Interplay between Na⁺/Ca²+ Exchangers and Mitochondria in Ca²+ Clearance at the Calyx of Held

Introduction

In presynaptic axon terminals, calcium plays a key role in activity-dependent changes in synaptic strength as well as for the release of neurotransmitter. Incoming Ca²⁺ during action potentials (APs) rapidly equilibrates with Ca²⁺ buffers and then decays by the concerted action of different Ca²⁺ clearance mechanisms (CCMs): plasma membrane Ca²⁺-ATPase (PMCA), Na⁺/Ca²⁺ exchanger (Na/CaX), smooth endoplasmic reticulum Ca²⁺-ATPase (SERCA), and mitochondria. Recent studies have shown that presynaptic CCMs have a strong influence on presynaptic residual calcium, which has been proposed as an underlying mechanism for short-term synaptic plasticity. It has been reported that presynaptic residual calcium after a period of high-frequency activity is caused by Ca²⁺ influx through a reverse-mode Na/Ca exchange or by a delayed Ca²⁺ release from mitochondria subsequent to the Ca²⁺ uptake (Tang and Zucker, 1997; Zhong et al., 2001). Investigating the quantitative role of different CCMs in nerve terminals will thus provide a basic understanding of the mechanisms underlying short-term plasticity.

There is evidence for presynaptic roles of PMCA in retinal bipolar cells (Zenisek and Matthews, 2000), for Na/CaX in cultured hippocampal neurons (Reuter and Porzig, 1995) and in cerebellar granule cells (Regehr, 1997), and for mitochondria in sympathetic ganglion (Peng, 1998). Previous studies on presynaptic CCMs aimed to determine the contribution of specific mechanisms but have not provided a comprehensive picture of how different CCMs interact with each other as a function of the cytosolic [Ca²⁺] attained after a stimulus. Considering that these mechanisms have different properties in terms of the affinity and the clearance capacity for Ca²⁺, intracellular calcium load might be one of the important factors that affect the relative contribution of CCMs (Fierro et al., 1998; Suzuki et al., 2002; Kim et al., 2003). In this respect, quantitative studies are required to elucidate the extent to which each CCM contributes depending on calcium load.

At the calyx of Held, a mammalian giant presynaptic terminal, [Ca²⁺]_i can be measured quantitatively using Ca²⁺ indicator dye introduced via a patch pipette. Recently, it has been reported that mitochondria contribute to the Ca²⁺ clearance at the calyx of Held and that mitochondrial Ca²⁺ uptake affects recovery from synaptic depression (Billups and Forsythe, 2002). However, Chuhma and Ohmori (2002) showed that the Ca²⁺ extrusion rate was reduced by inhibition of Na/CaX at the same preparation. Functional and immunocytochemical evidence indicates the existence of Na/CaX in the presynaptic terminals of other mammalian CNS synapses (Reuter et al., 1995; Regehr, 1997). Moreover, we recently described the role of a K⁺-dependent form of Na⁺/Ca²⁺ exchanger (NCKX) in peptidergic axon terminals and found that its distribution is polarized to the axon terminals in hypothalamic magnocellular neurons (Lee et al., 2002; Kim et al., 2003). The role of NCKX, however, has not yet been studied in CNS presynaptic terminals, probably because of the inaccessibility of most nerve terminals for intracellular ion exchange experiments. Here, we tested whether NCKX contributes to Ca²⁺ clearance at the calyx of Held and elucidated the dependence of the relative contributions of Na/CaX and other CCMs, especially mitochondria, on the intracellular calcium load.

Materials and Methods

Preparation of brainstem slices. Transverse brainstem slices containing the medial nucleus of the trapezoid body (MNTB) were prepared from 8- to 10-d-old Sprague Dawley rats (17 ± 4 g). Rats were decapitated, and brainstems were chilled in ice-cold low-calcium artificial CSF (aCSF) containing the following (in mm): 125 NaCl, 25 NaHCO₃, 2.5 KCl, 1.25 NaH₂PO₄, 2.5 MgCl₂, 0.5 CaCl₂, 25 glucose, 0.4 Na ascorbate, 3 myo-inositol, 2 Na pyruvate, at pH 7.4, when saturated with carbogen (95% O₂, 5% CO₂), and with an osmolarity of ∼320 mOsm. Isolated brainstems were glued onto the stage of a vibratome (VT1000S; Leica, Wetzlar, Germany), and 150- to 200-μm-thick transverse brainstem slices were cut from caudal to rostral in the same solution. Slices containing the MNTB (approximately four to five slices) were incubated at 37°C for 30 min in normal aCSF (as low-calcium aCSF above with 1 mm MgCl₂ and 2 mm CaCl₂) and thereafter maintained at room temperature (23-25°C) until required.

Electrophysiological recordings. Whole-cell patch-clamp recordings of calyces of Held were made under visual control using differential interference illumination in an upright microscope (BX50WI; Olympus, To-Tokyo, Japan). Calcium influx was evoked by applying depolarizing pulses in voltage-clamp mode. Patch pipettes with a resistance of 4.5-5.5 MΩ were used for recordings. The standard K⁺ pipette solution contained the following (in mm): 120 K gluconate, 30 KCl, 20 HEPES, 4 MgATP, 4 Na ascorbate, and 0.3 NaGTP at pH 7.3 (adjusted with KOH). For a K⁺-free pipette solution, K gluconate and KCl in the K⁺ pipette solution were replaced with equimolar tetraethylammonium (TEA)-Cl. Alternatively, to rule out the possibility of organic cations inhibiting the K⁺-independent form of Na/CaX (NCX), K⁺ ions in the K⁺ pipette solution were replaced with Li⁺ ions (Blaustein, 1977). Recordings were made in calyces of Held using an EPC-9 amplifier (HEKA Elektronik, Lambrecht, Germany). During recordings, series resistances were compensated up to 85% with a 10 μs lag time. Recordings were terminated when the series resistance exceeded 30 MΩ. Experiments were performed at 35 ± 1°C. All chemicals were obtained from Sigma (St. Louis, MO), except 5(6)-carboxyeosin diacetate (CE) and fura-2FF from Fluka (Buchs, Switzerland) and fura-4F from Molecular Probes (Eugene, OR).

Cytosolic Ca²⁺ measurements. The procedures for cytosolic Ca²⁺ measurement in slices have been described previously in detail (Kim et al., 2003). Ca²⁺ concentrations were measured by fluorescence imaging. Cells were loaded with fura-4F or fura-2FF (pentapotassium salts; 50 μm each) via patch electrodes. For fluorescence excitation, we used a polychromatic light source (xenon lamp based; Polychrome-II; TILL Photonics, Gräfelfing, Germany), which was coupled to the epiillumination port of an upright microscope (BX50; Olympus) via a quartz light guide and a UV condenser. Imaging was performed using a 60× water immersion objective (numerical aperture, 0.9; LUMPlanFl; Olympus) and an air-cooled slow-scan CCD camera (SensiCam; PCO, Kelheim, Germany). The monochromator and the CCD camera were controlled by a personal computer, running a custom-made software programmed with MicroSoft Visual C++ (version 6.0).

The ratio (R = F_iso/F₃₈₀) of fluorescence at the isosbestic wavelength (360 nm; F_iso) to that at 380 nm (F₃₈₀) was converted to [Ca²⁺]_i according to the following equation: (1)

Calibration parameters were determined by “in-cell” calibration as described by Lee et al. (2000). R_min values were measured using a Ca²⁺-free pipette solution containing 10 mm BAPTA. R_max values were obtained from in vitro measurements, because calyces of Held did not endure internal dialysis with high CaCl₂ (10 mm). The values for the calibration ratio at intermediate [Ca²⁺]_i were measured using a pipette solution containing 5 mm BAPTA and 3.5 mm CaCl₂ ([Ca²⁺]_i ≈ 540 nm) for fura-4F and a pipette solution containing 80 mm DPTA and 10 mm CaCl₂ ([Ca²⁺]_i ≈ 10 μm) for fura-2FF. The effective dissociation constant of fura-2 (K_eff) was calculated by measuring the fluorescence ratio at these intermediate [Ca²⁺]_i and by rearranging Equation 1 for K_eff. The K_d values of fura-4F and fura-2FF were calculated as 0.79 and 3.1 μm, respectively, from K_d = K_eff · (α + R_min)/(α + R_max), where α is the iso-coefficient (Zhou and Neher, 1993). To increase time resolution and minimize the photobleaching effect, we adopted a single-wavelength protocol (Helmchen et al., 1996; Lee et al., 2000) and pixels were binned by 8 × 8, which allowed exposure times of 5 ms. Images taken at 40 Hz with a single-wavelength excitation at 380 nm (F₃₈₀) were preceded and followed by images taken with excitation at isosbestic wavelengths (360 nm). F_iso (isosbestic fluorescence) values were linearly interpolated between points just before and after the period of excitation at 380 nm.

Calculation of total Ca²⁺ clearance rate. We measured the decay rate of free Ca²⁺ from the time derivative of the decay phase of a Ca²⁺ transient (d[Ca²⁺]_i/dt). Because genuine Ca²⁺ clearance rate of a cell is represented by the clearance rate of total calcium (d[Ca²⁺]_T/dt) rather than by decay rate of free calcium, d[Ca²⁺]_i/dt was converted to d[Ca²⁺]_T/dt using the following relationship: (2)

where κ_S and κ_B represent the calcium binding ratios of the endogenous and exogenous buffers, respectively (Neher and Augustine, 1992; Kim et al., 2003).

When the decay phase of a Ca²⁺ transient is fitted with the following biexponential function: (3)

the Ca²⁺ decay rate at the peak of the Ca²⁺ transient can be represented by a time derivative of the biexponential function estimated at t = 0 as follows: (4)

where A and λ represent amplitude and rate constant (inverse of time constant) of each component, respectively. In addition, according to Equation 2, the total Ca²⁺ clearance rate at the peak of a Ca²⁺ transient can be calculated by the following: (5)

Calculation of Ca²⁺ decay rate constant at the peak (λ_{t = 0}). Although the decay phase of Ca²⁺ transients in this study were best fitted with the biexponential function, each (fast or slow) component of the biexponential fit does not necessarily reflect the activity of a single entity of Ca²⁺ clearance mechanism, because the Ca²⁺ clearance caused by each CCM was not constant but is nonlinearly dependent on the [Ca²⁺]_i excursion from resting level (Δ[Ca²⁺]_i) (see Figs. 1 D,2 Bb, dotted lines). Thus, for statistical comparison of Ca²⁺ clearance measured from different cells and/or different experimental conditions, the decay rate at the peak was normalized by the peak Δ[Ca²⁺]_i level (Δ[Ca²⁺]_peak), resulting in a Ca²⁺ decay rate constant at the peak (λ_{t = 0}), which is defined as follows: (-d[Ca²⁺]_i/dt)_{t = 0}/Δ[Ca²⁺]_peak (Kim et al., 2003). In the particular case in which the Ca²⁺ decay is biexponential, λ_{t = 0} can be calculated by the following equation: (6)

Although the relationship between d[Ca²⁺]_i/dt and Δ[Ca²⁺]_i is not linear over the entire range of Δ[Ca²⁺]_i, the relationship is almost linear when Δ[Ca²⁺]_i is between 0.6 and 2.5 μm (see Figs. 1, 2, 3, 4, plots of d[Ca²⁺]_T/dt vs Δ[Ca²⁺]_i). In this range of Δ[Ca²⁺]_i, λ_{t = 0} is constant, independent of Δ[Ca²⁺]_i, especially when peak Δ[Ca²⁺]_i levels vary within a relatively narrow range. Thus, we adjusted the depolarizing pulse such that the peak Δ[Ca²⁺]_i level of a Ca²⁺ transient fell between 2 and 2.5 μm, and we used λ_{t = 0} as a parameter to statistically compare the Ca²⁺ clearance rates.

Calculation of relative contribution of a clearance mechanism. The contribution of a clearance mechanism, Φ, to the entire Ca²⁺ clearance at a given Δ[Ca²⁺]_i level has been described previously (Kim et al., 2003). From the time derivatives (d[Ca²⁺]_i/dt) of the decay phases of two Ca²⁺ transients before and after treatment with an inhibitor of Φ, total Ca²⁺ clearance rate (d[Ca²⁺]_T/dt) was calculated and plotted as a function of Δ[Ca²⁺]_i (Eq. 2). The difference between the polynomial fit (f_control) to the d[Ca²⁺]_T/dt curve under control condition and that (f_inhibitor) in the presence of the inhibitor was regarded as the contribution made by Φ, and the relative contribution made by Φ(R_Φ) to the entire Ca²⁺ clearance mechanism can be expressed as follows: (7)

Data analysis. Data were analyzed using IgorPro (version 4.1; WaveMetrics, Lake Oswego, OR). Statistical data are expressed as mean ± SEM, and n indicates the number of cells studied. The significance of differences between Ca²⁺ decay rate constants was evaluated using paired or nonpaired Student's t test using a significance level of 0.05.

Results

We investigated Ca²⁺ clearance mechanisms at the calyx of Held (Fig. 1A) by analyzing the decay of Ca²⁺ transients obtained from fluorescent Ca²⁺ imaging. Calyces of Held were loaded via a patch pipette with an internal solution containing 50 μm fura-4F or fura-2FF, and Ca²⁺ transients were evoked by applying a short depolarizing pulse (from a holding potential of -80 or -70 mV to 0 mV; 50 ms in duration). The decay phases of the Ca²⁺ transients were well fitted by biexponential functions (χ² < 0.05), and the fitting parameters in various experimental conditions are summarized in Table 1. The clearance is defined as a decay rate constant (τ^-1) in the case of a monoexponential decay. To obtain the parameter representing the Ca²⁺ clearance from biexponential Ca²⁺ decay rates, we adopted a weighted average of two decay rate constants (λ_{t = 0}) (see Materials and Methods), which is mathematically the same as the decay rate at the peak divided by the amplitude of a biexponential Ca²⁺ transient [(-d[Ca²⁺]_i/dt)_{t = 0}/Δ[Ca²⁺]_peak]. Thus, we refer to λ_{t = 0} as a Ca²⁺ decay rate constant at the peak (see Materials and Methods). Because the relative amplitude of the fast component was >80% in this study, the value for λ_t = 0 is quite close to τ_fast^-1 (Fig. 1C).

Na⁺/Ca²⁺ exchange is a major CCM at the calyx of Held To investigate the contribution of Na/CaXs to Ca²⁺ clearance, we examined the effect of [Na⁺]_o reduction (replacement of the 125 mm NaCl in the bathing solution with equimolar LiCl) on the Ca²⁺ decay. The calyces were stimulated by a depolarization pulse (50 ms) to 0 mV in voltage-clamp mode. With the standard K⁺ pipette solution (see Materials and Methods), [Na⁺]_o reduction significantly reduced the Ca²⁺ decay rate (Fig. 1Ba). Mean values for fast (τ_fast) and slow (τ_slow) time constants in control condition (Fig. 1Ba, black line) were 74.2 ± 4.1 and 975.4 ± 108.7 ms (n = 7), respectively. After [Na⁺]_o reduction (Fig. 1Ba, gray line), the increase in the fast time constant (τ_fast) was more significant (218.7 ± 13.5 ms; n = 7; paired t test, p < 0.01) than the increase in the slow time constant (τ_slow; 1428.2 ± 236.0 ms; n = 7; paired t test, p > 0.05) (Table 1). The preferential deceleration (approximately threefold) of the fast decay phase under the low [Na⁺]_o condition suggests that Na/CaX is a Ca²⁺ clearance mechanism of high-capacity and low-affinity at the calyx of Held.

We next tested whether the Na/CaX activity depends on intracellular K⁺. For this purpose, we recorded the Ca²⁺ transients using K⁺-free pipette solutions. Figure 1, Bb and Bc, shows the representative Ca²⁺ transients recorded with Li⁺ and TEA⁺ pipette solutions, respectively. The Ca²⁺ transients in normal [Na⁺]_o (black line) and after the Na⁺_o replacement (gray line) were superimposed for each recording condition. In normal [Na⁺]_o condition, the decay rate constants (λ_{t = 0}) of the Ca²⁺ transients recorded with Li⁺ and TEA⁺ pipettes were 7.18 ± 0.27 s^-1 (n = 6) and 6.69 ± 0.22 s^-1 (n = 3), respectively (Fig. 1C). These values are significantly lower than that with the K⁺ pipette (12.01 ± 0.72 s^-1; p < 0.01) (Fig. 1C). The slower decay rate constant in the K⁺-free internal condition suggests that NCKXs contribute to Ca²⁺ clearance at the calyx of Held. Under the K⁺-free internal condition, [Na⁺]_o reduction further decreased the Ca²⁺ decay rate (Fig. 1Bb,Bc, gray traces). When the Na⁺ gradient was reduced, the decay rate constants were reduced to 3.12 ± 0.17 s^-1 in the Li⁺ pipette condition and to 3.09 ± 0.25 s^-1 in the TEA⁺ pipette condition, which are similar to that in the K⁺ pipette condition (3.80 ± 0.37 s^-1; p > 0.05) (Fig. 1C). These results show that Na/CaX activity still remained even in the absence of internal K⁺, suggesting that both NCX and NCKX contribute to the calcium clearance at the calyx of Held.

To quantify the relative contributions of NCX and NCKX, the decrease in the Ca²⁺ decay rate constant (λ_{t = 0}) caused by low [Na⁺]_o in the K⁺ pipette condition was compared with those in the K⁺-free pipette condition. The relative contributions of NCKX according to the equations (λ_{t = 0,K_Na} - λ_{t = 0,Li_Na})/λ_{t = 0,K_Na} and (λ_{t = 0,K_Na} -λ_{t = 0,TEA_Na})/λ_{t = 0,K_Na} were estimated to be ∼41 and 45%, respectively (the first subscripts, K, Li, and TEA, represent major internal cations, and the second subscripts, Na and Li, represent major external cations). Similarly, the contributions of NCX according to (λ_{t = 0,Li_Na} -λ_{t = 0,K_Li})/λ_{t = 0,K_Na} and (λ_{t = 0,TEA_Na} - λ_{t = 0,K_Li})/λ_{t = 0,K_Na} were 28.2 and 24.0%, respectively. NCKX and NCX were responsible for ∼42 and 26% of total Ca²⁺ clearance, respectively. The overall contribution of Na/CaXs, including both NCX and NCKX, was calculated using (λ_{t = 0,K_Na} - λ_{t = 0,K_Li})/λ_{t = 0,K_Na} to be 68.6 ± 1.7%.

To estimate the relative contribution of Na/CaX to the entire Ca²⁺ clearance as a function of the Δ[Ca²⁺]_i level, the total Ca²⁺ clearance rate (-d[Ca²⁺]_T/dt) was calculated from the time derivative of the decay phase of a Ca²⁺ transient according to Equation 2, using a κ_S value of 40 (Helmchen et al., 1997) (Fig. 1D). From the polynomial fits to -d[Ca²⁺]_T/dt measured before (f_{K_Na}) (Fig. 1D, black solid line) and after (f_{K_Li}) (Fig. 1D, gray solid line) [Na⁺]_o reduction in the K⁺ pipette condition, we calculated the relative contribution of Na/CaX according to the equation (f_{K_Na} - f_{K_Li})/f_{K_Na} (Fig. 1D, open circles, right axis). The relative contribution of Na/CaX increased as Δ[Ca²⁺]_i increased to reach a maximum value (69%) at Δ[Ca²⁺]_i > 400 nm. We conclude that Na/CaX is the primary CCM in the calyx of Held in response to brief Δ[Ca²⁺]_i elevations in the range of 1-2 μm. Our experiments under different internal ionic conditions suggest that Na/CaX is a major Ca²⁺ clearance mechanism, and that both NCX and NCKX contribute to Na/CaX in the calyx of Held.

Calyces of Held in young rats [postnatal day 8 (P8) to P10] can follow discharge rates up to 200 Hz (Borst et al., 1995), and the calyx of Held in adult mice receives and follows higher input frequencies of up to 600 Hz (Wu and Kelly, 1993). We examined the role of Na/CaX in Ca²⁺ clearance when Ca²⁺ transients were evoked by repetitive depolarization pulses (2 ms in duration) at frequencies of 10, 20, or 50 Hz for 1 s. The Δ[Ca²⁺]_i during the 10 or 20 Hz stimulation underwent a fast rising phase and reached a plateau within 300 ms under control condition (Fig. 1E, black lines), but at 50 Hz stimulation, the fast rising phase was followed by a slower rising phase. Linear summation of Ca²⁺ transients predicts that the time required to reach the steady state is determined by the decay time constant of a single Ca²⁺ transient (Regehr et al., 1994; Helmchen et al., 1996). Consistently, after [Na⁺]_o reduction, the Δ[Ca²⁺]_i level continued to build up throughout the train of pulses (1 s), and thus Δ[Ca²⁺]_i at the end of the stimulation was greatly increased (Fig. 1E, gray lines). At a stimulation frequency of 50 Hz, the amplitudes of Δ[Ca²⁺]_i before and after [Na⁺]_o reduction were 1.88 ± 0.37 and 4.95 ± 0.19 μm, respectively (n = 3) (Fig. 1E). These results indicate that Na/CaXs dampen presynaptic Ca²⁺ load very efficiently. Such efficient Ca²⁺ clearance seems to be essential for presynaptic axon terminals to transduce high-frequency spike input to synaptic release of neurotransmitter and to maintain presynaptic Ca²⁺ homeostasis.

Discussion

Ca²⁺ signaling in axon terminals plays a crucial role in synaptic function. The Ca²⁺ clearance rate in axon terminals not only determines the duration and amplitude of a Ca²⁺ transient but also affects short-term plasticity by a mechanism involving residual calcium. In the present study, we quantified the contribution made by each putative CCM at the calyx of Held and demonstrate the role of NCKX in presynaptic Ca²⁺ clearance. In addition, we found that (1) Na/CaX, comprised of NCKX and NCX, is a major CCM in the calyx of Held; (2) PMCA is responsible for 23% of the Ca²⁺ clearance when Δ[Ca²⁺]_i is <1 μm; and (3) mitochondria contribute to Ca²⁺ clearance only when Na/CaX is inhibited or when the Ca²⁺ load is high enough to saturate Na/CaX capacity.