The sequence of events that underlie quantal transmission at central

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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: [email protected]
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
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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
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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
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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.
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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
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There is a tendency for scientists to become specialized,
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adequate biophysical methods and can be accounted for
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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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