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Department of Neuroscience, Erasmus University Medical Center Rotterdam, Rotterdam, The Netherlands
Submitted 22 April 2006; accepted in final form 4 August 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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) or decreased (short-term depression [STD]; Borst et al. 1995). In addition, after a high-frequency train of stimuli, both a several-minute–long decrease (posttetanic depression [PTD]; Forsythe et al. 1998) or increase (posttetanic potentiation [PTP]; Habets and Borst 2005; Korogod et al. 2005) in the synaptic responses may occur. The accessibility of this synapse to patch-clamp recordings has allowed great progress in identifying different underlying mechanisms that contribute to these forms of short-term plasticity (reviewed in von Gersdorff and Borst 2002). The four different forms of short-term plasticity that occur at the calyx have in common that they are largely presynaptic phenomena. A change in the output of a synapse can be the result of an increase in the size of the readily releasable pool (RRP), which is typically defined as the pool of vesicles that can be released by a very large stimulus, or of a change in the release probability of vesicles in the readily releasable pool. PTP is accompanied by a large increase in the release probability (Habets and Borst 2005; Korogod et al. 2005). In addition, we also observed a small increase in the RRP (Habets and Borst 2005). The PTP decayed with a similar time course as the presynaptic calcium increase. However, based on the measured affinity of the phasic calcium sensor (Bollmann et al. 2000; Lou et al. 2005; Schneggenburger and Neher 2000), by itself this increase in presynaptic calcium would be insufficient to cause a substantial increase in the activation of the phasic calcium sensor.
In this paper we further explore the mechanisms that underlie the increase in release probability during PTP at the calyx of Held synapse. We focus on possible changes in the calcium influx for several reasons. First, small changes in calcium influx lead to large changes in release. Release typically is proportional to the third or fourth power of the calcium influx (reviewed in Schneggenburger and Neher 2005). Therefore even relatively small changes in calcium influx would make a substantial contribution to PTP. Second, the calcium currents at the calyx of Held facilitate calcium dependently and this increase may contribute to PPF (Borst and Sakmann 1998b; Cuttle et al. 1998; Inchauspe et al. 2004; Ishikawa et al. 2005). Because PTP cannot be observed while the terminals are in whole cell patch-clamp configuration (Habets and Borst 2005; Korogod et al. 2005), we used a fluorometric method to investigate a possible contribution of changes in calcium influx to PTP.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Preparation of slices and electrophysiological measurements were done as described previously (Habets and Borst 2005). Animal procedures were in accordance with guidelines provided by the animal committee of the Erasmus MC. In brief, 7- to 10-day-old Wistar rats were decapitated without prior anesthesia. The brain stem was dissected and immersed in ice-cold saline containing (in mM): 125 NaCl, 2.5 KCl, 3 MgSO4, 0.1 CaCl2, 1.25 NaH2PO4, 0.4 ascorbic acid, 3 myo-inositol, 2 pyruvic acid, 25 D-glucose, and 25 NaHCO3 (Merck); pH 7.4. Transverse slices of 200 µm thickness were cut with a vibratome (Vibratome, St. Louis, MO).
Electrophysiological recordings
Normal Ringer solution had the same composition as the solution that was used for slicing, except that the concentration of CaCl2 and MgSO4 were 2 and 1 mM, respectively. Neurons were visualized with an upright microscope (BX-50; Olympus, Tokyo, Japan), equipped with infrared differential interference contrast optics. Axons originating from the cochlear nucleus were stimulated (0.5 ms, 0.03–0.5 mA) in the midline by a bipolar electrode (FHC, Bowdoinham, ME). Test frequency was increased compared with earlier experiments (0.5 vs. 0.1 Hz; Habets and Borst 2005) to allow collection of more fluorescence data in the same time period. At this frequency there was already some synaptic depression (von Gersdorff et al. 1997). PTP was elicited by a 5-min, 20-Hz tetanus. Cells were selected when extracellular recordings indicated postsynaptic action potential firing (Borst et al. 1995). Electrophysiological recordings were made at room temperature with an Axopatch 200B amplifier (Axon Instruments, Union City, CA). Pipette solutions contained (in mM): 125 K-gluconate, 20 KCl, 10 Na2-phosphocreatine, 4 MgATP, 0.3 Na2GTP, 10 HEPES (Sigma), and 0.05–0.2 calcium-sensitive dye (Molecular Probes, Eugene, OR) or 0.5 EGTA for pre- or postsynaptic recordings, respectively. Calcium currents were pharmacologically isolated by substituting 20 mM TEA-Cl (Fluka, Buchs, Switzerland) for 20 mM NaCl and adding 1 µM tetrodotoxin (TTX; Alomone Labs, Jerusalem, Israel) and 0.1 mM 3,4-diaminopyridine to the Ringer solution. In these experiments, the internal solution contained (in mM) 125 Cs-CH3SO3, 20 CsCl, 10 Na2-phosphocreatine, 4 MgATP, 0.3 Na2GTP, and 10 HEPES (Sigma). In some experiments Cs-CH3SO3 was replaced with Cs-Gluconate. Series resistance was 8–30 M
(compensated 80–98%, prediction was set to 80%). Leak subtraction was done with the P/-8 method. Holding potential in voltage-clamp experiments was –80 mV. Potentials were corrected for a –11-mV junction potential. Postsynaptic series resistance (<15 M) was electronically compensated by 80–98% with a lag of 5 µs. Signals were low-pass (10 kHz) filtered with a four-pole Bessel filter. Only cells with a membrane resistance >100 M were accepted for analysis. Signals were sampled at 20–50 kHz with a Digidata 1320A (Axon Instruments). Data acquisition and analysis were done with pClamp 8 (Axon Instruments) or Igor 5 (Wavemetrics, Lake Oswego, OR).
Imaging
Terminals were prefilled with fura-2, Fluo-4, rhod-dextran, or Oregon Green BAPTA-5N (OGB-5N) for 10 min via the patch pipette. Only cells in which a G outside-out patch formed after retraction were selected for analysis. The tissue was illuminated through a x40 objective (NA 0.8; Olympus, Tokyo, Japan) by a monochromator (Polychrome IV; 8-nm bandwidth, TILL Photonics, Martinsried, Germany). Excitation intensity was about 0.1 mW, when measured under the objective. Emission light was filtered through an appropriate band-pass filter and detected with a cooled photomultiplier tube (PMT, H7422-40; Hamamatsu, Hamamatsu City, Japan). Excess background fluorescence was removed by a 1-mm pinhole at the image plane of the microscope. PMT signals were amplified and low-pass filtered (2 kHz) with an eight-pole Bessel filter (Model 3382; Krohn-Hite, Brockton, MA) before digitization (Digidata 1320A; Axon Instruments). For fura-2, calcium concentrations were calculated as described in Habets and Borst (2005). For nonratiometric dyes, responses evoked by an action potential (
FAP) are given as a percentage of the basal fluorescence of the terminal, which was calculated as the difference between the fluorescence in the absence of stimuli and the fluorescence from a nearby region. In the case of PTP experiments, responses are expressed relative to the basal fluorescence before the tetanus (FAP/F0).
Data analysis
PTP. The amount of PTP was calculated as the percentage increase of the average amplitude of the first 10 excitatory postsynaptic currents (EPSCs) after tetanic stimulation relative to the average amplitude of the last 10 EPSCs before the tetanus.
APW TRAINS.
To test the relation between calcium influx and fluorescence changes, terminals were voltage clamped using a train of 10 action potential waveforms (APWs) at 100 Hz, as described earlier (Borst and Helmchen 1998). Because clearance will start as soon as the influx starts, we corrected the amplitude of fluorescence changes for clearance during the rising phase assuming a similar time course for clearance as that after the rising phase. In experiments in which terminals were loaded with a single dye, decay of the fluorescence between stimuli was fitted with a single exponential function. Its time constant was set to the time constant of a fit of the decay at the end of the train and its offset (value at t = 
) was set to the baseline level before the first stimulus. To calculate the amplitude of the fluorescence change triggered by an APW, the fit that described the decay of the calcium transient elicited by the preceding APW was extrapolated to the time point where the increase of the fluorescence arising from the actual APW was half-maximal. The fit of the decay after the actual APW was back-extrapolated, again to the time point where the increase of the fluorescence arising from the actual APW was half-maximal. The amplitude was then taken as the difference between both fits at that point. In experiments in which two dyes were loaded, decays were fit with a double-exponential function and fits were extrapolated to the peak value rather than the midpoint of the rising phase. Slow calcium-activated currents were subtracted before integration of the calcium currents.
Simulations
To assess the influence of the kinetics of the dye and the endogenous calcium buffer on the measured fluorescence transients, these transients were simulated using a single-compartment model (Helmchen et al. 1997). This model assumes spatial equilibrium at all time points. Even though diffusion of Ca2+ is disregarded, in the absence of significant buffer depletion, a single-compartment model may accurately describe the effect of the kinetics of calcium dyes on volume-averaged calcium transients at the calyx of Held (Helmchen et al. 1997; Meinrenken et al. 2003). We refer to Meinrenken et al. (2003) for a discussion of its limitations. Standard equations for buffering were solved numerically, by forward Euler finite difference using an adaptive step size. Calcium influx during an action potential was either provided by the response of a two-state Hodgkin–Huxley model of calyceal calcium currents to a previously recorded action potential (Borst and Sakmann 1998a) or the simultaneously measured calcium influx in presynaptic voltage-clamp recordings was used, after filtering, truncation of outward currents, and subtraction of slow calcium-activated currents. In the case of the modeled calcium influx, total Ca2+ influx during an action potential was 0.91 pC, leading to an increase of the total calcium concentration to 12 µM in the calyx volume of 0.4 pl (Helmchen et al. 1997). The standard model solution contained: free Ca2+ at a starting concentration of 50 nM; endogenous buffer concentration 1.3 mM, forward Ca2+ binding rate 5 x 108 per Ms (Klingauf and Neher 1997), off-rate 16,000 s–1 [calcium-binding ratio 40, calculated as described in Helmchen et al. (1997)]; no ATP and 50–200 µM of fura-2, on-rate 4 x 108 per Ms, off-rate 103 s–1; Fluo-4, on-rate 7.1 x 108 per Ms, off-rate 369 s–1 (Naraghi 1997); or Oregon Green BAPTA-5N (OGB-5N), on-rate 2.5 x 108 per Ms, off-rate 8,000 s–1 (Faas et al. 2005). Removal of Ca2+ from the cytoplasm was modeled as a linear, nonsaturable clearance mechanism. To match the experimentally observed decays, its rate constant was set to 800 (Fig. 1) or 400 s–1 (Fig. 4). In the simulations shown in Fig. 1, a concentration of 50 µM was assumed, to account for loss of dye into the axon during the PTP experiments.
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Data are given as means ± SE. Statistical comparisons were done using Student's t-test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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PTP at the calyx of Held synapse rapidly washes out during presynaptic whole cell recordings (Habets and Borst 2005; Korogod et al. 2005). To prevent washout of PTP we therefore switched to fluorometric methods and preloaded dyes into the terminal during whole cell recordings. Dyes were preloaded for 10 min to a final concentration of 200 µM (Fig. 1A), after which the presynaptic pipette was withdrawn. Release was estimated from the size of the glutamatergic EPSCs in postsynaptic whole cell recordings. We optimized fluorometric measurements by using dyes with a relatively low basal fluorescence and by using a cooled, high-quantum-efficiency PMT for detection. As a result, not only could the amplitude of the fluorescence transient that was triggered by an action potential (FAP) be accurately measured, but its rise time could also be resolved. The time course of the transients differed between dyes. Rise times of transients in the presence of OGB-5N, a low-affinity dye (Kd 32 µM), were 0.32 ± 0.03 ms. Their decay was well approximated by a single-exponential function with a time constant of 57 ± 8 ms (n = 5; Fig. 1B). In the presence of Fluo-4, a high-affinity calcium dye (Kd 350 nM), 20–80% rise times were 0.68 ± 0.06 ms and decay time constants were 295 ± 42 ms (n = 7; Fig. 1C). Even in the absence of a presynaptic whole cell recording, it was still possible to gather information about the timing of the presynaptic action potential using the prespike in the postsynaptic recordings. The prespike is the capacitatively coupled presynaptic action potential (Forsythe 1994) whose positive peak corresponds to the time when the speed of repolarization is maximal. For both dyes, the rise started around this time, which is shortly after the onset of the presynaptic calcium current (Borst and Sakmann 1998a).
Single-compartment model
The FAP transients shown in Fig. 1 were compared with the predicted transients for a single-compartment model that featured apart from the calcium dye, an endogenous calcium buffer, calcium influx, and a linear clearance mechanism (see METHODS for details). As long as the off-rate of the endogenous buffer was high (>5,000 s–1), the time course of the simulated and measured responses overlaid well for both the OGB-5N and the Fluo-4 transients (Fig. 1D). If the off-rate of the endogenous buffer was lowered to 
5,000 s–1, while adjusting its concentration to keep the endogenous binding ratio constant, the endogenous buffer was no longer able to "track" the calcium influx and a prominent overshoot in the simulated volume-averaged calcium concentration at the end of the repolarization phase became apparent in the simulations (Fig. 1D). This component resulted in a biphasic decay of the OGB-5N transients. At 5,000 s–1, the fast component was already sufficiently large (>5% of the peak amplitude) to be well above the detection threshold in the measured transients. Because such a component was not present, this suggests that the endogenous buffer has a high unbinding rate for Ca2+. This high off-rate implies that its Kd, which is the ratio of off-rate and on-rate, has to be large as well. Even if its on-rate were close to the diffusion limit, about 1.109 per Ms, its Kd would have to be >5 µM. At lower on-rates, the Kd would have to be correspondingly larger. These simulations therefore suggest that the endogenous buffer has a low affinity for calcium, enabling it to rapidly follow changes in the calcium concentration. The lack of a large overshoot in the volume-averaged calcium concentration, in combination with the rapid kinetics, means that the rise phase of the OGB-5N transients largely reflects the integral of the calcium currents (Sabatini and Regehr 1998). This is not the case for the Fluo-4 transients. Fluo-4 has a much smaller off-rate than that of OGB-5N and, as a result, the rising phase of the Fluo-4 transients probably largely reflects the transfer of calcium ions between the endogenous buffer and Fluo-4 (Sabatini and Regehr 1998). The nearly fivefold slower decay can be explained by the much larger calcium binding ratio of this high-affinity dye (Helmchen et al. 1997). Because of the inverse relation between total binding ratio (i.e., the sum of the contributions of the endogenous and the exogenous buffers) and the time constant of the decay of the fluorescence transients (Helmchen et al. 1997; Neher 1995), this indicates that on average Fluo-4 captures 
80% of inflowing calcium ions.
In summary, the simulations suggest that the large majority of the endogenous calcium buffer of the calyx of Held has low affinity for Ca2+ (Kd >5 µM) and that at the concentration used in the PTP experiments, Fluo-4 will capture most inflowing calcium ions.
Calcium transients during PTP
We used the fluorometric signals to test for a change in the calcium transients after the induction of PTP. Unfortunately, it was no longer possible to induce PTP after prolonged calcium imaging. Probably, this was the consequence of a phototoxic effect on the terminal. Only when light exposure was restricted to a short baseline period before the tetanus was it possible to induce PTP. After a 5-min, 20-Hz tetanus, the EPSCs in the terminals that had been preloaded with Fluo-4 increased by 98 ± 22% (n = 7), similar to intact terminals (Habets and Borst 2005). The PTP was accompanied by a clear increase in the amplitude of FAP (Fig. 2A). The amount of PTP and the increase in FAP were correlated (Fig. 2F). On average the fluorescence transient increased by 15 ± 4% (n = 7). There was a small increase (0.24 ± 0.11 ms) in the time to onset of FAP. A clear example is shown in Fig. 2B. This increase correlated well (r = 0.99) with an increase in the delay of the EPSCs in the same experiments (Fig. 2C), indicating that the increased delay after PTP induction was the result of an increased delay until presynaptic calcium channels opened, rather than changes downstream of Ca2+ entry. Apart from the small increase in the delay of FAP, its kinetics was very similar after the tetanus: both its rise time (P = 0.75; paired t-test) and its decay (P = 0.24) did not change significantly (Fig. 2C). The lack of a change in the kinetics of the fluorescence transients argues against a change in clearance after the induction of PTP. In most experiments, the decay of PTP matched the decay of the increase in FAP (Fig. 2, D and E). In the experiment shown in Fig. 2D the decay of PTP also matched the increase in basal fluorescence, as previously observed (Habets and Borst 2005; Korogod et al. 2005). However, in most experiments this relation could not be reliably assessed because basal fluorescence was generally not stable during the control period, probably resulting from washout of extracellular dye.
Surprisingly, in the presence of the low-affinity dyes rhod-dextran or OGB-5N, in only one of six synapses was a large increase in the size of the EPSC observed. This was accompanied by a clear increase in FAP. In the other five synapses, EPSCs increased by only 29 ± 3% and FAP changed little after the tetanus (–1 ± 4%).
Relation between calcium influx and fluorescence signal
The increase in the Fluo-4 transients after induction of PTP could be the result of an increase in calcium influx, an increase in the fraction of inflowing calcium ions that are captured by the dye, or a combination of the two. A possible increase in the captured fraction of inflowing calcium ions could be the result of either a decrease in the calcium clearance or a decrease in competing endogenous buffers. Our earlier conclusion that the endogenous buffer most likely has low affinity and that Fluo-4 captures most of the calcium ions at the concentration used in the PTP experiments suggests that an effect on calcium influx is more likely than a selective depletion of endogenous buffers. To test the relation between the size of the calcium influx and the calcium signals at different presynaptic Ca2+ levels, we voltage clamped the presynaptic terminals with trains of 10 action potential waveforms (APWs) at 100 Hz after pharmacologically isolating the calcium currents. This allowed us to directly compare the calcium influx per action potential with the fluorescence signals. Both in the presence of Fluo-4 and in the presence of OGB-5N, the calcium currents facilitated during the train (Fig. 3, A and B). In the presence of Fluo-4, the influx per APW increased by roughly 20% at the end of the train, in agreement with earlier results (Borst and Sakmann 1998b). In the presence of OGB-5N, the facilitation was more transient (Fig. 3D), which is in line with the lack of an increase in FAP after a tetanus in most of the PTP experiments in which OGB-5N was used.
The absence of calcium channel facilitation during long trains in the presence of OGB-5N may have contributed to the reduced PTP in the experiments described above. The train of APWs led to clearly resolvable fluorescent transients (FAPW), consisting of a rapid rising phase followed by an exponential decay (Fig. 3, A and B). The time constant of this decay changed little during the train, both for terminals filled with OGB-5N (200 µM) and with Fluo-4 (100 µM). The amplitude of the fluorescence increase after the first and the last APW were similar for the OGB-5N transients (Fig. 3A, inset), but the last APW evoked a smaller fluorescence transient than the first APW in the terminals filled with Fluo-4 (Fig. 3B, inset), despite the larger calcium influx (Fig. 3D). To take into account the changes in the calcium influx during the train, we calculated the ratio between fluorescence change and calcium influx (F/Q). In the presence of the low-affinity dye OGB-5N, F/Q largely remained the same, with a small increase during the first APWs and a small decrease toward the end of the train (Fig. 3D). In contrast, in the presence of the high-affinity dye Fluo-4, F/Q gradually decreased during the train, as Ca2+ accumulated. These experiments provide more evidence for our earlier conclusion that the large majority of the endogenous buffer has low affinity for Ca2+. As a consequence, the increase in the calcium concentration during the train leads to slight saturation of both the endogenous buffer and OGB-5N, resulting in only slight changes in F/Q. At the same time the gradual decrease in the availability of Fluo-4 leads to a gradual decrease in F/Q.
Although these experiments are in agreement with our earlier conclusion that the endogenous buffer has low affinity for Ca2+, they do not provide positive evidence that we would be able to detect the presence of a low concentration of an endogenous high-affinity calcium buffer. We therefore repeated these experiments in the presence of both OGB-5N (200 µM) and fura-2 (50 µM). The decay of the OGB-5N transients now clearly became biphasic (Fig. 3C). If the decay of the response to a single APW was fitted with two exponential functions, the fast time constant ranged between 1 and 2 ms, whereas the slow time constant was >100 ms (n = 4; not shown). Because of the different spectral properties of the two dyes, we could also measure the fura-2 fluorescence transients within the same experiment. The presynaptic calcium concentration increased from a basal level of about 100 to about 160 nM after the first APW (n = 4). The rising phase of the fura-2 transients was much slower than that of the OGB-5N transients and it largely matched the fast component in the decay of the OGB-5N transients (Fig. 3C). As calcium accumulated to a maximum level of about 1 µM at the end of the train, the relative contribution of the fast phase decreased from 81 ± 5 to 6 ± 3% (n = 4) after the last APW. The F/Q for OGB-5N showed a clear increase during the train, which we interpret as being a result of the saturation of the competing other exogenous calcium buffer fura-2 because F/Q of fura-2 became much smaller at the same time.
Train simulations
Our interpretation of the fluorescence signals during trains of APWs does not take diffusion or heterogeneities in calcium influx or calcium buffering into account. To test whether the binding kinetics and relative affinities of the different endogenous and exogenous calcium buffers provided a sufficient interpretation of the observed signals we repeated the simulations of the single-compartment model for trains of APWs. In these simulations we used the measured calcium currents illustrated in Fig. 3 (after filtering, truncation of outward currents, and subtraction of slow calcium-activated currents) as inputs and added the exogenous calcium buffers using the same low-affinity endogenous buffer as in Fig. 1. During these trains the volume-averaged calcium concentration rose to about 0.6 µM in the presence of a high-affinity calcium dye (Fluo-4, or fura-2 in combination with OGB-5N) and about 1.3 µM in the presence of OGB-5N. Both the simulated fluorescence transients (Fig. 4, A and C) and the resulting F/Q (Fig. 4D) were qualitatively similar to the data illustrated in Fig. 3. The simulations also confirmed the validity of the interpretation of the experiments in which we added both a high- and a low-affinity dye. The low-affinity dye is able to report how the calcium transfers from the low-affinity buffers to the high-affinity dye. Because of its slow equilibration, the high-affinity dye is not able to report subtle changes in the time course of the calcium influx, as may happen after PTP induction. The contribution of the high-affinity dye becomes less pronounced as calcium accumulates and the dye saturates. As a result, the low-affinity dye faces less competition and F/Q will increase during the train. The experiments and simulations with two different exogenous dyes confirm that even small concentrations (<50 µM) of a slowly equilibrating calcium buffer (off-rate <5,000 s–1) would lead to clear deviations from a single-exponential decay for the OGB-5N transients, in contrast to what was observed in Figs. 1 and 3A.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Helmchen et al. 1997; Schneggenburger et al. 1999). The signal-to noise ratio was also much higher than that in our earlier study, in which we used fura-2 (Habets and Borst 2005). This allowed us to compare the calcium transients evoked by an action potential before and after induction of PTP. We show that PTP at the calyx of Held synapse is accompanied by a clear increase in these calcium transients. The most likely cause for this increase is an increase in calcium influx during an action potential. We now review the evidence for this conclusion and discuss the significance of our findings for the mechanism of PTP at the calyx of Held synapse. Increased calcium transients during PTP
We observed a clear increase in the Fluo-4 transients after PTP induction. On average, the transients increased by roughly 15%. There are three possible causes for the observed increase in the Fluo-4 signal. It could be the result of either an increase in the calcium influx, a decrease in calcium clearance, or a decrease in the endogenous calcium buffers that compete with the Fluo-4. We have several arguments that support our conclusion that the increased Fluo-4 signal was a consequence of increased calcium influx. A decrease in Ca2+ clearance is unlikely because the time course of the Fluo-4 signal was not changed after PTP induction. After the induction of PTP, the presynaptic calcium concentration is elevated by about 100 nM and the decay of PTP largely matches the decay of this increase (Habets and Borst 2005). This increase in residual calcium could lead to a partial depletion of endogenous calcium buffers. At other terminals, fluorometric evidence for a high-affinity endogenous buffer component was obtained (Collin et al. 2005; Jackson and Redman 2003; Lin et al. 2005; Sinha et al. 1997) and partial depletion of the endogenous calcium buffer was proposed to be responsible for most of the short-term facilitation of release at the calyx of Held synapse (Felmy et al. 2003). We therefore analyzed the measured calcium transients in detail, to investigate whether under the conditions in which we observed the increase in calcium transients we could find fluorometric evidence for a high-affinity endogenous calcium buffer, whose depletion could be partially responsible for the observed increase in the calcium transients.
Analysis of calcium transients suggests low endogenous buffer affinity
A comparison of amplitude and time course of different calcium dyes with model predictions indicated that under our experimental conditions the large majority of endogenous buffers are characterized by low affinity (>5 µM) for Ca2+. The OGB-5N transients that were evoked by a single action potential decayed monophasically. Simulations (Fig. 1D) and experiments (Fig. 3C) suggested that the presence of even small amounts (<50 µM) of a high-affinity endogenous calcium buffer would lead to clear deviations from a monoexponential decay. In presynaptic voltage-clamp experiments we compared calcium influx during action-potential waveform stimuli and the resulting fluorescence changes at different Ca2+ concentrations. If the endogenous buffer has low affinity, both the amplitude and the time course of the low-affinity OGB-5N transients are predicted not to change, as long as the calcium clearance mechanism is linear and not saturated. The low-affinity buffer OGB-5N initially showed a small increase in the fraction of calcium ions that it captured per stimulus, followed by a small decrease as Ca2+ accumulated during the train (Fig. 3D). The increase of a few percent for the OGB-5N signal could be explained by saturation of a small component of the endogenous buffer with a higher affinity for Ca2+ than for the dye. Alternatively, it could arise from small errors in the quantification of the calcium influx, resulting from the incorrect estimate of calcium-activated currents or gating currents; or from small errors in the quantification of the fluorescence step, resulting from the incorrect correction for clearance during the rising phase of the transients. Apart from this small increase, both the amplitudes and the time course of the OGB-5N transients closely followed the prediction of the model with a low-affinity endogenous buffer and a linear clearance mechanism, up to a level of 1 µM. This concentration was much higher than the increase in residual calcium observed after PTP induction at the calyx of Held (Habets and Borst 2005; Korogod et al. 2005). A linear clearance mechanism was also observed at the crayfish neuromuscular junction (Tank et al. 1995).
In contrast, in the presence of a low-affinity endogenous buffer, a high-affinity dye like Fluo-4 is expected to show a gradual decrease in the fraction of inflowing calcium ions that it captures during the train, as less and less dye will be available to compete with the endogenous buffer. At other terminals it was not possible to directly measure the calcium influx during trains of action potentials, but nevertheless qualitatively similar results were obtained, a decrease in signals during the train for high-affinity dyes, but increases or no change for low-affinity dyes (David et al. 1997; Koester and Johnston 2005; Kreitzer and Regehr 2000).
From the analysis of the time course and amplitudes of the calcium transients we therefore conclude that the endogenous calcium buffer in these experiments had a low affinity for Ca2+. A similar conclusion was also reached along different lines by Bollmann and Sakmann (2005). At cerebellar terminals (Sabatini and Regehr 1998) or in chromaffin cells (Xu et al. 1997) the endogenous buffer also has low affinity for Ca2+. Our data cannot exclude the washout during dye loading of a high-affinity mobile buffer. The absence of PTP in experiments in which the terminals were loaded with low-affinity dye indicates that calcium buffering is important for the induction of PTP. Possibly these buffers interfered with PTP induction by limiting the maximal Ca2+ increase during the tetanus more effectively than the low-affinity buffer fluo-4. Alternatively, the low-affinity buffers shortened the decay time of the PTP to the extent that it was missed in our recordings, which did not start until more than 1 min after the tetanus to allow for pool recovery. At later developmental stages, high-affinity calcium-binding proteins such as calretinin or parvalbumin may make a larger contribution to calcium binding (Felmy and Schneggenburger 2004; Lohmann and Friauf 1996) and this might explain why longer stimulation is needed to induce PTP in older animals (Korogod et al. 2005).
Increased calcium influx during PTP
If the endogenous buffer is low, this means that the increase in the Fluo-4 transients after PTP induction was not the result of a selective depletion of a high-affinity endogenous calcium buffer. We therefore conclude that this increase must have arisen from an increase in calcium influx. Although the Fluo-4 concentrations we used were low (<200 µM) and comparable to the buffer concentrations we used in our previous study in which we focused on residual calcium (Habets and Borst 2005), because of the low endogenous buffer capacity of the calyx of Held at young ages (Helmchen et al. 1997), the calcium dye nevertheless captured most of the incoming calcium ions (>80%) in the PTP experiments. This means that in these experiments, the "overload" condition was almost reached, meaning that under these conditions the dye provided a sensitive indication for calcium influx (Neher 1995). Changes in the endogenous buffer will therefore have comparatively little effect on the measured Fluo-4 transients and it is hard to come up with a scenario for which these effects are greater than the effects that residual calcium will have on the availability of Fluo-4 after PTP induction. This means that the increase of 15% has to be viewed as a lower estimate for the increase in calcium influx.
What caused the increase in calcium influx?
The increase in calcium influx can be explained by a facilitation of the calcium currents (Borst and Sakmann 1998b; Cuttle et al. 1998) or by a change in the action potential shape. We did observe a change in the prespike (Fig. 2, A and B), suggesting that a broadening of the action potential may have contributed to the increased calcium influx (Borst and Sakmann 1999). We were not able to address this conclusively because of the inability to evoke PTP in presynaptic whole cell recordings and because the Fluo-4 transients were too slow to detect changes in the time course of the calcium influx. Facilitation of calcium currents very likely contributed to the observed changes in the calcium influx. A few action-potential waveforms were sufficient to induce a facilitation of the calcium influx that was comparable to the increases that we observed in the Fluo-4 signal after PTP induction (Fig. 3). In contrast, in the presence of OGB-5N, a train of action-potential waveforms did not give a sustained facilitation of the calcium influx and no increase in the calcium signals after a tetanus was observed in most current-clamp experiments that used OGB-5N as the dye. Because PTP was reduced in the presence of OGB-5N, whereas in the fluo-4 experiments it was comparable in size to the PTP observed in intact terminals, these experiments suggest that the increase of the calcium influx that we observed in the Fluo-4 voltage-clamp experiments best matches the situation in the undialyzed terminals. Interestingly, an increase of roughly 15% in calcium signals during action potential trains was also observed in granule cell terminals of the cerebellum (Kreitzer and Regehr 2000).
Our experiments provide further evidence for the key role that the modulation of calcium channels plays in the regulation of short-term plasticity. Facilitation of calcium currents is also important for short-term facilitation of transmitter release at the calyx of Held (Inchauspe et al. 2004; Ishikawa et al. 2005; Taschenberger et al. 2002) and inactivation of calcium currents contributes to synaptic depression (Forsythe et al. 1998; Xu and Wu 2005). The facilitation of calcium currents depends on an interaction with the high-affinity calcium-binding protein neuronal calcium sensor 1 (NCS-1; Tsujimoto et al. 2002) and this protein was also implicated in short-term facilitation of transmitter release at the neuromuscular junction or the hippocampus (Rivosecchi et al. 1994; Sippy et al. 2003), although as of yet there is no evidence that it exerts its effects by calcium channels at these synapses. The regulation of calcium channels by calcium-binding proteins like NCS-1 or other members of this family is complex and they may have both Ca2+-dependent and Ca2+-independent effects on both inactivation and facilitation of calcium channels (Burgoyne et al. 2004; Few et al. 2005). It would be interesting to test to what extent these differential effects could explain why after a long tetanus calcium currents inactivate during presynaptic whole cell recordings, leading to PTD (Forsythe et al. 1998), whereas the opposite appears to be true when synapses are intact.
The observed increase in calcium influx most likely played a significant role in the increase in release probability after the tetanus. A third power relation between volume-averaged calcium signals and release was observed in previous experiments at the calyx of Held synapse (Borst and Sakmann 1999; Wu et al. 1999) and combined with the pool size increase of nearly 30% that we observed in our earlier experiments after PTP induction (Habets and Borst 2005), this would be sufficient to account for most if not all of the PTP (Fig. 2F). This conclusion depends on the assumption that even if the time course of the calcium influx would change, the third power relation between influx and release would remain valid. Although this may be true for the changes in the time course of calcium influx arising from action potential changes during high-frequency trains (Borst and Sakmann 1999), both lower (Bollmann and Sakmann 2005) and higher (Fedchyshyn and Wang 2005) values for this power relation were also observed when the time course of the calcium transients was changed. Therefore our experiments cannot exclude that local buffer saturation or changes downstream of Ca2+ may provide additional contributions, as suggested by other experiments in the same preparation (Awatramani et al. 2005; Felmy et al. 2003; Lou et al. 2005).
In earlier experiments we observed that the potentiation of evoked release decayed similarly to residual calcium, whereas increases in the frequency of spontaneous release decayed more rapidly (Habets and Borst 2005). An attractive feature of the possibility that the increased calcium influx was a result of the interaction of a high-affinity calcium-binding protein such as NCS-1 with the presynaptic calcium channels is that it provides a simple explanation for this observation because a regulation of calcium influx by residual calcium will preferentially affect evoked release over spontaneous release. The more rapid decay of spontaneous release could be explained by supralinear effects of a direct activation of the calcium sensor for release by residual calcium (Habets and Borst 2005).
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Address for reprint requests and other correspondence: J.G.G. Borst, Erasmus MC, University Medical Center Rotterdam, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands (E-mail: g.borst{at}erasmusmc.nl)
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