Cell Biology International (2008) 32, 1136-1142 - Ruxin Li and others - Calcium near the release site is essential for basal ACh release in Xenopus

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Cell Biology International (2008) 32, 1136–1142 (Printed in Great Britain)

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Calcium near the release site is essential for basal ACh release in Xenopus

Ruxin Li, Qi Lei, Ge Song, Xiangping He and Zuoping Xie^*

Department of Biological Sciences and Biotechnology, National State Key Laboratory of Biomembrane and Membrane Biotechnology, Tsinghua University, Beijing 100084, PR China

Abstract

Extracellular calcium is essential for neurotransmitter release, but the detailed mechanism by which Ca²⁺ regulates basal synaptic release has not yet been fully explored. In this study, calcium imaging and the whole-cell patch-clamp technique were used to investigate the role of Ca²⁺ in basal acetylcholine (ACh) release in the Xenopus neuromuscular junction and in isolated myocytes exogenously loaded with ACh. Carried out in normal and Ca²⁺-free extracellular solution, the results indicate that Ca²⁺ near the release site is essential for basal neurotransmitter release.

Keywords: Calcium, Basal neurotransmitter release, Spontaneous synaptic currents.

^*Corresponding author. Tel.: +86 10 6278 8677; fax: +86 10 6277 2271.

1 Introduction

Neurotransmitter release is a Ca²⁺-dependent process (Pietrobon, 2005). Synaptic transmission is initiated when an action potential triggers release from a presynaptic terminal (Kato, 1969). Katz showed that a brief, transient Ca²⁺ influx induced by an action potential is directly required for release. Release is produced directly by Ca²⁺ interacting with a molecular Ca²⁺ sensor that enables vesicular exocytosis (Stevens, 2003). The depolarization of an action potential directly triggers phasic release when presynaptic intracellular Ca²⁺ concentration ([Ca²⁺]i) is raised. Calcium, therefore, not only triggers exocytosis but also seems to ‘prime’ the process, perhaps by mobilizing secretory granules to docking sites at the membrane (Zucker, 1993). Some studies conclude that external Ca²⁺ is the only immediate ionic requirement for depolarization to evoke transmitter release (Katz, 1971).

At rest, synapses have a finite but low probability of release, causing spontaneous exocytotic events (Sudhof, 2004). Spontaneous release is not simply a by-product of synaptic signaling but is important in its own right (Glitsch, 2008). At the Xenopus neuromuscular junction (NMJ), spontaneous synaptic currents (SSCs) are induced by spontaneous secretion of individual ACh-containing vesicles from motor nerve terminals independent of action potentials (Song et al., 1997). Since this may still depend on changes in [Ca²⁺]i, and hence cannot be considered to be spontaneous in the true meaning of the word, in this paper we refer to the neurotransmitter release that occurs in the absence of action potentials and without any apparent stimulus as ‘basal release’ (Fu and Huang, 1994; Glitsch, 2008). Some experiments indicate that transmembrane Ca²⁺ influx probably does not play a major role in basal release of quanta of neurotransmitter from neurons (Cummings et al., 1996). But intraterminal Ca²⁺ is associated with basal release; for example, at the frog NMJ, intraterminal Ca²⁺ is correlated with the frequency of miniature end plate potentials (Angleson and Betz, 2001). Increasing evidence shows that neurotransmitter discharge is modulated by changes in [Ca²⁺]i (Glitsch, 2007). The cellular and molecular complexities of the processes involved in intracellular Ca²⁺-dependent secretion are beginning to be unraveled in detail (Betz and Angleson, 1998).

In contrast to the complexity of central nervous system (CNS) synapses (He et al., 2000), the Xenopus NMJ offers a simple and easily accessible model to study the role of Ca²⁺ in basal ACh release. In this study, Ca²⁺ imaging and the whole-cell patch-clamp technique were used. SSCs were recorded from innervated myocytes in Xenopus nerve–muscle co-cultures. Further, SSCs from a non-neuronal preparation were also examined as the quantal secretion of ACh from isolated myocytes (autoreception) exogenously loaded with ACh. Most experiments were carried out in Ca²⁺-free extracellular solution with acute application of drugs to the bath. Our results indicate that Ca²⁺ near the release site is essential for basal neurotransmitter release.

2 Material and methods

2.1 Culture preparation

Xenopus nerve–muscle co-cultures were prepared according to an established procedure (Lu et al., 1992). In brief, the neural tube and associated myotomal tissue of Xenopus embryos at stages 20–22 were dissociated in Ca²⁺- and Mg²⁺-free saline supplemented with EDTA (67mM NaCl, 1.6mM KCl, 8mM Hepes, 1mM EDTA pH 7.8) for 15–20min. The cells were grown on glass coverslips for 24h at room temperature (20–22°C). The culture medium consisted (v/v) of 30% Leibovitz L-15 medium (Sigma), 1% FBS (Gibco), and 69% Ringer's solution (115mM NaCl, 2mM CaCl2, 2.5mM KCl, 10mM Hepes pH 7.4). In the isolated myocyte preparation, the neural tube was removed and the FBS in the culture medium was increased to 2%.

2.2 Calcium imaging

Xenopus nerve–muscle co-cultures were loaded with 4μmol/L Fluo-4-AM (Molecular Probes, USA) at room temperature for 30min, followed by three washes and a 15-min incubation for further de-esterification of the Fluo-4-AM before imaging. Cells grown on glass coverslips in culture dishes were directly imaged on an Olympus DSU (IX71) confocal microscope using a 40× numerical aperture. An Andor iXon DU897 EMCCD camera was used for bright-field and fluorescence image acquisition. Andor iQ imaging software was used for hardware control, image acquisition, and image analysis. The sampling rate was one frame every 2s, and the exposure time was 50ms. Changes in [Ca²⁺]i were calculated by measuring the Fluo-4 fluorescence intensity of a circle placed at the center of the cell body or the center of the NMJ. The intensity values were then subtracted from the background intensity, measured in cell-free regions. The intensity in a normal extracellular solution was set at 1, and the intensity in Ca²⁺-free extracellular solution and 5min after BAPTA-AM application (1,2-bis-o-aminophenoxy-ethane-N,N,-N′,N′-tetraacetic acid tetraacetoxy-methyl ester; BIOMOL, USA) were measured and normalized to the intensity in normal extracellular solution.

2.3 Electrophysiology

SSCs were recorded from myocytes innervated by spinal motoneurons using whole-cell voltage-clamp recording techniques (Lu et al., 1992). The solution inside the recording pipette contained 150mM KCl, 1mM NaCl, 1mM MgCl2, and 10mM Hepes buffer pH 7.2. For experiments performed in the absence of external Ca²⁺, the culture medium was replaced with a Ca²⁺-free extracellular solution containing (in mM) 115 NaCl, 2 MgCl2, 10 Hepes, 3 EGTA, and 0.1% BSA. In the experiments with single myocytes, the myocytes were loaded with ACh through a whole-cell pipette containing the internal solution supplemented with 25mmol/L ACh (Dan and Poo, 1992). Membrane currents in all recordings were monitored by a patch-clamp amplifier (Axon-200B, Axon, USA), with a current signal filter at 5kHz. The membrane potentials of the muscle cells were generally in the range of −55 to −75mV and were voltage-clamped at −70mV after measuring the membrane potential. The whole-cell electrode resistance was 3–8MΩ. All recordings were performed at room temperature. Data were stored in a PC computer using a Digidata 1320A interface and analyzed using pCLAMP 9.2 software (Axon, USA). To measure the changes in neurotransmitter release, the SSC frequency and amplitude in a 5-min period immediately before drug application were averaged as controls. The changes in SSC frequency and amplitude were measured and analyzed by averaging a 5-min period of recording starting from 1min after drug application. Data were analyzed for statistically significant differences using paired Student's t-test. Compiled data are expressed and graphed as mean±SEM, with “n” denoting the number of cells studied.

3 Results

3.1 Intraterminal Ca²⁺ accumulates in Ca²⁺-free extracellular solution

With the Ca²⁺ imaging technique, we continuously monitored the Fluo-4 labeled [Ca²⁺]i in the neuron soma, the myocyte and the NMJ. With Fluo-4-AM, an improved analog of the Ca²⁺ indicator, the green fluorescence intensity reflects the [Ca²⁺]i level. Each experiment was first performed in normal extracellular solution. Light green fluorescence was seen in the neuron soma, the myocyte and the NMJ (Fig. 1A and B). Five minutes later, the normal extracellular solution was exchanged for Ca²⁺-free extracellular solution. The fluorescence intensity of the neuron soma and the myocyte did not change. However, the intensity of the NMJ increased (Fig. 1A–C). Five minutes after applying the cell-permeable Ca²⁺ chelator BAPTA-AM (50μM), the intensity in the neuron soma, myocyte and NMJ decreased (Fig. 1A–C).

Fig. 1

Intraterminal Ca²⁺ accumulates in Ca²⁺-free extracellular solution. (A) Fluo-4-loaded cultured Xenopus NMJ. Scale bar=10μm. N: neuron. M: myocyte. NMJ: neuromuscular junction. BF: bright-field. Normal: in normal extracellular solution. Ca²⁺-free: in Ca²⁺-free extracellular solution. BAPTA-AM: bath application of BAPTA-AM at a final concentration of 50μM. The red circle shows the location of the NMJ. (B) Magnification of the NMJ area in (a). Scale bar=2μm. (C) Columns show the normalized fluorescence intensity of the neuron soma (black), myocyte (light grey) and NMJ (dark grey) in normal, Ca²⁺-free and BAPTA-AM-containing extracellular solution.

3.2 BAPTA-AM reduces SSC frequency and amplitude in Ca²⁺-free but not in normal extracellular solution

To investigate the particular relationship between [Ca²⁺]i and ACh release, we recorded SSCs from cultured innervated myocytes both in normal and in Ca²⁺-free extracellular solution (Fig. 2A). The mean frequency and amplitude of SSCs in normal extracellular solution were 13.28±3.26 events/min and 1233.45±104.58pA, mean±SD, n=8, respectively. The mean frequency and amplitude of SSCs in Ca²⁺-free extracellular solution were 5.85±0.96 events/min and 501.68±85.11pA, mean±SD, n=17, respectively. Without extracellular Ca²⁺, the frequency and amplitude of SSCs were reduced (P<0.05, t-test).

Fig. 2

BAPTA-AM reduces SSC frequency and amplitude in Ca²⁺-free but not normal extracellular solution. (A) Phase-contrast photomicrograph of a Xenopus myocyte innervated by a spinal motoneuron clamped by a patch-clamp pipette. Scale bar=10μm. (B) Two examples of SSCs recorded from cultured Xenopus myocytes innervated by spinal motoneurons in Ca²⁺-free (upper) and normal extracellular solution (lower) before and after BAPTA-AM application (50μM). BAPTA-AM was added to the bath 5min after the start of recording (the first minute of drug application was excluded from the statistics). (C) Normalized SSC frequency was reduced after BAPTA-AM application in Ca²⁺-free extracellular solution (dark grey, n=6, *P<0.05, **P<0.005), but did not change in normal extracellular solution (light grey, n=6). (D) Normalized SSC amplitude was reduced after BAPTA-AM application in Ca²⁺-free extracellular solution (dark grey, n=6, *P<0.05, **P<0.005), but did not change in normal extracellular solution (light grey, n=6).

In Ca²⁺-free extracellular solution, after BAPTA-AM application, the SSC frequency and amplitude were reduced (P<0.001 and P<0.05, respectively, t-test; Fig. 2B–D). But in normal extracellular solution, neither the frequency nor the amplitude of SSCs was affected by BAPTA-AM (Fig. 2B–D).

3.3 Mitochondrial Ca²⁺ does not affect SSC frequency and amplitude in Ca²⁺-free extracellular solution

Mitochondria can be considered as a Ca²⁺ store under some circumstances (Bootman et al., 2001). Application of the mitochondrial permeability transition pore (mPTP) blocker cyclosporin A (CSA, 10μM) (Bernardi, 1999), the mitochondrial Ca²⁺ depolarizer and depleter protonophore carbonyl cyanide 4-(trifluoromethoxy) phenyl hydrazone (FCCP, 1μM) (Yang et al., 2003), or nocodazole (15μM), which respectively stabilize and disrupt microtubules (MTs), and decrease the potential and Ca²⁺ in mitochondria (Mironov et al., 2005) (Fig. 3A), did not change the frequency (Fig. 3B) and amplitude (Fig. 3C) of SSCs in Ca²⁺-free conditions.

Fig. 3

Mitochondrial Ca²⁺ does not affect SSC frequency and amplitude in Ca²⁺-free extracellular solution. (A) Examples of SSCs recorded from cultured Xenopus myocytes innervated by spinal motoneurons in Ca²⁺-free extracellular solution before and after CSA (10μM), FCCP (1μM) or nocodazole (15μM) application. Drugs were added to the bath 5min after the start of recording (the first minute of drug application was excluded from the statistics). (B) Normalized SSC frequency did not change in Ca²⁺-free extracellular solution after application of CSA (black, n=4), FCCP (light grey, n=5) and nocodazole (dark grey, n=3). (C) Normalized SSC amplitude did not change in Ca²⁺-free extracellular solution after application of CSA (black, n=4), FCCP (light grey, n=5) and nocodazole (dark grey, n=3).

3.4 Ca²⁺ from endoplasmic reticulum does not affect SSC frequency and amplitude in Ca²⁺-free extracellular solution

The endoplasmic reticulum (ER) is a dynamic Ca²⁺ pool that plays an important role in cellular responses to both electrical and chemical signals. It is well known that inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) are major pathways of ER Ca²⁺ release (Collin et al., 2005; Simpson et al., 1995). Xestospongin C (XeC) is a potent, membrane-permeable IP3R blocker (Gafni et al., 1997). At high concentrations (100μmol/L), ryanodine acts as a RyR antagonist (He et al., 2000). In Ca²⁺-free extracellular solution, application of XeC, a high concentration ryanodine, or both (Fig. 4A) did not affect SSC frequency (Fig. 4B) and amplitude (Fig. 4C). These results indicate that basal ACh release in the cultured Xenopus NMJ is independent of both ER and mitochondrial Ca²⁺ stores in Ca²⁺-free extracellular solution.

Fig. 4

ER Ca²⁺ does not affect SSC frequency and amplitude in Ca²⁺-free extracellular solution. (A) Examples of SSCs recorded from cultured Xenopus myocytes innervated by spinal motoneurons in Ca²⁺-free extracellular solution before and after application of XeC (1μM), ryanodine (100μM) or their co-application. Drugs were added to the bath 5min after the start of recording (the first minute of drug application was excluded from the statistics). (B) Normalized SSC frequency did not change in Ca²⁺-free extracellular solution after application of XeC (black, n=5), ryanodine (light grey, n=5) and their co-application (dark grey, n=4). (C) Normalized SSC amplitude did not change in Ca²⁺-free extracellular solution after application of XeC (black, n=5), ryanodine (light grey, n=5) and their co-application (dark grey, n=4).

3.5 Ryanodine reduces ACh-loaded isolated myocyte SSC frequency but not amplitude in Ca²⁺-free extracellular solution

Basal ACh is released not only from the Xenopus NMJ but also from ACh-loaded single myocytes (Dan and Poo, 1992). In Ca²⁺-free extracellular solution, we recorded SSCs from ACh-loaded single myocytes (Fig. 5A and B), and the SSC frequency was reduced by application of 100μM ryanodine (Fig. 5B–D) (P<0.05, t-test). However, ryanodine did not affect the SSC amplitude.

Fig. 5

Ryanodine reduces ACh-loaded isolated myocyte SSC frequency but not amplitude in Ca²⁺-free extracellular solution. (A) Phase-contrast photomicrograph of an isolated Xenopus myocyte loaded with 10mM ACh and clamped by a patch-clamp pipette. Scale bar=10μm. (B) An example of SSCs recorded from an ACh-loaded Xenopus myocyte in Ca²⁺-free extracellular solution before and after ryanodine application (100μM). Ryanodine was added to the bath 5min after the start of recording (the first minute of drug application was excluded from the statistics). (C) Normalized SSC frequency was reduced after ryanodine application in Ca²⁺-free extracellular solution (n=3, *P<0.05, **P<0.005). (D) Normalized SSC amplitude did not change after ryanodine application (n=3).

4 Discussion

The major finding was that the Ca²⁺ near the release site is essential for basal neurotransmitter release, irrespective of where the Ca²⁺comes from (Ca²⁺ influx, NMJ terminal or isolated myocyte SR).

In cultured Xenopus NMJs, pulsatile current events (SSCs) represent the basal exocytosis of ACh-containing synaptic vesicles at the developing NMJs (Xie and Poo, 1986). The large-amplitude variability presumably results from immature filling of the synaptic vesicles (Evers et al., 1989). Our results showed that the frequency and amplitude of SSCs in Ca²⁺-free extracellular solution were lower than in normal extracellular solution, although the intraterminal [Ca²⁺]i in the Ca²⁺-free condition was higher (Fig. 2). A possible mechanism is that in the Ca²⁺-free condition, the instantaneous Ca²⁺ influx near the release site and Ca²⁺-induced Ca²⁺ release (CICR) were completely abolished. Others have also shown that changing the extracellular Ca²⁺ concentration influences spontaneous neurotransmitter release: increasing the extracellular Ca²⁺ concentration increases spontaneous neurotransmitter discharge whereas decreasing it reduces spontaneous exocytosis (Llano et al., 2000; Yamasaki et al., 2006).

BAPTA-AM combines with cytosolic Ca²⁺, including intraterminal Ca²⁺. It reduced the SSC frequency in Ca²⁺-free extracellular solution, indicating that cytosolic Ca²⁺ concentration is correlated with basal synaptic release. This result is in accord with previous studies (Angleson and Betz, 2001; Blochl and Thoenen, 1996; He et al., 2000; Tse et al., 1997). At rest, the cytosolic dissociated Ca²⁺ concentration is very low, about 100nM (Plieth, 2005). Without extracellular Ca²⁺, cytosolic dissociated Ca²⁺ is not sufficient to maintain basal neurotransmitter release. The Ca²⁺ accumulation in terminals was found (Fig. 1) in Ca²⁺-free extracellular solution contributed to basal neurotransmitter release.

In some cell types, it appears that mitochondria have sufficient Ca²⁺ at rest to participate in intracellular Ca²⁺ signaling. Propagating Ca²⁺ waves have been described following regenerative activation of permeability transition (Ichas and Mazat, 1998). But our results showed that blocking the mPTP Ca²⁺ release from mitochondria with CSA or by depleting mitochondrial Ca²⁺ with FCCP did not affect the amplitude and frequency of neurotransmitter release (Fig. 3). ER and mitochondria take up cytoplasmic Ca²⁺ and correspondingly release it into the cytoplasm via CICR or the mPTP. Mitochondria-bound MTs are depolymerized by nocodazole. Changes in MT structure promote opening of the mPTP, but do not induce activation of CICR, because the disruption of MTs spatially segregates ER from mitochondria (Mironov et al., 2005).

The ER is the largest single intracellular organelle, composed of an interconnected, internally continuous system of tubules and cisterns, which extend from the nuclear envelope to axons and presynaptic terminals, as well as to dendrites and dendritic spines. Calcium stored within the ER of neurons is an important source of signal Ca²⁺ that is released upon activation of either IP3Rs or RyRs (Henzi and MacDermott, 1992; Kostyuk and Verkhratsky, 1994). According to the previous studies, at high concentrations, ryanodine binding irreversibly inhibits channel opening (Serysheva, 1998). We applied a high (100μM) concentration of ryanodine in Ca²⁺-free extracellular solution, and the SSCs did not change detectably. Then we assessed whether the IP3R inhibitor XeC changed the SSCs' frequency. This treatment was still indistinguishable from control. Therefore, we inferred that these two channels may have complementary functions to some degree. However, when we used ryanodine and XeC at the same time, the SSC frequency and amplitude still did not differ from control. In a recent review, Verkhratsky (2005) considered that Ca²⁺ release channels in the ER membrane provide for synaptic excitability. But our surprising result, that basal synaptic release was independent of ER Ca²⁺, indicated that it is not necessary for basal transmitter release in the condition of Ca²⁺-free extracellular solution (Fig. 4).

At the NMJ, the myocyte is the post-synaptic cell, but in the ACh-loaded single myocyte model, the myocyte can be considered as both pre- and post-synaptic. It does not have a terminal structure, and the sarcoplasmic reticulum (SR) near the release site may provide Ca²⁺ for basal release. So, blocking the RyRs significantly reduced SSC frequency (Fig. 5). That there was no change in amplitude can be explained by quantal release (Dan and Poo, 1992).

To summarize, in a normal extracellular solution, external Ca²⁺ and strong Ca²⁺ influx near the release site maintained basal neurotransmitter release with high frequency and amplitude. In extracellular Ca²⁺-free conditions, basal neurotransmitter release depended only on [Ca²⁺]i near the release site, irrespective of where the Ca²⁺came from (NMJ terminal or isolated myocyte SR). These results indicate that Ca²⁺ near the release site is essential for basal neurotransmitter release.

Furthermore, consistent with Kano's group (Yamasaki et al., 2006), we showed that SSCs could still be recorded in the presence of an intracellular Ca²⁺ buffer (BAPTA-AM) and in the absence of external Ca²⁺ (Fig. 2), suggesting that this proportion of neurotransmitter release probably reflects truly spontaneous release.

Recent research indicates that Ca²⁺ is not only necessary for neurotransmitter exocytosis (Brose et al., 1992) but also plays an important role in endocytosis (Ceccarelli and Hurlbut, 1980). Moreover, of the two isoforms of synaptobrevin (a protein thought to play a central role in neurotransmitter release at synapses (Li and Chin, 2003; Sorensen, 2005; Sudhof, 2004)), synaptobrevin-1 appears to be associated with spontaneous release (Humeau et al., 2000). Current opinion is that there are two or more independent release machineries with different Ca²⁺ dependencies, responsible for action potential-evoked release and basal release (Glitsch, 2008). Thereby, the particular mechanism of Ca²⁺-dependent basal neurotransmitter release needs to be further investigated.

Acknowledgements

This work was supported by a grant from the Major State Basic Research Development Program of China (973 Program) (Grant No. 2005CB522503).

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Received 12 August 2007/28 January 2008; accepted 6 May 2008

doi:10.1016/j.cellbi.2008.05.001

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