Abstract
Ca2+-dependent transmitter release occurs in a fast and in a slow phase, but the differential roles of Ca2+ buffers and Ca2+ sensors in shaping release kinetics are still controversial. Replacing extracellular Ca2+ by Sr2+ causes decreased fast release but enhanced slow release at many synapses. Here, we established presynaptic Sr2+ uncaging and made quantitative Sr2+- and Ca2+-imaging experiments at the mouse calyx of Held synapse, to reveal the interplay between Ca2+ sensors and Ca2+ buffers in the control of fast and slow release. We show that Sr2+ activates the fast, Synaptotagmin-2 (Syt2) sensor for vesicle fusion with sixfold lower affinity but unchanged high cooperativity. Surprisingly, Sr2+ also activates the slow sensor that remains in Syt2 knock-out synapses with a lower efficiency, and Sr2+ was less efficient than Ca2+ in the limit of low concentrations in wild-type synapses. Quantitative imaging experiments show that the buffering capacity of the nerve terminal is markedly lower for Sr2+ than for Ca2+ (∼5-fold). This, together with an enhanced Sr2+ permeation through presynaptic Ca2+ channels (∼2-fold), admits a drastically higher spatially averaged Sr2+ transient compared with Ca2+. Together, despite the lower affinity of Sr2+ at the fast and slow sensors, the massively higher amplitudes of spatially averaged Sr2+ transients explain the enhanced late release. This also allows us to conclude that Ca2+ buffering normally controls late release and prevents the activation of the fast release sensor by residual Ca2+.
- calcium buffering capacity
- calcium sensor
- endogenous fixed buffer
- slow release sensor
- strontium
- synaptotagmin
Introduction
Ca2+-dependent transmitter release is a fundamental signaling process for fast information transfer between neurons. Release can be separated into a fast phase with a brief duration (<1 ms), followed by a slower phase of up to hundreds of milliseconds. There is good evidence that the fast phase of release is mediated by a rapidly acting Ca2+ sensor with a high Ca2+ cooperativity, requiring the binding of ∼4–5 Ca2+ ions before vesicle fusion occurs (Dodge and Rahamimoff, 1967; Goda and Stevens, 1994; Bollmann et al., 2000; Schneggenburger and Neher, 2000). Synaptotagmin-1 (Syt1) functions as the Ca2+ sensor for fast release at mouse forebrain synapses and in Drosophila (Geppert et al., 1994; Fernández-Chacón et al., 2001; Yoshihara and Littleton, 2002; Pang et al., 2006). Synaptotagmin-2 (Syt2) plays a homologous role as a Ca2+ sensor mediating the steeply Ca2+-dependent component of release at a large hindbrain synapses, the calyx of Held (Pang et al., 2006; Sun et al., 2007; Kochubey and Schneggenburger, 2011).
For the slow phase of release, the underlying mechanisms are less clear. Slow release is driven by the spatially averaged [Ca2+]i, which, because of its slower decay (∼50 ms), can summate during repeated AP activity. Relating slow release to the measured [Ca2+]i values in the nerve terminal has shown low apparent Ca2+ cooperativities (Delaney and Tank, 1994; Xu-Friedman and Regehr, 2000; Angleson and Betz, 2001), but nonlinear dependencies have been found in other synapses (Ravin et al., 1997; Kirischuk and Grantyn, 2003). Experiments at the calyx synapse have shown that release ∼<2 μm is caused by a more linearly acting Ca2+ sensor (Lou et al., 2005) that remains in Syt2 KO mice (Sun et al., 2007; Kochubey and Schneggenburger, 2011).
A characteristic feature of fast and slow release is their differential modulation when Ca2+ is exchanged by Sr2+, which causes decreased fast release, but increased slow release (Miledi, 1966; Goda and Stevens, 1994; Rumpel and Behrends, 1999; Xu-Friedman and Regehr, 2000). One study has postulated that Sr2+ preferentially activates a slow release sensor (Goda and Stevens, 1994). Another study, using imaging of axonal Ca2+ and Sr2+ transients, showed that Sr2+ transients were higher than Ca2+ transients and that Sr2+ decayed more slowly (Xu-Friedman and Regehr, 1999). They concluded that the differences in release were caused by the slower removal of Sr2+ from the nerve terminal (Xu-Friedman and Regehr, 2000). Nevertheless, the absolute values of spatially averaged [Sr2+]i transients have remained unknown; similarly, quantitative values of the buffering capacity of cells for Sr2+ ions are missing. Because there is good agreement that late release is strongly influenced by Ca2+ buffering (see above for references), we argue that quantitative measurements of alien divalent ion buffering, and sensor activation with presynaptic Sr2+ and Ca2+ uncaging, should yield significant new insights into how Ca2+ buffering and Ca2+ sensor activation normally shape fast and slow release.
Materials and Methods
Slice preparation and electrophysiology.
Acute brainstem slices at the level of MNTB were prepared as described previously (von Gersdorff et al., 1997), using C57BL/6J mice at postnatal days (P) 10–15, or Syt2−/− mice (Pang et al., 2006) bred on a C57BL6 background, at P12-P15. Mice of either sex were used throughout. Procedures of mouse breeding, handling, and killing before slice preparation were approved by the Veterinary office of the Canton of Vaud, Switzerland. Single or paired whole-cell recordings were made from MNTB principal neurons and/or calyces of Held nerve terminals at room temperature (22°C–24°C), using an EPC-10/2 patch-clamp amplifier (HEKA Elektronik). Series resistances (5–30 mΩ and 3–8 mΩ) were compensated up to 50% and 83% in presynaptic and postsynaptic recordings, respectively. The extracellular solution contained the following (in mm): 125 NaCl, 25 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 25 glucose, 1 MgCl2, 0.4 ascorbic acid, 3 myo-inositol, and 2 Na-pyruvate (all from Sigma-Aldrich/Fluka), continuously bubbled with 95% O2/5% CO2 (pH 7.4; 310 mosm), to which either 2 mm CaCl2 or 2 mm SrCl2 was added. During the afferent fiber stimulation experiments (see Fig. 2), 10 μm bicuculline methochloride (Biotrend) and 2 μm strychnine (Sigma) were added to suppress inhibitory synaptic currents. Afferent fibers were stimulated using a custom-built platinum/iridium bipolar electrode placed at the midline of the brainstem slice, with 2–10 V pulses of 0.1 ms length from an isolated stimulator (A-M Systems, model 2100). During paired recordings, the following chemicals were added to the extracellular solution: 10 mm tetraethylammonium chloride (TEA, Sigma), 1 μm TTX, 50 μm d-AP5, 2 mm γ-d-glutamylglycine, and 100 μm cyclothiazide (all from Biotrend). The postsynaptic pipette solution contained the following (in mm): 130 Cs-gluconate, 20 TEA, 10 HEPES, 5 Na2-phosphocreatine, 4 MgATP, 0.3 Na2GTP, 5 EGTA (pH 7.2 adjusted with CsOH). The presynaptic pipette solution for double recordings without imaging (see Fig. 3) was identical, except for a reduced concentration of EGTA (100 μm). The presynaptic intracellular solution for divalent ion uncaging and imaging experiments, referred to as “imaging solution” below, contained the following (in mm): 130 Cs-gluconate, 20 TEA, 20 HEPES, 5 Na2ATP, 0.3 Na2GTP (pH 7.2 adjusted with CsOH). To this, 1.5 or 1 mm DM-Nitrophen (DMN, Merck Chemicals), CaCl2 or SrCl2 to achieve 90% loading, and/or 100 μm of a fura2-like indicator were added.
Quantitative presynaptic divalent imaging and uncaging.
We established presynaptic quantitative Sr2+ uncaging by using DMN (Ellis-Davies, 2003) and ratiometric fura-2 and fura-4F indicators (Invitrogen) for postflash [Sr2+]i imaging (see Figs. 4 and 5). Titration experiments with 100 μm fura-2 to measure free Sr2+ (data not shown) gave a Kd value for Sr2+ binding to DMN of 160 nm (Table 1; ∼40 times higher than for Ca2+). Fura-2 and fura-4F were calibrated for Sr2+ binding in the presence of DMN (1.5 mm) using a 5-point calibration procedure (Fig. 1). For this, suitable free Sr2+ concentrations were set by mixtures of SrCl2 with CDTA, EGTA, or HEDTA using published Sr2+ affinities for these buffers (Martell and Smith, 1974) (Table 1), in the presence of 100 μm fura-4F and 1.5 mm DMN. Fluorescence spectra were then measured in the experimental setup using the same optical path and excitation parameters as in Sr2+ uncaging experiments, using 50 μm path length borosilicate glass capillaries (VitroCom). Figure 1 shows an example for Sr2+ calibration of fura4F, revealing a Kd of 31.9 μm. In all calibrations, at least one Sr2+ concentration was determined by two independent buffers (in the example of Fig. 1, EGTA and HEDTA for a free [Sr2+] close to 20 μm). All calibrations for Ca2+ and Sr2+ were performed using the imaging solution supplemented with 100 μm of the fura2-like dye, 1.5 mm DMN, and CaCl2 or SrCl2 DMN as appropriate. The amount of CaCl2 or SrCl2 to be added was calculated based on our estimates of the DMN stock purity (usually ∼65%).
For presynaptic Ca2+ and Sr2+ uncaging experiments, a xenon arc flash lamp (SP-20, Rapp OptoElectronic) with 395 nm short-pass filter was used to generate short (0.4–0.5 ms half-width) UV light flashes to induce DMN photolysis. The flashlight was fiber-coupled into the microscope together with the light from a monochromator (Polychrome IV, TILL Photonics) using an 85%/15% beamsplitter (TILL Photonics), and the flash intensity was modulated by neutral density filters (Rapp OptoElectronic). For imaging postflash [Ca2+]i and [Sr2+]i following weak flashes, we used fura-2, whereas fura-2FF (TEFLabs) or fura-4F was used for stronger flashes for Ca2+ and Sr2+, respectively (see Fig. 4C; Table 1). Ratiometric measurements of preflash and postflash [Ca2+]i and [Sr2+]i were made using alternating 5 ms excitation light pulses from the monochromator at 380 and 350 nm. In Sr2+ uncaging experiments, the extracellular solution contained 2 mm SrCl2 (no added CaCl2), to avoid the possibility of Ca2+ leak into the presynaptic cell. When DMN was used without added Sr2+ or Ca2+ in the presence of 2 mm extracellular Sr2+, neither ratiometric changes nor release responses were observed upon full intensity flashes (n = 3 cells). This suggests that these responses were caused by Sr2+ release from DMN and not by a possible Ca2+ contamination.
Presynaptic buffered loading of divalent ions.
To measure the divalent ion sensitivity of release close to the resting concentrations of Ca2+ or Sr2+ (see Fig. 6), we loaded calyx nerve terminals with buffered Ca2+ or Sr2+ solutions in paired presynaptic and postsynaptic recordings (Lou et al., 2005). For Sr2+, we used 0.3, 1.6, and 5 mm SrCl2 mixed with 10 mm CDTA and 100 μm fura-2 in the imaging solution, giving nominal free [Sr2+] values of 0.14, 0.83, and 4.3 μm, respectively. For Ca2+, we used buffered solutions with 10 mm EGTA and nominal [Ca2+] of 0.12, 0.31, and 0.85 μm. The effective presynaptic [Ca2+]i and [Sr2+]i was imaged continuously during the recording using fura-2 ratio measurements every 5 s, and the mEPSC frequency was simultaneously sampled by the postsynaptic recording.
Measurements of presynaptic Ca2+ and Sr2+ buffering capacity.
For measuring the endogenous Sr2+ buffering capacity, κS(Sr) (see Fig. 7), we attempted to compete with endogenous buffers by adding buffering capacity of the indicator dye (κB) during the loading phase of whole-cell recordings (Neher and Augustine, 1992). To do so, we used 75 and 200 μm of the high-affinity indicator fura-2 for the determination of κS for Ca2+ and Sr2+, respectively. At these concentrations and considering the binding affinities of fura-2 for Ca2+ and Sr2+ (Table 1), fura-2 will attain final κB values of ∼350 for Ca2+ and ∼50 for Sr2+. Measurements of κS were then performed as described previously (Neher and Augustine, 1992; Helmchen et al., 1997), using repeated presynaptic depolarizations of 3 ms to 0 mV (see Fig. 7A,B). Fura-2 ratios in response to depolarizations were measured at 380 nm wavelength and at the isosbestic wavelengths, which were 357 and 362 nm for Ca2+ and Sr2+, respectively, using alternating 5 ms monochromator light pulses. For the determination of κS (Sr), 2 mm extracellular Sr2+ was present throughout the experiment. The offline analysis started by extracting the ratio F380/F357 (or F380/F362) following suitable background correction. Ratio traces were then converted to [Ca2+]i or [Sr2+]i according to the equation of Grynkiewicz et al. (1985), using experimentally determined calibration constants Rmin, Rmax, and Keff. Endogenous buffering capacity (κS) and extrusion rates (γ) for Ca2+ and Sr2+ were estimated similarly as described before for Ca2+ (Neher and Augustine, 1992; Helmchen et al., 1997).
Imaging of physiological Ca2+ and Sr2+ transients.
For the experiments in Figure 8, we used the lowest-affinity Ca2+ indicator practical for ratiometric measurements, fura-6F (Invitrogen). Given the Kd values of fura-6F for Ca2+ and Sr2+ (Table 1), 100 μm fura-6F corresponds to κB values of ∼12 for Ca2+ and < 1 for Sr2+. Because these values are severalfold smaller than the cellular buffering capacities κS for each ion (see Fig. 7), fura-6F does not significantly influence the kinetics of Sr2+ and Ca2+ transients. Ratiometric measurements were made before, during, and after trains of 50 AP-like presynaptic depolarizations (1 ms to 45 mV at 100 Hz), using alternating 10 ms pulses of monochromator light at 380 and 350 nm at repetition rates of initially 8 Hz and then 41 Hz to follow fast changes of [Ca2+]i or [Sr2+]i (see Fig. 8). Fluorescence ratio traces were converted into [Ca2+]i or [Sr2+]i time courses as explained above. The extracellular recording solution contained 1 μm TTX and 10 mm TEA.
Data analysis.
Data analysis was done using custom-written routines in IgorPro 6.2 (WaveMetrics). Spontaneous mEPSC events were detected using a semiautomatic routine with a template-matching algorithm (Clements and Bekkers, 1997). EPSC deconvolution followed the methods introduced by Neher and Sakaba (2001), as described previously (Wölfel et al., 2007). The quantal size for deconvolution was set by the average mEPSC amplitude determined in each cell. Release rates were corrected for the predicted “spillover” current estimated using “template” protocols, which consisted of a few presynaptic depolarizations of different length applied at the beginning of paired recordings (Neher and Sakaba, 2001). In case of Syt2−/− recordings, template protocols were not helpful because of the absence of synchronous release; thus, the strength of residual current β was set to values between 10 and 20, similar to those observed in control synapses from the same age group. The cumulative release rate traces after uncaging were fitted with single-exponential, exponential plus line, double-exponential, double-exponential plus line, and triple-exponential functions with a fit routine (Wölfel et al., 2007; Kochubey et al., 2009). In wild-type synapses with uncaging steps > 4 μm [Ca2+]i or ∼>15 μm [Sr2+]i, the best fits usually contained two exponential components (see Fig. 4A,B, bottom; and Fig. 4E, open and closed symbols). In Syt2−/−, the best fit was usually monoexponential and much slower (∼200 ms; see Fig. 5A, bottom).
Data are reported as mean ± SEM, unless indicated. Paired or unpaired Student's t tests, as appropriate, were used to assess statistical significance.
Model calculations.
We modeled the Ca2+ and Sr2+ sensitivity of transmitter release obtained from uncaging experiments with the “2-sensor model” (see Fig. 4F) (Sun et al., 2007), or with an allosteric model of Ca2+ binding and vesicle fusion (Lou et al., 2005). For fitting the Ca2+ and Sr2+ uncaging data in Syt2−/− synapses (see Fig. 5), we assumed that release can be described by a slow sensor with a single divalent ion binding site (see Fig. 5F). The set of differential equations corresponding to each model was solved numerically with the fourth- or fifth-order Runge-Kutta method in IgorPro, calculating the accumulation into the various fused states for a series of Ca2+ and Sr2+ steps. Ca2+ and Sr2+ steps decayed by 30% per 100 ms similar to observed steps (see Fig. 4A,B). We derived the release rate traces by differentiation of the simulated cumulative release traces and then analyzed the peak release rate, release delay (time to release of 5 vesicles), and release time constants. The Ca2+ and Sr2+ sensitivities of peak release rate, release delay, and fast component of the release time constants were then globally fitted by the 2-sensor model (see Figs. 4C–E; Fig. 6B). We lowered the Ca2+ and Sr2+ binding affinities of the slow release sensor relative to the values found in Syt2 KO measurements (see Fig. 5), to account for “clamping” of the slow sensor by Syt2 in wild-type synapses (Table 2). The back-calculation of local [Ca2+]i and [Sr2+]i signals evoked by a single AP (see Fig. 9) was done as described previously (Kochubey et al., 2009).
The stochastic model of divalent ion influx and buffering (see Fig. 9E) was performed by the Monte Carlo simulator M-Cell (Kerr et al., 2008). The total simulated volume was a cube measuring 0.6 × 0.6 × 1.2 μm. The Sr2+ current through single Ca2+ channels was scaled to a factor of 1.8 that of Ca2+ flux (see Fig. 3); the Ca2+ current was 0.12 pA at 0 mV. Ca2+ channels were modeled using a Hodgkin–Huxley model (Borst and Sakmann, 1998) driven by an action potential with a half-width of 0.49 ms. We assumed the presence of the following buffers, with on-rates (kon) and off-rates (koff) for Ca2+ and Sr2+, respectively: (1) an endogenous immobile (“fixed”) Ca2+ buffer; kon 1 × 108 and 4 × 107 m−1s−1 and koff 1000 and 2500 s−1. The Kd values of the endogenous immobile buffer for Ca2+ and Sr2+ were 10 and 62.5 μm, respectively, achieved by using a 0.4-fold lower kon and a 2.5-fold higher koff for Sr2+ compared with Ca2+. Given the concentration of the buffer (400 μm), this corresponds to cellular buffer capacities κS of 40 and 6.4 for Ca2+ and Sr2+, respectively, close to the experimentally observed values (see Fig. 7). (2) ATP was present as a mobile Ca2+ buffer (Naraghi and Neher, 1997) at a concentration of 2 μM; kon 5 × 108/5 × 108 m−1s−1 (for Ca2+/Sr2+, respectively), koff 45,000/152,242 s−1. The lower affinity for Sr2+ was as found in previous experiments (Wilson and Chin, 1991) (Table 1). ATP also bound Mg2+ with kon of 5 × 108 m−1s−1 and koff of 22,500 s−1; free [Mg2+] was 300 μm. The diffusion constant of free Sr2+ was 2.2 × 10−6 cm2/s, and the basal free [Sr2+] was 100 nm.
Results
Sr2+ decreases AP evoked release probability but increases late asynchronous release
Previous studies have shown that phasic release during the presynaptic AP is reduced in the presence of Sr2+, whereas a late phase of release is increased (Miledi, 1966; Goda and Stevens, 1994; Rumpel and Behrends, 1999; Xu-Friedman and Regehr, 1999). Here, we used the ideal presynaptic accessibility of the calyx of Held synapse to perform direct measurements of the Sr2+ sensitivity of release and of Sr2+ buffering, to gain new insights into how Ca2+ buffering and Ca2+ sensor activation normally shape fast and slow release. We started by investigating the effects of Ca2+ replacement by Sr2+ on fiber-stimulation-evoked EPSCs. We used brief 100 Hz trains of 50 stimuli to study both AP-evoked fast release during the train and late asynchronous release after the train (Fig. 2A1, A2). When the extracellular Ca2+ was switched to Sr2+ (2 mm each), the first EPSC during 100 Hz trains was strongly reduced, from 20.4 ± 0.4 nA to 5.2 ± 0.5 nA (Fig. 2A,B; n = 5 cells; p < 0.01). The relative speed of depression was reduced (Fig. 2B), which is likely a consequence of a lower release probability prel in the presence of Sr2+. To quantify prel, we estimated the size of the readily releasable pool using high-frequency stimulation trains and cumulative EPSC amplitude plots (Fig. 2B) (Schneggenburger et al., 1999). The pool size was slightly, but significantly, decreased in the presence of Sr2+ (∼20%; Fig. 2C; p < 0.05). Dividing the first EPSC amplitude by the readily releasable pool showed that prel was strongly reduced in the presence of Sr2+ (Fig. 2C; p < 0.001). The cumulative EPSC amplitude plots also showed a reduced late slope, which corresponds to a reduced EPSC amplitude between the 30th and 50th stimuli (Fig. 2B, C). This could indicate a reduced Ca2+-dependent readily releasable pool recovery in the presence of Sr2+ (Wang and Kaczmarek, 1998; Sakaba and Neher, 2001), which was not further analyzed here. Together, replacing Ca2+ with Sr2+ decreased the EPSC amplitude, an effect that was caused by a reduced prel.
We next analyzed the late asynchronous release following the 100 Hz trains (Fig. 2A2). Late release, measured as mEPSC frequency, was significantly enhanced during the first 1 s interval after the train (2 mm Ca2+, 20.5 ± 6.8 Hz; 2 mm Sr2+; 143.5 ± 11.8 Hz; p < 0.01). During the second and third 1 s time intervals, release was still enhanced, although the difference was smaller (Fig. 2A2 bottom; p < 0.05 for both comparisons). This reveals a differential effect of Ca2+ replacement by Sr2+, with less phasic but enhanced late asynchronous release, as shown previously at other synapses.
Sr2+ decreases fast release rates despite carrying larger currents through Ca2+ channels
We next wished to investigate the mechanism that underlies the lower efficiency of extracellular Sr2+ in activating fast transmitter release. Using paired presynaptic and postsynaptic whole-cell recordings, we found that Sr2+ currents through presynaptic voltage-gated Ca2+ channels were increased (Fig. 3A), suggesting that Sr2+ is a better charge carrier than Ca2+ (Hagiwara and Ohmori, 1982). Despite the higher Sr2+ currents, pool-depleting 50 ms depolarizations caused smaller EPSCs, and deconvolution of EPSCs showed that peak release rates were smaller in the presence of Sr2+ (Fig. 3A, bottom, B). Fitting the cumulative release rates with double-exponential functions, we also found that the number of vesicles released in the fast phase was reduced, and the fast release time constant was slowed from an average value of 1.3 ms to ∼2.3 ms (Fig. 3C; p < 0.01 for both comparisons). These data show that, despite an increased flux of Sr2+ through Ca2+ channels, the fast release component in response to presynaptic depolarizations was activated less efficiently by Sr2+ than by Ca2+.
Sr2+ activates the fast release sensor with sixfold lower efficiency than Ca2+
We next wished to investigate the mechanisms that underlie a lower efficiency of extracellular Sr2+ in activating fast release. Because Sr2+ permeates more readily through Ca2+ channels (factor of ∼1.8-fold; see Fig. 3B), the microdomain intracellular Sr2+ concentration attained close to open Ca2+ channels should be higher in the presence of Sr2+ than Ca2+, as we will also show below (see Fig. 9). Therefore, Sr2+ might activate the Ca2+ sensor for fast release significantly less efficiently than Ca2+, a likely possibility that, however, has never been tested directly.
To study the intracellular Sr2+ sensitivity of transmitter release, we next established Sr2+ uncaging experiments at the calyx of Held synapse. This required an accurate calibration of all involved fura2-like Ca2+ indicators and of DMN for Sr2+ (see Materials and Methods; Fig. 1). For all chelators and indicators calibrated here, we found lower affinities for Sr2+ than for Ca2+ (Table 1) (Xu-Friedman and Regehr, 1999). The low affinity of fura-4F for Sr2+ (∼31 μm) turned out to be very useful for imaging [Sr2+]i in the range of 20–100 μm, following Sr2+ uncaging pulses. Figure 4A shows an example of a paired recording with 1.5 mm DMN (90% loaded with Sr2+) and 0.1 mm fura-4F in the presynaptic patch pipette. A full intensity flash led to a [Sr2+]i elevation of ∼50 μm; under similar conditions with 1.5 mm Ca2+-loaded DMN, smaller rises in free [Ca2+]i are observed (∼10–20 μm [Ca2+]i; Fig. 4A,B). We attribute this difference to the significantly lower Sr2+ buffering capacity of the nerve terminal (see also below, Fig. 7). These experiments show the feasibility to control presynaptic [Sr2+]i in Sr2+ uncaging experiments at a CNS nerve terminal.
Presynaptic Sr2+ uncaging experiments showed that, despite higher presynaptic [Sr2+]i steps, EPSCs and peak release rates were smaller compared with Ca2+ uncaging experiments (Fig. 4A,B), suggesting a lower efficiency of intracellular Sr2+ at the Ca2+ sensor for vesicle fusion. Plotting the peak release rates as a function of postflash [Ca2+]i and [Sr2+]i indeed revealed a clear, ∼6-fold rightward shift in the efficiency of Sr2+ in inducing transmitter release (Fig. 4C). Interestingly, the slopes in the steep parts of the double-logarithmic dose–response curves, determined by line fitting of the data points in double-logarithmic space, were similar for Ca2+ and Sr2+ uncaging (3.42 ± 0.37 and 3.50 ± 0.24 over the ranges 1–5 μm and 5–25 μm, respectively; error estimates are 95% confidence interval of the fits). Fits of the data with a modified 2-sensor model of release (Sun et al., 2007) (Fig. 4F) or with an allosteric release model (Lou et al., 2005) were consistent with an ∼6-fold rightward shift of the affinity of Sr2+ binding to the fast sensor (Fig. 4C, solid and dashed lines, respectively). The Sr2+ data were described well by 5 divalent ion binding sites and an unchanged cooperativity factor b (Table 2), in agreement with the finding of unchanged slopes in double-logarithmic coordinates.
We next analyzed the kinetic components of release induced by Sr2+ and Ca2+ uncaging to determine the kinetics of Sr2+ binding to the fast release sensor. The release delays with Sr2+ uncaging steps showed a clear rightward shift (Fig. 4D). Cumulative release rates in response to both Ca2+ and Sr2+ uncaging followed double-exponential functions (Fig. 4A,B, bottom) (Wölfel et al., 2007). The fast and the slow release time constants were significantly rightward shifted with Sr2+ compared with Ca2+ (Fig. 4E). Global fits of the 2-sensor model to the peak release rates, the release delays, and the fast release time constants (Fig. 4C–E) showed a decreased on-binding rate (kon), as well as an increased dissociation rate koff for Sr2+ relative to Ca2+, whereas the vesicle fusion rate γF of the fast sensor was left unchanged (Table 2). The parameters of the slow sensor (S in Fig. 4F) were fixed according to the data obtained at low [Sr2+]i and [Ca2+]i, which will be presented below (see Fig. 6). Taken together, presynaptic divalent ion uncaging shows that Sr2+ has a sixfold rightward shifted affinity at the fast release sensor compared with Ca2+. This is explained by a lower kon and by a higher koff for Sr2+ (Table 2). However, the intrinsic cooperativity of Ca2+ and Sr2+ ions in evoking fast release is unchanged.
Sr2+ is less efficient at the slow release sensor remaining in Syt2 KO mice
We have shown above that the fast Ca2+ sensor at the calyx of Held has a significantly lower affinity for Sr2+ compared with Ca2+. Because replacement of Ca2+ by Sr2+ causes decreased phasic release but enhanced asynchronous release (Fig. 2), it has been suggested that Sr2+ preferentially activates the slow release sensor (Goda and Stevens, 1994). To investigate this possibility directly, we made Sr2+ uncaging experiments in Syt2 KO mice. The low-cooperativity release component that remains in Syt2 KO mice is driven by a secondary release sensor, which might represent the slow release sensor in wild-type synapses (Sun et al., 2007; Kochubey and Schneggenburger, 2011) (see Discussion).
In Syt2 KO mice at P12-P15, Ca2+ uncaging stimuli evoked slowly rising EPSCs with small peak release rates of ∼1–20 ves/ms depending on postflash [Ca2+]i (Fig. 5A). Cumulative release rates in Syt2 KO mice showed a sigmoidal rise, and their relaxation after ∼50 ms could be fitted with single exponential functions with time constants ∼200 ms (Fig. 5A), much slower than in wild-type synapses (see above; Fig. 4). With Sr2+ uncaging, we found even lower peak release rates and slower release (Fig. 5B). When we plotted peak release rates, release time constants, and release delays as a function of divalent ion concentrations, we found a clear right shift with Sr2+ as compared to with Ca2+ (Fig. 5C–E). These measurements directly show that Sr2+ activates the slow sensor studied in Syt2 KO mice with lower efficiency compared with Ca2+.
To describe the Ca2+ and Sr2+ uncaging data in Syt2 KO mice quantitatively, we developed a kinetic model for the slow release sensor. For this, we first fitted the Ca2+ and Sr2+ dependencies of peak release rates in double-logarithmic coordinates by line functions, which revealed slopes of 0.85 ± 0.11 and 0.90 ± 0.14 (95% confidence intervals; see Fig. 5C, dashed lines). Because the cooperativity was <1, we developed a kinetic model with a single divalent ion binding site (Fig. 5F). A global fit of this model to the Sr2+ and Ca2+ dependencies of peak release rates, release time constants, and release delays indicated that Sr2+ elicited slow release with a 5.5-fold rightward shifted affinity compared with Ca2+ (Fig. 5C). The model fits indicated that the rightward shift was caused by a lower binding rate constant kon (∼4.8-fold; for parameters, see legend of Fig. 5). These experiments demonstrate that, even at the slow sensor, Sr2+ is less efficient than Ca2+, and the factor is similar to the one we found for Syt2 in wild-type mice (Fig. 4).
At low concentrations, Sr2+ also triggers release less efficiently than Ca2+
We have shown that Sr2+ has a significantly lower efficiency at the slow release sensor which remains in Syt2 KO synapses. However, in the absence of Syt2, slow release is increased, indicating that Syt2 normally “clamps” slow release (Yoshihara and Littleton, 2002; Kochubey and Schneggenburger, 2011). It is possible that the unclamped slow sensor in the absence of Syt2 has altered Ca2+- or Sr2+-binding properties. Therefore, we next measured the intracellular Sr2+ sensitivity of release in the low concentration range in wild-type mice. Because train stimulation elevates residual divalent ion concentrations to the low micromolar range (see below; Fig. 8), understanding how Ca2+ and Sr2+ regulate vesicle fusion in this concentration range is immediately relevant to explain late asynchronous release following trains of APs.
We loaded presynaptic calyx of Held nerve terminals with Sr2+-buffered solutions using CDTA (10 mm) as a Sr2+ buffer and various concentrations of SrCl2, to achieve elevated levels of presynaptic [Sr2+]i (see Materials and Methods). We then imaged the spatially averaged [Sr2+]i attained in calyces using 100 μm Fura-2 while simultaneously measuring the rate of spontaneous release (Fig. 6A) (Lou et al., 2005). These measurements showed that below ∼5 μm [Sr2+]i, the slope in the double-logarithmic plot of release rate versus [Sr2+]i progressively decreased (Fig. 6B, pink square symbols), similarly as shown previously for Ca2+ (Lou et al., 2005; Sun et al., 2007). The Sr2+ data fell below, and on the right of corresponding measurements with Ca2+ (Fig. 6B, gray square symbols). At low concentrations of each divalent ion in the range of 30–100 nm, release rates converged to the resting spontaneous release frequency, which is ∼1 Hz at the calyx of Held. These data suggest that Sr2+ activates the slow release sensor in wild-type synapses with lower efficiency compared with Ca2+.
The data obtained in the range of low intracellular Ca2+ and Sr2+ concentrations allowed us to fit the 2-sensor model to the Ca2+ and Sr2+ datasets (Fig. 6B). We assumed that the slow sensor has a single divalent ion binding site, based on the slope value of ∼1 revealed in the Syt2 KO experiments (see above; Fig. 5). Furthermore, for the fit of the wild-type data in Figure 6, we had to introduce “clamping” of the slow sensor because Syt2 suppresses the activation of the slow sensor (Kochubey and Schneggenburger, 2011). This was done by introducing an additional factor to decrease the affinity of the slow sensor for both Ca2+ and Sr2+ in wild-type synapses (Fig. 6; for the model parameters, see Table 2). The data and the fits clearly show that, in the range of high divalent ion cooperativity, which is dominated by Syt2, Sr2+ is ∼6-fold less efficient in triggering vesicle fusion than Ca2+. At lower divalent ion concentrations, this difference becomes smaller; but at all concentrations down to ∼100 nm, Sr2+ is less efficient than Ca2+ in driving release.
Using the 2-sensor model with the fitted parameters, we could predict the contributions of release occurring without previous divalent ion binding (spontaneous release), and release occurring after occupancy of the slow and the fast release sensor (Fig. 6C). Surprisingly, this indicates that late release measured after 100 Hz trains in the presence of Sr2+ (Fig. 2; 150 Hz) might be carried by a larger relative activation of the fast sensor. This is because release in the presence of extracellular Sr2+ extends more strongly into the steep part of the dose–response curve compared with Ca2+ (Fig. 6B, broken lines) (see Discussion).
The intracellular buffering capacity for Sr2+ is severalfold lower than for Ca2+
Because Sr2+ is less efficient at both the fast and the slow release sensors, the explanation for the significantly larger late release in the presence of Sr2+ must lie in a higher rise of spatially averaged Sr2+ compared with Ca2+. This might be caused by inefficient intracellular buffering of Sr2+ ions and/or by a slower decay of spatially averaged Sr2+, as concluded previously (Xu-Friedman and Regehr, 1999). However, quantitative information on how high spatially averaged Sr2+ rises in the nerve terminal, and on the Sr2+ buffering capacity of cellular cytosol has not been obtained. For this reason, we next measured the Sr2+ buffering capacity of the calyx nerve terminal, using the indicator overload method (Neher and Augustine, 1992; Helmchen et al., 1997). This again required careful calibration of fura-2 for Sr2+ (see Materials and Methods).
We loaded calyces with 200 μm fura-2 in the presence of 2 mm extracellular Sr2+ and stimulated the nerve terminals repeatedly with brief depolarizations (0 mV for 3 ms; Fig. 7A, inset). The first stimulus during whole-cell recordings, when only little fura-2 had entered the nerve terminal, induced large [Sr2+]i transients (Fig. 7A, arrow). During the loading phase of calyces with fura-2, the [Sr2+]i transients progressively decreased in amplitude, indicating increasing competition of fura-2 with the cellular buffering mechanism (Fig. 7A), similarly as previously demonstrated for Ca2+ (Neher and Augustine, 1992). Using the Kd value of Sr2+ binding to fura-2 as determined in calibration measurements (Kd ∼ 4.2 μm; Table 1), and the Sr2+-independent fura-2 fluorescence as a proxy of the intracellular fura-2 concentration, we calculated the exogenously added Sr2+ buffering capacity of fura-2, κB (Sr), for each stimulus. Plots of the inverse of the [Sr2+]i amplitude against κB (Sr) indicated an endogenous buffering capacity for Sr2+, κS (Sr), of 6 in this recording. The extrapolated [Sr2+]i transient at zero added exogenous buffer was ∼8 μm (Fig. 7C, arrows).
In parallel experiments, we measured the buffer capacity for Ca2+, κS (Ca), using 75 μm fura-2 in the patch pipette and 2 mm extracellular [Ca2+]. Despite similar divalent ion charge (Fig. 7A,B, insets), the free [Ca2+]i transients were significantly smaller, ∼≤0.5 μm. As a consequence, the inverse amplitudes of the free divalent concentration transients were significantly larger for Ca2+ than for Sr2+ (Fig. 7B,D). This indicates a higher buffering capacity for the nerve terminal for Ca2+ compared with Sr2+. On average, κS (Ca) was 46 ± 2.8 (n = 5 cells), whereas κS (Sr) was only 8.5 ± 0.6 (n = 11 cells; p < 0.001; Fig. 7F). This massive difference is most likely caused by a lower binding affinity of endogenous fixed buffers for Sr2+ compared with Ca2+ (see Discussion). The back-extrapolated amplitude of the [Sr2+]i transient was 12 ± 0.84 μm (n = 10), >10-fold higher than the back-extrapolated [Ca2+]i amplitude, which was 0.89 ± 0.05 μm (Fig. 7F, middle; n = 5, p < 0.001). This difference was not caused by larger currents carried by Sr2+ through Ca2+ channels because, in these particular experiments, the divalent ion charge was similar (Fig. 7F, right). These experiments are, to our knowledge, the first measurement of intracellular Sr2+ buffering capacity in any cell type. They show that the Ca2+ buffering system of the nerve terminal has an ∼5- to 6-fold lower capacity for Sr2+ compared with Ca2+. This admits a massively higher spatially averaged [Sr2+]i (Figs. 7A, arrow, and see Fig. 8).
We next analyzed the Sr2+ extrusion rate constant compared with Ca2+ using the fura-2 overload data in Figure 7. For this purpose, we plotted the decay time constants of the [Sr2+]i and [Ca2+]i transients as a function of the corresponding κB values for Ca2+ and Sr2+ (Fig. 7E). Interestingly, the extrapolated values of the [Ca2+]i and [Sr2+]i decay time constants for zero exogenous buffer were quite similar (∼90–100 ms; Fig. 7E, arrow; y-axis intercept). However, the slope of the plot of τ versus κB was much higher for Sr2+ than for Ca2+, which indicates a significantly lower extrusion rate γ for Sr2+ (Fig. 7E,G; ∼4.2-fold; p < 0.001). Thus, the similar extrapolated value of τdecay for [Sr2+]i and [Ca2+]i for no added exogenous buffers is caused by a compensation of the smaller extrusion rate constant γ by the significantly smaller endogenous buffering capacity for Sr2+, κS (Sr), resulting in similar time constants of decay. Taken together, fura-2 overload experiments establish that the endogenous divalent ion buffering capacity of the nerve terminals is drastically lower for Sr2+ than for Ca2+.
Spatially averaged [Sr2+]i elevations are much higher than [Ca2+]i
The significantly lower buffering capacity of the presynaptic cytosol for Sr2+ is expected to cause a drastically larger buildup of free Sr2+ compared with Ca2+. To investigate the Ca2+ and Sr2+ signals relevant for the late phase of release observed after high-frequency trains (Fig. 2), we performed quantitative Ca2+ and Sr2+ imaging experiments following 100 Hz trains of AP-like stimulations. We used a low-affinity indicator (fura-6F; 100 μm) to image physiologically relevant divalent ion transients while only minimally perturbing their amplitude or kinetics (see Materials and Methods).
In presynaptic voltage-clamp recordings, we first imaged [Ca2+]i in response to 100 Hz trains of AP-like voltage-clamp depolarizations (Fig. 8A) and then switched the extracellular solution to Sr2+ (2 mm in both cases; Fig. 8A, B). The peak [Sr2+]i transient reached at the end of the 100 Hz trains was 33.0 ± 3.5 μm, drastically higher than [Ca2+]i, which was 3.6 ± 0.4 μm (Fig. 8C, left; p < 0.001). Part of this difference was caused by the larger Sr2+ currents through Ca2+ channels (Fig. 8A,B, inset). The larger part of the ∼10-fold higher [Sr2+]i transients compared with [Ca2+]i must be caused by the significantly lower buffering capacity of the nerve terminal for Sr2+ compared with Ca2+ (∼5.5-fold; Fig. 7). We fitted the decay of the [Sr2+]i and [Ca2+]i transients with exponential plus line functions (Fig. 8A,B, dashed lines). This showed a significant difference of the decay time τ (Fig. 8C, middle; p < 0.01), but the values were close, consistent with the results of Figure 7E. The late residual [Sr2+]i signal, measured at a time of 3τ, was, however, >10-fold higher than the corresponding [Ca2+]i value (Fig. 8C, right; 4.6 ± 0.5 and 0.28 ± 0.03 μm, respectively; n = 5; p > 0.001). The large difference between the absolute values of [Sr2+]i and [Ca2+]i becomes clearer by direct overlay of two representative traces recorded in the same cell (Fig. 8B). Thus, the spatially averaged [Sr2+]i rises to ∼10-fold higher values than [Ca2+]i both during and after the train. This difference is, to a large part, caused by a significantly smaller buffering capacity κS of the nerve terminal for Sr2+.
The microdomain Sr2+ signal is not strongly affected by the weaker Sr2+ buffering
We finally wished to investigate whether the lower buffering capacity of the nerve terminal for Sr2+ would also affect the “local” Sr2+ signal relevant for phasic release. For this purpose, we first back-calculated the local Ca2+ and Sr2+ concentrations relevant for AP-driven fast release, based on the measured intracellular Ca2+ and Sr2+ sensitivities of release (Fig. 4) (for the back-calculation approach, see Schneggenburger and Neher, 2000). We recorded single EPSCs in response to afferent fiber stimulation in the presence of Ca2+ and then exchanged the bath solution to Sr2+. As expected, the EPSC amplitudes and peak release rates were smaller in the presence of Sr2+ (Fig. 9A,B). Nevertheless, the back-calculation approach returned larger local [Sr2+]i signals compared with Ca2+ (Fig. 9C), which reflects the lower efficiency of intracellular Sr2+ at the fast sensor (Fig. 4). On average, the amplitudes of the local Ca2+ and Sr2+ transients were 18.4 ± 1.98 μm and 38.9 ± 4.2 μm, respectively (n = 5 cells; p < 0.001; Fig. 9D), which corresponds to a relative increase of 2.12 ± 0.11-fold. This factor approximately reflects the increased Sr2+ flux through open Ca2+ channels (∼1.8-fold; Fig. 3B), suggesting that the lower buffering capacity measured for spatially averaged Sr2+ transients (Fig. 7) has only little influence on the brief “local” Sr2+ signal relevant for phasic release. This might be caused by the fact that endogenous fixed buffers will be rapidly saturated locally by incoming divalent ions (Roberts, 1994; Naraghi and Neher, 1997).
To validate the role of the divalent ion buffer in controlling the local Ca2+ and Sr2+ signals, we performed random-walk simulations of Ca2+ and Sr2+ influx and buffering at a release site (Fig. 9E), assuming that the κS-like buffer is represented by an endogenous fixed buffer with relatively low affinity (see Discussion). We used an array of several Ca2+ channels because release at the calyx of Held synapse is controlled by multiple Ca2+ channels (Meinrenken et al., 2002; Wang et al., 2009). With the chosen distances, a brief local Ca2+ transient with amplitude of ∼25 μm was found, in good agreement with the experimental observations (Fig. 9D) (Schneggenburger and Neher, 2000). To model the local Sr2+ signal, three modifications were made in the simulation, based on our experimental observations. First, the influx through Ca2+ channels was increased 1.8-fold. Second, the affinity of the endogenous immobile buffer was reduced 6.25-fold, as derived from our measurements of the reduced buffer capacity. Third, the divalent ion binding parameters for the mobile buffer ATP, which was also present in these simulations, were adapted to previously published Sr2+ values (see Materials and Methods). This resulted in a peak local Sr2+ signal of ∼60 μm; and in repeated simulations, the local Sr2+ signal was 2.38 ± 0.04-fold higher than Ca2+ (n = 95 repetitions), in good agreement with the back-calculation approach. We conclude that the lower affinity of the endogenous fixed buffer for Sr2+ does not have a large effect on the microdomain divalent ion concentration in the presence of Sr2+.
Discussion
We have used quantitative presynaptic Ca2+ and Sr2+ uncaging and imaging at the calyx of Held of wild-type and Syt2-deficient mice, to probe the divalent ion regulation of fast and slow transmitter release. Sr2+ uncaging showed that the highly cooperative Ca2+ sensor in wild-type synapses has a sixfold lower affinity for Sr2+ compared with Ca2+ but an unchanged steep slope value (Fig. 4). We show that Sr2+ also activates the slow sensor that remains in Syt2 KO synapses less efficiently than Ca2+ (Fig. 5) and that Sr2+ triggers release less efficiently than Ca2+ in the low concentration range (Fig. 6). Thus, the enhanced slow release in the presence of Sr2+ must be caused by a significantly higher spatially averaged [Sr2+]i in the nerve terminal, to overcome the lower efficiency of Sr2+ at both release sensors. Indeed, we found that spatially averaged [Sr2+]i rises to ∼10-fold higher values than [Ca2+]i (Fig. 8), in large part caused by a significantly lower buffering capacity of the nerve terminal for Sr2+ compared with Ca2+ (Fig. 7). Thus, replacing Ca2+ with Sr2+ illuminates the roles of Ca2+ sensing and Ca2+ buffering in controlling fast and slow release, and allows us to conclude that endogenous fixed buffers normally suppress asynchronous release in the nerve terminal.
Using presynaptic Sr2+ uncaging experiments, we showed that Sr2+ activates fast release with a drastically rightward shifted affinity, but unchanged cooperativity compared with Ca2+ (Fig. 4). Although it was somewhat expected that Sr2+ has a lower affinity at the fast sensor given the reduced amount of fast release in the presence of Sr2+, this premise has not been shown directly in intracellular ion uncaging experiments at synapses (but see Kishimoto et al., 2001). A previous study used measurements of EPSCs in hippocampal autaptic neurons cultured from Syt1 mutant mice and wild-type mice in the presence of Ca2+ and Sr2+, and additional biochemical experiments, and concluded that the Sr2+ action in fast release is caused by Sr2+ binding to the C2B domain (Shin et al., 2003). This view agrees with mutational analysis at various preparations, including the calyx of Held, which showed that neutralization of aspartate residues in the C2B domain abolished the steeply Ca2+-dependent release component (Mackler et al., 2002; Nishiki and Augustine, 2004; Kochubey and Schneggenburger, 2011). Interestingly, the unchanged cooperativity that we demonstrated here for the action of Sr2+ suggests that Sr2+ binding could be used to study mechanisms by which Syt1 and Syt2 mediate the steeply Ca2+-dependent component of release, which is a hallmark of fast transmitter release at synapses.
Using Sr2+ uncaging in Syt2 KO mice, we showed that the remaining slow release sensor is also activated less efficiently by Sr2+ than Ca2+ (∼5- to 6-fold). This shows that the slow sensor does not preferentially bind Sr2+, opposite to a previous postulate (Goda and Stevens, 1994). Rather, enhanced late release in the presence of extracellular Sr2+ must be caused by a significantly reduced Sr2+ buffering compared with Ca2+. This conclusion significantly extends beyond the previous axonal imaging studies with extracellular Sr2+, which concluded that free Sr2+ rises to ∼1.7-fold higher values than Ca2+ and decayed more slowly (Xu-Friedman and Regehr, 1999). However, an ∼1.7- to 2-fold increase in free Sr2+ compared with Ca2+ is expected by only considering the increased Sr2+ ion flux through open channels, which we found to be ∼1.8-fold for presynaptic Ca2+ channels, in good agreement with previous work in cell cultures (Hagiwara and Ohmori, 1982). Because, in addition, the cellular buffering capacity is fivefold lower for Sr2+ than for Ca2+ (Fig. 7), we expect an overall 10-fold higher spatially averaged Sr2+ signal compared with Ca2+. This large factor was borne out in independent imaging experiments with low-affinity indicators (Fig. 8). Finally, we also show that the decay of Sr2+ is not strongly prolonged compared with Ca2+, at least in conditions of low exogenously added buffers (Figs. 7 and 8). The latter observation was explained by a near-perfect match of the lower Sr2+ extrusion rate γ and by the lower Sr2+ buffering capacity κS (Fig. 7F,G).
Endogenous Ca2+ buffer(s) limit the rise of spatially averaged [Ca2+]i following Ca2+influx and the dissipation of Ca2+ gradients; the buffer capacity κS is defined by the ratio of bound over free Ca2+ ions (Neher and Augustine, 1992; Neher, 1998). We found a κS value for Ca2+ of ∼45, in good agreement with a previous study (Helmchen et al., 1997). This value implies that only ∼2.5% of all Ca2+ ions remain free after equilibration with the buffer. This Ca2+ buffering system is largely represented by immobile buffers because whole-cell recording with patch pipettes does not lead to a major loss of buffer capacity (Zhou and Neher, 1993; Müller et al., 2007). The lower buffering capacity for Sr2+ (κS ∼ 8), corresponds to ∼12% of all Sr2+ ions remaining free. The ∼5-fold lower buffer capacity for Sr2+ compared with Ca2+ suggests that the Sr2+ binding affinity at the endogenous fixed buffer is lower by a similar factor since κS can be approximated as κS = [S]/Kd, with [S], concentration of buffer, and Kd, its dissociation constant for the divalent ion (Neher, 1998).
Although the lower affinity of Sr2+ at the endogenous fixed buffer caused a drastically larger spatially averaged [Sr2+]i signal, this mechanism was less relevant for the local Ca2+ and Sr2+ concentrations that drive phasic release (Fig. 9). Back-calculating the local Ca2+ and Sr2+ signals for phasic release as well as random walk simulations predicted an only 2.1- to 2.4-fold increase in microdomain Sr2+ compared with Ca2+ (Fig. 9). This factor can be largely accounted for by the increased flux of Sr2+ through Ca2+ channels (Fig. 3). This suggests that the endogenous fixed buffer does not strongly contribute to buffering the microdomain divalent ion signal, probably because immobile buffers are rapidly saturated (Roberts, 1994; Naraghi and Neher, 1997). Thus, our study also suggests a divergent action of the endogenous fixed buffer in the control of microdomain versus spatially averaged Ca2+ concentration.
It has long been postulated that two separate release sensors mediate fast and asynchronous release, based on the phenotypes of Syt1 KO mice (Geppert et al., 1994), and based on observations with Ca2+ and Sr2+ (Goda and Stevens, 1994). Similarly, work at the calyx of Held synapse has shown that, in Syt2 KO mice, a release component with low cooperativity remains (Fig. 5) (Sun et al., 2007; Kochubey and Schneggenburger, 2011). We have therefore used the 2-sensor model (Sun et al., 2007) to fit the Ca2+ and Sr2+ data, with the modifications that only a single binding site was assumed for the slow sensor (Fig. 5) and that “clamping” of the slow sensor was provided for in the presence of Syt2 (Kochubey and Schneggenburger, 2011) (for details, see Methods and Materials).
Using the refined 2-sensor model fitted to both the Ca2+ and the Sr2+ uncaging data, we can predict the contribution of the fast and the slow sensors over a wide range of Ca2+ and Sr2+ concentrations (Fig. 6C). Below 0.6 μm [Ca2+]i and below 3 μm [Sr2+]i, the model predicts that >50% of release, is triggered by the slow sensor; above these concentrations, the fast sensor takes over, as is also apparent by the steep slope values above these concentrations (Fig. 6B). Given the measured values of late release rates of ∼15 and 150 Hz in the presence of Ca2+ and Sr2+, respectively (Fig. 2), which correspond to free [Ca2+]i and [Sr2+]i values of ∼0.5 and 5 μm, we can see that the late release in the presence of Sr2+ falls into a steeper part of the dose–response curve than late release with Ca2+ (Fig. 6B, red and black dashed lines, respectively). This indicates that reduced Sr2+ buffering results in a larger activation of the fast sensor despite the lower sensitivity of the latter for Sr2+ as compared to Ca2+. Thus, the activation of fast and slow sensors is more complex than initially thought (Goda and Stevens, 1994), and slow release in the presence of Sr2+ can be carried following the activation of the fast, Syt2-like sensor.
These conclusions from detailed fitting of the Ca2+ and Sr2+ sensitivities of release at the calyx synapse are relevant to other CNS synapses. A steep phase of release has also been observed in Ca2+ uncaging experiments at cerebellar inhibitory synapses (Sakaba, 2008) and at hippocampal autaptic synapses (Burgalossi et al., 2010). In addition, the latter study has shown that Syt1 KO leads to a more shallow Ca2+-dependency of release, again demonstrating the persistence of a more linear release sensor in the absence of the main Ca2+ sensor, Syt1/2. Some brain synapses, such as GABAergic synapses made by specific interneuron types, are distinguished by slow release kinetics and large asynchronous release (Hefft and Jonas, 2005; Best and Regehr, 2009; Daw et al., 2009). These synapses might both have active zone architectures with longer coupling distances between Ca2+ channels and vesicles implying a larger relative importance of global Ca2+ signaling; they might also have different mechanisms of intrinsic Ca2+ sensing and less clamping of a secondary Ca2+ sensor. Future work could investigate the molecular mechanisms that lead to the specification of fast and slow release kinetics at defined types of synapses.
Footnotes
This work was supported by Swiss National Science Foundation 31003A_138320/1 to R.S. We thank Heather Murray for expert technical assistance, and Erwin Neher for discussing a previous version of the manuscript.
The authors declare no competing financial interests.
- Correspondence should be addressed to Dr. Ralf Schneggenburger, Laboratory of Synaptic Mechanisms, Brain Mind Institute, School of Life Science, École Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland. ralf.schneggenburger{at}epfl.ch