Articles in PresS. J Neurophysiol (February 4, 2015). doi:10.1152/jn.00879.2014

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Transmitter release is evoked with low probability predominately by calcium flux through single channel openings at the frog neuromuscular junction Fujun Luo1,2*, Markus Dittrich1,3,4*, Soyoun Cho1, Joel R. Stiles1,3,4† and Stephen D. Meriney1,2 * Contributed equally † author is deceased

Running head: Single Ca2+ channels trigger release with low probability

1

Department of Neuroscience, Center for Neuroscience, University of Pittsburgh, Pittsburgh, PA 15260. 2 Center for the Neural Basis of Cognition, Pittsburgh, PA 15213. 3 Biomedical Applications Group, Pittsburgh Supercomputing Center, Carnegie Mellon University, Pittsburgh, PA 15213. 4 Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA 15260.

Correspondence should be addressed to Stephen D. Meriney, Department of Neuroscience, Center for Neuroscience, University of Pittsburgh, A210 Langley Hall, Pittsburgh, PA 15260. E-mail: [email protected].

Copyright © 2015 by the American Physiological Society.

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Abstract The quantitative relationship between presynaptic calcium influx and transmitter

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release critically depends on the spatial coupling of presynaptic calcium channels to

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synaptic vesicles. When there is a close association between calcium channels and

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synaptic vesicles, the flux through a single open calcium channel may be sufficient to

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trigger transmitter release. With increasing spatial distance, however, a larger number

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of open calcium channels might be required to contribute sufficient calcium ions to

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trigger vesicle fusion. Here we used a combination of pharmacological calcium channel

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block, high-resolution calcium imaging, postsynaptic recording, and 3D Monte Carlo

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reaction-diffusion simulations in the adult frog neuromuscular junction, to show that

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release of individual synaptic vesicles is predominately triggered by calcium ions

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entering the nerve terminal through the nearest open calcium channel. Furthermore,

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calcium ion flux through this channel has a low probability of triggering synaptic vesicle

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fusion (~6%), even when multiple channels open in a single active zone. These

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mechanisms work to control the rare triggering of vesicle fusion in the frog

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neuromuscular junction from each of the tens of thousands of individual release sites at

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this large model synapse.

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Key words:

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Neuromuscular junction, calcium channels, active zone, synapse, MCell

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Introduction

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Neurotransmitter release is triggered by action potential-evoked Ca2+ influx through

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voltage-gated Ca2+ channels. The magnitude and time-course of transmitter release is

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variable and tightly dependent on the coupling between Ca2+ channels and synaptic

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vesicles (Augustine and Neher, 1992; Meinrenken et al., 2002). At some synapses, the

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summed Ca2+ flux through many open channels appears to be required for synaptic

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vesicle fusion, suggesting a loose coupling between Ca2+ channels and vesicle fusion

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machinery (Borst and Sakmann, 1996; Wu et al., 1999; Meinrenken et al.,

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2002). However, at other synapses a small number of Ca2+ channels are often

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sufficient to trigger vesicle fusion (Brandt et al., 2005; Bucurenciu et al., 2010; Jarsky et

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al., 2010; Schmidt et al., 2013). In fact, it has been argued that the Ca2+ flux through

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only one or two open Ca2+ channels triggers vesicle fusion at some synapses

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(Yoshikami et al., 1989; Stanley, 1993; Scimemi and Diamond, 2012). Such variable

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stoichiometric relationships are likely determined by the spatial organization of Ca2+

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channels and synaptic vesicles into either microdomain or nanodomain-controlled

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release sites (Eggermann et al., 2012; Tarr et al., 2013).

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The frog neuromuscular junction (NMJ) has been commonly used to study

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presynaptic mechanisms that control transmitter release (Bennett, 1996; Grinnell 1995;

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Harlow et al, 2001; Meriney and Dittrich, 2013). This synapse features hundreds of

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linear active zones that are each characterized by double rows of presynaptic ion

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channels and synaptic vesicles (Heuser et al., 1974; Pawson et al.,

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1998). Experimentally, it has been estimated that individual active zones release on

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average a single vesicle following every other action potential stimulus (Dittrich et al.,

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2013). An earlier study by Shahrezaei et al. (2006) assumed 200 Ca2+ channels per

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active zone. However, a recent statistical analysis of high spatial and temporal

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resolution Ca2+ imaging data (Luo et al., 2011) showed that active zones contain

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relatively few Ca2+ channels (20-40) among the total of 200-250 presynaptic

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transmembrane protein particles observed in freeze fracture electron microscopy, and

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that during an action potential these Ca2+ channels open with relatively low probability

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(~0.2). These data suggest that the number of Ca2+ channels in each active zone is

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roughly equivalent to the number of docked synaptic vesicles (Wachman et al., 2004;

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Luo et al., 2011). Consistent with this hypothesis, previous studies have suggested that

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transmitter release at the frog NMJ is controlled by Ca2+ influx through very few,

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perhaps only one or two channels (Yoshikami et al., 1989; Shahrezaei et al., 2006). The

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resulting approximate 1:1 Ca2+ channel-vesicle stoichiometry significantly impacts how

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we think about the structure-function relationship in this system. Here, using a

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combination of high-resolution Ca2+ imaging, postsynaptic recording, pharmacological

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channel block, and 3D Monte Carlo reaction-diffusion simulations of a realistic active

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zone model, we present evidence that vesicle fusion is a rare event triggered primarily

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by the Ca2+ flux through a single open Ca2+ channel. Further, we show that Ca2+ flux

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through a single open Ca2+ channel triggers fusion of a closely-associated vesicle with

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only low probability (5-6%). Based on this insight, we propose that in the frog NMJ the

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observed low probability of release from individual single vesicle release sites within

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active zones is governed both by a low probability of Ca2+ channel opening and a low

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probability that flux through an open channel will trigger vesicle fusion.

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Materials and Methods

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Tissue preparation

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Adult frogs (Rana pipiens) were decapitated and pithed following anesthesia in 0.6%

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tricaine methane sulfonate solution as approved by the University of Pittsburgh

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Institutional Animal Care and Use Committee. The cutaneous pectoris nerve-muscle

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preparation was dissected bilaterally and bathed in normal frog Ringer (NFR; in mM:

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116 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES, pH 7.4) as previously described

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(Wachman et al., 2004; Luo et al., 2011).

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Dye loading

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The Ca2+-sensitive dye Calcium Green-1 (3000 MW dextran conjugate; Molecular

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Probes) was loaded through the cut end of the nerve as previously described

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(Wachman et al., 2004; Luo et al., 2011). Briefly, the nerve was immersed in a drop of

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30 mM dye dissolved in distilled water for 6-8 hours at room temperature. After rinsing

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in NFR, the preparation was stored at 4 °C for 2-3 hours.

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Ca2+ imaging and processing

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These procedures were similar to those described by Wachman et al. (2004) and Luo et

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al. (2011). The muscle was pinned over an elevated Sylgard (Dow Corning) platform in

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a 35 mm dish mounted on a microscope stage. The postsynaptic acetylcholine

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receptors were labeled by exposure to 2 μg/ml Alexa 594-conjugated α-bungarotoxin (α-

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BTX) for 10 minutes. α-BTX staining was used to locate and focus the postsynaptic

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receptor bands, which are positioned directly opposite from the presynaptic active

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zones, and evaluate the z-axis drift over the course of data collection. Superficially

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positioned nerve terminals, the majority of which were in a single focal plane as judged

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by α-BTX staining, were chosen for study. All Ca2+ imaging was performed in NFR

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(except as noted) with 10 μM curare added to prevent nerve-evoked muscle

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contractions that were not completely blocked by the α-BTX.

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An acousto-optic tunable filter (AOTF; ChromoDynamics, Inc.) was employed to

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select wavelengths and gate the laser illumination with sub-millisecond time resolution

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(Krypton-Argon laser; Innova 70 Spectrum, Coherent). The laser was fiber-coupled into

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the epi-illumination port of an upright fluorescence microscope equipped with a 100x

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water-immersion long working-distance objective with 1.0 NA (Lumplan/FL IR, Olympus).

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Calcium Green-1 was excited at 488 nm and emission light was collected through a 530

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± 20 nm filter. Alexa 594-α-BTX was excited at 567 nm and emission light was

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collected through a 620 ± 30 nm filter. The timing of laser illumination was triggered

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with nerve stimulation following a 1.5 msec delay (for nerve conduction), and dye was

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illuminated for only 1 msec during action potential invasion of the nerve terminal to limit

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spatial diffusion of Ca2+ entering through voltage gated Ca2+ channels. Images were

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recorded on a liquid nitrogen-cooled, back-thinned CCD camera (LN1300B, Roper

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Scientific), which provided the high-sensitivity low-noise detection necessary for the

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measurement of fluorescence changes sampled during brief 1 msec time windows.

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Raw resting photoelectron counts ranged between 1200 and 4000 in different dye-

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loaded nerve terminals, and fluorescence changes during nerve stimulation (using a

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suction electrode at a current intensity of 5X threshold) were significantly above resting

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fluctuations (dominated by shot noise). Images were collected at 0.5 Hz in sets of 20;

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the first 10 images with stimulator off (background) and the second 10 images with

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stimulator on (nerve-evoked signals). Images were processed on a Pentium-based

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computer using MATLAB (MathWorks). Prior to analysis, images were co-registered to

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correct for slight fluctuation in the lateral position of the preparation during data

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collection using custom software written by Greg Hood (Pittsburgh Supercomputer

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Center). Differences in fluorescence above rest were determined for individual images

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by subtracting mean resting fluorescence (generated by averaging the 10 background

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images). The resulting “difference images” represented the relative fluorescence

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changes, computed as ΔF/F0 = (F - F0)/F0, and were displayed in pseudo-color. In

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multiple trial experiments, 5-10 sets of Ca2+ images (each set consisting of 10 Ca2+

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images with stimulation off and 10 Ca2+ images with stimulation on as described above)

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were collected for each well-focused nerve terminal and co-registered to the first image

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of the first dataset. Between each set, we confirmed/adjusted focus onto the active

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zone in reference to α-BTX staining and discarded image sets from analysis if they

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showed noticeable z-axis drift.

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Imaging analysis

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We restricted our quantitative analysis to the nerve terminal regions as defined by a

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mask based on the resting fluorescence intensity. The blocking effect of titrating ω-

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conotoxin GVIA (CgTX) on action potential-evoked Ca2+ influx was measured by

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calculating the ratio of total Ca2+ entry into active zones before and after CgTX

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treatment from the same terminal regions. The total Ca2+ entry was estimated by the

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sum of pixel fluorescence intensities above background (ΔF/F0, see above). Only pixels

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that reported significant action potential-evoked fluorescence change under control

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condition (i.e., before drug treatment, t-test, p < 0.01) were included.

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Exposure to relatively high concentrations of CgTX greatly reduced action potential-

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evoked Ca2+ entry and therefore fluorescence change (e.g. by 92% in 600 nM CgTX).

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We have previously shown that the calcium signal reported using this approach is

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linearly related to calcium entry (Luo et al., 2011). In order to reliably identify Ca2+ entry

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under this condition, we used a per-pixel criterion that required that stimulated

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fluorescence change above rest (ΔF) should exceed 3 times the standard deviation

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(3xSD) of the resting fluorescence. This criterion was chosen based on the following

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reasons. First, the use of 2xSD as a criterion was too sensitive and yielded the selection

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of a large number of pixels between active zones, and a significant number of false

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positives selected even under unstimulated conditions. On the other hand, the use of

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4xSD was too stringent and eliminated so many pixels from the selection that there

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were almost none detected within active zone regions. Second, in testing a variety of

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criteria (2, 3, or 4 SD), we compared the number of pixels that were chosen to the

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expected number of channels remaining based on the use of such a high concentration

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of CgTX (600 nM). This CgTX treatment decreased total average Ca2+ entry (as

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measured by the decrease in Ca2+-sensitive fluorescent signal) into the nerve terminal

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by 92% (see table 1 below). In a previous study of untreated control synapses, we

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estimated that there are 20-40 Ca2+ channels in each active zone, and that each open

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during an action potential with a probability (po) of ~0.2 (see Luo at al., 2011).

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Therefore, a 92% reduction in total Ca2+ entry would correspond to ~2 Ca2+ channels

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remaining in each active zone. Calculating the probability of at least one of these two

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channels A and B opening during an action potential yields a probability of po(A or B) =

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po(A) + po(B) – po(AB) = 0.36. Using the 3xSD criterion led to a detection of a number

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of Ca2+ entry sites that was roughly consistent with this expectation for po (see Fig. 4 &

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5), providing further support for this measure.

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For statistical analysis, the number of detected openings was normalized (as

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openings per active zone per action potential) for each terminal by dividing the total

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number of openings by the number of active zones and the number of stimulation trials.

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Electrophysiology

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Intracellular recordings from the adult frog cutaneous pectoris nerve-muscle preparation

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were performed as described previously (Meriney & Grinnell, 1991; Douthitt et al.,

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2011). In brief, the nerve of the cutaneous pectoris muscle was stimulated via a suction

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electrode at 5X the threshold intensity required to elicit muscle twitch. Intracellular

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micropipettes were made from glass pipettes (Warner Instruments, filament glass

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catalog #64-0787 O.D. 1mm I.D. 0.58mm) and had a resistance of 25-50 MΩ after filling

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with 3M potassium acetate. Surface muscle fibers were penetrated close to nerve

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endings under visual control with a long working distance water-immersion objective

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(40x, 3mm working distance). Only muscles with a resting potential more

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hyperpolarized than -70mV were recorded for analysis. Spontaneous miniature

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endplate potentials (mEPPs) and action potential-evoked endplate potentials (EPPs)

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were recorded. To calculate the number of synaptic vesicles released following each

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action potential, quantal content was calculated using the direct method (EPP

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amplitude/mEPP amplitude). Data were amplified using a Dagan BBC 700 amplifier and

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acquired using Clampex 9 software (Axon Instruments, Foster City, CA). Clampfit 9.2

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was used for data analysis.

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Drug treatment

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CgTX is known to block N-type Ca2+ channels in a manner that is essentially irreversible

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(Stocker and Tsien, 1997). For titrating channel block, the frog nerve-muscle

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preparation was incubated with various doses of CgTX for 45-60 minutes and then

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washed in NFR. Superficially positioned nerve terminals and muscle fibers were

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chosen for study to avoid variability in toxin access to nerve terminals positioned at

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different depths of the preparation. Blocking effects on transmitter release and Ca2+

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influx were determined in separate experiments to optimize the health of the terminal for

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imaging experiments and to avoid potential complications due to the buffering effects of

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Ca2+-sensitive dye during recordings of transmitter release.

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Monte Carlo simulations

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All computational modeling was carried out with version 3.1 of the MCell software

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developed in our lab (Kerr et al., 2006; www.mcell.org). The active zone model used in

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this paper was identical to our recently developed spatially realistic 3-D model of the

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frog presynaptic active zone (Dittrich et al., 2013) and used here without modification.

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This model is heavily constrained by a wealth of anatomical, physiological, and

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biochemical data that has been collected over decades of study on the frog NMJ and

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other model synapses, as well as more recent work in our laboratory. The geometry of

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our active zone model was based on published ultrastructural data from the adult frog

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NMJ (Heuser et al., 1974; Pawson et al., 1998; Harlow et al., 2001) and contained 26

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synaptic vesicles arranged in two double rows (Figure 1B). In addition, 26 N-type

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voltage-gated Ca2+ channels were positioned in a one-to-one topographic relationship

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with each docked synaptic vesicle at an average distance from the vesicle of 35 nm

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(Figure 1B, see also Dittrich et al, 2013). The total number of Ca2+ channels and their

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opening probability following single action potentials were constrained by our recent

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Ca2+ imaging experiments (Luo et al., 2011). During each simulation, an invading

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action potential led to the stochastic opening of a varying number of voltage gated Ca2+

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channels based on their open probability (p ~ 0.2). Once open, Ca2+ channels would

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permit Ca2+ ions to enter the presynaptic space according to the instantaneous driving

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force and the experimentally known channel conductance. Presynaptic, freely diffusing

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Ca2+ ions could then either bind to Ca2+ sensor sites on synaptic vesicles

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(synaptotagmin) or 2 mM fixed buffer molecules with kinetic rates as reported in Dittrich

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et al. (2013). Synaptic vesicle release was determined via our excess-calcium-binding-

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site model (Dittrich et al., 2013), which unifies recent biochemical evidence regarding

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the number, arrangement and properties of available synaptotagmin molecules on

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synaptic vesicles (our model has 8 synaptotagmin molecules near the base of the

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vesicle with 5 low affinity Ca2+ binding sites each, see Figure 1A) and classical

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physiology such as the 4th order calcium-release relationship. The number and

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positioning of synaptotagmin molecules in the model was chosen to be consistent with

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previously published proteomic studies (Takamori et al., 2006; Mutch et al., 2011),

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recently hypothesized orientations of these proteins (Kummel et al., 2011; Wang et al.,

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2014), and the best fit of the physiology data with these model parameters (Dittrich et al.,

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2013). In particular, the detailed arrangement of synaptotagmin sites on the bottom of

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synaptic vesicles does not impact the qualitative features of our model as we have

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recently shown for second sensor sites within an extension of the model presented in

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this paper (Ma et al., 2015). Our model captures the well-known narrow distribution of

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release latencies at the frog NMJ (Katz and Miledi, 1965). In contrast to earlier

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computational studies (Shahrezaei et al, 2006) our model is significantly more detailed

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and includes the previously determined estimated number of presynaptic voltage-gated

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Ca2+ channels and their opening probability (Luo et al., 2011), proper timing of

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stochastic channel opening driven by an action potential waveform and the resulting

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narrow latency distribution of synaptic vesicle release. To simulate the experimental

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titration with increasing concentrations of ω-CgTX GVIA we randomly removed

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increasing numbers of voltage gated Ca2+ channels from the model and then measured

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the resulting synaptic vesicle release.

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For each model condition we performed 10,000 statistically independent runs on a

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computer cluster at the Pittsburgh Supercomputing Center (codon.psc.edu, a cluster of

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23 dual processor machines, each with two 1.6 GHz AMD Opteron processors and 8

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gigabytes of memory). Data analysis was performed with custom written scripts.

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Results

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The relationship between presynaptic Ca2+ entry domains and triggering of

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transmitter release

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In order to explore the quantitative relationship between Ca2+ channel openings

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and transmitter release at the frog NMJ, which predominantly expresses N-type Ca2+

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channels, we titrated the concentration of CgTX to block different fractions of Ca2+

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channels, and thus presynaptic Ca2+ entry. Although similar experiments have been

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performed previously (Shahrezaei et al., 2006), our study improves on these in two

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significant ways. First, we determined changes in Ca2+ influx and transmitter release in

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separate experiments to avoid potential buffering effects of Ca2+-sensitive dye on our

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measures of transmitter release. Second, we imaged Ca2+ entry with fast spatial and

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temporal resolution to detect restricted sites of Ca2+ entry as opposed to volume

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averaged signals within the whole nerve terminal. Furthermore, we compare these data

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to our computer simulations presented below.

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When the nerve was stimulated at low frequency (0.5 Hz), strong Ca2+-

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dependent fluorescence signals were detected within presynaptic active zones

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(Wachman et al., 2004; Luo et al., 2011). An example of averaged action potential-

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evoked fluorescence change (normalized as ΔF/F0) over 10 stimulus trials is shown in

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Figure 2A. When compared to control, exposure to 100 nM CgTX caused a dramatic

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decrease in action potential-evoked fluorescence signals at the nerve terminal. The ratio

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of action potential-evoked-Ca2+ influx before and after drug treatment was used to

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represent the blocking percentage of Ca2+ channels by various concentrations of CgTX

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(see Table 1).

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We next examined the effects of the same concentrations of CgTX on the

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magnitude of neurotransmitter release using intracellular recording of endplate

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potentials (EPPs). EPPs were recorded from the same muscle fiber before and after

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incubation with CgTX. For these recordings, 3-7 µM curare (for partial block of

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acetylcholine receptors) and 2 µM µ-conotoxin PIIIA (a muscle-specific sodium channel

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blocker) were added to the Ringer bathing the preparation to block muscle contraction.

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The concentration of curare was titrated to reduce EPP amplitude to less than 10 mV to

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avoid significant effects of nonlinear summation of quantal events underlying the EPP,

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while still permitting the EPP to be measured both before and after exposure to various

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concentrations of CgTX. Figure 2B shows an example of averaged EPP traces before

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and after exposure to 100 nM CgTX and the summary data are reported in Table 1.

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We estimated the stoichiometric relationship between Ca2+ entry and transmitter

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release by plotting the blocking effects of titrating CgTX on both Ca2+ entry and

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transmitter release on a log-log scale. We have previously demonstrated that our

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imaged Ca2+ signals are linearly related to Ca2+ influx under these experimental

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conditions (Luo et al., 2011). As shown in Figure 2C, the resulting relationship is not

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completely linear over the range of CgTX doses examined. At low doses of CgTX (25-

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50 nM) the relationship appeared to fall on a line with a slope of ~2, but as the

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concentration of CgTX was increased, this relationship approached a slope closer to 1.

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These results suggest that under control conditions, only a small number of Ca2+

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channels contribute Ca2+ to individual vesicle release events, consistent with previous

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observations at this synapse (Yoshikami et al., 1989; Shahrezaei et al., 2006).

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However, as presynaptic Ca2+ channels are blocked, release events depend

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increasingly on single open channels.

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We then tested whether our spatially realistic excess-Ca2+-binding-site model of

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the frog NMJ active zone (Dittrich et al., 2013) was able to reproduce the effect of

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titrating CgTX to block presynaptic Ca2+ entry on transmitter release. This model has

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been shown to reproduce many properties of neuromuscular transmission faithfully

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including the normal release rate, the 4th power relationship between vesicle release

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and extracellular Ca2+ concentration, and synaptic delay (Dittrich et al., 2013). To

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model CgTX titration, an increasing number of Ca2+ channels were randomly removed

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from the active zone and the resulting release probability following single action

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potential stimulation was simulated. As shown in Figure 2D and Table 2, the model

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reproduced accurately the experimental observations (compare with Fig. 2C and Table

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1), and thus could be used to aid in interpreting these data.

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Since our experimental data indicated that very few, but likely more than one

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Ca2+ channel contributed Ca2+ ions to triggering single vesicle release from control

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terminals, we used our MCell results to estimate the percentage of released vesicles

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that bound Ca2+ ions from one, two or more Ca2+ channels (Fig. 2E). The simulations

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showed that 5.2 ± 0.3 Ca2+ channels opened in each active zone following a single

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action potential stimulation. Interestingly, we found that about 34% of synaptic vesicles

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that were triggered to fuse bound only Ca2+ ions that entered the nerve terminal through

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a single open Ca2+ channel, and 41% of vesicle fusion events were triggered by Ca2+

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ions derived from only two open Ca2+ channels (see also Dittrich et al, 2013). Vesicle

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fusion triggered by Ca2+ ions from more than two channels openings was less likely. On

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average, synaptic vesicle fusion was triggered by Ca2+ ions derived from 1.9 Ca2+

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channels, consistent with our experimental CgTX titration data. The spatial distribution

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of the average contribution of specific Ca2+ channels to a given vesicle fusion event is

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graphically depicted in Fig. 3B. As expected, this relationship changed when we

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decreased the number of available Ca2+ channels in the model active zone (Fig. 2F).

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When 80% of Ca2+ channels were blocked, the average number of Ca2+ channels that

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open was reduced to 1.0 ± 0.2 and the average number of vesicles released per single

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active zone was reduced to 0.06 ± 0.007. Under these conditions, using large

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simulation runs (10,000) to increase the reliability of our estimates, we determined that

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460 out of 550 (84%) released vesicles were bound by Ca2+ ions from a single Ca2+

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channel. Therefore, the model predicts that vesicle fusion will still occur with only a

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small number of active Ca2+ channels in the active zone and that these events will be

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triggered almost exclusively by Ca2+ ions derived from a single open Ca2+ channel.

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Single Ca2+ channel openings predominate in contributing Ca2+ ions to vesicle

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fusion even when more than one channel opens in the active zone

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To further understand the quantitative contribution of Ca2+ channels to triggering

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vesicle release, we tracked the pattern of Ca2+ channel opening and Ca2+ ion binding for

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each given fused vesicle using our recently published excess-calcium-binding-site

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model of the frog NMJ active zone (Dittrich et al., 2013). Using this approach, Dittrich et

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al. (2013) showed that known properties of action potential-evoked transmitter release

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at the frog NMJ could be best fit if synaptic vesicle fusion was triggered by at least two

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Ca2+ ions binding to at least three of the modeled synaptotagmin molecules on a given

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synaptic vesicle. In fact, on average, 7-8 Ca2+ ions bound to a synaptic vesicle (among

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the 40 total Ca2+ binding sites available, see Methods; Dittrich et al., 2013) at the time of

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vesicle fusion. Here, we extended our use of this model to evaluate the Ca2+ channel of

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origin for these bound Ca2+ ions. Interestingly, we found that when a particular vesicle

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fused, 94% of the time the nearest, closely-associated Ca2+ channel opened (Fig. 3A).

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For these cases, the Ca2+ ions that bound to the fused vesicle were found 82% of the

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time to originate from the single nearest Ca2+ channel (Fig. 3B, left panel). Therefore,

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our model suggests that of the average 7-8 Ca2+ ions that bind to trigger vesicle fusion,

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6-7 were derived from the closest Ca2+ channel and only 1-2 Ca2+ ions originate from

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other open Ca2+ channels (Fig. 3B, right panel). On the other hand, when the nearest,

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closely-associated Ca2+ channel did not open, a synaptic vesicle rarely fused (only 6%

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of total fusion events). Under these rare conditions, the Ca2+ ions that bound to the

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fused synaptic vesicle derived predominately from a nearby cluster of 3-5 Ca2+ channels,

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with each of these channels contributing 1-2 Ca2+ ions to the synaptic vesicle Ca2+

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binding sites (Fig. 3C). In conclusion, our simulations predict that action potential-

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evoked transmitter release at the frog NMJ is controlled by the Ca2+ ions that derive

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predominantly from the opening of one closely-associated Ca2+ channel, even when

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several Ca2+ channels open in the active zone (Fig. 3A and B).

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Experimental evidence that single channel openings can trigger synaptic vesicle

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release

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Our previous Ca2+ imaging data (Wachman et al., 2004; Luo et al., 2011) have

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shown that each active zone in the adult frog NMJ has relatively few Ca2+ channels (20-

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40), and that each Ca2+ channel has a relatively low opening probability during an action

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potential (~0.2). Therefore, only a few of these Ca2+ channels would be predicted to

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open (4-8) within each active zone during a single action potential. Thus, we attempted

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to reduce the number of functional Ca2+ channels so that a single action potential would

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open at most one Ca2+ channel within each active zone. To block most Ca2+ channels,

404

we exposed the preparation to 600 nM CgTX for 60 minutes. We imaged action

405

potential-evoked Ca2+ influx at active zones and found that the total Ca2+ entry was

406

reduced by 92 ± 1.7% (Fig. 4A-C, n = 3). Following a reduction of this magnitude, the

407

number of available Ca2+ channels within individual active zones is expected to be very

408

small (~2), and given the low probability of opening (~0.2), the likelihood that more than

409

one channel would open in the same active zone during a single action potential

410

stimulus would be very small (~0.04). In agreement with this expectation, the Ca2+-

411

sensitive fluorescent signals detected by each imaging pixel in our imaging system had

412

a large coefficient of variation (CV = 1.6 ± 0.5) after 600 nM CgTX treatment, as

413

compared to control conditions (CV = 0.5 ± 0.3), consistent with a large reduction in the

414

number of channels. To increase the total number of Ca2+ signals detected for these

415

experiments, we collected 70-100 trials of stimulus-evoked Ca2+ images after CgTx

416

treatment. After exposure to 600 nM CgTX, we identified discrete, sparse and spatially

417

distributed signals evoked by single action potentials within well-focused regions of the

418

nerve terminal (Fig. 4C). As shown in Figure 4D, under 600 nM CgTX block the

419

histogram distribution of the fluorescence intensity detected by individual pixels

420

underlying Ca2+ channel openings takes a log-normal form, similar to the distribution of

421

the single channel current integral (see figure 2C&D in Dittrich et al., 2013). This

422

distribution is generated as single channels open with variable mean open time under

423

conditions of rapidly changing driving force at different times during the action potential

424

waveform. The similarity of the fluorescence and single channel current histogram

425

distributions is consistent with our prediction that at most one Ca2+ channel opens in

426

each active zone under these conditions. A similar lognormal distribution was observed

427

after exposure to 400 nM CgTX, which reduced total action potential-evoked Ca2+ influx

428

evoked by 84.7 ± 3.3%. In contrast, for control terminals, the histogram distribution of

429

signal intensities from individual pixels was significantly shifted to higher magnitude and

430

took a more Gaussian shape (Fig. 4D). We hypothesize that exposure to either 400 or

431

600 nM CgTX blocked sufficient numbers of Ca2+ channels in each active zone that

432

imaged Ca2+ signals were derived predominantly from a single open channel. Under

433

these conditions, we predict that transmitter release would be rare, and when it

434

occurred, it would be triggered by single channel openings.

435

In order to test this hypothesis, we carried out postsynaptic recording of EPPs

436

from muscle fibers after exposure to either 600 nM or 400 nM CgTX. As shown in

437

Figure 4E, we still detected significant transmitter release from nerve terminals after

438

exposure to either 400 or 600 nM CgTX, despite the rare Ca2+ channel openings in

439

each active zone as predicted under these conditions. After exposure to 400 nM CgTX

440

quantal content measured 15.2 ± 8.8, while after exposure to 600 nM CgTX quantal

441

content was only 7.6 ± 4.9. These values are obviously much lower than what has been

442

reported in untreated control nerve terminals (quantal content ~350; Katz and Miledi,

443

1979; Dittrich et al., 2013), but support the hypothesis that isolated single Ca2+ channel

444

openings can trigger transmitter release from the frog NMJ.

445 446

Estimating the probability of transmitter release triggered by the Ca2+ flux through

447

a single Ca2+ channel opening

448

How reliably can single Ca2+ channel openings trigger synaptic vesicle fusion? In

449

order to evaluate the vesicle fusion probability due to single Ca2+ channel openings, we

450

measured the frequency of observing Ca2+ channel openings at the single active zone

451

level after exposure to different concentrations of CgTX. We then compared the channel

452

opening frequency with the release probability of a single active zone under each

453

treatment condition. To calculate Ca2+ channel openings per active zone, we limited our

454

analysis to those nerve terminal regions where we could clearly identify individual active

455

zones (see Fig. 4A). For example, 31 active zones can be distinguished within the

456

region of interest identified in the representative nerve terminal shown in Figure 4A.

457

After exposure to 600 nM CgTX, we collected imaging data from 100 stimulus trials and

458

calculated that each action potential evoked an average of 0.23 Ca2+ channel openings

459

per active zone. Similar results were obtained from 8 nerve terminals and the mean

460

number of openings evoked by a single action potential was estimated to be 0.18 ± 0.07

461

per active zone (Fig. 5A). This value is very close to our estimate of the average

462

opening probability of individual Ca2+ channels (Luo et al., 2011), which provides further

463

support for our hypothesis that the number of functional Ca2+ channels within individual

464

active zones is reduced to approximately one after exposure to 600 nM CgTX. We also

465

calculated the number of detected Ca2+ entry sites for nerve terminals treated with 400

466

nM CgTX. In these experiments, we found that the number of Ca2+ channel openings

467

per active zone was 0.40 ± 0.11. This value is roughly double the value observed after

468

treatment with 600 nM CgTX and consistent with a doubling in the total Ca2+ entry that

469

remained after block (15.3% vs 7% of control for 400 and 600 nM respectively).

470

Next we calculated the average number of released vesicles from individual

471

active zones during an action potential. Because the quantal content indicates the total

472

number of synaptic vesicles released from the entire nerve terminal, which on average

473

has about 700 active zones at the frog NMJ (Dittrich et al., 2013), the number of

474

released vesicles per active zone can be calculated by dividing the quantal content by

475

the total number of active zones. As shown above, our measured quantal content was

476

7.6 ± 4.9 (n = 42) and 15.1 ± 8.8 (n = 42) after exposure to 600 nM and 400 nM CgTX,

477

respectively. Therefore, the release probability per active zone during an action

478

potential is estimated to be ~0.01 (7.6/700) and ~0.02 (15.1/700), respectively, after 600

479

nM and 400 nM CgTX treatment (Fig. 5B). This is much lower than the release

480

probability of control terminals (~0.5, Katz and Miledi, 1979; Dittrich et al., 2013).

481

Finally, the probability that a single Ca2+ channel opening triggers synaptic

482

vesicle fusion can be estimated by dividing the average number of release events per

483

active zone by the average number of single Ca2+ channel openings in each active zone.

484

Using this approach, the probability that Ca2+ flux through a single open Ca2+ channel

485

can trigger vesicle fusion was calculated to be ~6% (Fig. 5C).

486

We then used our Monte Carlo simulation approach to validate these results by

487

examining the release probability of synaptic vesicles in the model when the number of

488

Ca2+ channels in the active zone was greatly reduced. Under the extreme condition

489

when very few Ca2+ channels were available, the probability that only a single Ca2+

490

channel opened clearly followed the binomial distribution. For example, if the total

491

number of Ca2+ channels within the active zone was reduced to 3, the probability that

492

only one channel opened during an action potential was predicted as

493

0.37 (for P equal to 0.19; see Luo et al., 2011). Indeed, we observed 3823 single

494

channel opening events out of 10,000 modeling runs (generated by distinct random

495

number seeds) and 221 out of these released a synaptic vesicle. Therefore, our model

! !× !

(1 − ) =

496

predicted that the release probability per opening was equal to 5.8% (221/3823; Fig.

497

5C), very close to our experimental measurement (~ 6%). We then varied the number

498

of available Ca2+ channels in the active zone from 1 to 10 and determined that the

499

release probability was 5.3 ± 0.3% (mean ± SD, n = 10) for release events in which only

500

one Ca2+ channel opened during an action potential (see Table 3).

501

We then used our MCell model to ask whether additional open Ca2+ channels in

502

the active zone would enhance the release probability per opening. If each vesicle

503

release event is mainly triggered by a single open channel, the release probability per

504

opening should not change significantly regardless how many channels open in the

505

active zone. On the other hand, if several open channels cooperate to trigger fusion of a

506

given vesicle, the release probability per opening should increase with more open

507

channels, perhaps even in a supra-linear fashion. With the full complement of Ca2+

508

channels available in our active zone model (26), the average number of channels that

509

opened with each action potential was ~5, but even under this condition, the probability

510

of vesicle release per opening was only 6-7%. As shown in Figure 5D and Table 3, our

511

model predicted that the release probability per Ca2+ channel opening was little affected

512

by the total number of open Ca2+ channels in the active zone. These data underscore

513

the predominance of vesicle fusion events triggered by Ca2+ flux through a single open

514

channel, even when several Ca2+ channels open in the active zone.

515 516 517 518

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

519

overwhelmingly derived from the single Ca2+ channel most tightly associated with the

520

fused vesicle, with contributions from a small number of additional channels nearby. In

521

fact, we show that a single open Ca2+ channel is able to trigger synaptic vesicle release

522

from presynaptic active zones at the adult frog NMJ. Beyond what was previously

523

reported in the literature (Shahrezaei et al., 2006), using realistic estimates of the

524

channel-vesicle stoichiometry and channel open probability (Luo et al, 2011), we

525

provide an estimate for the release probability of a synaptic vesicle triggered by the

526

Ca2+ flux through a single open Ca2+ channel, which is only about 6%. Thus, despite a

527

tight 1:1 coupling of Ca2+ channels to synaptic vesicles, open Ca2+ channels trigger

528

release unreliably. These data advance our understanding of presynaptic active zone

529

function at a model synapse (the frog NMJ).

530 531 532

The role of Ca2+ channel cooperativity in triggering transmitter release An ongoing debate centers on whether release of individual synaptic vesicles is

533

triggered by Ca2+ flux through a single open Ca2+ channel or through multiple open Ca2+

534

channels in the active zone (Tarr et al., 2013). Since the spatial relationship between

535

docked synaptic vesicles and presynaptic Ca2+ channels is not known at most synapses,

536

previous studies typically relied on measuring the power relationship between

537

transmitter release and Ca2+ entry by altering the number of available Ca2+ channels

538

(Yoshikami et al., 1989; Augustine, 1990; Mintz et al., 1995; Wu et al., 1999;

539

Shahrezaei et al., 2006; Scimemi and Diamond, 2012). Conceptually, the effectiveness

540

of gradual Ca2+ channel blockade on transmitter release is determined by the spatial

541

relationship between Ca2+ channels and synaptic vesicles, and therefore reflects the

542

cooperative coupling (denoted by m) of Ca2+ channels in controlling vesicle secretion.

543

The value of m has been shown to vary for different synapses and different subtypes of

544

Ca2+ channels at a single synapse. For example, at cerebellar parallel fiber synapses,

545

Mintz et al. (1995) determined that transmitter release triggered by N-type Ca2+

546

channels had an m value of 2.5, whereas transmitter release triggered by P/Q type Ca2+

547

channels had an m value of 4.0. Similarly, at the young calyx of Held synapse,

548

transmitter release triggered by P/Q type Ca2+ channels has a different m value (m = 3.7)

549

than release triggered by N-type Ca2+ channels (m = 1.3; Wu et al., 1999). As the

550

calyces mature, the m value for the P/Q type Ca2+ channels decreases (Fedchyshyn

551

and Wang, 2005), suggesting that the spatial coupling between Ca2+ channels and

552

docked vesicles is tightened during development. At the chick ciliary ganglion calyx,

553

where N-type channels trigger release, m has been reported to be 1.3 (Gentile and

554

Stanley, 2005). Such a large variability in the cooperative coupling of Ca2+ channels

555

with neurotransmitter release suggests a wide variety of spatial organization of Ca2+

556

channels within individual release sites at different synapses. Lower values of m (1-2)

557

suggest that a small number of open Ca2+ channels are sufficient for triggering the

558

fusion of a synaptic vesicle and which may thus be tightly associated with those open

559

channels. In contrast, higher values of m (4-5) indicate that many Ca2+ channels need

560

to open simultaneously to contribute the necessary Ca2+ ions to trigger the release of a

561

single synaptic vesicle. Under conditions where m values are high, the Ca2+ channel

562

cooperativity measurement is governed by the molecular cooperativity of the Ca2+

563

sensor (Dodge & Rahamimoff, 1967) as titrating channel block is essentially similar to

564

changing extracellular Ca2+ concentration at these synapses (Meinrenken et al., 2002).

565

In agreement with previous studies (Yoshikami et al., 1989; Shahrezaei et al

566

2006), we have shown at the adult frog NMJ that Ca2+ channels have a low

567

cooperativity in triggering transmitter release (m = 1-2). In contrast to previous reports,

568

our studies are built upon our recent high resolution Ca2+ imaging data that support the

569

hypothesis that there are relatively few functional Ca2+ channels in each frog NMJ active

570

zone (Luo et al., 2011). These data predict a 1:1 relationship between presynaptic Ca2+

571

channels and docked synaptic vesicles and allow our current study to make detailed

572

predictions regarding the probability with which individual channel openings trigger

573

vesicle fusion (see below). The small m value we report here further supports the close

574

association between individual docked synaptic vesicles and a single Ca2+ channel. As

575

evidenced by our Monte Carlo simulations, Ca2+ ions that bound to released synaptic

576

vesicles were primarily derived from the nearest open Ca2+ channel. This dominant

577

control of vesicle fusion by a single Ca2+ channel (nanodomain coupling) becomes more

578

pronounced when a majority of Ca2+ channels are blocked. On the other hand, a

579

significant number of vesicle release events involved small contributions from

580

neighboring Ca2+ channels and it will be interesting to investigate possible functional

581

implications of this finding in future work.

582 583

Functional organization of single vesicle release sites within active zones of the

584

frog NMJ

585

At individual release sites in a variety of synapses the measured Ca2+ channel-

586

release site cooperativity (the m value) for triggering transmitter release has been used

587

to infer the stoichiometric relationship between Ca2+ channels and docked synaptic

588

vesicles. However, there has been no direct evidence on how reliable the Ca2+ flux

589

through a single open Ca2+ channel can trigger synaptic vesicle fusion. Past studies

590

have at most determined the approximate number of Ca2+ channels contributing ions to

591

vesicle release (Shahrezaei et al., 2006). By greatly reducing the number of Ca2+

592

channels in each active zone using drug treatments combined with high-resolution Ca2+

593

imaging techniques we were able to capture sparsely-distributed Ca2+ influx from single

594

Ca2+ channel openings. In addition we conducted Monte Carlo simulations of a realistic

595

active zone model with small numbers of active Ca2+ channels and estimated the

596

coupling between single Ca2+ channel openings and vesicle fusion. Analysis of our data

597

supports the conclusion that after block of 85 and 92% of Ca2+ influx within active zones

598

(using 400 or 600 nM CgTX, respectively), we were able to image the Ca2+ flux through

599

single open Ca2+ channels in individual active zones during single action potential

600

stimulation. Further, our results show that Ca2+ flux through single open voltage-gated

601

Ca2+ channels triggered synaptic vesicle fusion with a probability of only ~6%.

602

Importantly, even under control conditions with an average of five Ca2+ channel

603

openings per action potential stimulus, the vesicle release probability per open channel

604

remained approximately constant at ~6% (Table 3). This finding provides a mechanistic

605

explanation for the observation that action potential stimulation only unreliably triggers

606

vesicle fusion at each single vesicle release site in the frog NMJ. Importantly, however,

607

these highly unreliable single vesicle release sites (composed of one synaptic vesicle

608

and its closely-associated Ca2+ channel) can be assembled to build a strong and

609

reliable synapse using thousands of such units. Such assembly of unreliable single

610

vesicle release sites into active zones and nerve terminals is important for conserving

611

presynaptic resources, and allows both strength and reliability of synaptic transmission

612

at the entire NMJ during repetitive nerve activity (Tarr et al., 2013).

613

Even though vesicle fusion is predominately triggered by the closest Ca2+

614

channel during single activation events, the long, linear arrangement of Ca2+ channels

615

at frog active zones might lead to significant contributions of adjacent channels during

616

trains of action potentials. Indeed, an evaluation of short-term synaptic plasticity

617

mechanisms at the frog NMJ combining physiological data and MCell computer

618

simulations showed that the contribution of adjacent Ca2+ channels to vesicle release

619

increased during subsequent stimuli (Ma et al., 2015). Clearly, the precise relationship

620

between active zone structural organization (especially with respect to Ca2+ channels

621

and synaptic vesicles) and physiological function requires further examination.

622 623 624

625

Acknowledgements

626

We thank Greg Hood for writing the image alignment routine, and Tyler Tarr for

627

feedback during the preparation of this manuscript. This work was supported by grants

628

from the National Institutes of Health R01 NS043396 (to S.D.M.), R01 NS090644 (to

629

S.D.M and M.D.), R01 GM068630, P41 RR06009, and P41 GM103712 (to J.R.S. and

630

M.D.), and The National Science Foundation (0844174 to M.D., 0844604 to S.D.M., and

631

1249546 to M.D. and S.D.M.).

632 633 634 635 636 637 638 639 640 641 642 643 644 645 646

Competing financial interests: The authors declare no competing financial interests.

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) %Ca2+ entry blocked (mean ± SD) %vesicle release blocked (mean ± SD)

25

50

75

100

400

600

22 ± 1.0

41 ± 2.6

59 ± 4.6

70 ± 5.9

85 ± 8.0

92 ± 1.7

34 ± 2.4

64 ± 2.9

79 ± 5.0

84 ± 1.8

96 ± 2.5

98 ± 1.4

647 648 649

Table 2. The effect of titrating blockade of Ca2+ channels on action potential-evoked neurotransmitter release using Monte Carlo simulation. Active zone channel 26 20 15 8 4 2 number control 23 42 69 85 92 % channel blocked % vesicle release control 32 57 79 91 96 blocked 650 651 652

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 Total release probability Release probability per channel opening 653 654

1

2

3

4

5

5.2 ± 0.3

11.4 ± 0.8

18.6 ± 0.9

24.0 ± 6.0

29.0 ± 5.5

5.2 ± 0.3

5.7 ± 0.4

6.2 ± 0.3

6.0 ± 1.5

5.8 ± 1.1

655

Figure legends

656

Figure 1. Diagram of the MCell model representation of synaptic vesicles and their

657

arrangement into an active zone. A. Schematic view of the bottom of a docked synaptic

658

vesicle used in our MCell model. In this view, the 40 Ca2+ binding sites in groups of 5

659

are depicted in various shades of gray and each represent a synaptotagmin molecule.

660

B. View of our MCell frog AZ model encompassing a single frog AZ including 26 docked

661

synaptic vesicles and their closely associated presynaptic Ca2+ channels (VGCCs).

662

During an action potential, only a small subset of these Ca2+ channels are predicted to

663

open. This diagram shows a sample single model run in which open channels are

664

represented as white pentagons and closed channels as black pentagons.

665 666 667 668

Figure 2. Titrating ω-CgTX GVIA blockade and comparing block of Ca2+ entry with

669

block of vesicle fusion reveals a low Ca2+ channel cooperativity in transmitter release. A.

670

CgTX (100 nM) significantly reduced single action potential-evoked Ca2+ entry into the

671

nerve terminals. B. EPP amplitude decreased after exposure to 100 nM CgTX. C. A log-

672

log plot of fractional Ca2+ entry and neurotransmitter release after treatment with various

673

concentrations of CgTX (25, 50, 75, 100, 400, and 600 nM) reveals a relationship with a

674

slope between 1 and 2 (lines with indicated slopes are represented in black, green, blue,

675

and yellow). The dashed green line is a fit to the first few data points during only a small

676

fractional block of Ca2+ channels. This dashed green line has a slope close to 2 (similar

677

to the solid green line). However, after a large fraction block of Ca2+ channels the data

678

are fit using a dashed black line and show a relationship with a slope close to 1 (similar

679

to the solid black line). D. MCell computer simulations reproduce experimental data

680

and predict that each vesicle fusion event is triggered by the Ca2+ flux through very few

681

Ca2+ channels. A log-log plot compares changes in Ca2+ entry and simulated

682

neurotransmitter release after randomly removing increasing numbers of Ca2+ channels

683

from the modeled active zone (which mimics the CgTX titration shown in C). The

684

simulated data fall on a slope between 1 and 2; similar to experimental data shown in C.

685

As in C, the data collected after the removal of only a few calcium channels are fit using

686

a dashed green line which has a slope close to 2, while the data collected after the

687

removal of many calcium channels are fit using a dashed black line which has a slope

688

close to 1. E. Histogram of the fraction of released synaptic vesicles that bound Ca2+

689

ions contributed by various numbers of Ca2+ channels under the control condition (26

690

Ca2+ channels in the AZ). F. Histograms of the fraction of released synaptic vesicles

691

that bound Ca2+ ions that originated from various numbers of Ca2+ channels when most

692

of the AZ Ca2+ channels were removed from the simulation (8 channels present; 70%

693

block, and 2 channels present; 92% block). With increasing removal of AZ Ca2+

694

channels, vesicle fusion increasingly is triggered by the Ca2+ flux through a single Ca2+

695

channel (dark blue bar).

696

Figure 3. Ca2+ flux through a single open Ca2+ channel primarily provides the trigger for

697

vesicle fusion in our simulated active zone model, even when other channels open

698

nearby. A. Given a particular vesicle fusion event in the AZ (red circle), the average

699

probability that individual Ca2+ channels have opened is represented by the red area in

700

the pie chart and the corresponding percentage associated with each channel icon. B.

701

left panel: For the majority of vesicle fusion cases in which the closely-associated Ca2+

702

channel has opened (94% as represented in A above), the number in each Ca2+

703

channel circle icon indicates the percentage of Ca2+ ions contributed by each Ca2+

704

channel to the total number of ions bound by the vesicle at the time of fusion (red circle).

705

right panel: Representative model schematic depicting the 40 Ca2+ binding sites (gray

706

areas) at the bottom of synaptic vesicles. The colored dots indicate Ca2+ ions (color

707

coded by their channel of origin) bound to different synaptotagmin binding sites at the

708

time of vesicle fusion for a representative single model run under the condition that the

709

closely-associated Ca2+ channel has opened (94% of fusion events). C. left panel: For

710

the minority of vesicle fusion cases in which the closely-associated Ca2+ channel has

711

not opened (6% as represented in A above), the number in each Ca2+ channel circle

712

icon indicates the percentage of Ca2+ ions contributed by each Ca2+ channel to total

713

ions bound by the vesicle at the time of fusion (red circle). right panel: Representative

714

model schematic of the bound Ca2+ ions on vesicular synaptotagmin binding sites at the

715

time of fusion for a representative single model run under the conditions that the

716

closely-associated Ca2+ channel has not opened (6% of fusion events). Schematic is

717

organized as described above for B.

718

Figure 4. Imaging Ca2+ entry through single Ca2+ channel openings in the frog NMJ

719

active zone. A. Postsynaptic labeling of acetylcholine receptors (Alexa 594-conjugated

720

α-bungarotoxin) is used for estimating the number of active zones within the imaged

721

regions of the nerve terminal. For visual clarity, offset yellow ovals show schematic

722

outlines of the 31 AZs identified in this fluorescence image. B. Average Ca2+ signal for

723

a representative control nerve terminal (100 stimuli at 0.5 Hz). C. Average Ca2+ signal

724

remaining after block of presynaptic Ca2+ channels using a 60 minute exposure to 600

725

nM CgTX (100 stimuli at 0.5 Hz). D. Histogram distribution of single pixel fluorescence

726

intensity under resting (unstimulated), stimulated control, and stimulated CgTX

727

treatment conditions. Exposure to 400 or 600 nM CgTX resulted in a distribution that

728

took a lognormal form suggesting that individual pixels sampled single Ca2+ channel

729

openings. This was distinct from the Gaussian-like distribution of resting intensities, and

730

the distribution of intensities following stimulation under control conditions (no toxin

731

block). E. Representative recordings of mEPPs from a muscle fiber (top), and averaged

732

EPP response evoked by nerve stimulation (bottom), after exposure to 600 nM CgTX.

733

Using these recordings, quantal content (QC) was calculated (QC = average EPP

734

amplitude / average mEPP amplitude) as listed in the text. Scale bars = 2 µm.

735

736

Figure 5. The coupling of Ca2+ influx to vesicle fusion events after exposure to a high

737

concentration of CgTX. A. Shown is the number of detected Ca2+ channel openings per

738

active zone and action potential after exposure to either 400 nM or 600 nM CgTX.

739

These data are obtained by dividing the number of imaged Ca2+ entry sites per action

740

potential by the number of active zones within an imaged area (see Fig. 3). B.

741

Normalized release probability per active zone and action potential after exposure to

742

400 nM or 600 nM CgTX determined by dividing the average QC by the average

743

number of active zones in the nerve terminal of adult frog cutaneous pectoris NMJs

744

(700). C. Experimental estimate of release probability per Ca2+ channel opening is

745

similar (5-6%) when calculated following exposure to 400 nM or 600 nM CgTX (plot

746

generated using the data in panels A and B). D. MCell simulations predict that release

747

probability following a single Ca2+ channel opening is small (5-6%) and comparable to

748

our experimental estimate shown in C. Further, the release probability per Ca2+ channel

749

opening remains relatively constant even as the number of open channels within an

750

active zone is increased in our simulation.

751 752 753 754 755 756 757

758

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Figure 1

A

B

VGCCs closed

70 nm

50 nm

open

Synaptic Vesicle

Figure 2

C

Single AP-evoked calcium entry 2 'F/F(%) 0 CgTX

Ctrl

B

EPP

1 mV 10 ms

Ctrl

fraction of transmitter release

A

(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

1.0

0. 8

0. 8

0. 6

0. 6

0.1

Fraction of calcium entry

1

Reduced # channels 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 26 AZ Ca2+ channels

3

2

0

8 AZ channels 2 AZ channels

# Channels contributing bound ions to vesicle

Figure 3

A SV VGCC 4%

15%

94%

15%

5%

3%

9%

14%

9%

4%

70nm 35nm

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

Figure 4

B

C

D

BTX labeling

control

600 nM CgTX

10 8 6 4 'F/F(%) 2 0

1 0

20

resting 600 nM CgTX 400 nM CgTX control

15 10 5 0 -20 -10 0 10 20 30 40 50

'F/F(%)

E mEPPs

3 2

25

frequency (%)

A

1 mV 100 ms

1 mV 10 ms

'F/F(%) EPP

Figure 5

B

Pr/o(%)

C

0.4 0.2 0

0.04

release probability per AZ per AP

0.6

10

D 10

8

8

6

6

4 2

0.03 0.02 0.01 0

600 nM 400 nM CgTX CgTX

Pr/o(%)

# channel openings per AZ per AP

A

600 nM 400 nM CgTX CgTX

4 2

0 600 nM 400 nM CgTX CgTX

0

1

2

3

4

number of open channels

5