J Neurophysiol 113: 2480 –2489, 2015.
First published February 4, 2015; doi:10.1152/jn.00879.2014.
Transmitter release is evoked with low probability predominately by calcium
flux through single channel openings at the frog neuromuscular junction
Fujun Luo,1,2* Markus Dittrich,1,3,4* Soyoun Cho,1 Joel R. Stiles,1,3,4† and Stephen D. Meriney1,2
1
Department of Neuroscience, Center for Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania; 2Center for the
Neural Basis of Cognition, Pittsburgh, Pennsylvania; 3Biomedical Applications Group, Pittsburgh Supercomputing Center,
Carnegie Mellon University, Pittsburgh, Pennsylvania; and 4Department of Computational and Systems Biology, University of
Pittsburgh, Pittsburgh, Pennsylvania
Submitted 7 November 2014; accepted in final form 31 January 2015
neuromuscular junction; calcium channels; active zone; synapse;
MCell
NEUROTRANSMITTER RELEASE is triggered by action potentialevoked Ca2⫹ influx through voltage-gated Ca2⫹ channels. The
magnitude and time course of transmitter release is variable
and tightly dependent on the coupling between Ca2⫹ channels
and synaptic vesicles (Augustine and Neher 1992; Meinrenken
et al. 2002). At some synapses, the summed Ca2⫹ flux through
many open channels appears to be required for synaptic vesicle
fusion, suggesting a loose coupling between Ca2⫹ channels
and vesicle fusion machinery (Borst and Sakmann 1996; Meinrenken et al. 2002; Wu et al. 1999). However, at other synapses
a small number of Ca2⫹ channels are often sufficient to trigger
vesicle fusion (Brandt et al. 2005; Bucurenciu et al. 2010;
Jarsky et al. 2010; Schmidt et al. 2013). In fact, it has been
argued that the Ca2⫹ flux through only one or two open Ca2⫹
channels triggers vesicle fusion at some synapses (Scimemi
* F. Luo and M. Dittrich contributed equally to this work.
† Deceased 20 May 2011.
Address for reprint requests and other correspondence: S. D. Meriney, Dept.
of Neuroscience, Center for Neuroscience, Univ. of Pittsburgh, A210 Langley
Hall, Pittsburgh, PA 15260 (e-mail: [email protected]).
2480
and Diamond 2012; Stanley 1993; Yoshikami et al. 1989).
Such variable stoichiometric relationships are likely determined by the spatial organization of Ca2⫹ channels and synaptic vesicles into either microdomain- or nanodomain-controlled release sites (Eggermann et al. 2012; Tarr et al. 2013).
The frog neuromuscular junction (NMJ) has been commonly
used to study presynaptic mechanisms that control transmitter
release (Bennett 1996; Grinnell 1995; Harlow et al. 2001;
Meriney and Dittrich 2013). This synapse features hundreds of
linear active zones that are each characterized by double rows
of presynaptic ion channels and synaptic vesicles (Heuser et al.
1974; Pawson et al. 1998). Experimentally, it has been estimated that individual active zones release on average a single
vesicle following every other action potential stimulus
(Dittrich et al. 2013). An earlier study by Shahrezaei et al.
(2006) assumed 200 Ca2⫹ channels per active zone. However,
a recent statistical analysis of high spatial and temporal resolution Ca2⫹ imaging data (Luo et al. 2011) showed that active
zones contain relatively few Ca2⫹ channels (20 – 40) among
the total of 200 –250 presynaptic transmembrane protein particles observed in freeze fracture electron microscopy, and that
during an action potential these Ca2⫹ channels open with
relatively low probability (⬃0.2). These data suggest that the
number of Ca2⫹ channels in each active zone is roughly
equivalent to the number of docked synaptic vesicles (Luo et
al. 2011; Wachman et al. 2004). Consistent with this hypothesis, previous studies have suggested that transmitter release at
the frog NMJ is controlled by Ca2⫹ influx through very few,
perhaps only one or two channels (Shahrezaei et al. 2006;
Yoshikami et al. 1989). The resulting approximate 1:1 Ca2⫹
channel-vesicle stoichiometry significantly impacts how we
think about the structure-function relationship in this system.
Here, using a combination of high-resolution Ca2⫹ imaging,
postsynaptic recording, pharmacological channel block, and
3D Monte Carlo reaction-diffusion simulations of a realistic
active zone model, we present evidence that vesicle fusion is a
rare event triggered primarily by the Ca2⫹ flux through a single
open Ca2⫹ channel. Further, we show that Ca2⫹ flux through a
single open Ca2⫹ channel triggers fusion of a closely associated vesicle with only low probability (5– 6%). Based on this
insight, we propose that in the frog NMJ the observed low
probability of release from individual single vesicle release
sites within active zones is governed both by a low probability
of Ca2⫹ channel opening and a low probability that flux
through an open channel will trigger vesicle fusion.
0022-3077/15 Copyright © 2015 the American Physiological Society
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Luo F, Dittrich M, Cho S, Stiles JR, Meriney SD. Transmitter
release is evoked with low probability predominately by calcium flux
through single channel openings at the frog neuromuscular junction. J
Neurophysiol 113: 2480 –2489, 2015. First published February 4,
2015; doi:10.1152/jn.00879.2014.—The quantitative relationship between presynaptic calcium influx and transmitter release critically
depends on the spatial coupling of presynaptic calcium channels to
synaptic vesicles. When there is a close association between calcium
channels and synaptic vesicles, the flux through a single open calcium
channel may be sufficient to trigger transmitter release. With increasing spatial distance, however, a larger number of open calcium
channels might be required to contribute sufficient calcium ions to
trigger vesicle fusion. Here we used a combination of pharmacological calcium channel block, high-resolution calcium imaging, postsynaptic recording, and 3D Monte Carlo reaction-diffusion simulations in the adult frog neuromuscular junction, to show that release of
individual synaptic vesicles is predominately triggered by calcium
ions entering the nerve terminal through the nearest open calcium
channel. Furthermore, calcium ion flux through this channel has a low
probability of triggering synaptic vesicle fusion (⬃6%), even when
multiple channels open in a single active zone. These mechanisms
work to control the rare triggering of vesicle fusion in the frog
neuromuscular junction from each of the tens of thousands of individual release sites at this large model synapse.
SINGLE Ca2⫹ CHANNELS TRIGGER RELEASE WITH LOW PROBABILITY
MATERIALS AND METHODS
during data collection using custom software written by Greg Hood
(Pittsburgh Supercomputer Center). Differences in fluorescence above
rest were determined for individual images by subtracting mean
resting fluorescence (generated by averaging the 10 background
images). The resulting “difference images” represented the relative
fluorescence changes, computed as ⌬F/F0 ⫽ (F ⫺ F0)/F0, and were
displayed in pseudo-color. In multiple trial experiments, 5–10 sets of
Ca2⫹ images (each set consisting of 10 Ca2⫹ images with stimulation
off and 10 Ca2⫹ images with stimulation on as described above) were
collected for each well-focused nerve terminal and coregistered to the
first image of the first dataset. Between each set, we confirmed/
adjusted focus onto the active zone in reference to ␣-BTX staining
and discarded image sets from analysis if they showed noticeable
z-axis drift.
Imaging analysis. We restricted our quantitative analysis to the
nerve terminal regions as defined by a mask based on the resting
fluorescence intensity. The blocking effect of titrating ␻-conotoxin
GVIA (CgTX) on action potential-evoked Ca2⫹ influx was measured
by calculating the ratio of total Ca2⫹ entry into active zones before
and after CgTX treatment from the same terminal regions. The total
Ca2⫹ entry was estimated by the sum of pixel fluorescence intensities
above background (⌬F/F0, see above). Only pixels that reported
significant action potential-evoked fluorescence change under control
condition (i.e., before drug treatment, t-test, P ⬍ 0.01) were included.
Exposure to relatively high concentrations of CgTX greatly reduced action potential-evoked Ca2⫹ entry and therefore fluorescence
change (e.g., by 92% in 600 nM CgTX). We have previously shown
that the calcium signal reported using this approach is linearly related
to calcium entry (Luo et al. 2011). In order to reliably identify Ca2⫹
entry under this condition, we used a per-pixel selection criterion that
required that stimulated fluorescence change above rest (⌬F) should
exceed 3 times the standard deviation (3 ⫻ SD) of the resting
fluorescence. This criterion was chosen based on the following reasons. First, the use of 2 ⫻ SD as a criterion was too sensitive and
yielded the selection of a large number of pixels between active zones,
and a significant number of false positives selected even under
unstimulated conditions. On the other hand, the use of 4 ⫻ SD was too
stringent and eliminated so many pixels from the selection that there
were almost none detected within active zone regions. Second, in
testing a variety of criteria (2, 3, or 4 SD), we compared the number
of pixels that were chosen to the expected number of channels
remaining based on the use of such a high concentration of CgTX
(600 nM). This CgTX treatment decreased total average Ca2⫹ entry
(as measured by the decrease in Ca2⫹-sensitive fluorescent signal)
into the nerve terminal by 92% (see Table 1). In a previous study of
untreated control synapses, we estimated that there are 20 – 40 Ca2⫹
channels in each active zone, and that each open during an action
potential with a probability (po) of ⬃0.2 (see Luo at al. 2011).
Therefore, a 92% reduction in total Ca2⫹ entry would correspond to
⬃2 Ca2⫹ channels remaining in each active zone. Calculating the
probability of at least one of these two channels A and B opening
during an action potential yields a probability of po(A or B) ⫽ po(A) ⫹
po(B) - po(AB) ⫽ 0.36. Using the 3 ⫻ SD criterion led to a detection
of a number of Ca2⫹ entry sites that was roughly consistent with this
expectation for po (see Figs. 4 and 5), providing further support for
this measure.
Table 1. The effect of CgTX titration on single action potential-evoked Ca2⫹ entry and neurotransmitter release (as measured by
changes in quantal content)
[CgTX], nM:
25
50
75
100
400
600
%Ca2⫹ entry blocked
%Vesicle release blocked
22 ⫾ 1.0
34 ⫾ 2.4
41 ⫾ 2.6
64 ⫾ 2.9
59 ⫾ 4.6
79 ⫾ 5.0
70 ⫾ 5.9
84 ⫾ 1.8
85 ⫾ 8.0
96 ⫾ 2.5
92 ⫾ 1.7
98 ⫾ 1.4
Values are means ⫾ SD.
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Tissue preparation. Adult frogs (Rana pipiens) were decapitated
and pithed following anesthesia in 0.6% tricaine methane sulfonate
solution as approved by the University of Pittsburgh Institutional
Animal Care and Use Committee. The cutaneous pectoris nervemuscle preparation was dissected bilaterally and bathed in normal
frog Ringer (NFR; in mM: 116 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 5
HEPES, pH 7.4) as previously described (Luo et al. 2011; Wachman
et al. 2004).
Dye loading. The Ca2⫹-sensitive dye Calcium Green-1 (3,000 MW
dextran conjugate; Molecular Probes) was loaded through the cut end
of the nerve as previously described (Luo et al. 2011; Wachman et al.
2004). Briefly, the nerve was immersed in a drop of 30 mM dye
dissolved in distilled water for 6 – 8 h at room temperature. After
rinsing in NFR, the preparation was stored at 4°C for 2–3 h.
Ca2⫹ imaging and processing. These procedures were similar to
those described by Wachman et al. (2004) and Luo et al. (2011). The
muscle was pinned over an elevated Sylgard (Dow Corning) platform
in a 35-mm dish mounted on a microscope stage. The postsynaptic
acetylcholine receptors were labeled by exposure to 2 ␮g/ml Alexa
594-conjugated ␣-bungarotoxin (␣-BTX) for 10 min. ␣-BTX staining
was used to locate and focus the postsynaptic receptor bands, which
are positioned directly opposite from the presynaptic active zones, and
evaluate the z-axis drift over the course of data collection. Superficially positioned nerve terminals, the majority of which were in a
single focal plane as judged by ␣-BTX staining, were chosen for
study. All Ca2⫹ imaging was performed in NFR (except as noted)
with 10 ␮M curare added to prevent nerve-evoked muscle contractions that were not completely blocked by the ␣-BTX.
An acousto-optic tunable filter (AOTF; ChromoDynamics) was
employed to select wavelengths and gate the laser illumination with
sub-millisecond time resolution (Krypton-Argon laser; Innova 70
Spectrum, Coherent). The laser was fiber-coupled into the epi-illumination port of an upright fluorescence microscope equipped with a
⫻100 water-immersion long working-distance objective with 1.0 NA
(Lumplan/FL IR, Olympus). Calcium Green-1 was excited at 488 nm
and emission light was collected through a 530 ⫾ 20 nm filter. Alexa
594-␣-BTX was excited at 567 nm and emission light was collected
through a 620 ⫾ 30 nm filter. The timing of laser illumination was
triggered with nerve stimulation following a 1.5-ms delay (for nerve
conduction), and dye was illuminated for only 1 ms during action
potential invasion of the nerve terminal to limit spatial diffusion of
Ca2⫹ entering through voltage-gated Ca2⫹ channels. Images were
recorded on a liquid nitrogen-cooled, back-thinned CCD camera
(LN1300B, Roper Scientific), which provided the high-sensitivity
low-noise detection necessary for the measurement of fluorescence
changes sampled during brief 1-ms time windows.
Raw resting photoelectron counts ranged between 1,200 and 4,000
in different dye-loaded nerve terminals, and fluorescence changes
during nerve stimulation (using a suction electrode at a current
intensity of 5⫻ threshold) were significantly above resting fluctuations (dominated by shot noise). Images were collected at 0.5 Hz
in sets of 20: the first 10 images with stimulator off (background)
and the second 10 images with stimulator on (nerve-evoked signals). Images were processed on a Pentium-based computer using
MATLAB (MathWorks). Prior to analysis, images were coregistered
to correct for slight fluctuation in the lateral position of the preparation
2481
SINGLE Ca2⫹ CHANNELS TRIGGER RELEASE WITH LOW PROBABILITY
2482
A
B
number of Ca2⫹ channels and their opening probability following
single action potentials were constrained by our recent Ca2⫹ imaging
experiments (Luo et al. 2011). During each simulation, an invading
action potential led to the stochastic opening of a varying number of
voltage gated Ca2⫹ channels based on their open probability (p ⬃
0.2). Once open, Ca2⫹ channels would permit Ca2⫹ ions to enter the
presynaptic space according to the instantaneous driving force and the
experimentally known channel conductance. Presynaptic, freely diffusing Ca2⫹ ions could then either bind to Ca2⫹ sensor sites on
synaptic vesicles (synaptotagmin) or 2 mM fixed buffer molecules
with kinetic rates as reported in Dittrich et al. (2013). Synaptic vesicle
release was determined via our excess-calcium-binding-site model
(Dittrich et al. 2013), which unifies recent biochemical evidence
regarding the number, arrangement, and properties of available synaptotagmin molecules on synaptic vesicles (our model has 8 synaptotagmin molecules near the base of the vesicle with 5 low-affinity
Ca2⫹ binding sites each, see Fig. 1A) and classical physiology such as
the 4th-order calcium-release relationship. The number and positioning of synaptotagmin molecules in the model was chosen to be
consistent with previously published proteomic studies (Mutch et al.
2011; Takamori et al. 2006), recently hypothesized orientations of
these proteins (Kummel et al. 2011; Wang et al. 2014), and the best
fit of the physiology data with these model parameters (Dittrich et al.
2013). In particular, the detailed arrangement of synaptotagmin sites
on the bottom of synaptic vesicles does not impact the qualitative
features of our model as we have recently shown for second sensor
sites within an extension of the model presented in this paper (Ma et
al. 2015). Our model captures the well-known narrow distribution of
release latencies at the frog NMJ (Katz and Miledi 1965). In contrast
to earlier computational studies (Shahrezaei et al. 2006) our model is
significantly more detailed and includes the previously determined
estimated number of presynaptic voltage-gated Ca2⫹ channels and
their opening probability (Luo et al. 2011), proper timing of stochastic
channel opening driven by an action potential waveform, and the
resulting narrow latency distribution of synaptic vesicle release. To
simulate the experimental titration with increasing concentrations of
␻-CgTX GVIA we randomly removed increasing numbers of voltagegated Ca2⫹ channels from the model and then measured the resulting
synaptic vesicle release.
For each model condition we performed 10,000 statistically independent runs on a computer cluster at the Pittsburgh Supercomputing
Center (codon.psc.edu, a cluster of 23 dual processor machines, each
with two 1.6-GHz AMD Opteron processors and 8 gigabytes of
memory). Data analysis was performed with custom-written scripts.
RESULTS
The relationship between presynaptic Ca2⫹ entry domains
and triggering of transmitter release. In order to explore the
quantitative relationship between Ca2⫹ channel openings and
transmitter release at the frog NMJ, which predominantly
VGCCs
closed
open
Synaptic Vesicle
70 nm
50 nm
Fig. 1. Diagram of the MCell model representation of synaptic vesicles and their arrangement into an active zone. A: schematic view of the bottom of a docked
synaptic vesicle used in our MCell model. In this view, the 40 Ca2⫹ binding sites in groups of 5 are depicted in various shades of gray and each represent a
synaptotagmin molecule. B: view of our MCell frog neuromuscular junction (NMJ) active zone (AZ) model including 26 docked synaptic vesicles and their
closely associated presynaptic Ca2⫹ channels (VGCCs). During an action potential, only a small subset of these Ca2⫹ channels are predicted to open. Depicted
is a sample single model run in which open channels are represented as white pentagons and closed channels as black pentagons.
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For statistical analysis, the number of detected openings was
normalized (as openings per active zone per action potential) for each
terminal by dividing the total number of openings by the number of
active zones and the number of stimulation trials.
Electrophysiology. Intracellular recordings from the adult frog
cutaneous pectoris nerve-muscle preparation were performed as described previously (Douthitt et al. 2011; Meriney and Grinnell 1991).
In brief, the nerve of the cutaneous pectoris muscle was stimulated via
a suction electrode at 5⫻ the threshold intensity required to elicit
muscle twitch. Intracellular micropipettes were made from glass
pipettes (Warner Instruments, filament glass catalog no. 64-0787;
O.D. 1 mm; I.D. 0.58 mm) and had a resistance of 25–50 M⍀ after
filling with 3 M potassium acetate. Surface muscle fibers were
penetrated close to nerve endings under visual control with a long
working distance water-immersion objective (⫻40, 3 mm working
distance). Only muscles with a resting potential more hyperpolarized
than ⫺70 mV were recorded for analysis. Spontaneous miniature
endplate potentials (mEPPs) and action potential-evoked endplate
potentials (EPPs) were recorded. To calculate the number of synaptic
vesicles released following each action potential, quantal content was
calculated using the direct method (EPP amplitude/mEPP amplitude).
Data were amplified using a Dagan BBC 700 amplifier and acquired
using Clampex 9 software (Axon Instruments, Foster City, CA).
Clampfit 9.2 was used for data analysis.
Drug treatment. CgTX is known to block N-type Ca2⫹ channels in
a manner that is essentially irreversible (Stocker et al. 1997). For
titrating channel block, the frog nerve-muscle preparation was incubated with various doses of CgTX for 45– 60 min and then washed in
NFR. Superficially positioned nerve terminals and muscle fibers were
chosen for study to avoid variability in toxin access to nerve terminals
positioned at different depths of the preparation. Blocking effects on
transmitter release and Ca2⫹ influx were determined in separate
experiments to optimize the health of the terminal for imaging
experiments and to avoid potential complications due to the buffering
effects of Ca2⫹-sensitive dye during recordings of transmitter release.
Monte Carlo simulations. All computational modeling was carried
out with version 3.1 of the MCell software developed in our lab (Kerr
et al. 2008; www.mcell.org). The active zone model used in this paper
was identical to our recently developed spatially realistic 3-D model
of the frog NMJ active zone (Dittrich et al. 2013) and used here
without modification. This model is heavily constrained by a wealth of
anatomical, physiological, and biochemical data that have been collected over decades of study on the frog NMJ and other model
synapses, as well as more recent work in our laboratory. The geometry
of our active zone model was based on published ultrastructural data
from the adult frog NMJ (Harlow et al., 2001; Heuser et al. 1974;
Pawson et al. 1998) and contained 26 synaptic vesicles arranged in
two double rows (Fig. 1B). In addition, 26 N-type voltage-gated Ca2⫹
channels were positioned in a one-to-one topographic relationship
with each docked synaptic vesicle at an average distance from the
vesicle of 35 nm (Fig. 1B, see also Dittrich et al. 2013). The total
SINGLE Ca2⫹ CHANNELS TRIGGER RELEASE WITH LOW PROBABILITY
C
Single AP-evoked calcium entry
10
ΔF/F(%)
0
CgTX
Ctrl
B
EPP
1 mV
10 ms
Ctrl
fraction of transmitter release
A
trials is shown in Fig. 2A. When compared with control,
exposure to 100 nM CgTX caused a dramatic decrease in
action potential-evoked fluorescence signals at the nerve terminal. The ratio of action potential-evoked-Ca2⫹ influx before
and after drug treatment was used to represent the blocking
percentage of Ca2⫹ channels by various concentrations of
CgTX (see Table 1).
We next examined the effects of the same concentrations of
CgTX on the magnitude of neurotransmitter release using
intracellular recording of endplate potentials (EPPs). EPPs
were recorded from the same muscle fiber before and after
incubation with CgTX. For these recordings, 3–7 ␮M curare
(for partial block of acetylcholine receptors) and 2 ␮M ␮-conotoxin PIIIA (a muscle-specific sodium channel blocker) were
added to the Ringer bathing the preparation to block muscle
contraction. The concentration of curare was titrated to reduce
EPP amplitude to less than 10 mV to avoid significant effects
of nonlinear summation of quantal events underlying the EPP,
(R)elease =
1
log (R) =
E
fraction of fusion events
Fraction of transmitter release
m log (Ch)
2
3 4
0. 01
0. 01
0. 1
1
fraction of calcium entry
D
1
1
2
3
4
1.0
F
Control
Reduced # channels
1.0
0. 8
0. 8
0. 6
0. 6
1
1
2
0. 4
0. 4
1
2
3
0. 2
0. 2
4
0.01
0.01
m
1
0. 1
CgTX
0.1
(Ch)annel
0.1
Fraction of calcium entry
1
0
26 AZ Ca2+ channels
3
2
0
8 AZ channels 2 AZ channels
# Channels contributing bound ions to vesicle
Fig. 2. Titrating ␻-conotoxin (CgTX) GVIA blockade and comparing block of Ca2⫹ entry with block of vesicle fusion reveals a low Ca2⫹ channel cooperativity
in transmitter release. A: CgTX (100 nM) significantly reduced single action potential-evoked Ca2⫹ entry into the nerve terminals. B: EPP amplitude decreased
after exposure to 100 nM CgTX. C: a log-log plot of fractional Ca2⫹ entry and neurotransmitter release after treatment with various concentrations of CgTX (25,
50, 75, 100, 400, and 600 nM) reveals a relationship with a slope between 1 and 2 (lines with indicated slopes are represented in black, green, blue, and yellow).
The dashed green line is a fit to the first few data points during only a small fractional block of Ca2⫹ channels. This dashed green line has a slope close to 2
(similar to the solid green line). However, after a large fraction block of Ca2⫹ channels the data are fit using a dashed black line and show a relationship with
a slope close to 1 (similar to the solid black line). D: MCell computer simulations reproduce experimental data and predict that each vesicle fusion event is
triggered by the Ca2⫹ flux through very few Ca2⫹ channels. A log-log plot compares changes in Ca2⫹ entry and simulated neurotransmitter release after randomly
removing increasing numbers of Ca2⫹ channels from the modeled active zone (which mimics the CgTX titration shown in C). The simulated data fall on a slope
between 1 and 2, similar to experimental data shown in C. As in C, the data collected after the removal of only a few calcium channels are fit using a dashed
green line, which has a slope close to 2, while the data collected after the removal of many calcium channels are fit using a dashed black line, which has a slope
close to 1. E: histogram of the fraction of released synaptic vesicles that bound Ca2⫹ ions contributed by various numbers of Ca2⫹ channels under the control
condition (26 Ca2⫹ channels in the AZ). F: histograms of the fraction of released synaptic vesicles that bound Ca2⫹ ions that originated from various numbers
of Ca2⫹ channels when most of the AZ Ca2⫹ channels were removed from the simulation (8 channels present; 70% block, and 2 channels present; 92% block).
With increasing removal of AZ Ca2⫹ channels, vesicle fusion increasingly is triggered by the Ca2⫹ flux through a single Ca2⫹ channel (dark blue bar).
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expresses N-type Ca2⫹ channels, we titrated the concentration
of CgTX to block different fractions of Ca2⫹ channels, and
thus presynaptic Ca2⫹ entry. Although similar experiments
have been performed previously (Shahrezaei et al. 2006), our
study improves on these in two significant ways. First, we
determined changes in Ca2⫹ influx and transmitter release in
separate experiments to avoid potential buffering effects of
Ca2⫹-sensitive dye on our measures of transmitter release.
Second, we imaged Ca2⫹ entry with fast spatial and temporal
resolution to detect restricted sites of Ca2⫹ entry as opposed to
volume averaged signals within the whole nerve terminal.
Furthermore, we compare these data to our computer simulations presented below.
When the nerve was stimulated at low frequency (0.5 Hz),
strong Ca2⫹-dependent fluorescence signals were detected
within presynaptic active zones (Luo et al. 2011; Wachman et
al. 2004). An example of averaged action potential-evoked
fluorescence change (normalized as ⌬F/F0) over 10 stimulus
2483
SINGLE Ca2⫹ CHANNELS TRIGGER RELEASE WITH LOW PROBABILITY
Table 2. The effect of titrating blockade of Ca2⫹ channels on
action potential-evoked neurotransmitter release using Monte Carlo
simulation
Active Zone Channel Number:
26
20
15
8
4
2
%Channels removed
%Vesicle release blocked
Control
Control
23
32
42
57
69
79
85
91
92
96
A
SV
VGCC
4%
15%
94%
15%
5%
3%
9%
14%
9%
4%
B
SV
1
4
82
4
1
1
2
3
2
1
VGCC
C
SV
2
31
---
27
2
2
6
16
8
2
VGCC
Fig. 3. Ca2⫹ flux through a single open Ca2⫹ channel primarily provides the
trigger for vesicle fusion in our simulated active zone model, even when other
channels open nearby. A: given a particular vesicle fusion event in the AZ (red
circle), the average probability that individual Ca2⫹ channels have opened is
represented by the red area in the pie chart and the corresponding percentage
associated with each channel icon. B, left: for the majority of vesicle fusion
cases in which the closely-associated Ca2⫹ channel has opened (94% as
represented in A above), the number in each Ca2⫹ channel circle icon indicates
the percentage of Ca2⫹ ions contributed by each Ca2⫹ channel to the total
number of ions bound by the vesicle at the time of fusion (red circle). B, right:
representative model schematic depicting the 40 Ca2⫹ binding sites (gray
areas) at the bottom of synaptic vesicles (SV). The colored dots indicate Ca2⫹
ions (color coded by their channel of origin) bound to different synaptotagmin
binding sites at the time of vesicle fusion for a representative single model run
under the condition that the closely associated Ca2⫹ channel has opened (94%
of fusion events). C, left: for the minority of vesicle fusion cases in which the
closely associated Ca2⫹ channel has not opened (6% as represented in A
above), the number in each Ca2⫹ channel circle icon indicates the percentage
of Ca2⫹ ions contributed by each Ca2⫹ channel to total ions bound by the
vesicle at the time of fusion (red circle). C, right: representative model
schematic of the bound Ca2⫹ ions on vesicular synaptotagmin binding sites at
the time of fusion for a representative single model run under the conditions
that the closely associated Ca2⫹ channel has not opened (6% of fusion events).
Schematic is organized as described above for B.
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while still permitting the EPP to be measured both before and
after exposure to various concentrations of CgTX. Figure 2B
shows an example of averaged EPP traces before and after
exposure to 100 nM CgTX, and the summary data are reported
in Table 1.
We estimated the stoichiometric relationship between Ca2⫹
entry and transmitter release by plotting the blocking effects of
titrating CgTX on both Ca2⫹ entry and transmitter release on a
log-log scale. We have previously demonstrated that our imaged Ca2⫹ signals are linearly related to Ca2⫹ influx under
these experimental conditions (Luo et al. 2011). As shown in
Fig. 2C, the resulting relationship is not completely linear over
the range of CgTX doses examined. At low doses of CgTX
(25–50 nM) the relationship appeared to fall on a line with a
slope of ⬃2, but as the concentration of CgTX was increased,
this relationship approached a slope closer to 1. These results
suggest that under control conditions, only a small number of
Ca2⫹ channels contribute Ca2⫹ to individual vesicle release
events, consistent with previous observations at this synapse
(Shahrezaei et al. 2006; Yoshikami et al. 1989). However, as
presynaptic Ca2⫹ channels are blocked, release events depend
increasingly on single open channels.
We then tested whether our spatially realistic excess-Ca2⫹binding-site model of the frog NMJ active zone (Dittrich et al.
2013) was able to reproduce the effect of titrating CgTX to
block presynaptic Ca2⫹ entry on transmitter release. This
model has been shown to reproduce many properties of neuromuscular transmission faithfully including the normal release
rate, the 4th power relationship between vesicle release and
extracellular Ca2⫹ concentration, and synaptic delay (Dittrich
et al. 2013). To model CgTX titration, an increasing number of
Ca2⫹ channels were randomly removed from the active zone,
and the resulting amount of vesicle release following single
action potential stimulation was simulated. As shown in Fig.
2D and Table 2, the model reproduced accurately the experimental observations (compare with Fig. 2C and Table 1), and
thus could be used to aid in interpreting these data.
Since our experimental data indicated that very few, but
likely more than one Ca2⫹ channel contributed Ca2⫹ ions to
triggering single vesicle release from control terminals, we
used our MCell results to estimate the percentage of released
vesicles that bound Ca2⫹ ions from one, two, or more Ca2⫹
channels (Fig. 2E). The simulations showed that 5.2 ⫾ 0.3
Ca2⫹ channels opened in each active zone following a single
action potential stimulation. Interestingly, we found that about
34% of released synaptic vesicles bound only Ca2⫹ ions that
entered the nerve terminal through a single open Ca2⫹ channel,
and 41% of vesicle fusion events were triggered by Ca2⫹ ions
derived from only two open Ca2⫹ channels (see also Dittrich et
al. 2013). Vesicle fusion triggered by Ca2⫹ ions from more
than two channel openings was less likely. On average, synaptic vesicle fusion was triggered by Ca2⫹ ions derived from
1.9 Ca2⫹ channels, consistent with our experimental CgTX
titration data. The spatial distribution of the average contribution of specific Ca2⫹ channels to a given vesicle fusion event
is graphically depicted in Fig. 3B. As expected, this relation-
70nm 35nm
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SINGLE Ca2⫹ CHANNELS TRIGGER RELEASE WITH LOW PROBABILITY
B
C
D
BTX labeling
10
8
6
4 ΔF/F(%)
2
0
control
600 nM CgTX
1
0
2⫹
20
15
10
5
0
-20 -10 0 10 20 30 40 50
ΔF/F(%)
E
mEPPs
3
2
resting
600 nM CgTX
400 nM CgTX
control
25
frequency (%)
A
the time to originate from the single nearest Ca2⫹ channel (Fig.
3B, left panel). Therefore, our model suggests that of the
average 7– 8 Ca2⫹ ions that bind to trigger vesicle fusion, 6 –7
were derived from the closest Ca2⫹ channel and only 1–2 Ca2⫹
ions originate from other open Ca2⫹ channels (Fig. 3B, right
panel). On the other hand, when the nearest, closely-associated
Ca2⫹ channel did not open, a synaptic vesicle rarely fused
(only 6% of total fusion events). Under these rare conditions,
the Ca2⫹ ions that bound to the fused synaptic vesicle derived
predominately from a nearby cluster of 3–5 Ca2⫹ channels,
with each of these channels contributing 1–2 Ca2⫹ ions to the
synaptic vesicle Ca2⫹ binding sites (Fig. 3C). In conclusion,
our simulations predict that action potential-evoked transmitter
release at the frog NMJ is controlled by the Ca2⫹ ions that
derive predominantly from the opening of one closely associated Ca2⫹ channel, even when several Ca2⫹ channels open in
the active zone (Fig. 3, A and B).
Experimental evidence that single channel openings can
trigger synaptic vesicle release. Our previous Ca2⫹ imaging
data (Luo et al. 2011; Wachman et al. 2004) have shown that
each active zone in the adult frog NMJ has relatively few Ca2⫹
channels (20 – 40), and that each Ca2⫹ channel has a relatively
low opening probability during an action potential (⬃0.2).
Therefore, only a few of these Ca2⫹ channels would be
predicted to open (4 – 8) within each active zone during a single
action potential. Thus we attempted to reduce the number of
functional Ca2⫹ channels so that a single action potential
would open at most one Ca2⫹ channel within each active zone.
To block most Ca2⫹ channels, we exposed the preparation to
600 nM CgTX for 60 min. We imaged action potential-evoked
Ca2⫹ influx at active zones and found that the total Ca2⫹ entry
was reduced by 92 ⫾ 1.7% (Fig. 4, A–C; n ⫽ 3). Following a
reduction of this magnitude, the number of available Ca2⫹
channels within individual active zones is expected to be very
1 mV
100 ms
1 mV
10 ms
ΔF/F(%)
EPP
2⫹
Fig. 4. Imaging Ca entry through single Ca channel openings in the frog NMJ active zone. A: postsynaptic labeling of acetylcholine receptors [Alexa
594-conjugated ␣-bungarotoxin (BTX)] is used for estimating the number of active zones within the imaged regions of the nerve terminal. For visual clarity,
offset yellow ovals show schematic outlines of the 31 AZs identified in this fluorescence image. B: average Ca2⫹ signal for a representative control nerve terminal
(100 stimuli at 0.5 Hz). C: average Ca2⫹ signal remaining after block of presynaptic Ca2⫹ channels using a 60-min exposure to 600 nM CgTX (100 stimuli at
0.5 Hz). D: histogram distribution of single pixel fluorescence intensity under resting (unstimulated), stimulated control, and stimulated CgTX treatment
conditions. Exposure to 400 or 600 nM CgTX resulted in a distribution that took a lognormal form suggesting that individual pixels sampled single Ca2⫹ channel
openings. This was distinct from the Gaussian-like distribution of resting intensities, and the distribution of intensities following stimulation under control
conditions (no toxin block). E: representative recordings of miniature endplate potentials (mEPPs) from a muscle fiber (top), and averaged EPP response evoked
by nerve stimulation (bottom), after exposure to 600 nM CgTX. Using these recordings, quantal content (QC) was calculated (QC ⫽ average EPP
amplitude/average mEPP amplitude) as listed in the text. Scale bars ⫽ 2 ␮m.
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ship changed when we decreased the number of available Ca2⫹
channels in the model active zone (Fig. 2F). When 80% of
Ca2⫹ channels were blocked, the average number of Ca2⫹ channels that open was reduced to 1.0 ⫾ 0.2 and the average number
of vesicles released per single active zone was reduced to 0.06 ⫾
0.007. Under these conditions, using large simulation runs
(10,000) to increase the reliability of our estimates, we determined that 460 of 550 (84%) released vesicles were bound by
Ca2⫹ ions from a single Ca2⫹ channel. Therefore, the model
predicts that vesicle fusion will still occur with only a small
number of active Ca2⫹ channels in the active zone and that
these events will be triggered almost exclusively by Ca2⫹ ions
derived from a single open Ca2⫹ channel.
Single Ca2⫹ channel openings predominate in contributing
Ca2⫹ ions to vesicle fusion even when more than one channel
opens in the active zone. To further understand the quantitative
contribution of Ca2⫹ channels to triggering vesicle release, we
tracked the pattern of Ca2⫹ channel opening and Ca2⫹ ion
binding for each given fused vesicle using our recently published excess-calcium-binding-site model of the frog NMJ
active zone (Dittrich et al. 2013). Using this approach, Dittrich
et al. (2013) showed that known properties of action potentialevoked transmitter release at the frog NMJ could be best fit if
synaptic vesicle fusion was triggered by at least two Ca2⫹ ions
binding to at least three of the modeled synaptotagmin molecules on a given synaptic vesicle. In fact, on average, 7– 8 Ca2⫹
ions bound to a synaptic vesicle (among the 40 total Ca2⫹
binding sites available, see MATERIALS AND METHODS; Dittrich et
al. 2013) at the time of vesicle fusion. Here, we extended our
use of this model to evaluate the Ca2⫹ channel of origin for
these bound Ca2⫹ ions. Interestingly, we found that when a
particular vesicle fused, 94% of the time the nearest, closelyassociated Ca2⫹ channel opened (Fig. 3A). For these cases, the
Ca2⫹ ions that bound to the fused vesicle were found 82% of
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SINGLE Ca2⫹ CHANNELS TRIGGER RELEASE WITH LOW PROBABILITY
treatment condition. To calculate Ca2⫹ channel openings per
active zone, we limited our analysis to those nerve terminal
regions where we could clearly identify individual active zones
(see Fig. 4A). For example, 31 active zones can be distinguished within the region of interest identified in the representative nerve terminal shown in Fig. 4A. After exposure to 600
nM CgTX, we collected imaging data from 100 stimulus trials
and calculated that each action potential evoked an average of
0.23 Ca2⫹ channel openings per active zone. Similar results
were obtained from 8 nerve terminals and the mean number of
openings evoked by a single action potential was estimated to
be 0.18 ⫾ 0.07 per active zone (Fig. 5A). This value is very
close to our estimate of the average opening probability of
individual Ca2⫹ channels (Luo et al. 2011), which provides
further support for our hypothesis that the number of functional
Ca2⫹ channels within individual active zones is reduced to
approximately one after exposure to 600 nM CgTX. We also
calculated the number of detected Ca2⫹ entry sites for nerve
terminals treated with 400 nM CgTX. In these experiments, we found
that the number of Ca2⫹ channel openings per active zone was
C
B
0.6
0.4
0.2
0
0.03
0.02
0.01
0
600 nM 400 nM
CgTX CgTX
600 nM 400 nM
CgTX CgTX
D
10
10
8
8
6
6
Pr/o(%)
Pr/o(%)
0.04
release probability
per AZ per AP
A
4
2
4
2
0
600 nM 400 nM
CgTX CgTX
0
1
2
3
4
5
number of open channels
Fig. 5. The coupling of Ca2⫹ influx to vesicle fusion events after exposure to
a high concentration of CgTX. A: shown is the number of detected Ca2⫹
channel openings per active zone and action potential after exposure to either
400 nM or 600 nM CgTX. These data are obtained by dividing the number of
imaged Ca2⫹ entry sites per action potential by the number of active zones
within an imaged area (see Fig. 3). B: normalized release probability per active
zone and action potential after exposure to 400 nM or 600 nM CgTX
determined by dividing the average QC by the average number of active zones
in the nerve terminal of adult frog cutaneous pectoris NMJs (700). C:
experimental estimate of release probability per Ca2⫹ channel opening [Pr/
o(%)] is similar (5– 6%) when calculated following exposure to 400 nM or 600
nM CgTX (plot generated using the data in A and B). D: MCell simulations
predict that release probability following a single Ca2⫹ channel opening is
small (5– 6%) and comparable to our experimental estimate shown in C.
Further, the release probability per Ca2⫹ channel opening remains relatively
constant even as the number of open channels within an active zone is
increased in our simulation.
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small (1⬃2), and given the low probability of opening (⬃0.2),
the likelihood that more than one channel would open in the
same active zone during a single action potential stimulus
would be very small (⬃0.04). In agreement with this expectation, the Ca2⫹-sensitive fluorescent signals detected by each
imaging pixel in our imaging system had a large coefficient of
variation (CV ⫽ 1.6 ⫾ 0.5) after 600 nM CgTX treatment,
compared with control conditions (CV ⫽ 0.5 ⫾ 0.3), consistent
with a large reduction in the number of channels. To increase
the total number of Ca2⫹ signals detected for these experiments, we collected 70 –100 trials of stimulus-evoked Ca2⫹
images after CgTx treatment. After exposure to 600 nM CgTX,
we identified discrete, sparse, and spatially distributed signals
evoked by single action potentials within well-focused regions
of the nerve terminal (Fig. 4C). As shown in Fig. 4D, under
600 nM CgTX block the histogram distribution of the fluorescence intensity detected by individual pixels underlying Ca2⫹
channel openings takes a log-normal form, similar to the
distribution of the single channel current integral (see figure 2,
C and D, in Dittrich et al. 2013). This distribution is generated
as single channels open with variable mean open time under
conditions of rapidly changing driving force at different times
during the action potential waveform. The similarity of the
fluorescence and single channel current histogram distributions
is consistent with our prediction that at most one Ca2⫹ channel
opens in each active zone under these conditions. A similar
lognormal distribution was observed after exposure to 400 nM
CgTX, which reduced total action potential-evoked Ca2⫹ influx evoked by 84.7 ⫾ 3.3%. In contrast, for control terminals,
the histogram distribution of signal intensities from individual
pixels was significantly shifted to higher magnitude and took a
more Gaussian shape (Fig. 4D). We hypothesize that exposure
to either 400 or 600 nM CgTX blocked sufficient numbers of
Ca2⫹ channels in each active zone that imaged Ca2⫹ signals
were derived predominantly from a single open channel. Under
these conditions, we predict that transmitter release would be
rare, and when it occurred, it would be triggered by single
channel openings.
In order to test this hypothesis, we carried out postsynaptic
recording of EPPs from muscle fibers after exposure to either
600 nM or 400 nM CgTX. As shown in Fig. 4E, we still
detected significant transmitter release from nerve terminals
after exposure to either 400 or 600 nM CgTX, despite the rare
Ca2⫹ channel openings in each active zone as predicted under
these conditions. After exposure to 400 nM CgTX quantal
content measured 15.2 ⫾ 8.8, while after exposure to 600 nM
CgTX quantal content was only 7.6 ⫾ 4.9. These values are
obviously much lower than what has been reported in untreated
control nerve terminals (quantal content ⬃350; Dittrich et al.
2013; Katz and Miledi 1979), but support the hypothesis that
isolated single Ca2⫹ channel openings can trigger transmitter
release from the frog NMJ.
Estimating the probability of transmitter release triggered
by the Ca2⫹ flux through a single Ca2⫹ channel opening. How
reliably can single Ca2⫹ channel openings trigger synaptic
vesicle fusion? In order to evaluate the vesicle fusion probability due to single Ca2⫹ channel openings, we measured the
frequency of observing Ca2⫹ channel openings at the single
active zone level after exposure to different concentrations of
CgTX. We then compared the channel opening frequency with
the release probability of a single active zone under each
# channel openings
per AZ per AP
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SINGLE Ca2⫹ CHANNELS TRIGGER RELEASE WITH LOW PROBABILITY
mainly triggered by a single open channel, the release probability per opening should not change significantly regardless
how many channels open in the active zone. On the other hand,
if several open channels cooperate to trigger fusion of a given
vesicle, the release probability per opening should increase
with more open channels, perhaps even in a supralinear fashion. With the full complement of Ca2⫹ channels available in
our active zone model (26), the average number of channels
that opened with each action potential was ⬃5, but even under
this condition, the probability of vesicle release per opening
was only 6 –7%. As shown in Fig. 5D and Table 3, our model
predicted that the release probability per Ca2⫹ channel opening
was little affected by the total number of open Ca2⫹ channels
in the active zone. These data underscore the predominance of
vesicle fusion events triggered by Ca2⫹ flux through a single
open channel, even when several Ca2⫹ channels open in the
active zone.
DISCUSSION
We have provided experimental and computational evidence
that at the adult frog NMJ vesicle release is triggered by a
spatially localized nanodomain of Ca2⫹ ions overwhelmingly
derived from the single Ca2⫹ channel most tightly associated
with the fused vesicle, with contributions from a small number
of additional channels nearby. In fact, we show that a single
open Ca2⫹ channel is able to trigger synaptic vesicle release
from presynaptic active zones at the adult frog NMJ. Beyond
what was previously reported in the literature (Shahrezaei et al.
2006), using realistic estimates of the channel-vesicle stoichiometry and channel open probability (Luo et al. 2011), we
provide an estimate for the release probability of a synaptic
vesicle triggered by the Ca2⫹ flux through a single open Ca2⫹
channel, which is only about 6%. Thus, despite a tight 1:1
coupling of Ca2⫹ channels to synaptic vesicles, open Ca2⫹
channels trigger release unreliably. These data advance our
understanding of presynaptic active zone function at a model
synapse (the frog NMJ).
The role of Ca2⫹ channel cooperativity in triggering transmitter release. An ongoing debate centers on whether release
of individual synaptic vesicles is triggered by Ca2⫹ flux
through a single open Ca2⫹ channel or through multiple open
Ca2⫹ channels in the active zone (Tarr et al. 2013). Since the
spatial relationship between docked synaptic vesicles and presynaptic Ca2⫹ channels is not known at most synapses, previous studies typically relied on measuring the power relationship between transmitter release and Ca2⫹ entry by altering the
number of available Ca2⫹ channels (Augustine 1990; Mintz et
al. 1995; Scimemi and Diamond 2012; Shahrezaei et al. 2006;
Wu et al. 1999; Yoshikami et al. 1989). Conceptually, the
effectiveness of gradual Ca2⫹ channel blockade on transmitter
release is determined by the spatial relationship between Ca2⫹
Table 3. Release probability from a single active zone. The numbers present averages over active zone models that had 1 to 10
available Ca2⫹ channels
Number of Ca2⫹ Channel Openings:
1
2
3
4
5
Total release probability
Release probability per channel opening
5.2 ⫾ 0.3
5.2 ⫾ 0.3
11.4 ⫾ 0.8
5.7 ⫾ 0.4
18.6 ⫾ 0.9
6.2 ⫾ 0.3
24.0 ⫾ 6.0
6.0 ⫾ 1.5
29.0 ⫾ 5.5
5.8 ⫾ 1.1
Values are means ⫾ SD. The numbers present averages over active zone models that had 1 to 10 available Ca2⫹ channels.
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0.40 ⫾ 0.11. This value is roughly double the value observed
after treatment with 600 nM CgTX and consistent with a
doubling in the total Ca2⫹ entry that remained after block
(15.3% vs. 7% of control for 400 and 600 nM, respectively).
Next we calculated the average number of released vesicles
from individual active zones during an action potential. Because the quantal content indicates the total number of synaptic
vesicles released from the entire nerve terminal, which on
average has about 700 active zones at the frog NMJ (Dittrich
et al. 2013), the number of released vesicles per active zone can
be calculated by dividing the quantal content by the total
number of active zones. As shown above, our measured quantal content was 7.6 ⫾ 4.9 (n ⫽ 42) and 15.1 ⫾ 8.8 (n ⫽ 42)
after exposure to 600 nM and 400 nM CgTX, respectively.
Therefore, the release probability per active zone during an
action potential is estimated to be ⬃0.01 (7.6/700) and ⬃0.02
(15.1/700), respectively, after 600 nM and 400 nM CgTX
treatment (Fig. 5B). This is much lower than the release
probability of control terminals (⬃0.5, Dittrich et al. 2013;
Katz and Miledi 1979).
Finally, the probability that a single Ca2⫹ channel opening
triggers synaptic vesicle fusion can be estimated by dividing
the average number of release events per active zone by the
average number of single Ca2⫹ channel openings in each active
zone. Using this approach, the probability that Ca2⫹ flux
through a single open Ca2⫹ channel can trigger vesicle fusion
was calculated to be ⬃6% (Fig. 5C).
We then used our Monte Carlo simulation approach to
validate these results by examining the release probability of
synaptic vesicles in the model when the number of Ca2⫹
channels in the active zone was greatly reduced. Under the
extreme condition when very few Ca2⫹ channels were available, the probability that only a single Ca2⫹ channel opened
clearly followed the binomial distribution. For example, if the
total number of Ca2⫹ channels within the active zone was
reduced to 3, the probability that only one channel opened
during an action potential was predicted as [(3!)/(1! ⫻ 2!)]P1(1 ⫺ P)2 ⫽ 0.37 (for P equal to 0.19; see Luo et al. 2011).
Indeed, under these conditions, we observed 3,823 single
channel opening events out of 10,000 modeling runs (generated by distinct random number seeds) and 221 of these
released a synaptic vesicle. Therefore, our model predicted that
the release probability per opening was equal to 5.8% (221/
3,823; Fig. 5C), very close to our experimental measurement
(⬃6%). We then varied the number of available Ca2⫹ channels
in the active zone from 1 to 10 and determined that the release
probability was 5.2 ⫾ 0.3% (mean ⫾ SD) for release events in
which only one Ca2⫹ channel opened during an action potential (see Table 3).
We then used our MCell model to ask whether additional
open Ca2⫹ channels in the active zone would enhance the
release probability per opening. If each vesicle release event is
2487
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SINGLE Ca2⫹ CHANNELS TRIGGER RELEASE WITH LOW PROBABILITY
Ca2⫹ channels and docked synaptic vesicles. However, there
has been no direct evidence on how reliably the Ca2⫹ flux
through a single open Ca2⫹ channel can trigger synaptic
vesicle fusion. Past studies have at most determined the approximate number of Ca2⫹ channels contributing ions to vesicle release (Shahrezaei et al. 2006). By greatly reducing the
number of Ca2⫹ channels in each active zone using drug
treatments combined with high-resolution Ca2⫹ imaging techniques, we were able to capture sparsely distributed Ca2⫹
influx from single Ca2⫹ channel openings. In addition we
conducted Monte Carlo simulations of a realistic active zone
model with small numbers of active Ca2⫹ channels and estimated the coupling between single Ca2⫹ channel openings and
vesicle fusion. Analysis of our data supports the conclusion
that after block of 85 and 92% of Ca2⫹ influx within active
zones (using 400 or 600 nM CgTX, respectively), we were able
to image the Ca2⫹ flux through single open Ca2⫹ channels in
individual active zones during single action potential stimulation. Further, our results show that Ca2⫹ flux through single
open voltage-gated Ca2⫹ channels triggered synaptic vesicle
fusion with a probability of only ⬃6%. Importantly, even
under control conditions with an average of five Ca2⫹ channel
openings per action potential stimulus, the vesicle release
probability per open channel remained approximately constant
at ⬃6% (Table 3). This finding provides a mechanistic explanation for the observation that action potential stimulation only
unreliably triggers vesicle fusion at each single vesicle release
site in the frog NMJ. Importantly, however, these highly
unreliable single vesicle release sites (composed of one synaptic vesicle and its closely associated Ca2⫹ channel) can be
assembled to build a strong and reliable synapse using thousands of such units. Such assembly of unreliable single vesicle
release sites into active zones and nerve terminals is important
for conserving presynaptic resources and allows both strength
and reliability of synaptic transmission at the entire NMJ
during repetitive nerve activity (Tarr et al. 2013).
Even though vesicle fusion is predominately triggered by the
closest Ca2⫹ channel during single activation events, the long,
linear arrangement of Ca2⫹ channels at frog active zones might
lead to significant contributions of adjacent channels during
trains of action potentials. Indeed, an evaluation of short-term
synaptic plasticity mechanisms at the frog NMJ combining
physiological data and MCell computer simulations showed
that the contribution of adjacent Ca2⫹ channels to vesicle
release increased during subsequent stimuli (Ma et al. 2015).
Clearly, the precise relationship between active zone structural
organization (especially with respect to Ca2⫹ channels and
synaptic vesicles) and physiological function requires further
examination.
ACKNOWLEDGMENTS
We thank G. Hood for writing the image alignment routine and T. Tarr for
feedback during the preparation of this manuscript.
GRANTS
This work was supported by National Institutes of Health Grants R01-NS043396 (to S. D. Meriney), R01-NS090644 (to S. D. Meriney and M. Dittrich),
R01-GM-068630, P41-RR-06009, and P41-GM-103712 (to J. R. Stiles and M.
Dittrich) and by National Science Foundation Grants 0844174 (to M. Dittrich),
0844604 (to S. D. Meriney), and 1249546 (to M. Dittrich and S. D. Meriney).
J Neurophysiol • doi:10.1152/jn.00879.2014 • www.jn.org
Downloaded from http://jn.physiology.org/ by 10.220.33.3 on July 12, 2017
channels and synaptic vesicles, and therefore reflects the cooperative coupling (denoted by m) of Ca2⫹ channels in controlling vesicle secretion. The value of m has been shown to
vary for different synapses and different subtypes of Ca2⫹
channels at a single synapse. For example, at cerebellar parallel
fiber synapses, Mintz et al. (1995) determined that transmitter
release triggered by N-type Ca2⫹ channels had an m value of
2.5, whereas transmitter release triggered by P/Q type Ca2⫹
channels had an m value of 4.0. Similarly, at the young calyx
of Held synapse, transmitter release triggered by P/Q type
Ca2⫹ channels has a different m value (m ⫽ 3.7) than release
triggered by N-type Ca2⫹ channels (m ⫽ 1.3; Wu et al. 1999).
As the calyces mature, the m value for the P/Q type Ca2⫹
channels decreases (Fedchyshyn and Wang 2005), suggesting
that the spatial coupling between Ca2⫹ channels and docked
vesicles is tightened during development. At the chick ciliary
ganglion calyx, where N-type channels trigger release, m has
been reported to be 1.3 (Gentile and Stanley 2005). Such a
large variability in the cooperative coupling of Ca2⫹ channels
with neurotransmitter release suggests a wide variety of spatial
organization of Ca2⫹ channels within individual release sites at
different synapses. Lower values of m (1–2) suggest that a
small number of open Ca2⫹ channels are sufficient for triggering the fusion of a synaptic vesicle and which may thus be
tightly associated with those open channels. In contrast, higher
values of m (4 –5) indicate that many Ca2⫹ channels need to
open simultaneously to contribute the necessary Ca2⫹ ions to
trigger the release of a single synaptic vesicle. Under conditions where m values are high, the Ca2⫹ channel cooperativity
measurement is governed by the molecular cooperativity of the
Ca2⫹ sensor (Dodge and Rahamimoff 1967) as titrating channel block is essentially similar to changing extracellular Ca2⫹
concentration at these synapses (Meinrenken et al. 2002).
In agreement with previous studies (Shahrezaei et al. 2006;
Yoshikami et al. 1989), we have shown at the adult frog NMJ
that Ca2⫹ channels have a low cooperativity in triggering
transmitter release (m ⫽ 1–2). In contrast to previous reports,
our studies are built upon our recent high-resolution Ca2⫹
imaging data that support the hypothesis that there are relatively few functional Ca2⫹ channels in each frog NMJ active
zone (Luo et al. 2011). These data predict a 1:1 relationship
between presynaptic Ca2⫹ channels and docked synaptic vesicles and allow our current study to make detailed predictions
regarding the probability with which individual channel openings trigger vesicle fusion (see below). The small m value we
report here further supports the close association between
individual docked synaptic vesicles and a single Ca2⫹ channel.
As evidenced by our Monte Carlo simulations, Ca2⫹ ions that
bound to released synaptic vesicles were primarily derived
from the nearest open Ca2⫹ channel. This dominant control of
vesicle fusion by a single Ca2⫹ channel (nanodomain coupling)
becomes more pronounced when a majority of Ca2⫹ channels
are blocked. On the other hand, a significant number of vesicle
release events involved small contributions from neighboring
Ca2⫹ channels, and it will be interesting to investigate possible
functional implications of this finding in future work.
Functional organization of single vesicle release sites within
active zones of the frog NMJ. At individual release sites in a
variety of synapses the measured Ca2⫹ channel-release site
cooperativity (the m value) for triggering transmitter release
has been used to infer the stoichiometric relationship between
SINGLE Ca2⫹ CHANNELS TRIGGER RELEASE WITH LOW PROBABILITY
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: F.L., M.D., S.C., J.R.S., and S.D.M. conception and
design of research; F.L., M.D., S.C., and S.D.M. performed experiments; F.L.,
M.D., S.C., and S.D.M. analyzed data; F.L., M.D., and S.D.M. interpreted
results of experiments; F.L., M.D., and S.D.M. prepared figures; F.L., M.D.,
and S.D.M. drafted manuscript; F.L., M.D., and S.D.M. edited and revised
manuscript; F.L., M.D., S.C., and S.D.M. approved final version of manuscript.
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