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The calcium sensor synaptotagmin 7 is required for synaptic facilitation

It has been known for over 70 years that synaptic strength is dynamically regulated in a use-dependent manner1. At synapses with a low initial release probability, closely spaced presynaptic action potentials can result in facilitation, a short-term form of enhancement where each subsequent action potential evokes greater neurotransmitter release2. Facilitation can enhance neurotransmitter release manyfold and profoundly influence information transfer across synapses3, but the underlying mechanism remains a mystery. Among the proposed mechanisms is that a specialized calcium sensor for facilitation transiently increases the probability of release2,4 and is distinct from the fast sensors that mediate rapid neurotransmitter release. Yet such a sensor has never been identified, and its very existence has been disputed5,6. Here we show that synaptotagmin 7 (syt7) is a calcium sensor that is required for facilitation at multiple central synapses. In syt7 knockout mice, facilitation is eliminated even though the initial probability of release and presynaptic residual calcium signals are unaltered. Expression of wild-type syt7 in presynaptic neurons restored facilitation, whereas expression of a mutated syt7 with a calcium-insensitive C2A domain did not. By revealing the role of syt7 in synaptic facilitation, these results resolve a longstanding debate about a widespread form of short-term plasticity, and will enable future studies that may lead to a deeper understanding of the functional importance of facilitation.

longer-lasting Ca 2+ signals between action potentials 4 . Under one scenario, this sensor modulates Ca 2+ channels to produce use-dependent increases in Ca 2+ influx 10 . Several canditate proteins have been proposed to act in this manner 11,12 , but increased Ca 2+ influx cannot account for facilitation at most synapses 13 . Alternatively, an unidentified Ca 2+ sensor could mediate facilitation by directly increasing the probability of release (p).
Syt7 is located presynaptically, and binds Ca 2+ with high affinity and slow kinetics [14][15][16] , making it a promising candidate sensor for the modest increases in residual Ca 2+ that mediate facilitation. Previous studies suggest that syt7 contributes to a slow phase of transmission known as asynchronous release 17,18 , and to Ca 2+ -dependent recovery from depression 19 , but the role of syt7 in facilitation was not examined because these studies employed synapses with prominent depression that obscures facilitation. We therefore examined synaptic transmission at four facilitating synapses: Schaffer collateral synapses between hippocampal CA3 and CA1 pyramidal cells 9 (Fig. 1a), thalamocortical synapses between layer 6 cortical pyramidal cells and thalamic relay cells 20 (Fig. 1b), mossy fiber synapses between dentate granule and CA3 cells 9 (Fig. 1c), and perforant path synapses between layer II/III cells of the entorhinal cortex and dentate granule cells 21 (Fig. 1d). Immunohistochemistry shows that syt7 is present in regions where these synapses are located (Extended Data Figs. 2 and 3). Facilitation is often assessed using pairs of closelyspaced stimuli. In slices from wild-type (WT) mice, paired-pulse facilitation resulted in ~2fold enhancement of neurotransmitter release lasting several hundred milliseconds ( Fig. 1ad, black traces). In syt7 knockout (KO) mice, paired-pulse facilitation was eliminated ( Fig.  1a-d, red traces). Sustained high frequency activation produces up to 10-fold enhancement in wild-type animals, but in knockouts facilitation is eliminated at all synapses except for mossy fiber synapses, where the remaining enhancement is consistent with use-dependent spike broadening that occurs at this synapse 22 (Fig. 1e-h, Extended Data Fig. 4).
The loss of facilitation in syt7 knockouts cannot be accounted for by slowed recovery from depression reported with syt7 deletion 19 , because recovery from depression is too slow to strongly influence rapid facilitation, nor can it produce the large increase in release associated with facilitation. There are several possible explanations for the loss of facilitation in knockouts: (1) the presynaptic Ca 2+ signal that induces facilitation could be altered, (2) the probability of release (p) for synaptic vesicles could be increased, which by promoting vesicle depletion would indirectly reduce facilitation, or (3) the mechanism for facilitation could be disrupted directly. We assessed these possibilities at the CA3→CA1 synapse.
Action-potential-evoked increases in presynaptic Ca 2+ consist of a large, brief localized Ca 2+ signal that activates the low-affinity Ca 2+ sensor synaptotagmin 1 to trigger neurotransmitter release 23 , and a small residual Ca 2+ signal (Ca res ) that persists for tens of milliseconds and has been implicated in facilitation 2 . It is difficult to measure local Ca 2+ signals that trigger release, but Ca res is readily measured. We used a low-affinity Ca 2+ indicator to measure the time course of Ca res in CA3 presynaptic terminals, because facilitation can be attenuated by the accelerated decay of Ca res 4 . Ca res decayed similarly in wild-type and syt7 knockout animals (Fig. 2a), indicating that the loss of facilitation in knockouts is not a consequence of accelerated Ca res decay. We also used Ca res as a measure of Ca influx to determine whether there are use-dependent manner changes in Ca 2+ entry. However, each of two closely spaced stimuli evoked the same incremental increase in Ca res in both wild-types and knockouts (Fig. 2b), indicating that use-dependent changes in total Ca influx cannot account for facilitation. This suggests that if changes in Ca influx contribute to facilitation at this synapse, they must be restricted to the small subset of presynaptic calcium channels that evoke neurotransmitter release. We repeated the experiment using a highaffinity Ca 2+ indicator, where the degree of saturation during paired stimuli can be used to measure the magnitude of Ca res evoked by the first stimulus (see Methods). We conclude that Ca influx evoked by the first stimulus is the same in wild-type and knockout animals (Fig.  2c).
We further explored the role of Ca 2+ in facilitation by examining the Ca 2+ -dependence of excitatory postsynaptic currents (EPSC) and facilitation. Raising extracellular Ca 2+ leads to a steep increase in EPSC amplitude (Fig. 2d), but a decrease in facilitation (Fig. 2e Fig. 5). These findings suggest that the loss of facilitation in knockouts is not a consequence of higher initial p, because facilitation was absent even when the initial p was strongly attenuated by reducing external Ca 2+ .
To further test whether initial p is elevated in syt7 knockouts, we measured how field excitatory postsynaptic potentials (fEPSPs) scaled with stimulus intensity 24 (Fig. 3a). The slope of the fEPSP vs. presynaptic volley gives a relative measure of p (see Methods), which was unchanged in knockouts (Fig. 3b). Moreover, the fEPSP to presynaptic volley ratio changed steeply with extracellular Ca 2+ , showing that this method is sensitive to p (Fig. 3c, d). We also assessed p using pharmacological blockade of synaptically-activated NMDARs by the use-dependent blocker MK801 25 ( Fig. 3e-g). This approach is widely-used to detect changes in p: An increase in p leads to more glutamate release, more activation of NMDARs, and a more rapid blockade of NMDA receptors, while a decrease in p leads to a slower blockade (Extended Data Fig. 6). The rate of blockade of NMDA-mediated field EPSPs (NMDA-fEPSP) was unaffected by syt7 deletion (Fig. 3e), indicating similar initial p. However, when we evoked NMDA-fEPSPs with trains of 3 stimuli 25 , amplitudes decayed more rapidly in wild-types ( Fig. 3f, g), suggesting that syt7 is required to increase p for the second and third stimuli. Thus, initial p and presynaptic Ca 2+ signaling are unaffected by syt7 deletion, but knockouts lack the use-dependent increase in p that underlies facilitation. This suggests that the mechanism underlying facilitation is directly impaired by syt7 deletion.
Syt7 is implicated in neuroendocrine release 16 , insulin secretion 26 , and exocytosis of lysosomes 27 , which could all indirectly influence synaptic transmission in global syt7 knockouts. Therefore, to determine whether syt7 controls facilitation by acting in presynaptic neurons in a cell autonomous manner, we tested whether viral expression of syt7 in CA3 pyramidal cells of syt7 knockouts rescued facilitation. This approach is complicated by our inability to virally transduce all CA3 pyramidal cells, which prohibits the use of extracellular stimulation that would activate some presynaptic cells that express syt7 and others that do not. We overcame this problem with an adeno-associated virus (AAV) that drove bicistronic expression of both ChR2 and syt7, allowing optical stimulation of only those fibers expressing syt7.
Using conditions we have previously shown allow facilitation to be studied with optogenetic stimulation (see Methods), we confirmed that when ChR2 alone was expressed, optical and electrical stimulation produced similar facilitation in wild-types (Fig. 4a,e,f), and similar depression in knockouts (Fig. 4b,e,f). We next used a bicistronic vector to express both ChR2 and wild-type syt7 in knockout animals. Light-evoked responses exhibited facilitation, whereas electrically-evoked responses did not (Fig. 4c,e,f). This suggests that bicistronic expression of ChR2 along with a presynaptic protein of interest offers a powerful new approach to characterize the effect of gene manipulation on presynaptic function within intact neural circuits. When syt7 was expressed in wild-type animals the peak facilitation was unaffected (Fig. 4e,f, Extended Data Fig. 7a). Thus, expressing syt7 in CA3 pyramidal cells rescued facilitation in a cell autonomous manner, with facilitation restored only at synapses expressing syt7 and ChR2.
To determine whether Ca 2+ -binding by syt7 is important for facilitation, we assessed whether facilitation is rescued by syt7 with a mutated Ca 2+ -insensitive C2A domain (syt7 C2A*). Previous studies established that Ca 2+ -binding to the C2A domain of syt7 is required for syt7 to mediate asynchronous release 18 . We found that syt7 C2A* did not rescue facilitation in knockouts ( Fig. 4d-f). Moreover, in wild-type animals, syt7 C2A* expression strongly attenuated facilitation (Fig. 4e,f, Extended Data Fig. 7b), suggesting that syt7 C2A* competes with native syt7 to suppress facilitation.
Our results indicate that facilitation requires Ca 2+ -binding to the C2A domain of syt7, and also provide insight into the role of syt7 in facilitation. We conclude that syt7 does not produce facilitation by altering the amplitude and time course of Ca res (Fig. 2), by increasing initial p (Fig. 3), by acting as a Ca 2+ buffer (Extended Data Fig. 8), or through usedependent increases in the total Ca influx (Extended Data Fig. 1b, Fig. 2). The observation that initial p is unaltered in syt7 knockouts indicates that local Ca influx , is unaffected for the first stimulus, but it is difficult to rule out the possibility that syt7 mediates a use-dependent increase in Ca influx through the subset of channels that trigger vesicle fusion. There is, however, no evidence for syt7 associating with or regulating calcium channels. In contrast, syt7 is known to interact with syt1 and can mediate vesicle fusion [16][17][18] . The most parsimonious explanation is that syt7 acts as the hypothesized specialized Ca 2+ sensor to elevate p during facilitation. Facilitated release exhibits rapid kinetics, suggesting that syt7 somehow increases the probability of syt1-dependent vesicle fusion. Whether this is through a direct interaction of syt7 with a fast synaptotagmin isoform such as syt1 remains an open question. It is also unclear whether the recently described interaction between syt7 and calmodulin that promotes vesicle replenishment 19 is similarly required for facilitation. Finally, it is possible that at other synapses facilitation is mediated by additional specialized Ca 2+ sensors or involves other mechanisms. Further studies are needed to clarify these issues.
Based primarily on theoretical considerations, facilitation is thought to profoundly influence both information transfer and network dynamics. In the hippocampus, the high-pass filtering imposed by facilitating synapses may account for the burst firing in place cells that encode spatial information 28 . In the auditory pathway, facilitation is proposed to counteract shortterm depression in order to maintain linear transmission of rate-coded sound intensity 29 . It has even been suggested that facilitation forms the basis of short-term memory, as facilitating recurrent connections within cortical networks could support the persistent activity states associated with working memory 30 . In future studies the selective elimination of syt7 from specific cell types could allow the first direct tests of the impact of facilitation on neural circuits and behavior.

Animals and viruses
All mice were handled in accordance with NIH guidelines and protocols approved by Harvard Medical School. Syt7 KO mice 31 (Jackson Laboratory) and WT littermates of either sex were used. Statistical tests were not used to predetermine sample size. Blinding and randomization were not performed. AAV2/9-hSyn-hChR2(H134R)-EYFP and its pAAV backbone (Addgene 26973) were obtained from the University of Pennsylvania Vector Core. cDNA encoding the rat syt7 WT α isoform and C2A* mutant (D225A, D227A, D233A) 18 were generously provided by Taulant Bacaj and Thomas Sudhof. For rescue experiments involving syt7 with mutated Ca 2+ binding domains, we used the mutated C2A* version instead of the C2A*C2B* double mutant, as mutation of both C2 domains leads to lower levels of expression. The porcine teschovirus-1 2A (P2A) cleavage sequence 32 and syt7 were inserted after the ChR2 C-terminus in the pAAV backbone (Genscript). Plasmid-driven expression of ChR2-YFP and syt7 was confirmed in HEK cells by syt7 immunostaining and patch-clamp recording of ChR2 photocurrents. AAVs were produced and purified from HEK cells as previously described 33 .

Electrophysiology
For ChR2 stimulation, 160 mW/mm 2 laser pulses (0.2-0.5 ms) from a 100 mW 473 nm laser (OptoEngine, MBL-III) were focused through the 60X objective of the microscope (Olympus, BX51WI) to produce a 80 μm diameter spot over the stratum radiatum, >500 μm from the recorded cell to avoid activating ChR2 in presynaptic boutons, which can artificially raise the probability of release and obscure facilitation 34 . Extracellular stimulation was performed with a stimulus isolation unit (WPI, A360) using glass monopolar electrodes (0.5-1 MΩ) filled with ACSF. Stimulus electrodes were positioned ~500 μm from the recording electrode in the stratum radiatum (Schaffer collaterals), the internal capsule (corticothalamic), the hillus adjacent to the dentate granule cell layer (mossy fibers), and the outer molecular layer (lateral performant path). To ensure that mossy fiber responses were not contaminated by associational/commissural inputs the metabotropic glutamate receptor agonist DCG-IV (1 μM) was applied at the end of experiments to selectively block mossy fiber responses 35 . Data were included only if responses were reduced by more than 80% (average reduction was 88 ± 1 % in WT and 90 ± 2% in syt7 KO mice), and the amplitude of mossy fiber responses was measured after subtracting the response remaining in the presence of DCG-VI. Stimulus trials were repeated at 0.1 Hz (0.033 Hz at mossy fibers to avoid potentiation), and artifacts were deleted for display. Recordings were acquired using an amplifier (Axon Instruments, Multiclamp 700B) controlled by custom software written in IgorPro (provided by Matthew Xu-Friedman, SUNY Buffalo), and low-pass filtered at 2 kHz. Whole-cell recordings were obtained using borosilicate patch pipettes (2-5 MΩ) pulled with a horizontal puller (Sutter P-97). The internal recording solution contained (in mM): 150 Cs-gluconate, 3 KCl, 10 HEPES, 0.5 EGTA, 3 MgATP, 0.5 NaGTP, 5 phosphocreatine-tris, and 5 phosphocreatine-Na. pH = 7.2. Cells were held at −70 mV, and series resistance was monitored during recordings. fEPSPs were recorded in current-clamp mode with ACSF-filled patch pipettes (0.5-1 MΩ). Inhibition was blocked with picrotoxin (50 μM), and during fEPSP recordings, CPP (2 μM) and CGP (3 μM) was added to the bath. 4-10 trials were conducted for each stimulus frequency, and recordings were averaged over trials. Data in all figures represent the mean ± SEM. Average responses are displayed with double exponential or polynomial curves fit in IgorPro. Unless stated otherwise, statistical significance was assessed by unpaired two-tailed Student's t-test, or one-way ANOVA followed by Tukey's post hoc test.

Probability of release
To record NMDA-EPSCs cells were voltage clamped at +40 mV, and the internal solution contained (in mM): 85 Cs-methanesulfonate, 4 NaCl, 10 HEPES, 0.2 EGTA, 30 BAPTA, 2 MgATP, 0.4 NaGTP, 10 phosphocreatine-Na, 25 TEA, 5 QX-314, pH = 7.3. For recording NMDA-fEPSPs, Mg 2+ was excluded from ACSF to relieve Mg 2+ block of NMDA receptors. Picrotoxin (100 μM) and NBQX (5 μM) were added to the bath, and stimulation was conducted at 0.1 Hz (unless otherwise indicated) for 5 minutes to obtain a baseline response. Stimulation was halted for 10 minutes while (+)-MK801 (40 μM) was added and allowed to equilibrate. For experiments involving fEPSPs vs. presynaptic volley, the postsynaptic response was measured by the slope of the fEPSP, while the amplitude of the presynaptic volley was used to determine the number of activated fibers. If p increases, the same number of activated presynaptic fibers will produce a larger fEPSP. The ratio between fEPSP and volley was determined by line fits to the linear regime of the input-output curve of individual experiments (20-80 μA stimuli).
The study of probability of release is complicated because many people use p to refer to the probability of release of a vesicle (p v ) and others refer to probability of release from an active zone (p synapse ) that contains N vesicles in its readily releasable pool (RRP). Thus, an increase in the size of the RRP for an active zone can increase p synapse even if p v is unaltered. Although MK801 blockade 25 and fEPSPs vs. presynaptic volley 24 are both widely-used methods to detect changes in the probability of release, for both approaches it is conceivable (though unlikely) that increases in p v could be obscured by a perfectly balanced decrease in the RRP size. However, the relationship between EPSC amplitude and extracellular Ca 2+ is similar in WT and syt7 KO animals. This suggests there is no increase in p v , which would cause this curve to saturate at lower values of Ca e for syt7 KO animals. Moreover, the large differences in facilitation in WT and syt7 KO animals were even more pronounced when the probability of release was reduced 10-fold by lowering Ca e from 2 mM to 0.5 mM, which is incompatible with an increase in p v obscuring facilitation by depleting vesicles.

Ca 2+ measurements
Ca 2+ was measured as described previously 4 . Briefly, CA3 fibers were labelled for three minutes using an ACSF-filled pipette containing either magnesium green AM or fura-2 AM (240 μM) and 1% fast green, placed into the border of the CA3-CA1 field. A vacuum pipette placed above the loading site removed excess indicator. Slices were incubated for at least 1 hour and imaging was performed in stratum radiatum of CA1 at least 500 μm from the injection site using a 60X objective and custom-built photodiode. Excitation was achieved using a tungsten (magnesium green) or xenon lamp (fura-2). Schaffer collaterals were stimulated using a glass electrode placed at least 300 μm from the imaging site. To prevent recurrent excitation, experiments were performed in the presence of NBQX (10 μM), CPP (2 μM) and picrotoxin (50 μM).
Magnesium green is a low-affinity calcium indicator 36 (K D =7 μM) that provides an approximately linear measure of Ca res 37 . As such it is well suited to measuring the time course of presynaptic Ca res ( Fig. 2A) and detecting changes in Ca influx during successive stimulations (Fig. 2B). However, with the bulk loading approach the size of the fluorescence change is proportional to the number of stimulated fibers, so the absolute Ca res signal is not readily quantified with magnesium green. In contrast, fura-2 has a high affinity for calcium 38,39 (K D = 131 nM) so it provides a saturating sublinear response to increases in Ca res [40][41][42] . This can be used to test for changes in the absolute size of Ca influx because a change in the Ca influx per stimulus would change the ratio between the fluorescence change produced by the first and second stimuli.

Immunohistochemistry
Two to four weeks after AAV injection, mice were anesthetized with ketamine and transcardially perfused with 4% PFA in PBS. The brain was removed and post-fixed for 24 hours. Slices (50 μm thick) were permeabilized (PBS + 0.4% Triton X-100) for

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Extended Data
Extended Data Figure 1

. Possible mechanisms for synaptic facilitation
It is established that calcium plays an important role in synaptic facilitation, and a number of mechanisms have been proposed that involve different aspects of calcium signaling 2 . Here we discuss the calcium signals that evoke rapid vesicle fusion, and also those thought to be involved in facilitation (a), and 3 mechanisms of facilitation are presented schematically 43 (b-d). a, To understand the mechanisms that have been proposed to account for facilitation, it is important to appreciate different aspects of presynaptic calcium signaling. Calcium signals are complex, but can be approximated by 2 components. An action potential opens calcium channels for less than a millisecond, and near open channels the calcium levels reach tens of micromolar. Release sites near calcium channels experience high local calcium levels (Ca local ) that are highly dependent on the distance from open calcium channels. Ca local can be reduced by high concentrations of fast calcium buffers that rapidly bind calcium. In addition there is a residual calcium signal (Ca res ) that results from calcium equilibrating within presynaptic terminals, before calcium is gradually removed over tens to hundreds of milliseconds. The amplitude of Ca res (and also total influx of Ca 2+ , Ca influx ) is determined by all of the calcium channels that open, not only those that produce Ca local that drives release, and after initial equilibration Ca res is roughly uniform throughout the presynaptic bouton. It is generally accepted that fast synaptic transmission is produced by calcium binding to syt1, syt2 or syt9 which have low-affinity binding sites, fast kinetics, and require the binding of multiple calcium ions 7,44 . The time course of release follows the time course of calcium channel opening, but with a brief delay (< 1 ms). Ca res after a single stimulus is much smaller than Ca local . Typical fluorescence-based approaches to measure calcium readily detect Ca res , but are insensitive to Ca local which is too localized and shortlived to measure. Note the y-axis is logarithmic to show both Ca local and Ca res in (a), but not in (b-d). b, For one mechanism of facilitation a fast calcium buffer is present in presynaptic terminals that binds calcium and reduces Ca local . Stimulating twice in rapid succession results in the same calcium influx for both stimuli. If there is no fast presynaptic buffer, the amplitudes of Ca local and the EPSCs are the same for both stimuli (red traces). If a fast highaffinity buffer is present (black traces), it reduces the initial Ca local and reduces the amplitude of the initial EPSC, but if enough calcium enters and binds to the buffer, it reduces its ability to buffer calcium. As a result the second stimulus produces larger Ca local than the first, and the EPSC is facilitated. c, A second possible mechanism is that more calcium enters for the second stimulus, and as a result there is more neurotransmitter release. This could arise from a spike broadening, or from the modulation of calcium channels. It is possible that influx through all calcium channels in the presynaptic terminal would be increased, in which case both Ca res and Ca local would be increased. It is also possible that the only calcium channels that are modulated are the subset that produce Ca local that triggers release, in which case Ca res would not be significantly increased. d, Finally, it is possible that there is a specialized calcium sensor that produces facilitation that is distinct from syt1 2,4,45 . Previous studies have shown that such a sensor would need to be sensitive to Ca res based on the observation that facilitation is altered at some synapses by manipulations that affect Ca res without affecting Ca local . According to this scheme, release is mediated by syt1 but calcium binding to a second sensor would increase p. The sensor is sufficiently slow that it does not influence release evoked by the first stimulus, but it able to influence release evoked by a second stimulus. Notably, syt7 expression in WT animals was higher in the LPP, where synapses exhibit facilitation, compared to the MPP, where synapses exhibit depression. Scale bar, 50 μm. The presence of syt7 labeling in regions containing CA3→CA1 synapses, layer 6 to thalamus synapses, MF synapses and LPP→granule cell synapses that are also colabeled with antibodies to the presynaptic marker for glutamatergic synapses vGlut1, suggests that syt7 is located presynaptically at these synapses. It is, however, difficult to obtain sufficient resolution with confocal microscopy in brain slices to unambiguously establish that syt7 is located presynaptically at these synapses. Importantly, the Allen Brain atlas suggests that the presynaptic cells for these synapses contain mRNA for syt7 46 . Lastly, immunoelectron microscopy revealed selective staining of presynaptic boutons in the CA1 region of the hippocampus 16 .

Extended Data Figure 8. Evidence suggests that syt7 does not produce facilitation by acting as a local calcium buffer at the CA3→CA1 synapse
This graph illustrates the general relationship between PPR and external calcium for synapses in which buffer saturation produces facilitation (green) and for facilitation observed at the CA3→CA1 synapse and many other synapses (black) 9 . It has been shown previously that the for buffer saturation mechanism (Extended Data Fig. 1B) the amplitude of facilitation is reduced when Ca influx is reduced by lowering external calcium 9 . This can be understood by considering that this form of facilitation is thought to require sufficient Ca influx to saturate the endogenous buffer, and thereby reduce its ability to buffer calcium for subsequent stimuli. If Ca influx is low then there is insufficient calcium entry to bind very much of the endogenous buffer, and little facilitation would result. In addition, as shown in Extended Data Figure 1 for a calcium buffer to produce facilitation it would need to buffer calcium sufficiently that it would reduce initial p. We have shown, however, that p is unaltered in syt7 knockouts. This is perhaps not surprising in light of the fact that syt7 is thought to be located on the plasma membrane, and in cases where this type of facilitation has been observed it is associated with high concentrations of a fast cytosolic buffer 9 .
Extended Data Table 1 Number of electrophysiological recordings from WT and syt7 KO animals.  Train (20-50 Hz) KO 13 3 Extended Data Table 2 Number of experiments related to the Ca 2+ -dependence of probability of release.