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Cell Biology International (2008) 32, 11361142 (Printed in Great Britain)
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
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.
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+]
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+]
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 (67
2.2 Calcium imaging
Xenopus nerve–muscle co-cultures were loaded with 4
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 150
3.1 Intraterminal Ca2+ accumulates in Ca2+-free extracellular solution
With the Ca2+ imaging technique, we continuously monitored the Fluo-4 labeled [Ca2+]
Intraterminal Ca2+ accumulates in Ca2+-free extracellular solution. (A) Fluo-4-loaded cultured Xenopus NMJ. Scale bar
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+]
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
In Ca2+-free extracellular solution, after BAPTA-AM application, the SSC frequency and amplitude were reduced (P
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, 10
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 (10
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 (IP
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 (1
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 100
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 10
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+]
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 100
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 IP
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+]
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.
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 2008doi:10.1016/j.cellbi.2008.05.001