 μs; and the
conformational transition of the channel to the open
state requires ~100 μs.
A hotspot of AMPA channel activation. Only a fraction of the average synapse area is actually involved in
generating a quantal response. This is because there are
large concentration gradients in the cleft, and the mM
glutamate concentration necessary to open channels is
only achieved near (within ~100 nm) the site of vesicle
release. As a result, the 20 or so channels that open
during a quantal event are mostly within a hotspot
(with an area of 0.03 μm2) that is only ~25% of the
area of the average CA1 synapse; this area is an even
smaller fraction at large mushroom spines (FIG. 3d).
These findings are consistent with previous evidence
that the average quantal current is far below saturation82–84. Importantly, it follows that quantal size is not
a good indicator of AMPA channel number (adding
AMPA channels to enlarge the synapse will have little
NATURE REVIEWS | NEUROSCIENCE
effect). Rather, the main postsynaptic determinant of
quantal size is AMPA channel density77,85.
Fusion pore diameter affects AMPA channel
activation. We now turn to the question of how the size
of the fusion pore affects AMPA channel activation. This
issue was recently analysed86, and FIG. 4c shows related
calculations. If the fusion pore is at least 4 nm in diameter, the channels are efficiently activated. This activation is not sensitive to the exact pore diameter, because
the diffusion bottleneck is the cleft itself. On the other
hand, when the fusion pore is less than 2 nm in diameter,
diffusion through the pore becomes limiting; indeed, at
1 nm, the concentration of glutamate in the cleft becomes
so low that the mEPSC amplitude is insignificant compared with the background noise. However, even small
pores generate a normal N-methyl-d-aspartate (NMDA)
response, because these channels have a much higher
glutamate affinity than AMPA channels. A synapse with
small pores will therefore produce a ‘whispering’ form
of ‘silent synapse’ (in contrast to synapses that are silent
because they lack AMPA channels87). Experimental
evidence for whispering synapses and the possible
role of fusion pore modulation in synaptic plasticity is
discussed in BOX 2.
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Box 3 | Structural and functional properties of AMPA channels
a Resting
Structural model of activation and desensitization
AMPA channels are tetramers composed of combinations
of channel subunits (GluR1–4) arranged in a dimer-ofdimers configuration163,164. Each subunit can bind
glutamate; as illustrated in the figure part a, binding
of glutamate (Glu) to the upper element is followed by
an upward tilt of the lower element. The force of this tilt
is conveyed to the channel through a linker region,
causing the channel to open165–167. Desensitization occurs
if glutamate binding causes a downward tilt of the binding
domain; under these conditions, upward tilt of the lower
element fails to produce channel opening168. Figure
reproduced with permission from REF. 167 © (2006)
Elsevier Sciences.
Coefficient of variation
(CV). The standard deviation
divided by the mean. The CV is
thus a convenient measure of
the relative variability of a
quantity. For instance, a CV of
0.2 would mean that most
measurements were within plus
or minus 20% of the mean.
Amplitude histogram
An amplitude histogram of the
EPSC is made by defining
amplitude bins and then
plotting the number of
occurrences that fall within
each bin as a function of
amplitude. For responses
comprising a variable number
of elementary quantal units,
the histogram should show
multiple peaks at integral
multiples of quantal size. In
practice, the peaks become
smeared because of the CV
of quantal size and because of
measurement noise.
Kinetic model of an AMPA channel
As illustrated in the state diagram of figure b, the channel
undergoes a transition between states (horizontal
transitions) when glutamate binds to each of the four
subunits. The number of glutamates bound varies from left
to right. From these close states, there may be
conformational changes resulting in the open (O) or
desensitized (D) states. The rate constant at physiological
temperature for opening or desensitizing is shown, as is
the average channel conductance. Both rate constant and
channel conductance vary with the number of glutamates
bound169,170. In this scheme, the opening rate increases
threefold with each additional glutamate bound
(multiplicative gating), in line with strong evidence
for multiplicative gating in other channels171,172. With these
factors known, the model parameters77 were generated by
fitting responses to controlled glutamate applications to
outside-out patches of CA1 dendritic membranes98. Early
models of AMPA channel gating91,173–175 assumed only a
single conductance state, reached upon the binding of two
glutamate molecules (other ligand states were not
considered). Consideration of multiple conductance levels
and all liganded states is necessary to predict the 0.1 ms
rise of mEPSCs77 at CA1 synapses, to predict the
temperature dependence of EPSCs at the Calyx of Held176
and to account for the desensitization properties of AMPA
channels177. AMPA, α-amino-3-hydroxy-5-methylisoxazole4-proprionic acid; CA, Cornu Ammonis; mEPSC, miniature
excitatory postsynaptic current.
How quantal is the quantal response?
Neuronal computations often rely on small differences in
the summed excitatory input from many synapses, and it
is therefore important to minimize the variability of the
responses from each contributing synapse. The elementary unit of transmission at the synapse is the quantal
response. This term was developed at the NMJ to indicate that the EPSC is composed of stereotyped subunits,
but the size of quantal responses is not always exactly
the same. This variability is quantified by the coefficient
of variation (CV), which is 0.3 at the NMJ. Variation of
this magnitude still allows the quantal peaks in amplitude
histograms to be detected.
The extent of variability of the quantal response at
central synapses is controversial. High CVs (~0.6) have
been found at single synapses of hippocampal neurons in
culture88 and in hippocampal slices89. However, analysis
of amplitude histograms at other synapses indicates lower
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Bound
12.7 Å
D1
Glu
D2
31.9 Å
Open
Desensitized
22.0 Å
13.1 Å
14°
21°
14°
21°
26.5 Å
37.4 Å
b
5pS
7pS
12pS
O2
O3
O4
1200
R0
R1
3600
R2
1080
D1
R3
1440
D2
10800
D3
R4
1920
D4
CV values: a CV of 0.4 at the Calyx of Held, 0.22 at the
CA3 mossy fibre synapses (FIG. 5a) and 0.23 at synapses
on cerebellar granule cells 82,90,91. Most measurements of
mEPSCs have been made from groups of synapses, but
one study used a suction pipette to measure the current
from a single synaptic spine (which generally contains a
single synapse) and found a CV of 0.28 (REF. 81). Taken
together, these lines of evidence leave little doubt that
nature has found a way of making quantal variability low,
at least at some central synapses.
Origin of high CVs. How can the larger CVs (~0.6) be
explained? It has been suggested that variation in vesicle
volume (a CV of 0.3 measured in fast frozen material53)
produces variation in vesicle glutamate content92–94, and
that this, together with other factors (channel noise
and intrasynaptic variation in AMPA channel density)
accounts for high CV values. However, it is unclear how
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40
20
c
80
60
Rise time (μs)
b
50
Number of
particles per synapse
Number of events
a
40
400
300
200
20
100
0
0
40 pA
1 ms
0
0
10
20
30
Peak EPSC (pA)
40
50
0
0.05
0.1
0.15
0.2
0.25 0.3
Synaptic area (μm2)
0
60
120
Amplitude (pA)
Figure 5 | Determinants of quantal transmission a | A multi-peaked amplitude histogram of EPSCs evoked at a CA3
mossy fibre synapse. In this case (but not all), the data could be fitted as the sum of three Gaussian curves, with amplitudes
corresponding to one, two or three released vesicles. The narrow half-width of the Gaussian indicates a low coefficient of
variation (CV); after correction for noise, the CV of quantal size was 0.22. b | The AMPA receptor labelling of CA3 mossy
fibre synapses, as determined by electron microscopy, immunogold labelling and reconstruction methods. Content varies
linearly with synapse area, indicating that AMPA receptors are present at fixed density. c | Larger mEPSCs at CA1
hippocampal synapses have slower rise-times (inset shows mEPSCs). Responses were evoked by the application of sucrose
to a local dendritic region and were recorded intradendritically from that region. CA, Cornu Ammonis; mEPSC, miniature
excitatory postsynaptic current. Part a reproduced with permission from REF. 91 © (1993) Cambridge Univ. Press. Part b
reproduced with permission from REF. 113 © (1998) Cell Press. Part c reproduced with permission from REF. 80 © (2003)
Cambridge Univ. Press.
the importance of glutamate content can be reconciled
with the observation that larger mEPSCs have slower
rise-times80,95,96 (FIG. 5c). If the larger responses are due to
a higher glutamate content in the vesicles, it should be
possible to reproduce the slower rise-time by artificially
elevating vesicle glutamate. When this is done in simulations77 or experimentally97, mEPSCs do get larger but not
slower (and they may even get faster).
A different explanation of high CVs is that larger
mEPSCs are generated by multiple vesicles. According to
this view, the rise-time of multiquantal mEPSCs is slower
because of jitter in the time of vesicle release. Consistent
with this explanation, there have been many reports of
mEPSC amplitude distributions that have more than
one peak95,96,98–105. In some cases, the release of multiple
vesicles appears to be due to spontaneous ‘sparks’ of Ca2+
coming from intracellular stores106–108. Notably, altering
Ca2+ stores alters the amplitude of mEPSCs106,109–111. These
results strongly suggest that in some cases the high CV of
mEPSCs arises because the mEPSCs consist of a variable
number of quantal responses. However, this need not
be the only explanation; there are indications that the
mechanisms responsible for low quantal variability (see
next section) may simply not be present in young animals, and may instead develop over time95. Indeed, high
CV and low CV synapses can coexist in the hippocampi
of young animals89.
Attaining a low CV. The fact that a low CV can be
achieved is remarkable given the potential sources of
variation112. Vesicles have to be made and filled in a
reproducible way, variation in fusion pore properties
must be minimized and the stochastic noise attributable
to the gating of a small number of AMPA channels must
be low. A low CV (0.22) at CA3 mossy fibre spines is particularly remarkable given that the recorded events are
due to multiple synapses that vary considerably in their
NATURE REVIEWS | NEUROSCIENCE
size and AMPA channel content113 (FIG. 5b). However, this
becomes more understandable in view of the fact that it
is channel density (which is nearly constant), not channel
number, that is the primary postsynaptic determinant of
quantal size (see above).
A major presynaptic determinant of quantal size is the
number of glutamate transporters in the vesicle114, but
it is not known how this number is regulated. A recent
study surprisingly showed that quantal size is not related
to vesicle volume, but that it does depend on vesicle glutamate content115. Much remains to be learned about how
the size and content of synaptic vesicles is controlled in a
way that minimizes the variability of quantal size.
Binary or graded transmission?
The NMJ, CA3 mossy fibre boutons and the Calyx of Held
contain multiple synapses (active zones and postsynaptic
densities), each of which can contribute to vesicle release.
However, most excitatory connections in the CNS occur
on single spines that contain only a single synapse (FIG. 3a).
When considering the information transmission through
such synapses, it is crucial to understand whether evoked
transmission is univesicular, and therefore binary, or
multivesicular, and therefore graded.
Electrical recordings of transmission at identified
mushroom spines have been difficult to achieve: the
single existing recording at a large mushroom spine
indicated multivesicular release on almost every trial;
EPSCs were highly variable and the largest were much
larger than mEPSCs116,117. Optical measurements of
NMDA-mediated Ca2+ entry118 also provide evidence
for multivesicular release. More evidence is provided by
experiments using low-affinity antagonists; increasing
multi-vesicular release (and thus glutamate concentration) reduces the inhibitory action of the competing
antagonist119–121. At the Calyx of Held, high Ca2+ levels,
generated by uncaging, release the readily releasable
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pool (1,500 vesicles) with a time-constant of 0.6 ms40.
As there are ~500 synapses contributing122, each must
release multiple vesicles (see also REF. 71). Related
findings have been made at inhibitory synapses123.
However, some studies indicate that release at certain
synapses is always univesicular. Procedures that increase
the probability of release produced no change in the
size of successful responses (potency)124–126, contrary
to what would be observed if multiple quanta sometimes summated. Similar constancy was found during
depression89. Other experiments have shown that when
release is enhanced, there is no reduction in the efficacy
of low affinity antagonist, arguing against multiquantal
transmission127.
Much of this conflicting evidence has been obtained
at hippocampal synapses and so cannot be attributed
to regional variation. However, because of technical
issues, different experiments probably sampled synapses
of different sizes. One resolution may be that synapses are
made of modules128,129; indeed, FIG. 3c could be interpreted
as indicating a modular organization of release. Perhaps,
then, each module contains only a single release site (possibly the triangular particle), which provides a structural
basis for univesicular release. Small synapses may contain
a single module, and thus be restricted to univesicular
release, whereas larger synapses would be capable of multivesicular release. Consistent with this idea, the average
synapse area at the Calyx of Held, where multivesicular
release occurs, is 0.1 μm2 (REF. 122) — considerably larger
than synapse areas at the cerebellar synapses (0.024 μm2),
which show univesicular release130. This general idea is
1.
Katz, B. Neural transmitter release: from quantal
secretion to exocytosis and beyond. The Fenn Lecture.
J. Neurocytol. 25, 677–686 (1996).
2.
Heuser, J. E. et al. Synaptic vesicle exocytosis
captured by quick freezing and correlated with quantal
transmitter release. J. Cell Biol. 81, 275–300
(1979).
3.
Heuser, J. E. & Reese, T. S. Structural changes after
transmitter release at the frog neuromuscular
junction. J. Cell Biol. 88, 564–580 (1981).
4.
Stiles, J. R., Van Helden, D., Bartol, T. M. Jr,
Salpeter, E. E. & Salpeter, M. M. Miniature endplate
current rise times less than 100 microseconds from
improved dual recordings can be modeled with passive
acetylcholine diffusion from a synaptic vesicle.
Proc. Natl Acad. Sci. USA 93, 5747–5752 (1996).
5.
Borst, J. G., Helmchen, F. & Sakmann, B. Pre- and
postsynaptic whole-cell recordings in the medial
nucleus of the trapezoid body of the rat. J. Physiol.
489, 825–840 (1995).
6.
Geiger, J. R. & Jonas, P. Dynamic control of
presynaptic Ca2+ inflow by fast-inactivating K+
channels in hippocampal mossy fiber boutons. Neuron
28, 927–939 (2000).
7.
Llinas, R., Steinberg, I. Z. & Walton, K. Relationship
between presynaptic calcium current and postsynaptic
potential in squid giant synapse. Biophys. J. 33,
323–351 (1981).
8.
Augustine, G. J., Charlton, M. P. & Smith, S. J. Calcium
entry and transmitter release at voltage-clamped
nerve terminals of squid. J. Physiol. 367, 163–181
(1985).
9.
Wu, L. G., Westenbroek, R. E., Borst, J. G.,
Catterall, W. A. & Sakmann, B. Calcium channel types
with distinct presynaptic localization couple
differentially to transmitter release in single calyx-type
synapses. J. Neurosci. 19, 726–736 (1999).
10. Iwasaki, S., Momiyama, A., Uchitel, O. D. & Takahashi, T.
Developmental changes in calcium channel types
mediating central synaptic transmission. J. Neurosci.
20, 59–65 (2000).
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
consistent with the model of Bolshakov et al.131, which
is based on their observation that the late phase of longterm potentiation, a process associated with synapse
growth132, can convert synapses from uniquantal EPSCs
and mEPSCs to multiquantal ones131.
In summary, it seems safe to conclude that multivesicular release, though not universal, is common, and
that analysis of information transmission at synapses
should consider the enhancements made possible by
graded release.
Conclusion
There is a tendency for scientists to become specialized,
and the field of synaptic physiology is no different; there
are presynaptic specialists and postsynaptic specialists.
In this Review, we have attempted to bridge the cleft that
divides these fields. We have given a brief overview of a
broad range of topics in synaptic transmission, and tried
to make clear the necessity of understanding presynaptic
and postsynaptic events in an integrated way. We hope
that the reader has gained an appreciation for the complexities of synaptic transmission, but also an appreciation
of the tremendous progress that has been made. Although
there remain aspects of the problem that are still quite
mysterious, many aspects have been rigorously studied by
adequate biophysical methods and can be accounted for
by well-constrained computational models. The increasing
understanding of the biophysics of synaptic transmission
at central synapses will provide a sound framework for
determining the molecular basis of transmission and for
elucidating how synapses are modified during learning.
Reid, C. A., Bekkers, J. M. & Clements, J. D.
Presynaptic Ca2+ channels: a functional patchwork.
Trends Neurosci. 26, 683–687 (2003).
Momiyama, A. & Takahashi, T. Development of
inhibitory synaptic currents in rat spinal neurons.
Ann. NY Acad. Sci. 707, 447–448 (1993).
Mintz, I. M., Sabatini, B. L. & Regehr, W. G. Calcium
control of transmitter release at a cerebellar synapse.
Neuron 15, 675–688 (1995).
Sabatini, B. L. & Regehr, W. G. Timing of
neurotransmission at fast synapses in the mammalian
brain. Nature 384, 170–172 (1996).
A study that provides a different view of the timing
of steps leading to vesicle release. This study
suggests that release can occur before the falling
phase of the action potenital, making a much
shorter synaptic delay possible.
Sabatini, B. L. & Regehr, W. G. Timing of synaptic
transmission. Annu. Rev. Physiol. 61, 521–542 (1999).
McDonough, S. I., Mintz, I. M. & Bean, B. P. Alteration
of P-type calcium channel gating by the spider toxin
ω-Aga-IVA. Biophys. J. 72, 2117–2128 (1997).
Llinas, R., Sugimori, M. & Silver, R. B. Microdomains
of high calcium concentration in a presynaptic
terminal. Science 256, 677–679 (1992).
Serulle, Y., Sugimori, M. & Llinas, R. R. Imaging
synaptosomal calcium concentration microdomains
and vesicle fusion by using total internal reflection
fluorescent microscopy. Proc. Natl Acad. Sci. USA
104, 1697–1702 (2007).
Stanley, E. F. The calcium channel and the organization
of the presynaptic transmitter release face. Trends
Neurosci. 20, 404–409 (1997).
Harlow, M. L., Ress, D., Stoschek, A., Marshall, R. M.
& McMahan, U. J. The architecture of active zone
material at the frog’s neuromuscular junction. Nature
409, 479–484 (2001).
Pumplin, D. W., Reese, T. S. & Llinás, R. Are the
presynaptic membrane particles the calcium
channels? Proc. Natl Acad. Sci. USA 78, 7210–7213
(1981).
606 | ADVANCE ONLINE PUBLICATION
22. Shahrezaei, V., Cao, A. & Delaney, K. R. Ca2+ from one
or two channels controls fusion of a single vesicle at
the frog neuromuscular junction. J. Neurosci. 26,
13240–13249 (2006).
23. Ohana, O. & Sakmann, B. Transmitter release
modulation in nerve terminals of rat neocortical
pyramidal cells by intracellular calcium buffers.
J. Physiol. 513, 135–148 (1998).
24. Burnashev, N. & Rozov, A. Presynaptic Ca2+ dynamics,
Ca2+ buffers and synaptic efficacy. Cell Calcium 37,
489–495 (2005).
25. Schneggenburger, R. & Neher, E. Intracellular calcium
dependence of transmitter release rates at a fast
central synapse. Nature 406, 889–893 (2000).
An elegant study that measured the release
attributable to defined Ca2+ changes in the
presynaptic terminal.
26. Bollmann, J. H., Sakmann, B. & Borst, J. G. Calcium
sensitivity of glutamate release in a calyx-type
terminal. Science 289, 953–957 (2000).
27. Bollmann, J. H. & Sakmann, B. Control of synaptic
strength and timing by the release-site Ca2+ signal.
Nature Neurosci. 8, 426–434 (2005).
A clever strategy was used to mimic the brief Ca2+
elevation produced by an action potential and to
study the resulting transmitter release.
28. Bennett, M. K., Calakos, N. & Scheller, R. H. Syntaxin:
a synaptic protein implicated in docking of synaptic
vesicles at presynaptic active zones. Science 257,
255–259 (1992).
29. Sheng, Z. H., Rettig, J., Cook, T. & Catterall, W. A.
Calcium-dependent interaction of N-type calcium
channels with the synaptic core complex. Nature 379,
451–454 (1996).
30. Jarvis, S. E. & Zamponi, G. W. Masters or slaves?
Vesicle release machinery and the regulation of
presynaptic calcium channels. Cell Calcium 37,
483–488 (2005).
31. Meinrenken, C. J., Borst, J. G. & Sakmann, B. Local
routes revisited: the space and time dependence
of the Ca2+ signal for phasic transmitter release at
www.nature.com/reviews/neuro
© 2007 Nature Publishing Group
REVIEWS
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
the rat calyx of Held. J. Physiol. 547, 665–689
(2003).
Meinrenken, C. J., Borst, J. G. & Sakmann, B. Calcium
secretion coupling at calyx of held governed by
nonuniform channel-vesicle topography. J. Neurosci.
22, 1648–1667 (2002).
Wadel, K., Neher, E. & Sakaba, T. The coupling
between synaptic vesicles and Ca2+ channels
determines fast neurotransmitter release. Neuron 53,
563–575 (2007).
Fedchyshyn, M. J. & Wang, L. Y. Developmental
transformation of the release modality at the calyx of
Held synapse. J. Neurosci. 25, 4131–4140 (2005).
Zhai, R. G. & Bellen, H. J. The architecture of the
active zone in the presynaptic nerve terminal.
Physiology (Bethesda) 19, 262–270 (2004).
Zampighi, G. A. et al. Conical electron tomography of a
chemical synapse: vesicles docked to the active zone
are hemi-fused. Biophys. J. 91, 2910–2918 (2006).
Phillips, G. R. et al. The presynaptic particle web:
ultrastructure, composition, dissolution, and
reconstitution. Neuron 32, 63–77 (2001).
Felmy, F., Neher, E. & Schneggenburger, R. The timing
of phasic transmitter release is Ca2+-dependent and
lacks a direct influence of presynaptic membrane
potential. Proc. Natl Acad. Sci. USA 100,
15200–15205 (2003).
Dodge, F. A. Jr & Rahamimoff, R. Co-operative action
of calcium ions in transmitter release at the
neuromuscular junction. J. Physiol. 193, 419–432
(1967).
Wolfel, M. & Schneggenburger, R. Presynaptic
capacitance measurements and Ca2+ uncaging reveal
submillisecond exocytosis kinetics and characterize
the Ca2+ sensitivity of vesicle pool depletion at a fast
CNS synapse. J. Neurosci. 23, 7059–7068 (2003).
Zucker, R. S. & Regehr, W. G. Short-term synaptic
plasticity. Annu. Rev. Physiol. 64, 355–405 (2002).
Bischofberger, J., Engel, D., Frotscher, M. & Jonas, P.
Timing and efficacy of transmitter release at mossy
fiber synapses in the hippocampal network. Pflugers
Arch. 453, 361–372 (2006).
Korogod, N., Lou, X. & Schneggenburger, R.
Presynaptic Ca2+ requirements and developmental
regulation of posttetanic potentiation at the calyx of
Held. J. Neurosci. 25, 5127–5137 (2005).
Isaacson, J. S. & Walmsley, B. Counting quanta: direct
measurements of transmitter release at a central
synapse. Neuron 15, 875–884 (1995).
Diamond, J. S. & Jahr, C. E. Asynchronous release of
synaptic vesicles determines the time course of the
AMPA receptor-mediated EPSC. Neuron 15,
1097–1107 (1995).
Millet, O., Bernado, P., Garcia, J., Rizo, J. & Pons, M.
NMR measurement of the off rate from the first
calcium-binding site of the synaptotagmin I C2A
domain. FEBS Lett. 516, 93–96 (2002).
Collin, T. et al. Developmental changes in parvalbumin
regulate presynaptic Ca2+ signaling. J. Neurosci. 25,
96–107 (2005).
Sudhof, T. C. The synaptic vesicle cycle. Annu. Rev.
Neurosci. 27, 509–547 (2004).
Geppert, M. et al. Synaptotagmin I: a major Ca2+
sensor for transmitter release at a central synapse.
Cell 79, 717–727 (1994).
Nishiki, T. & Augustine, G. J. Dual roles of the C2B
domain of synaptotagmin I in synchronizing Ca2+dependent neurotransmitter release. J. Neurosci. 24,
8542–8550 (2004).
Nishiki, T. & Augustine, G. J. Synaptotagmin I
synchronizes transmitter release in mouse
hippocampal neurons. J. Neurosci. 24, 6127–6132
(2004).
Fernandez-Chacon, R. et al. Synaptotagmin I functions
as a calcium regulator of release probability. Nature
410, 41–49 (2001).
Takamori, S. et al. Molecular anatomy of a trafficking
organelle. Cell 127, 831–846 (2006).
Stewart, B. A., Mohtashami, M., Trimble, W. S. &
Boulianne, G. L. SNARE proteins contribute to calcium
cooperativity of synaptic transmission. Proc. Natl
Acad. Sci. USA 97, 13955–13960 (2000).
Hua, Y. & Scheller, R. H. Three SNARE complexes
cooperate to mediate membrane fusion. Proc. Natl
Acad. Sci. USA 98, 8065–8070 (2001).
Montecucco, C., Schiavo, G. & Pantano, S.
SNARE complexes and neuroexocytosis: how many,
how close? Trends Biochem. Sci. 30, 367–372 (2005).
Xu, Y., Zhang, F., Su, Z., McNew, J. A. & Shin, Y. K.
Hemifusion in SNARE-mediated membrane fusion.
Nature Struct. Mol. Biol. 12, 417–422 (2005).
58. Reese, C., Heise, F. & Mayer, A. Trans-SNARE pairing
can precede a hemifusion intermediate in intracellular
membrane fusion. Nature 436, 410–414 (2005).
59. Giraudo, C. G., Eng, W. S., Melia, T. J. & Rothman, J. E.
A clamping mechanism involved in SNARE-dependent
exocytosis. Science 313, 676–680 (2006).
60. Tang, J. et al. A complexin/synaptotagmin 1 switch
controls fast synaptic vesicle exocytosis. Cell 126,
1175–1187 (2006).
This paper provides insights into the steps that
make the vesicle primed for release.
61. Schaub, J. R., Lu, X., Doneske, B., Shin, Y. K. &
McNew, J. A. Hemifusion arrest by complexin is
relieved by Ca2+–synaptotagmin I. Nature Struct. Mol.
Biol. 13, 748–750 (2006).
Direct measurements of membrane mixing provide
unique insights into the fusion process.
62. Zimmerberg, J., Akimov, S. A. & Frolov, V.
Synaptotagmin: fusogenic role for calcium sensor?
Nature Struct. Mol. Biol. 13, 301–303 (2006).
63. Jackson, M. B. & Chapman, E. R. Fusion pores and
fusion machines in Ca2+-triggered exocytosis. Annu.
Rev. Biophys. Biomol. Struct. 35, 135–160 (2006).
An excellent review of the issues regarding the
mechanism by which the fusion pore is generated.
64. Han, X., Wang, C. T., Bai, J., Chapman, E. R. &
Jackson, M. B. Transmembrane segments of syntaxin
line the fusion pore of Ca2+-triggered exocytosis.
Science 304, 289–292 (2004).
65. Han, X. & Jackson, M. B. Electrostatic interactions
between the syntaxin membrane anchor and
neurotransmitter passing through the fusion pore.
Biophys. J. 88, L20–L22 (2005).
66. Almers, W. Exocytosis. Annu. Rev. Physiol. 52,
607–624 (1990).
67. He, L., Wu, X. S., Mohan, R. & Wu, L. G. Two modes
of fusion pore opening revealed by cell-attached
recordings at a synapse. Nature 444, 102–105
(2006).
A technical breakthrough allows direct
measurements of vesicle fusion and fusion pore
formation at the secretory face of a central synapse.
68. Chen, X., Wang, L., Zhou, Y., Zheng, L. H. & Zhou, Z.
‘Kiss-and-run’ glutamate secretion in cultured and
freshly isolated rat hippocampal astrocytes.
J. Neurosci. 25, 9236–9243 (2005).
69. Klyachko, V. A. & Jackson, M. B. Capacitance steps
and fusion pores of small and large-dense-core vesicles
in nerve terminals. Nature 418, 89–92 (2002).
70. Harata, N. C., Aravanis, A. M. & Tsien, R. W. Kiss-andrun and full-collapse fusion as modes of exoendocytosis in neurosecretion. J. Neurochem. 97,
1546–1570 (2006).
71. Harata, N. C., Choi, S., Pyle, J. L., Aravanis, A. M. &
Tsien, R. W. Frequency-dependent kinetics and
prevalence of kiss-and-run and reuse at hippocampal
synapses studied with novel quenching methods.
Neuron 49, 243–256 (2006).
72. Aravanis, A. M., Pyle, J. L. & Tsien, R. W. Single synaptic
vesicles fusing transiently and successively without
loss of identity. Nature 423, 643–647 (2003).
73. Ceccarelli, B., Hurlbut, W. P. & Mauro, A. Turnover of
transmitter and synaptic vesicles at the frog
neuromuscular junction. J. Cell Biol. 57, 499–524
(1973).
74. Gandhi, S. P. & Stevens, C. F. Three modes of synaptic
vesicular recycling revealed by single-vesicle imaging.
Nature 423, 607–613 (2003).
75. Pawlu, C., DiAntonio, A. & Heckmann, M. Postfusional
control of quantal current shape. Neuron 42,
607–618 (2004).
76. Burger, P. M. et al. Synaptic vesicles immunoisolated
from rat cerebral cortex contain high levels of
glutamate. Neuron 3, 715–720 (1989).
77. Raghavachari, S. & Lisman, J. E. Properties of quantal
transmission at CA1 synapses. J. Neurophysiol. 92,
2456–2467 (2004).
This paper used Monte-Carlo simulations to show
how the glutamate released from a vesicle diffuses
in the cleft and activates AMPA channels.
78. Tanaka, J. et al. Number and density of AMPA
receptors in single synapses in immature cerebellum.
J. Neurosci. 25, 799–807 (2005).
79. Nielsen, T. A., DiGregorio, D. A. & Silver, R. A.
Modulation of glutamate mobility reveals the
mechanism underlying slow-rising AMPAR EPSCs and
the diffusion coefficient in the synaptic cleft. Neuron
42, 757–771 (2004).
This paper uses a clever combination of
experimental manipulation and modelling to
discern the diffusion coefficient of the
NATURE REVIEWS | NEUROSCIENCE
80.
81.
82.
83.
84.
85.
86.
87.
88.
89.
90.
91.
92.
93.
94.
95.
96.
97.
98.
neurotransmitter glutamate in the synpaptic cleft.
This is an important parameter in determining the
amplitude of synaptic responses.
Smith, M. A., Ellis-Davies, G. C. & Magee, J. C.
Mechanism of the distance-dependent scaling of
Schaffer collateral synapses in rat CA1 pyramidal
neurons. J. Physiol. 548, 245–258 (2003).
Forti, L., Bossi, M., Bergamaschi, A., Villa, A. &
Malgaroli, A. Loose-patch recordings of single quanta
at individual hippocampal synapses. Nature 388,
874–878 (1997).
A direct measurement of the quantal response at
putatively single synapses showing that quantal
responses have small variability.
Silver, R. A., Cull-Candy, S. G. & Takahashi, T. NonNMDA glutamate receptor occupancy and open
probability at a rat cerebellar synapse with single and
multiple release sites. J. Physiol. 494, 231–250
(1996).
Liu, G., Choi, S. & Tsien, R. W. Variability of
neurotransmitter concentration and nonsaturation
of postsynaptic AMPA receptors at synapses in
hippocampal cultures and slices. Neuron 22,
395–409 (1999).
McAllister, A. K. & Stevens, C. F. Nonsaturation of
AMPA and NMDA receptors at hippocampal synapses.
Proc. Natl Acad. Sci. USA 97, 6173–6178 (2000).
Franks, K. M., Bartol, T. M. Jr. & Sejnowski, T. J.
A Monte Carlo model reveals independent signaling
at central glutamatergic synapses. Biophys. J. 83,
2333–2348 (2002).
Choi, S., Klingauf, J. & Tsien, R. W. Fusion pore
modulation as a presynaptic mechanism contributing
to expression of long-term potentiation. Philos. Trans.
R. Soc. Lond. B Biol. Sci. 358, 695–705 (2003).
A review of experiments pointing to the importance
of the mode of vesicle release in AMPA channel
activation. Experiments suggesting a role for this
process in synaptic plasticity are also reviewed.
Liao, D., Hessler, N. A. & Malinow, R. Activation of
postsynaptically silent synapses during pairinginduced LTP in CA1 region of hippocampal slice.
Nature 375, 400–404 (1995).
Bekkers, J. M., Richerson, G. B. & Stevens, C. F.
Origin of variability in quantal size in cultured
hippocampal neurons and hippocampal slices. Proc.
Natl Acad. Sci. USA 87, 5359–5362 (1990).
Hanse, E. & Gustafsson, B. Quantal variability at
glutamatergic synapses in area CA1 of the rat
neonatal hippocampus. J. Physiol. 531, 467–480
(2001).
Sahara, Y. & Takahashi, T. Quantal components of the
excitatory postsynaptic currents at a rat central
auditory synapse. J. Physiol. 536, 189–197 (2001).
Jonas, P., Major, G. & Sakmann, B. Quantal
components of unitary EPSCs at the mossy fibre
synapse on CA3 pyramidal cells of rat hippocampus.
J. Physiol. 472, 615–663 (1993).
Franks, K. M., Stevens, C. F. & Sejnowski, T. J.
Independent sources of quantal variability at single
glutamatergic synapses. J. Neurosci. 23, 3186–3195
(2003).
Karunanithi, S., Marin, L., Wong, K. & Atwood, H. L.
Quantal size and variation determined by vesicle size
in normal and mutant Drosophila glutamatergic
synapses. J. Neurosci. 22, 10267–10276 (2002).
Schikorski, T. & Stevens, C. F. Quantitative
ultrastructural analysis of hippocampal excitatory
synapses. J. Neurosci. 17, 5858–5867 (1997).
Wall, M. J. & Usowicz, M. M. Development of the
quantal properties of evoked and spontaneous
synaptic currents at a brain synapse. Nature
Neurosci. 1, 675–682 (1998).
Ulrich, D. & Luscher, H. R. Miniature excitatory
synaptic currents corrected for dendritic cable
properties reveal quantal size and variance.
J. Neurophysiol. 69, 1769–1773 (1993).
Yamashita, T., Ishikawa, T. & Takahashi, T.
Developmental increase in vesicular glutamate content
does not cause saturation of AMPA receptors at the
calyx of held synapse. J. Neurosci. 23, 3633–3638
(2003).
Magee, J. C. & Cook, E. P. Somatic EPSP amplitude is
independent of synapse location in hippocampal
pyramidal neurons. Nature Neurosci. 3, 895–903
(2000).
This paper combines local dendritic recording and
focal sucrose application to measure the mEPSC
with minimal dendritic filtering. This provides the
most constrained measurement of the time-course
of the mEPSC.
ADVANCE ONLINE PUBLICATION | 607
© 2007 Nature Publishing Group
REVIEWS
99. Bornstein, J. C. Spontaneous multiquantal release at
synapses in guinea-pig hypogastric ganglia: evidence
that release can occur in bursts. J. Physiol. 282,
375–398 (1978).
100. Paulsen, O. & Heggelund, P. The quantal size at
retinogeniculate synapses determined from
spontaneous and evoked EPSCs in guinea-pig thalamic
slices. J. Physiol. 480, 505–511 (1994).
101. Paulsen, O. & Heggelund, P. Quantal properties of
spontaneous EPSCs in neurones of the guinea-pig
dorsal lateral geniculate nucleus. J. Physiol. 496,
759–772 (1996).
102. Min, M. Y. & Appenteng, K. Multimodal distribution of
amplitudes of miniature and spontaneous EPSPs
recorded in rat trigeminal motoneurones. J. Physiol.
494, 171–182 (1996).
103. Dennis, M. J., Harris, A. J. & Kuffler, S. W. Synaptic
transmission and its duplication by focally applied
acetylcholine in parasympathetic neurons in the heart
of the frog. Proc. R. Soc. Lond. B Biol. Sci. 177,
509–539 (1971).
104. Liu, G. & Feldman, J. L. Quantal synaptic transmission
in phrenic motor nucleus. J. Neurophysiol. 68,
1468–1471 (1992).
105. Edwards, F. A., Konnerth, A. & Sakmann, B. Quantal
analysis of inhibitory synaptic transmission in the
dentate gyrus of rat hippocampal slices: a patch-clamp
study. J. Physiol. 430, 213–249 (1990).
106. Llano, I. et al. Presynaptic calcium stores underlie largeamplitude miniature IPSCs and spontaneous calcium
transients. Nature Neurosci. 3, 1256–1265 (2000).
107. Melamed, N., Helm, P. J. & Rahamimoff, R. Confocal
microscopy reveals coordinated calcium fluctuations
and oscillations in synaptic boutons. J. Neurosci. 13,
632–649 (1993).
108. Emptage, N. J., Reid, C. A. & Fine, A. Calcium stores in
hippocampal synaptic boutons mediate short-term
plasticity, store-operated Ca2+ entry, and spontaneous
transmitter release. Neuron 29, 197–208 (2001).
109. Simkus, C. R. & Stricker, C. The contribution of
intracellular calcium stores to mEPSCs recorded in
layer II neurones of rat barrel cortex. J. Physiol. 545,
521–535 (2002).
110. Galante, M. & Marty, A. Presynaptic ryanodinesensitive calcium stores contribute to evoked
neurotransmitter release at the basket cell–Purkinje
cell synapse. J. Neurosci. 23, 11229–11234 (2003).
111. Sharma, G. & Vijayaraghavan, S. Modulation of
presynaptic store calcium induces release of glutamate
and postsynaptic firing. Neuron 38, 929–939 (2003).
112. Jack, J. J., Larkman, A. U., Major, G. & Stratford, K. J.
Quantal analysis of the synaptic excitation of CA1
hippocampal pyramidal cells. Adv. Second Messenger
Phosphoprotein Res. 29, 275–299 (1994).
113. Nusser, Z. et al. Cell type and pathway dependence of
synaptic AMPA receptor number and variability in the
hippocampus. Neuron 21, 545–559 (1998).
114. Wilson, N. R. et al. Presynaptic regulation of quantal
size by the vesicular glutamate transporter VGLUT1.
J. Neurosci. 25, 6221–6234 (2005).
115. Wu, X. S. et al. The origin of quantal size variation:
vesicular glutamate concentration plays a significant
role. J. Neurosci. 27, 3046–3056 (2007).
116. Conti, R. & Lisman, J. The high variance of AMPA
receptor- and NMDA receptor-mediated responses at
single hippocampal synapses: evidence for
multiquantal release. Proc. Natl Acad. Sci. USA 100,
4885–4890 (2003).
117. Conti, R. & Lisman, J. A large sustained Ca2+ elevation
occurs in unstimulated spines during the LTP pairing
protocol but does not change synaptic strength.
Hippocampus 12, 667–679 (2002).
118. Oertner, T. G., Sabatini, B. L., Nimchinsky, E. A. &
Svoboda, K. Facilitation at single synapses probed
with optical quantal analysis. Nature Neurosci. 5,
657–664 (2002).
This paper uses two-photon calcium imaging to
infer that multivesicular release can occur at single
synapses.
119. Wadiche, J. I. & Jahr, C. E. Multivesicular release at
climbing fiber–Purkinje cell synapses. Neuron 32,
301–313 (2001).
120. Foster, K. A., Kreitzer, A. C. & Regehr, W. G.
Interaction of postsynaptic receptor saturation with
presynaptic mechanisms produces a reliable synapse.
Neuron 36, 1115–1126 (2002).
121. Christie, J. M. & Jahr, C. E. Multivesicular release at
Schaffer collateral–CA1 hippocampal synapses.
J. Neurosci. 26, 210–216 (2006).
122. Satzler, K. et al. Three-dimensional reconstruction of a
calyx of Held and its postsynaptic principal neuron in
123.
124.
125.
126.
127.
128.
129.
130.
131.
132.
133.
134.
135.
136.
137.
138.
139.
140.
141.
142.
143.
144.
145.
146.
the medial nucleus of the trapezoid body. J. Neurosci.
22, 10567–10579 (2002).
Biro, A. A., Holderith, N. B. & Nusser, Z. Quantal size
is independent of the release probability at
hippocampal excitatory synapses. J. Neurosci. 25,
223–232 (2005).
Stevens, C. F. & Wang, Y. Facilitation and depression at
single central synapses. Neuron 14, 795–802 (1995).
Bolshakov, V. Y. & Siegelbaum, S. A. Regulation of
hippocampal transmitter release during development
and long-term potentiation. Science 269,
1730–1734 (1995).
Chen, G., Harata, N. C. & Tsien, R. W. Paired-pulse
depression of unitary quantal amplitude at single
hippocampal synapses. Proc. Natl Acad. Sci. USA 101,
1063–1068 (2004).
Silver, R. A., Lubke, J., Sakmann, B. & Feldmeyer, D.
High-probability uniquantal transmission at excitatory
synapses in barrel cortex. Science 302, 1981–1984
(2003).
Shapira, M. et al. Unitary assembly of presynaptic
active zones from Piccolo-Bassoon transport vesicles.
Neuron 38, 237–252 (2003).
Lisman, J. & Raghavachari, S. A unified model of the
presynaptic and postsynaptic changes during LTP at
CA1 synapses. Science’s STKE [online] http://stke.
sciencemag.org/cgi/content/full/sigtrans;2006/356/
re11 (2006).
Cathala, L., Holderith, N. B., Nusser, Z., DiGregorio, D. A.
& Cull-Candy, S. G. Changes in synaptic structure
underlie the developmental speeding of AMPA
receptor-mediated EPSCs. Nature Neurosci. 8,
1310–1318 (2005).
Bolshakov, V. Y., Golan, H., Kandel, E. R. &
Siegelbaum, S. A. Recruitment of new sites of synaptic
transmission during the cAMP-dependent late phase
of LTP at CA3–CA1 synapses in the hippocampus.
Neuron 19, 635–651 (1997).
Ostroff, L. E., Fiala, J. C., Allwardt, B. & Harris, K. M.
Polyribosomes redistribute from dendritic shafts into
spines with enlarged synapses during LTP in
developing rat hippocampal slices. Neuron 35,
535–545 (2002).
Tsien, R. W., Ellinor, P. T. & Horne, W. A. Molecular
diversity of voltage-dependent Ca2+ channels. Trends
Pharmacol. Sci. 12, 349–354 (1991).
Catterall, W. A. Structure and function of voltagegated sodium and calcium channels. Curr. Opin.
Neurobiol. 1, 5–13 (1991).
Dunlap, K., Luebke, J. I. & Turner, T. J. Exocytotic Ca2+
channels in mammalian central neurons. Trends
Neurosci. 18, 89–98 (1995).
Nowycky, M. C., Fox, A. P. & Tsien, R. W. Three types
of neuronal calcium channel with different calcium
agonist sensitivity. Nature 316, 440–443 (1985).
Hirning, L. D. et al. Dominant role of N-type Ca2+
channels in evoked release of norepinephrine from
sympathetic neurons. Science 239, 57–61 (1988).
Stanley, E. F. & Goping, G. Characterization of a
calcium current in a vertebrate cholinergic presynaptic
nerve terminal. J. Neurosci. 11, 985–993 (1991).
Stanley, E. F. Single calcium channels on a cholinergic
presynaptic nerve terminal. Neuron 7, 585–591
(1991).
Llinás, R., Sugimori, M., Hillman, D. E. & Cherksey, B.
Distribution and functional significance of the P-type,
voltage-dependent Ca2+ channels in the mammalian
central nervous system. Trends Neurosci. 15,
351–355 (1992).
Sather, W. A. et al. Distinctive biophysical and
pharmacological properties of class A (BI) calcium
channel α1 subunits. Neuron 11, 291–303 (1993).
Randall, A. & Tsien, R. W. Pharmacological dissection
of multiple types of Ca2+ channel currents in rat
cerebellar granule neurons. J. Neurosci. 15,
2995–3012 (1995).
Takahashi, T. & Momiyama, A. Different types of
calcium channels mediate central synaptic
transmission. Nature 366, 156–158 (1993).
Luebke, J. I., Dunlap, K. & Turner, T. J. Multiple
calcium channel types control glutamatergic synaptic
transmission in the hippocampus. Neuron 11,
895–902 (1993).
Wheeler, D. B., Randall, A. & Tsien, R. W. Roles of
N-type and Q-type Ca2+ channels in supporting
hippocampal synaptic transmission. Science 264,
107–111 (1994).
Forsythe, I. D. Direct patch recording from identified
presynaptic terminals mediating glutamatergic EPSCs
in the rat CNS in vitro. J. Physiol. 479, 381–387
(1994).
608 | ADVANCE ONLINE PUBLICATION
147. Ertel, E. A. et al. Nomenclature of voltage-gated
calcium channels. Neuron 25, 533–535 (2000).
148. Olivera, B. M., Miljanich, G. P., Ramachandran, J. &
Adams, M. E. Calcium channel diversity and
neurotransmitter release: the ω-conotoxins and
ω-agatoxins. Annu. Rev. Biochem. 63, 823–867 (1994).
149. Campbell, K. P., Leung, A. T. & Sharp, A. H.
The biochemistry and molecular biology of the
dihydropyridine-sensitive calcium channel. Trends
Neurosci. 11, 425–430 (1988).
150. Scholz, K. P. & Miller, R. J. Developmental changes in
presynaptic calcium channels coupled to glutamate
release in cultured rat hippocampal neurons.
J. Neurosci. 15, 4612–4617 (1995).
151. Tsien, R. W., Hess, P., McCleskey, E. W. & Rosenberg,
R. L. Calcium channels: mechanisms of selectivity,
permeation, and block. Annu. Rev. Biophys. Biophys.
Chem. 16, 265–290 (1987).
152. Sather, W. A. & McCleskey, E. W. Permeation and
selectivity in calcium channels. Annu. Rev. Physiol. 65,
133–159 (2003).
153. Zhou, Y. D., Turner, T. J. & Dunlap, K. Enhanced G
protein-dependent modulation of excitatory synaptic
transmission in the cerebellum of the Ca2+ channelmutant mouse, tottering. J. Physiol. 547, 497–507
(2003).
154. Colecraft, H. M., Patil, P. G. & Yue, D. T. Differential
occurrence of reluctant openings in G-protein-inhibited
N- and P/Q-type calcium channels. J. Gen. Physiol. 115,
175–192 (2000).
155. Pietrobon, D. Calcium channels and channelopathies
of the central nervous system. Mol. Neurobiol. 25,
31–50 (2002).
156. Miljanich, G. P. Ziconotide: neuronal calcium channel
blocker for treating severe chronic pain. Curr. Med.
Chem. 11, 3029–3040 (2004).
157. Choi, S., Klingauf, J. & Tsien, R. W. Postfusional
regulation of cleft glutamate concentration during LTP
at ‘silent synapses’. Nature Neurosci. 3, 330–336
(2000).
158. Montgomery, J. M., Pavlidis, P. & Madison, D. V.
Pair recordings reveal all-silent synaptic connections
and the postsynaptic expression of long-term
potentiation. Neuron 29, 691–701 (2001).
159. Renger, J. J., Egles, C. & Liu, G. A developmental
switch in neurotransmitter flux enhances synaptic
efficacy by affecting AMPA receptor activation. Neuron
29, 469–484 (2001).
160. Krupa, B. & Liu, G. Does the fusion pore contribute to
synaptic plasticity? Trends Neurosci. 27, 62–66
(2004).
161. Photowala, H., Blackmer, T., Schwartz, E., Hamm, H. E.
& Alford, S. G protein βγ-subunits activated by
serotonin mediate presynaptic inhibition by regulating
vesicle fusion properties. Proc. Natl Acad. Sci. USA 103,
4281–4286 (2006).
162. Zakharenko, S. S., Zablow, L. & Siegelbaum, S. A.
Altered presynaptic vesicle release and cycling during
mGluR-dependent LTD. Neuron 35, 1099–1110
(2002).
163. Nakagawa, T., Cheng, Y., Sheng, M. & Walz, T.
Three-dimensional structure of an AMPA receptor
without associated stargazin/TARP proteins. Biol.
Chem. 387, 179–187 (2006).
164. Madden, D. R. The structure and function of
glutamate receptor ion channels. Nature Rev.
Neurosci. 3, 91–101 (2002).
165. Armstrong, N., Sun, Y., Chen, G. Q. & Gouaux, E.
Structure of a glutamate-receptor ligand-binding
core in complex with kainate. Nature 395, 913–917
(1998).
166. Armstrong, N. & Gouaux, E. Mechanisms for
activation and antagonism of an AMPA-sensitive
glutamate receptor: crystal structures of the GluR2
ligand binding core. Neuron 28, 165–181 (2000).
167. Sun, Y. et al. Mechanism of glutamate receptor
desensitization. Nature 417, 245–253 (2002).
168. Armstrong, N., Jasti, J., Beich-Frandsen, M. &
Gouaux, E. Measurement of conformational changes
accompanying desensitization in an ionotropic
glutamate receptor. Cell 127, 85–97 (2006).
A structural study of AMPA channels that
provides clear insight into the mechanism of
desensitization.
169. Rosenmund, C., Stern-Bach, Y. & Stevens, C. F.
The tetrameric structure of a glutamate receptor
channel. Science 280, 1596–1599 (1998).
An important paper demonstrating the tetrameric
structure of AMPA channels and the fact that
different channel occupancy by glutamate leads to
different single-channel conductance.
www.nature.com/reviews/neuro
© 2007 Nature Publishing Group
REVIEWS
170. Smith, T. C. & Howe, J. R. Concentration-dependent
substate behavior of native AMPA receptors. Nature
Neurosci. 3, 992–997 (2000).
171. Ruiz, M. & Karpen, J. W. Opening mechanism of a
cyclic nucleotide-gated channel based on analysis of
single channels locked in each liganded state. J. Gen.
Physiol. 113, 873–895 (1999).
172. Ruiz, M. L. & Karpen, J. W. Single cyclic nucleotidegated channels locked in different ligand-bound states.
Nature 389, 389–392 (1997).
173. Raman, I. M. & Trussell, L. O. The mechanism of
α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate
receptor desensitization after removal of glutamate.
Biophys. J. 68, 137–146 (1995).
174. Hausser, M. & Roth, A. Estimating the time course of
the excitatory synaptic conductance in neocortical
pyramidal cells using a novel voltage jump method.
J. Neurosci. 17, 7606–7625 (1997).
175. Diamond, J. S. & Jahr, C. E. Transporters buffer
synaptically released glutamate on a submillisecond
time scale. J. Neurosci. 17, 4672–4687 (1997).
176. Postlethwaite, M., Hennig, M. H., Steinert, J. R.,
Graham, B. P. & Forsythe, I. D. Acceleration of AMPA
receptor kinetics underlies temperature-dependent
changes in synaptic strength at the rat calyx of Held.
J. Physiol. 579, 69–84 (2007).
177. Robert, A. & Howe, J. R. How AMPA receptor
desensitization depends on receptor occupancy.
J. Neurosci. 23, 847–858 (2003).
178. Rostaing, P. et al. Analysis of synaptic ultrastructure
without fixative using high-pressure freezing and
tomography. Eur. J. Neurosci. 24, 3463–3474 (2006).
179. Siksou, L. et al. Three-dimensional architecture of
presynaptic terminal cytomatrix. J. Neurosci. 27,
6868–6877 (2007).
Acknowledgements
The authors would like to thank M. Jackson, J. Zimmerberg,
A. Silver, S. Marty, L.-G. Wu, Z. Nusser, R. Schneggenburger,
G. Fain and E. Marder for useful discussions. J.E.L. has been
supported by the US National Institutes of Health (NIH)
grants R01 NS27337 and R01 NS50944 as part of the
NATURE REVIEWS | NEUROSCIENCE
Collaborative Research in Computational Neuroscience
Program. R.W.T. has been supported by NIH grants NS24067
and MH64070 and generous support from the Mathers
Foundation. S.R. has been supported by the National Science
Foundation grant 0642000.
Competing interests statement
The authors declare no competing financial interests.
DATABASES
The following terms in this article are linked online to:
Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=gene
Cav2.1 | Cav2.2 | Cav2.3
FURTHER INFORMATION
John Lisman’s homepage:
http://www.bio.brandeis.edu/lismanlab/
Synapse Web: http://synapse-web.org/
Access to this links box is available online.
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