INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The medial nucleus of the trapezoid body (MNTB) synapse mediates fast, high-fidelity transmission that is crucial for auditory processing (Goldberg and Brown 1968; Guinan and Li 1990; Oertel 1999). Synchronized release develops during early postnatal life. At postnatal days 4-5 (P4-5), when the calyx-like structure of the presynaptic terminal begins to form (Kandler and Friauf 1993), the excitatory postsynaptic current (EPSC) is followed by many miniature EPSC (mEPSC)-like currents (Chuhma and Ohmori 1998). These mEPSC-like currents are a part of the evoked response, as they do not arise spontaneously, appearing only following evoked EPSCs (Chuhma et al. 2001). They disappear after P9 (Chuhma and Ohmori 1998). Synchronization of transmitter release progresses in parallel with maturation of Ca²⁺ extrusion and Ca²⁺ buffering capacities in the presynaptic terminal (Chuhma et al. 2001), which are likely to reduce the lifetime of Ca²⁺ transients in the presynaptic terminal (Mironov et al. 1993; Roberts 1994; Reuter and Porzig 1995). Sequential expressions of some Ca²⁺ binding proteins were observed in these postnatal days in the brain stem auditory nuclei (Friauf 1993; Lohmann and Friauf 1996).

If the nature of Ca²⁺ extrusion and buffering capacities are essential for synchronized transmitter release, then manipulations to prolong the lifetime of Ca²⁺ transients should desynchronize transmitter release in the MNTB synapse (after P9). In agreement with this hypothesis, Sr²⁺ is known to produce delayed asynchronous release (Goda and Stevens 1994) and is reported to have a long lifetime in the presynaptic terminal (Xu-Friedman and Regehr 2000). In this report, we tested this hypothesis with inhibitors of Ca²⁺ clearance to modulate Ca²⁺ lifetime in the terminal.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Preparations of brain slices

Most experiments were performed in brain-stem slices containing the MNTB prepared from P9-12 Wister rats as described previously (Chuhma and Ohmori 1998). A part of the records shown in Fig. 1 was obtained from P4-5 rats. Briefly, rats were deeply anesthetized with ether and decapitated. Brain-stem slices were cut 200-300 µM thick using a vibratome (DTK-2000; Dosaka, Kyoto, Japan). After incubation at 36°C for 1 h in high-glucose artificial cerebrospinal fluid (high-glucose ACSF; concentrations in mM as follows unless otherwise noted: 75 NaCl, 2.5 KCl, 26 NaHCO₃, 1.25 NaH₂PO₄, 0.7 CaCl₂, 2 MgCl₂, and 100 glucose, pH 7.4) saturated with 95% O₂-5% CO₂, slices were maintained in the same high-glucose ACSF at room temperature until they were used. Experiments were performed at room temperature (20-25°C).

View larger version (24K): [in this window] [in a new window]   Fig. 1. Sr2+-generated delayed releases in the medial nucleus of the trapezoid body (MNTB) synapse. A: evoked excitatory postsynaptic current (EPSCs) (P11). Six traces are superimposed in each panel. Insets indicate 10-20 trace-averaged miniature EPSCs (mEPSCs). In this and subsequent figures, EPSCs and mEPSCs were recorded from neurons voltage-clamped at 70 mV. a: P11 EPSC in control. b: P11 EPSC after replacement of external Ca2+ with Sr2+ recorded from the same synapse as in a. c: immature P5 EPSC in control. B: event time histograms made from 30 consecutive traces (in a, b) or 50 consecutive traces (in c) of EPSC. Each histogram was made from the same EPSC traces as indicated in A. C: asynchronicity indexes of P11 EPSC in control (open bar), after replacement with Sr2+ (hatched bar) and immature P5 EPSC (filled bar).

Recordings of EPSCs

As described previously (Chuhma and Ohmori 1998), EPSCs were recorded from MNTB principal neurons superfused with ACSF (125 NaCl, 2.5 KCl, 26 NaHCO₃, 1.25 NaH₂PO₄, 2 CaCl₂, 1 MgCl₂, and 17 glucose, pH 7.4, saturated with 95% O₂-5% CO₂) and supplemented with 20 µM strychnine (Sigma), 10 µM bicuculline (Sigma), and 50 µM D-2-amino-5-phosphonovalerate (APV; Tocris). The pipette solution was Cs⁺-based (136 Cs-glucuronate, 14 CsCl, 10 HEPES, 5 EGTA, pH 7.4) with the addition of 5 mM N-(2,6-dimethylphenylcarbamoylmethyl) triethylammonium bromide (QX-314; Alomone Labs, Jerusalem, Israel). In some experiments, CaCl₂ was replaced with SrCl₂ (2 mM) (Sr²⁺-ACSF).

Neurons were voltage clamped at 70 mV (Axopatch 200A; Axon Instruments). Corrections were made for the liquid junction potential (approximately 10 mV). Pipette resistances were approximately 2-5 M. Series resistances were 9-20 M and compensated by 70-80%. Presynaptic nerve fibers were electrically stimulated (0.5-8V, 100-µs duration) every 5 s using bipolar tungsten electrode.

Ca²⁺ overloading

Ca²⁺ clearance was reduced in the presynaptic terminal by several procedures: 1) replacement of external Na⁺ with Li⁺, 2) bath application of eosin Y (0.2 mM, Sigma), 3) La³⁺ (1 mM), 4) thapsigargin (10 µM, Sigma), 5) carbonyl cyanide m-chlorophenyl hydrazone (CCCP, 2-10 µM, Sigma), and 6) tetraphenyl phosphonium (TPP, 100 µM, Sigma). When Na⁺ was replaced with Li⁺, Li⁺-ACSF was used (125 LiCl, 2.5 KCl, 26 NaHCO₃, 1.25 NaH₂PO₄, 2 CaCl₂, 1 MgCl₂, 17 glucose pH 7.4). La³⁺ was added to HEPES-buffered external solution (138 NaCl, 2.5 KCl, 10 HEPES, 2 CaCl₂, 1 MgCl₂, 17 glucose, pH 7.4, saturated with 100% O₂).

Partial block of Ca²⁺ channels

Cd²⁺ (3 µM) or -Agatoxin IVA (50 nM, Peptide Institute, Osaka, Japan) was applied to block a fraction of presynaptic Ca²⁺ channels. Cd²⁺ reduced the size of EPSCs with a K_d of 1.77 µM and a cooperativity of 1.76 (Chuhma et al. 2001). These concentrations of Cd²⁺ or -Agatoxin reduced the EPSC size to 20-30% of the control.

Measurement of presynaptic [Ca²⁺]_i with fura-2 and Mg-fura2

The Ca²⁺ transient and basal Ca²⁺ level in the presynaptic terminal were monitored with a high-affinity Ca²⁺ indicator fura-2 (K_d 145 nM) or a low-affinity indicator Mg-fura-2 (K_d 25 µM) loaded directly through the patch electrode. Pipette solution was K⁺-based (120 K-gluconate, 20 KCl, 10 HEPES, pH 7.4) with 0.1 mM fura-2 pentapotassium salt (fura-2-5K, Molecular Probes), 5 Mg-ATP, 5 creatine phosphate. When [Ca²⁺]_i was measured with Mg-fura-2, the same pipette solution was used but with 0.4 mM Mg-fura-2-4K (Molecular Probes) in the place of fura-2. Ca²⁺ transients measured with fura-2 were induced by a train of five action potentials to improve the signal-to-noise ratio or by a single action potential (see Fig. 3 and Chuhma et al. 2001), with depolarizing current injection through the patch electrode by using EPC-7 (List). In measurements with Mg-fura-2, Ca²⁺ transients were induced by a train of 20 action potentials because fluorescence changes were small and <1% by a single action potential (see Helmchen et al. 1997). Membrane potential was maintained at approximately 70 mV. Pipette resistances were 5-10 M. Because of the limitation of the current clamp speed of EPC-7 (Magestretti et al. 1996), the time course of the presynaptic action potential, which was recorded during the fluorescence measurement (Fig. 3A), was slightly slower than those published (half-amplitude width of 0.5 ms in Borst et al. 1995). A wavelength pair of 340 and 380 nm alternately excited fura-2 or Mg-fura-2, and fluorescences (f340 and f380) were sampled by a photomultiplier (OSP-3, Olympus) through a 500-nm-long pass filter. The fluorescence ratio (R = f340/f380) of the presynaptic terminal area was calculated on-line without background subtraction. The time resolution was 100-200 ms. The ratio was converted to [Ca²⁺]_i by the equation determined through in situ calibration done on MNTB neurons as described in Neher (1989): for fura-2, [Ca²⁺]_i = 2.7(R  0.7)/(5.3 - R), and for Mg-fura-2, [Ca²⁺]_i = 12.4(R  0.47)/(0.71  R). The decay time constant (_decay) of Ca²⁺ transients was determined by fitting a single exponential function to the data. Because a train of five action potentials for fura-2 and 20 action potentials for Mg-fura-2 was used to induce the Ca²⁺ transient, the decay time course was likely prolonged more than the case generated by a single action potential (Chuhma and Ohmori 2001). However, the time constant of decay we observed at room temperature (1 s for fura-2 and 0.4 s for Mg-fura-2) was within the range reported by using fura-2 (0.1 mM; Helmchen et al. 1997) and the low-affinity Ca²⁺ indicator dyes (0.4 mM Mg-fura-2 or 0.2 mM Calcium Green-5N; Borst et al. 1995; Helmchen et al. 1997).

Data analysis

Data were stored and analyzed as described previously (Chuhma and Ohmori 1998). The occurrence time of mEPSC-like currents was determined off-line by registering visually identified peaks and scored in a histogram (event time histogram). The frequency of mEPSC-like currents was quantified as an asynchronicity index, obtained by dividing the total counts of mEPSC-like currents during a 60-ms time window (from 20 to 80 ms in 100-ms records) by the number of traces (30-50 traces). Data are given as the mean ± SE (number of cells) unless otherwise noted.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Sr²⁺ desynchronized transmitter release

In the mature (P9-P12) synapse, delayed mEPSC-like currents were rarely observed (Fig. 1Aa). We have scored the occurrence time of EPSC or mEPSC-like currents from 10 ms before stimulation of the presynaptic fiber to 100 ms after (Fig. 1B). In the control event time histogram, a large peak corresponding to the main EPSC was seen (Fig. 1Ba), and the asynchronicity index (defined in METHODS) was 0.07 ± 0.03 (n = 3). In Sr²⁺-ACSF, many mEPSC-like currents followed the EPSC (Fig. 1Ab) and the amplitude of EPSC was reduced to 24.8 ± 6.4% (n = 4) of the control. The event time histogram showed many late counts reflecting mEPSC-like currents (Fig. 1Bb) and the asynchronicity index was 1.03 ± 0.16 (n = 3, P < 0.05, paired t-test, Fig. 1C). In the immature P4-5 synapse, similar delayed mEPSC-like currents were observed in the control ACSF (Fig. 1, Ac and Bc). The asynchronicity index was 1.34 ± 0.21 (n = 11, Fig. 1C). The amplitude and the decay time constant of the mEPSC-like currents were almost the same for all conditions [P = 0.70 for amplitude; P = 0.14 for decay time constant, analysis of variance (ANOVA)] as follows: P4-5 synapse (30.9 ± 1.8 pA, 1.48 ± 0.10 ms, n = 3), P9-12 synapse in Sr²⁺-ACSF (29.4 ± 2.5 pA, 1.76 ± 0.02 ms, n = 3), or in 5 mM Ba²⁺-enriched ACSF (26.6 ± 0.5 pA, 1.70 ± 0.11 ms, n = 3).

Effects of reduced Ca²⁺ clearance capacities

During the early stage of postnatal development (P4-9), the delayed mEPSC-like currents disappeared and presynaptic Ca²⁺ currents increased twofold (Chuhma and Ohmori 1998); at the same time, both presynaptic Ca²⁺ buffering and Ca²⁺ clearance capacities were increased (Chuhma et al. 2001). Delayed asynchronous transmitter release observed in Sr²⁺-ACSF seems to have a close relation to the prolonged lifetime of Sr²⁺ in the presynaptic terminal (Xu-Friedman and Regehr 2000). These observations about Ca²⁺ dynamics and Sr²⁺ effects suggest a close relationship between delayed asynchronous release in immature MNTB synapses and the slow decay time course of Ca²⁺ transients in the presynaptic terminal. To test this hypothesis, we first prolonged the lifetime of the Ca²⁺ transient in the synapse after P9. Of the two major factors that determine the lifetime of the Ca²⁺ transient, it is practically impossible to reduce the Ca²⁺ binding capacity of soluble Ca²⁺ binding proteins in the presynaptic terminal, so we chose to reduce Ca²⁺ clearance.

1) Replacement of Na⁺ in ACSF with Li⁺ is expected to reduce Ca²⁺ extrusion through the Na⁺-Ca²⁺ exchanger. When [Na⁺]_o was reduced by replacement with Li⁺, the amplitude of the evoked EPSC increased and the decay time course was slowed (Fig. 2A). The decay time course of the mature EPSC was well-fit by a combination of three exponential functions in the control and in Li⁺-ACSF (Fig. 2B). We are not certain what caused each of the three components of decay (see Otis et al. 1996 for one possibility for delayed clearance of transmitter). However, in Li⁺-ACSF, the relative amplitude of the third (slowest) component was reduced (2.1 ± 1.3%) from that in the control (5.0 ± 1.3%, n = 5), and the decay was nearly fit by a combination of only two exponentials (Fig. 2B, c and d). Both the first (fastest) and the second (intermediate) decay time constants were increased in Li⁺-ACSF, but the relative amplitude of each component was not significantly different from the control (Table 1). These effects of Li⁺ could be reversed by washing, implying that these effects were not due to deterioration of the synapse. The amplitude and decay time constant of mEPSC recorded in Li⁺-ACSF were the same as those in the control (Fig. 2A, insets; 27.3 ± 2.8 pA, 1.5 ± 0.1 ms in control and 24.9 ± 3.0 pA, 1.6 ± 0.2 ms in Li⁺-ACSF, n = 4, P = 0.57 and 0.88, at 70 mV). However, delayed mEPSC-like currents were not observed in Li⁺-ACSF (Fig. 2A).

View larger version (20K): [in this window] [in a new window]   Fig. 2. Effects of external Li+ in MNTB synapse. A: evoked EPSCs (P11) recorded in the control (a) and after replacement of external Na+ with Li+ (b). Five traces are superimposed. Broken lines indicate baselines. Ten to twenty trace-averaged mEPSCs are indicated in insets. B: two-exponential fits (a, c) and three-exponential fits (b, d) of the decay phase of the ensemble-averaged EPSC in control (a, b) and in Li+-ACSF (c, d). Open circles indicate the averaged EPSC of 10 traces from the same records shown in A. Smooth lines are the fitted curves. The time constants of the fitted exponentials are indicated in each panel.

The lifetime of the Ca²⁺ transient in the presynaptic terminal was prolonged in Li⁺-ACSF. The Ca²⁺ transient measured by fura-2 rose quickly and decayed exponentially (Fig. 3B). The decay time constant was 1.2 ± 0.1 s (n = 5) in the control, 4.2 ± 1.0 s (n = 5) in Li⁺-ACSF, and 1.8 ± 0.2 s (n = 3) after washing. When an action potential was generated every 5 s in the control solution, a small Ca²⁺ transient was generated (Fig. 3C) and basal [Ca²⁺]_i was increased slightly (approximately 20 nM). After replacement of Na⁺ with Li⁺, the basal Ca²⁺ level was increased by 0.1 µM (0.14 ± 0.05 µM, n = 4, Fig. 3C) and remained about 50 nM higher than the control. During exposure to Li⁺-ACSF, the half-amplitude width of the presynaptic action potential was not affected significantly (1.3 ± 0.1 ms in control, 1.4 ± 0.1 ms in Li⁺, n = 3 cells, Fig. 3A). When Ca²⁺ transients were measured with a low-affinity Ca²⁺ indicator Mg-fura-2, the decay time constant was faster than it was when measured by fura-2; however, the same threefold increase in the time constant was seen in Li⁺-ACSF (0.40 ± 0.02 s in control, 1.25 ± 0.20 s in Li⁺, n = 3 cells).

View larger version (31K): [in this window] [in a new window]   Fig. 3. Effects of replacement of external Na+ by Li+ on [Ca2+]i in the presynaptic terminal. A: trains of presynaptic action potentials to induce Ca2+ transients in B. Resting potential was maintained near 66 mV. B: Ca2+ transients under control condition (left), after replacement with Li+ (middle), and after washing (right) in P11 presynaptic terminals. Decay time constant of each trace is indicated. C: time course of change of presynaptic basal [Ca2+]i after solution change to Li+-ACSF. Noisy signals starting at 40 s are due to Ca2+ transients induced by individual action potentials.

2) We then examined the effects of eosin Y, an inhibitor of the plasma membrane Ca²⁺-ATPase (Gatto and Milanick 1993). Application of eosin Y (0.2 mM) to the external solution increased the peak amplitude and the first and the second decay time constants of the EPSC (Table 1). The amplitude (27.1 ± 1.9 pA in control vs. 24.6 ± 1.9 pA in eosin Y, P = 0.46) and decay time constant (1.9 ± 0.3 ms in control vs. 1.9 ± 0.3 ms in eosin Y, P = 0.37) of the mEPSC were not significantly affected (n = 4). Delayed mEPSC-like currents were not observed. We could not visualize the expected increase in presynaptic [Ca²⁺]_i after eosin Y because of its fluorescence. La³⁺ is reported to inhibit plasma membrane Ca²⁺-ATPase (Carafoli 1991; Zenisek and Matthews 2000). However, 1 mM La³⁺ progressively reduced EPSC amplitude until it blocked transmission completely (n = 3 cells). When the first and second decay time constants and the asynchronicity index were compared between control and during the course of block, when the EPSC was reduced to 20%, there was no change in the EPSC parameters [1st decay time constant 0.93 ± 0.16 ms in control, 0.77 ± 0.17 ms at 20% amplitude (P < 0.01); 2nd decay time constant 3.67 ± 0.56 ms in control, 3.99 ± 1.18 ms at 20% amplitude (P = 0.75); asynchronicity index 0.06 ± 0.03 in control, 0.25 ± 0.12 at 20% amplitude (P = 0.18); n = 3 cells]. Lower concentrations of La³⁺ (100 and 10 µM) had no effect on EPSC (n = 3 cells).

3) We then tested thapsigargin (10 µM, n = 4), an inhibitor of Ca²⁺ uptake by the endoplasmic reticulum (ER; Jackson et al. 1988). There was no effect on the EPSC even after 30 min incubation (Table 1).

4) We examined two agents affecting mitochondrial Ca²⁺ dynamics: CCCP (Gunter and Pfeiffer 1990; Herrington et al. 1996) and TPP (Tang and Zucker 1997). Both 2-10 µM CCCP and 100 µM TPP progressively reduced the EPSC amplitude until it was blocked completely. The time course of the effect of 100 µM TPP on the EPSC is shown in Fig. 4. When the peak amplitude of the EPSC was reduced to 20% of that of the control, there was no difference in the first and second decay time constants or the asynchronicity index [1st decay time constant 1.34 ± 0.40 ms in control, 1.24 ± 0.36 ms at 20% amplitude (P = 0.74); 2nd decay time constant 4.34 ± 0.83 ms in control, 4.52 ± 1.30 ms at 20% amplitude (P = 0.81); asynchronicity index 0.03 ± 0.02 in control, 0.05 ± 0.03 at 20% amplitude (P = 0.48); n = 3 cells]. However, in two cells, many spontaneous mEPSC-like currents appeared after complete block of the evoked EPSC (Fig. 4Ad). This suggests that TPP increased the basal Ca²⁺ level. TPP effects on evoked EPSC were probably masked by its inhibitory effects on ATP production (Nguyen et al. 1997). CCCP (2-10 µM) gradually blocked synaptic transmission similarly. The decay time constants and the asynchronicity index were not different from the control when EPSC amplitude was reduced to 20% [1st decay time constant 1.43 ± 0.18 ms in control, 2.25 ± 0.89 ms at 20% amplitude (P = 0.42); 2nd decay time constant 5.01 ± 1.11 ms in control, 5.94 ± 2.21 ms at 20% amplitude (P = 0.78); asynchronicity index 0.03 ± 0.02 in control, 0.01 ± 0.01 at 20% amplitude (P = 0.42); n = 5 cells].

View larger version (17K): [in this window] [in a new window]   Fig. 4. Effects of tetraphenyl phosphonium (TPP) on synaptic transmission. A: sample traces of EPSC (P12) at the points indicated in B. Five traces are superimposed in each panel. a: control. b, c, d: after application of 100 µM TPP. The arrow in d indicates stimulus artifact. B: time course of effects of TPP on the peak amplitude of EPSC. The hatched bar indicates the period TPP was applied.

Li⁺ reduced presynaptic Ca²⁺ influx and generated delayed mEPSC-like currents

MEPSC-like currents were not observed in the late phase of EPSCs even though the lifetime of Ca²⁺ transient was prolonged (Fig. 2A). Since Ca²⁺ currents were smaller in immature P5-6 terminals than in the synapse after P10 (Chuhma and Ohmori 1998), we examined the combined effects of reduced Ca²⁺ influx and prolonging the Ca²⁺ transient.

After reducing Ca²⁺ influx with -Agatoxin IVA (50 nM), mEPSC-like currents started to appear following the EPSC, but the asynchronicity index was still small (0.23 ± 0.09, n = 5), as previously reported (Chuhma et al. 2001 and Fig. 5Ab). When external Na⁺ was replaced with Li⁺ in the presence of 50 nM -Agatoxin, the frequency of mEPSC-like currents was further increased, and many late counts emerged in the event time histogram (Fig. 5Ac). The asynchronicity index was 1.22 ± 0.31 (n = 5, Fig. 5Ba) and was significantly different from the control (P < 0.05). This level of the asynchronicity index was close to that of the immature synapse (1.34 ± 0.21, n = 11). The induction of delayed mEPSC-like currents by a combination of reduced Ca²⁺ influx and replacement of ACSF with Li⁺-ACSF did not depend on the method of reducing Ca²⁺ influx (Fig. 5B). Cd²⁺ (3 µM) induced delayed mEPSC-like currents (Fig. 5Bb), and the frequency markedly increased after addition of Li⁺ (1.22 ± 0.31, n = 5, P < 0.01). When [Ca²⁺]_o was reduced from 2 to 0.7 mM, the asynchronicity index was not increased. However, in Li⁺-ACSF with reduced [Ca²⁺]_o, the asynchronicity index was markedly increased (0.79 ± 0.18, n = 3, P = 0.085; Fig. 5Bc). The delayed mEPSC-like currents disappeared after returning to control solution.

View larger version (30K): [in this window] [in a new window]   Fig. 5. Combination of reduced Ca2+ extrusion and reduced Ca2+ influx generated asynchronous release. A: effects of replacement of Na+ with Li+ after partial block of Ca2+ channels with -Agatoxin 50 nM (P9). Sample traces are shown; control (a), after application of -Agatoxin (b), and after replacement of external Na+ with Li+ in the presence of -Agatoxin (c). All traces were obtained from a single neuron sequentially. Six traces are superimposed. Insets indicate the event time histograms made from 30 consecutively recorded traces. B: asynchronicity indexes after combined reduction of Ca2+ influx and Ca2+ extrusion. The indexes were calculated from 30 EPSC traces in P9-12 synapses. a-c: Ca2+ extrusion was reduced by replacement of Na+ with Li+. Ca2+ influx was reduced 3 ways: application of 50 nM -Agatoxin (a, n = 5), of 3 µM Cd2+ (b, n = 4), and reduction of [Ca2+]o to 0.7 mM (c, n = 3). d: Ca2+ influx was reduced by 50 nM -Agatoxin, and Ca2+ extrusion was reduced by application of 0.2 mM eosin Y. Each panel shows the index in the control (open bar) in reduced Ca2+ influx (hatched bar) and in reduced Ca2+ extrusion combined with reduced Ca2+ influx (filled bar).

Instead of Li⁺ replacement, we asked whether adding eosin Y to reduce Ca²⁺ extrusion would affect the EPSC. We found that the combination of eosin Y with -Agatoxin increased mEPSC-like currents (the asynchronicity index was increased from 0.11 ± 0.02 in -Agatoxin to 0.72 ± 0.17 in -Agatoxin and eosin Y, n = 3, P < 0.05, paired t-test, Fig. 4Bd).

	DISCUSSION

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Synchronization of transmitter release at the MNTB synapse depends on a rapid and robust Ca²⁺ influx, which is quickly removed by active extrusion. Solely reduced Ca²⁺ influx, when Ca²⁺ channels were partially blocked, produced only a slight increase of asynchronous delayed release (Fig. 5B). Similarly, reduced Ca²⁺ extrusion alone prolonged the decay phase of EPSC, but asynchronous delayed releases were not observed (Figs. 2 and 3). This indicates that cooperative increase of both Ca²⁺ influx and Ca²⁺ clearance capacities in the presynaptic terminal are required to make a sharper Ca²⁺ spike for synchronized transmitter release at the MNTB synapse. Sharpening of presynaptic action potentials during development in MNTB is compatible with this idea (Taschenberger and von Gersdorff 2000).

Ca²⁺ clearance mechanisms in the presynaptic terminal

Presynaptic [Ca²⁺]_i is reduced by uptake by the ER and mitochondria, or extrusion by the Na⁺-Ca²⁺ exchanger or the plasma membrane Ca²⁺-ATPase (Rosenberger and Triggle 1978). In the MNTB synapse, replacement of external Na⁺ with Li⁺ prolonged the lifetime of the Ca²⁺ transient and increased EPSC amplitude and decay time constants (Figs. 2 and 3). Eosin Y, an inhibitor of the plasma membrane Ca²⁺-ATPase (Gatto and Milanick 1993), had similar effects. Although La³⁺, another inhibitor of plasma membrane Ca²⁺-ATPase (Carafoli 1991; Zenisek and Matthews 2000), blocked synaptic transmission completely, this is probably due to the effect of Ca²⁺ channel block by La³⁺ (Hagiwara and Takahashi 1967). Thapsigargin, an inhibitor of Ca²⁺ uptake to ER (Jackson et al. 1988), had little effect on the Ca²⁺ transient and EPSCs (Table 1). TPP and CCCP, both inhibitors of Ca²⁺ uptake by mitochondria (Gunter and Pfeiffer 1990; Herrington et al. 1996; Tang and Zucker 1997), blocked EPSCs (Fig. 4). This is probably a consequence of the inhibition of ATP production by these agents (Parsons et al. 1995; Rothman 1994). From these results, it is suggested that Ca²⁺ extrusion to the outside of the terminal may be more important than Ca²⁺ uptake and storage within organelles. A similar conclusion was also reached for hippocampal presynaptic boutons (Reuter and Porzig 1995) and for retinal bipolar cells (Kobayashi and Tachibana 1995), and was suggested for the presynaptic terminal of the MNTB synapse (Helmchen et al. 1997).

Our results on TPP and CCCP are different from observations by others. In the crayfish neuromuscular junction, Tang and Zucker (1997) reported that inhibition of mitochondrial Ca²⁺ uptake with either TPP or CCCP enhanced the elevation of [Ca²⁺]_i induced by tetanus and increased the amplitude of excitatory junctional potential within 10 min after application. Similar results were observed in the terminal of the ribbon synapse of the bipolar cell in the goldfish retina (Zenisek and Matthews 2000); furthermore, these authors reported that the contribution of Na⁺-Ca²⁺ exchanger to the terminal Ca²⁺ clearance was negligible. In our experiments with MNTB presynaptic terminals, these inhibitors blocked synaptic transmission (Fig. 4). We did not see any increase in the amplitude nor prolongation of the EPSC preceding the block. However, in some experiments of TPP, the frequency of occurrence of mEPSC-like currents was increased after the block of evoked synaptic responses (Fig. 4Ad). This suggests mitochondrial Ca²⁺ uptake may still have some contribution to keep the basal Ca²⁺ level low in the terminal.

Effects of Li⁺

Li⁺ is a popular substitute of Na⁺ to reduce Ca²⁺ extrusion through the Na⁺-Ca²⁺ exchanger while maintaining the excitability of nerve fibers. When external Na⁺ was replaced with Li⁺ without reducing Ca²⁺ influx, the first and the second decay time constants of the EPSC were prolonged (Fig. 2B). These prolongations might be expected from the increased asynchronous release; however, mEPSC-like currents were not seen. This is probably because individual mEPSC-like currents were buried in the large number of delayed release events, so we could not detect them. There is also the possibility that Li⁺ might have affected the desensitization of postsynaptic AMPA receptors (Karakanias and Papke 1999). Cyclothiazide (CTZ) had a Li⁺-like effect on the EPSC. Both Li⁺ (Fig. 2A) and 100 µM CTZ (Barnes-Davies and Forsythe 1995) increased the evoked EPSC amplitude and prolonged the decay time course. However, it is unlikely that the effects on desensitization were the major Li⁺ effects in this synapse, because Li⁺ did not affect the amplitude nor the decay time course of the mEPSC (Fig. 2A, insets).

Li⁺ has other activities, such as reducing PI turnover (Berridge and Irvine 1989) and inhibition of the glutamate transporter (Dixon and Hokin 1998). Both suppression of Ca²⁺ extrusion through the Na⁺-Ca²⁺ exchanger and suspension of PI turnover should increase intracellular Ca²⁺ concentrations. Inhibition of the glutamate transporter with Li⁺ was, if anything, minor, because application of 200 µM D,L-threo--hydroxyaspartic acid (THA), a glutamate transporter inhibitor, did not affect synaptic transmission during a 1 h application (unpublished observation). There is the further possibility that suspension of PI turnover might affect transmitter release via synaptotagmin (Mikoshiba et al. 1999), which we cannot exclude as a possibility. However, we believe that the asynchronous release induced by Li⁺-ACSF with reduced presynaptic Ca²⁺ influx is a consequence of the prolonged lifetime of the Ca²⁺ transient, because eosin Y, the other inhibitor of Ca²⁺ extrusion, also induced robust asynchronous release under similar conditions (Fig. 4Bd). In summary, both the rapid rise of the Ca²⁺ transient and the subsequent clearance of Ca²⁺ appear to underlie synchronous transmitter release at the MNTB synapse.