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Control of the calcium concentration involved in acetylcholine release and its facilitation: an additional role for synaptic vesicles?
Pergamon
PII:
Neuroscience Vol. 85, No. 1, pp. 85–91, 1998 Copyright 1998 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/98 $19.00+0.00 S0306-4522(97)00591-5
CONTROL OF THE CALCIUM CONCENTRATION INVOLVED IN ACETYLCHOLINE RELEASE AND ITS FACILITATION: AN ADDITIONAL ROLE FOR SYNAPTIC VESICLES? P. FOSSIER,* M.-F. DIEBLER, J.-P. MOTHET, M. ISRAEL, L. TAUC and G. BAUX Laboratoire de Neurobiologie Cellulaire et Mole´culaire, C.N.R.S., 91198 Gif sur Yvette cedex, France Abstract––2,5-Diterbutyl-1,4-benzohydroquinone, a specific blocker of Ca2+-ATPase pumps, increased acetylcholine release from an identified synapse of Aplysia, as well as from Torpedo and mouse caudate nucleus synaptosomes. Because 2,5-diterbutyl-1,4-benzohydroquinone does not change the presynaptic Ca2+ influx, the enhancement of acetylcholine release could be due to an accumulation of Ca2+ in the terminal. This possibility was further checked by studying the effects of 2,5-diterbutyl-1,4benzohydroquinone on twin pulse facilitation, classically attributed to residual Ca2+. While preventing the fast sequestration of Ca2+ by presynaptic organelles, 2,5-diterbutyl-1,4-benzohydroquinone magnified both twin pulse facilitation observed under low extracellular Ca2+ concentration and twin pulse dysfacilitation observed under high extracellular Ca2+ concentration. Thus, it is concluded that 2,5-diterbutyl-1,4-benzohydroquinone, by preventing Ca2+ buffering near transmitter release sites, modulates acetylcholine release. As 2,5-diterbutyl-1,4-benzohydroquinone was also shown to decrease by 50% the uptake of 45Ca2+ by isolated synaptic vesicles, we propose that synaptic vesicles can control the presynaptic Ca2+ concentration triggering the release of neurotransmitter. 1998 IBRO. Published by Elsevier Science Ltd. Key words: acetylcholine release, synaptic vesicles, Ca2+, facilitation, Ca2+-ATPase pumps.
vesicles.4,5,16,28,33 Third, they were shown to have an ATP-dependent Ca2+ uptake system, with a low Km for Ca2+, which allows them to be effective in the normal physiological control of the concentration of Ca2+ in the terminal.18,22,27 The aim of the present work was to check whether a modulation of synaptic transmission could be achieved by a presynaptic Ca2+ store located in the vicinity of transmitter release sites. For this purpose, we have suppressed the ability of these stores to accumulate Ca2+ by using 2,5-diterbutyl-1,4benzohydroquinone (tBuBHQ), a specific blocker of Ca2+-ATPase pumps,23,29,31 and have checked whether this treatment induces changes in transmitter release at different cholinergic synapses. We indeed observed an increase in acetylcholine (ACh) release from various identified cholinergic synapses of Aplysia, as well as from Torpedo and mouse caudate nucleus synaptosomes. The next step was to identify the presynaptic structures which may be involved. As synaptic vesicles are present at the terminals and are known to accumulate Ca2+, we have investigated the action of tBuBHQ on the uptake of 45Ca2+ by isolated synaptic vesicles.
Calcium transients due to the opening of voltagegated Ca2+ channels are well known to play a primary role in triggering transmitter release. In addition, Ca2+ storage organelles could also play a crucial role in controlling Ca2+ concentration, because they can perform as sinks as well as sources for free calcium.38 The endoplasmic reticulum and its specializations are present in almost all parts of the neuron, including synaptic terminals. These Ca2+ storage organelles can act as Ca2+ buffering systems through their Ca2+-ATPases, which enable rapid sequestration of cytoplasmic calcium,32,34 and they are able to release Ca2+ in the cytosol via the activation of inositol trisphosphate receptors or ryanodine receptors.15,34,38,42 However, little is known on the nature of the intracellular structures participating in the fast buffering of Ca2+ ions in the immediate vicinity of release sites. Synaptic vesicles are good candidates for participating in Ca2+ buffering for three reasons. First, they accumulate at the nerve terminals and occupy strategic positions at the active zone.8 Second, ultrastructural studies have demonstrated the accumulation of Ca2+ within *To whom correspondence should be addressed. Abbreviations: ACh, acetylcholine; ASW, artificial seawater; EGTA, ethyleneglycolbis(aminoethyl ether)tetra-acetate; EPSC, excitatory postsynaptic current; IPSC, inhibitory postsynaptic current; tBuBHQ, 2,5diterbutyl-1,4-benzohydroquinone.
EXPERIMENTAL PROCEDURES
Materials . Aplysia californica were purchased from Marinus (Long Beach, CA, U.S.A.) and Torpedo marmorata from the 85
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Marine Stations of Arcachon and Roscoff (France). Concentrated solutions of tBuBHQ (Aldrich) and thapsigargin (Sigma) were made in dimethylsulfoxide and diluted as necessary. The highest concentration of dimethylsulfoxide attained in the incubation medium was 1‰; and, although it had no effect on synaptic transmission in Aplysia or ACh release from synaptosomes, it was added to all controls. 45 CaCl2 (5–50 mCi/mg Ca2+) was from Amersham. All other chemicals were of the highest purity from common commercial sources. Electrophysiological methods Inhibitory postsynaptic currents (IPSCs) were recorded in the postsynaptic neuron (B3 or B6) following the stimulation of the cholinergic presynaptic neuron (B4 or B5) in the buccal ganglion of Aplysia. Excitatory postsynaptic currents (EPSCs) were elicited in the cell R15 (abdominal ganglion) by stimulation of the presynaptic axon located in the right pleuro-abdominal connective. This axon forms a cholinergic synapse on R15. The protective tissue sheath covering the neurons was removed with fine forceps and the preparation was pinned to the bottom of an experimental chamber. During the experiment, the ganglion was continuously perfused with artificial seawater (ASW; 460 mM NaCl, 10 mM KCl, 11 mM CaCl2, 25 mM MgCl2, 28 mM MgSO4, 10 mM Tris–HCl buffer, pH 7.8) at 0.5–1 ml/min flow rate. Neurons were each impaled with two microelectrodes filled with 3 M KCl. The presynaptic neuron was polarized at 50 mV with a manual current device and the action potential was evoked by a constant depolarizing step. The consecutive Cl-dependent IPSC was recorded in the postsynaptic neuron clamped at 80 mV. The use of lowresistance, KCl-filled electrodes led to a leakage of Cl ions from the electrode, resulting in the loading of the postsynaptic neuron with Cl ions and thus in a shift in the Cl equilibrium potential that would increase the size of the postsynaptic response. To take into account this problem, the reversal potential of the response was regularly reassessed throughout the experiments and the amplitude of the response was expressed as a conductance. Facilitation of the postsynaptic response was measured in paired-pulse experiments with a 15-ms time interval between the two stimuli. Facilitation was quantified by measuring the ratio a2/a1, where a1 is the amplitude of the postsynaptic response evoked by the first action potential and a2 the amplitude of the second postsynaptic response. a2 was measured 15 ms after the peak of the first response by extrapolating the shape of the first response beyond the beginning of the second response according to its monoexponential decay.12 Values are expressed either as meanS.D. from at least 20 values normalized to 100% taken from at least four independent experiments or as means with their 95% confidence intervals calculated from at least 20 individual values, using Prism Software. Biochemical methods Measurement of evoked acetylcholine release by synaptosomes. Synaptosomes were purified from the electric organ of Torpedo, according to the procedure of Israe¨l et al.21 and Morel et al.30 A crude synaptosomal fraction from mouse caudate nucleus was prepared as follows. Slices of the caudate nucleus from one to two mouse brains were washed in a large volume (200 ml) of mammalian Krebs for about 60 min at room temperature. They were then homogenized in 200–300 µl of Krebs medium by repetitive sucking through a blue Eppendorf tip. After a 10-fold dilution with Krebs, this homogenate was filtered through a 50-µm nylon gaze and centrifuged at 4342g for 20 min at 4C. The
resulting pellet (crude P2 synaptosomal fraction) was then resuspended in 500 µl of Krebs medium. ACh release from synaptosomes was monitored by continuous recording, using the choline oxidase chemiluminescent procedure described by Israe¨l and Lesbats.19,20 Release was triggered by addition of 50 mM KCl and 5 mM Ca2+. 45 Ca uptake by synaptic vesicles. Synaptic vesicles from the electric organ of Torpedo were purified on a sucrose sedimentation gradient as described by Israe¨l et al.22 Vesicles were preincubated in the presence of tBuBHQ or vehicle for 5 min at room temperature before initiating 45Ca uptake. The ATP-dependent uptake reaction was performed as described previously by Israe¨l et al.22 in the presence of 1 mM ATP, 2 mM MgCl2, 80 µM CaCl2 and 45Ca (40 d.p.m./pmol, final specific activity). Incubation was stopped after 5 or 10 min by vacuum filtration on GF/C glass fiber filters (Whatman), followed by a 3-ml wash (2 mM EGTA in 400 mM KCl, 10 mM Tris buffer, pH 7.1). Radioactivity bound to the filters was measured by scintillation counting in 3 ml Ready Protein (Beckman). Nonspecific uptake measured in the absence of ATP was subtracted from total uptake.
RESULTS
Effect of 2,5-diterbutyl-1,4-benzohydroquinone on evoked postsynaptic responses in Aplysia ganglia Bath application of tBuBHQ (5 µM) increased the amplitude of the IPSC evoked by a presynaptic action potential in the buccal ganglion. The effect of tBuBHQ appeared after a 20-min delay and was maximal after 45 min of continuous application. At the plateau, the amplitude of the IPSC was increased by 25.27.3% (Fig. 1A) compared to the control value. This potentiating effect remained at the same level for at least 90 min (i.e. the duration of the experiment) and persisted when the preparation was washed out with normal ASW. Identical studies were carried out on an excitatory cholinergic synapse in the abdominal ganglion (Fig. 1B). The application of 5 µM tBuBHQ resulted in an increase of the EPSC amplitude by 28.26.6% with respect to controls.
Effect of 2,5-diterbutyl-1,4-benzohydroquinone on acetylcholine release from isolated nerve endings Presynaptic action of tBuBHQ was further studied on vertebrate isolated cholinergic nerve endings, using synaptosomes purified from the Torpedo electric organ and from the mouse caudate nucleus. Figure 2 shows that, in both preparations, tBuBHQ had a stimulatory effect on the release of ACh evoked by KCl depolarization. This effect was dose dependent and reached a maximum at 10 µM tBuBHQ. ACh release was then increased about four-fold in Torpedo synaptosomes and three-fold in mouse synaptosomes, with respect to control preparations. The stimulating action of tBuBHQ on ACh release by synaptosomes was reduced or absent when ACh release was evoked by a Ca2+ ionophore, A23187
Intracellular calcium concentration and acetylcholine release
Fig. 1. tBuBHQ increases the amplitude of evoked postsynaptic responses. (A) Typical potentiation of the IPSC recorded in the buccal ganglion as a function of time of exposure to 5 µM tBuBHQ. Insets: IPSC (upper trace) evoked by a presynaptic action potential (lower trace) in control conditions (a) and after the complete effect of tBuBHQ (b). Calibrations: horizontal bar=20 ms; vertical bars=200 nS (upper trace) and 20 mV (lower trace). (B) Typical potentiation of the EPSC recorded in the abdominal ganglion. Insets show EPSC recordings in control conditions (a) and after application of the drug (b). Calibrations: horizontal bar=20 ms; vertical bar=100 nS.
(data not shown), which induced a large and non-localized entry of Ca2+ into the synaptosomes. Calcium dependency of the effect of 2,5-diterbutyl-1,4benzohydroquinone In order to assess the dependency of the synaptic effects of tBuBHQ on the Ca2+ concentration within the terminal, three series of experiments were conducted on the inhibitory synaptic couple of Aplysia. The influx of Ca2+ into the terminal during the action potential was varied by manipulating the Ca2+ and Mg2+ concentrations in the seawater bath. The effect of tBuBHQ on the amplitude of the IPSC was recorded in the presence of normal seawater (ASW), high Mg2+ seawater (MgCl2 was increased from 25 to 50 mM; 2Mg2+ ASW) and high Ca2+ seawater (33 mM Ca2+; 3Ca2+ ASW). The percentage increases of IPSCs induced by 5 µM tBuBHQ were 37.59.2% in 2Mg2+ ASW, 25.21.2% in ASW and 7.91.8% in 3Ca2+ ASW (95% confidence interval).
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Fig. 2. Dose-dependent increase of ACh release from Torpedo synaptosomes (A) and mouse caudate nucleus synaptosomes (B) by tBuBHQ. ACh release elicited by addition of 50 mM KCl and 5 mM Ca2+ was measured by the chemiluminescent method of Israe¨l and Lesbats.19,20 The slope of the rising phase of the signal was used to estimate the ACh release. Calibration was obtained by injecting ACh standards at the end of release. In A, two independent experiments are plotted. In B, each point is the mean of three determinations from one experiment. (C) The effect of tBuBHQ on ACh release monitored from caudate nucleus synaptosomes. The left trace is a control release. The right trace shows the increased release after drug action.
Effect of 2,5-diterbutyl-1,4-benzohydroquinone on twin pulse facilitation Facilitation of transmitter release observed during paired-pulse stimulation is believed to be due to residual Ca2+ in the terminal.39,45 Using the inhibitory cholinergic synapse in the buccal ganglion of Aplysia, we have shown previously that tBuBHQ potentiates quantal ACh release by increasing the intraterminal concentration of calcium,13 and we report above that the intensity of the tBuBHQ effect is related to the Ca2+ concentration in the terminal. These observations prompted us to investigate the effects of tBuBHQ on facilitation at various
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Fig. 4. Specificity of tBuBHQ action. tBuBHQ (5 µM) had no additional facilitatory effect on IPSC amplitude after the increase induced by thapsigargin (2.5 µM), another inhibitor of the reticulum ATP-dependent Ca2+ pump. Fig. 3. Effects of 5 µM tBuBHQ on the amplitude of IPSCs evoked by paired pulses at a 15-ms time interval. IPSCs are shown in the control condition and after a 45-min application of tBuBHQ under 2Mg2+ ASW or 3Ca2+ ASW. Table 1. Facilitation or dysfacilitation of the second inhibitory postsynaptic current with respect to the first, calculated by the ratio a2/a1, as shown in Fig. 3
a2/a1 2Mg2+ ASW a2/a1 3Ca2+ ASW
Control
tBuBHQ
2.000.16*
2.430.16*
0.380.01*
0.3420.006*
*95% confidence interval. In 2Mg2+ ASW, this ratio was increased, whereas it was decreased in 3Ca2+ ASW.
extracellular Ca2+ concentrations. Figure 3 shows that facilitation in a 15-ms paired pulse was dependent on the influx of Ca2+. It was important in conditions where the Ca2+ influx was reduced (2Mg2+ ASW), but it was no longer observed when the presynaptic Ca2+ influx was potentiated in 3Ca2+ ASW (Table 1). Indeed, in the latter condition, the amplitude of the second IPSC was depressed with respect to the first. In the presence of 5 µM tBuBHQ, facilitation in 2Mg2+ ASW and depression in 3Ca2+ ASW were both enhanced (Table 1). Specificity of the blockade of the ATPdependent calcium pumps by 2,5-diterbutyl-1,4benzohydroquinone To relate the calcium-dependent potentiating effect of tBuBHQ on ACh release with a specific action on ATP-dependent Ca2+ pumps, two types of experiment were carried out. First, the well-known inhibitor of these Ca2+ pumps, thapsigargin,43 had a similar effect as tBuBHQ on IPSC amplitude. Thapsigargin (2.5 µM) increased the IPSC amplitude by 28.88.1% (95% confidence interval), and sub-
sequent addition of 5 µM tBuBHQ in the bath did not produce any additional increase of the IPSC amplitude (Fig. 4). Second, in the presence of 5 mM NaCN, which inhibits ATP synthesis and hence the accumulation of Ca2+ by ATP-dependent Ca2+ pumps, the amplitude of the IPSC increased to 118.64.3% (95% confidence interval), and the subsequent application of tBuBHQ did not induce any additional effect (117.24.2%; 95% confidence interval). Effect of 2,5-diterbutyl-1,4-benzohydroquinone on 45 Ca2+ uptake by isolated cholinergic synaptic vesicles Cholinergic synaptic vesicles were shown previously to take up Ca2+ in the presence of ATP18,22,27 and, more recently, the presence of a Ca2+-ATPase has been reported.14 In order to determine whether vesicles are involved in regulating the Ca2+ concentration triggering ACh release, we investigated the effects of tBuBHQ on the ATP-dependent Ca2+ uptake by pure fractions of synaptic vesicles isolated from the Torpedo electric organ. Figure 5 shows that tBuBHQ inhibits the ATP-dependent calcium uptake by vesicles in a concentration range similar to that effective on ACh release from synaptosomes (see Fig. 2). Fifty percent inhibition was reached at about 5 µM tBuBHQ. DISCUSSION
The present experiments aimed towards the further understanding of the relationship between transmitter release and the intraterminal Ca2+ concentration. Inhibition of ATP-dependent Ca2+ pumps by thapsigargin or tBuBHQ results in an increase of cytosolic free Ca2+ concentration.17,35,41 We have introduced tBuBHQ in the field of cholinergic synaptic transmission13 in order to see whether it can interfere with Ca2+-dependent ACh release. Using
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cannot be discarded. Nevertheless, it does not seem likely, because in the Aplysia synaptic preparations we have shown that, in the presence of EGTA, the inactivation of the nifedipine-resistant Ca2+ channels triggering ACh release does not depend on intracellular Ca2+ concentration.44 Several possibilities can then be envisaged:
Fig. 5. Inhibition of ATP-dependent 45Ca uptake by Torpedo cholinergic synaptic vesicles by tBuBHQ. Synaptic vesicles were pretreated for 5 min with the indicated concentrations of the drug before initiating the Ca2+ uptake reaction. Calcium uptake was stopped after 5 or 10 min, as described in Experimental Procedures. The ATP-dependent 45 Ca uptake was determined after subtracting the nonspecific uptake measured in the absence of ATP. Each point (duplicate or triplicate determinations) is the mean (S.E.M.) from duplicate or triplicate determinations on three different preparations. Data are expressed as a percentage of control.
the inhibitory cholinergic neurons of the buccal ganglion of Aplysia, we showed that tBuBHQ induces an increase of the evoked Ca2+-dependent ACh release from the presynaptic neuron as a result of an increased cytoplasmic Ca2+ concentration without changing the presynaptic Ca2+ influx.13 We report here that tBuBHQ also enhanced ACh release from vertebrate isolated nerve endings. tBuBHQ (5 µM) and thapsigargin (2.5 µM) have a similar potentiating effect on ACh release in Aplysia. As the effects of these two drugs were not additive, this suggests that they act on a common target site. However, 2 µM thapsigargin has also been reported to block high-voltage-activated Ca2+ channels.6,36 The present studies have thus focused on tBuBHQ, which exerts no action on Ca2+ channels, as a pharmacological probe to identify the subcellular compartment participating in discrete Ca2+ buffering related to the modulation of transmitter release. Increasing the external Ca2+ concentration affects both the magnitude of the Ca2+ influx during depolarization and the level of internal resting Ca2+ in such a way that an increase of transmitter release can be expected. The present experiments demonstrated that the potentiating effects of tBuBHQ on ACh release are more efficient when the calcium entry is small. Facilitation experiments using twin pulses in the absence or in the presence of tBuBHQ showed that a transient increase in Ca2+ concentration in the terminal can facilitate the release of transmitter elicited by the second pulse, up to a maximum level beyond which any additional increase in Ca2+ concentration will lead to a reduced transmitter release. To explain the observed dysfacilitation of the second response in twin pulse protocols applied in the presence of high external Ca2+ concentration, the desensitization of the presynaptic Ca2+ channels
(1) Our results on twin pulse facilitation in control conditions, as well as in the presence of tBuBHQ, are in agreement with the model proposed by Bertram et al.3 on the basis of former observations in which a fourth power relationship between transmitter release and external Ca2+ concentration1,11 or internal Ca2+ concentration24 has been demonstrated. In this model, transmitter release is proposed to be activated by the binding of Ca2+ to four acceptors of low and high affinities, and facilitation is due to residual Ca2+ still bound to one or more of four sites of the mechanism triggering ACh release after the first pulse. However, in contrast to total release, the amplitude of the second response evoked by a twin pulse is a decreasing function of Ca2+ concentration, and a dysfacilitation is observed while the release elicited by the first pulse is increased. One possible explanation is the saturation of the Ca2+ acceptors by high Ca2+ entering during the first pulse, thus reducing the release elicited by the second pulse. Indeed, increasing the presynaptic Ca2+ influx increases the amplitude of the IPSC, but leads to a dysfacilitation of the second response, a phenomenon which was dramatized by application of tBuBHQ, which increased the intracellular Ca2+ concentration. Interestingly, although the identity of the Ca2+ binding protein(s) which trigger(s) ACh release is still under investigation, synaptotagmin, a protein of the vesicle membrane which is very often presented as a ‘‘negative clamp’’ of synaptic transmission, can bind four calcium ions.9 In view of this, the model of Bertram et al.,3 which is in agreement with our observations on twin pulse facilitation, could fit with two low-affinity binding sites for Ca2+ of this protein associated with two highaffinity binding sites of another, still unknown protein. (2) An alternative model in which the number of releasing sites plays a major role can also explain our present results on facilitation. In this model, at low external Ca2+ concentration, a single stimulus would recruit a limited number of releasing sites, while a second stimulus causes other releasing sites to be activated. In the presence of a Ca2+ uptake inhibitor such as tBuBHQ, the cytoplasmic Ca2+ concentration reaches a higher level and both pulses become more efficient because many releasing sites are still available. At high extracellular Ca2+
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concentration, it can be expected that a large number of releasing sites will already be recruited by the first pulse, and the second stimulus can only activate a limited number of sites which have recovered during the time interval after the first one. The observations of Llinas et al.26 showed that the high increase in cytoplasmic Ca2+ concentration (of the order of several hundred micromoles) generated by the opening of presynaptic voltage-gated Ca2+ channels,25 and which triggers the transmitter release, occurs in microdomains and falls off steeply away from the cytoplasmic mouth of the channel.7,37,47 This implies that tBuBHQ, which facilitates ACh release, should act on Ca2+ stores located close to the release sites, allowing the unsequestered Ca2+ to participate in the building up of the Ca2+ concentration triggering ACh release in the active zone. Due to the short latency between Ca2+ influx and transmitter release (200 µs) and the restricted diffusion of Ca2+, Llinas et al.25 proposed a distance within 100 nm between Ca2+ channels and Ca2+ binding sites triggering transmitter release, an estimate which has been largely substantiated.2,46 It is obvious that the only organelles situated in such a strategic location and having the required mechanisms are the synaptic vesicles, or at least a specialized fraction of them. The finding in chick ciliary ganglia that Ca2+ influx through a single opened channel may be sufficient to
gate a transmitter release mechanism supports a highly structured model in which the synaptic vesicle release mechanism is closely tethered to one or more presynaptic Ca2+ channels.40 The accumulation of Ca2+ by synaptic vesicles via an ATP-dependent Ca2+ pump has been shown biochemically22,27 and histochemically.10,28 Our observation that tBuBHQ inhibits Ca2+ uptake by a purified preparation of synaptic vesicles from Torpedo electric organ, and is effective in the same concentration range as on ACh release, supports the view that synaptic vesicles play a role as Ca2+ buffers in the nerve terminal. A possible contamination by the endoplasmic reticulum can be discarded because of the absence of sarcoplasmic/ endoplasmic reticulum ATPase in isolated vesicles fractions from Torpedo.14
CONCLUSION
We present evidence here that synaptic vesicles can control the presynaptic Ca2+ concentration triggering ACh release and in this way modulate the release of neurotransmitter.
Acknowledgements—This work was partly supported by grants from Association Franc¸aise contre les Myopathies (Myasthe´nie no. 4664 to P.F. and no. 4297 to G.B.) and from Direction des Recherches Etudes et Techniques (95/ 141) to P.F. and L.T.
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J. 160, 113–115. Israe¨l M., Manaranche R., Marsal J., Meunier F. M., Morel N., Frachon P. and Lesbats B. (1980) ATP-dependent calcium uptake by cholinergic synaptic vesicles isolated from Torpedo electric organ. J. Membrane Biol. 54, 115–126. Kass G. E. N., Duddy S. K., Moore G. A. and Orrenius S. (1989) 2,5-Di-(tert-butyl)-1,4-benzohydroquinone rapidly elevates cytosolic Ca2+ concentration by mobilizing the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool. J. biol. Chem. 264, 15192–15198. Lando L. and Zucker R. S. (1994) Ca2+ cooperativity in neurosecretion measured using photolabile Ca2+ chelators. J. Neurophysiol. 72, 825–830. Llinas R., Steinberg I. Z. and Walton K. (1981) Relationship between presynaptic calcium current and postsynaptic potential in squid giant synapse. Biophys. J. 33, 323–352. Llinas R., Sugimori M. and Silver R. B. (1992) Microdomains of high calcium concentration in a presynaptic terminal. Science 256, 677–679. Michaelson D. M., Ophir I. and Angel I. (1980) ATP-stimulated Ca2+ transport into cholinergic Torpedo synaptic vesicles. J. Neurochem. 35, 116–124. Mizuhira V., Hasegawa H. and Notoya M. (1994) Microwave fixation and localization of calcium in synaptic vesicles. J. Neurosci. Meth. 55, 125–136. Moore G. A., Kass G. E. N., Duddy S. K., Farrell G. C., Llopis J. and Orrenius S. (1990) 2,5-Di-(tert-butyl)-1,4benzohydroquinone: a novel mobilizer of the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool. Free Rad. Res. Commun. 8, 337–345. Morel N., Israe¨l M., Manaranche R. and Mastour-Frachon P. (1977) Isolation of pure cholinergic nerve endings from Torpedo electric organ. Evaluation of their metabolic properties. J. Cell Biol. 75, 43–55. Oldershaw K. A. and Taylor C. W. (1990) 2,5-Di-(tert-butyl)-1,4-benzohydroquinone mobilizes inositol 1,4,5trisphosphate-sensitive and -insensitive Ca2+ stores. Fedn Eur. biochem. Socs Lett. 274, 214–216. Plessers L., Eggermont J. A., Wuytack F. and Casteels R. (1991) A study of the organellar Ca2+ transport ATPase isozymes in pig cerebellar Purkinje neurones. J. Neurosci. 11, 650–656. Politoff A. L., Rose S. and Pappas G. D. (1974) The calcium binding sites of synaptic vesicles of the frog sartorius neuromuscular junction. J. Cell Biol. 61, 818–823. Pozzan T., Rizzuto R., Volpe P. and Meldolesi J. (1994) Molecular and cellular physiology of intracellular calcium stores. Pharmac. Rev. 74, 595–635. Razani-Boroujerdi S., Partridge L. D. and Sopori M. L. (1994) Intracellular calcium signaling induced by thapsigargin in excitable and inexcitable cells. Cell Calcium 16, 467–474. Shmigol A., Kostyuk P. and Verkhratsky A. (1995) Dual action of thapsigargin on calcium mobilization in sensory neurons: inhibition of Ca2+ uptake by caffeine-sensitive pools and blockade of plasmalemmal Ca2+ channels. Neuroscience 65, 1109–1118. Simon S. M. and Llinas R. R. (1985) Compartmentalization of the submembrane calcium activity during calcium influx and its significance in transmitter release. Biophys. J. 48, 485–498. Simpson P. B., Challiss R. A. J. and Nahorski S. R. (1995) Neuronal Ca2+ stores: activation and function. Trends Neurosci. 18, 299–306. Stanley E. F. (1986) Decline in calcium cooperativity as the basis of facilitation at the squid giant synapse. J. Neurosci. 6, 782–789. Stanley E. F. (1993) Single calcium channels and acetylcholine release at a presynaptic nerve terminal. Neuron 11, 1007–1011. Takemura H., Ohshika H., Yokosawa N., Oguma K. and Thastrup O. (1991) The thapsigargin-sensitive intracellular Ca2+ pool is more important in plasma membrane Ca2+ entry than the IP3-sensitive intracellular Ca2+ pool in neuronal cell lines. Biochem. biophys. Res. Commun. 180, 1518–1526. Taylor C. W. and Traynor D. (1995) Calcium and inositol trisphosphate receptors. J. Membrane Biol. 145, 109–118. Thastrup O., Cullen P. J., Drobak B. K., Hanley M. R. and Dawson A. P. (1990) Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2+ ATPase. Proc. natn. Acad. Sci. U.S.A. 87, 2466–2470. Trudeau L.-E., Baux G., Fossier P. and Tauc L. (1993) Transmitter release and calcium currents at an Aplysia buccal ganglion synapse—I. Characterization. Neuroscience 53, 571–580. Winslow J. L., Duffy S. N. and Charlton M. P. (1994) Homosynaptic facilitation of transmitter release in crayfish is not affected by mobile calcium chelators: implications for the residual ionized calcium hypothesis from electrophysiological and computational analyses. J. Neurophysiol. 72, 1769–1793. Yoshikami D., Bagaboldo Z. and Olivera B. M. (1989) The inhibitory effects of omega-conotoxins on Ca channels and synapses. Ann. N. Y. Acad. Sci. 560, 230–248. Zucker R. S. and Fogelson A. L. (1986) Relationship between transmitter release and presynaptic calcium influx when calcium enters through discrete channels. Proc. natn. Acad. Sci. U.S.A. 83, 3032–3036. (Accepted 3 November 1997)
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Neuroscience Vol. 85, No. 1, pp. 85–91, 1998 Copyright 1998 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/98 $19.00+0.00 S0306-4522(97)00591-5
CONTROL OF THE CALCIUM CONCENTRATION INVOLVED IN ACETYLCHOLINE RELEASE AND ITS FACILITATION: AN ADDITIONAL ROLE FOR SYNAPTIC VESICLES? P. FOSSIER,* M.-F. DIEBLER, J.-P. MOTHET, M. ISRAEL, L. TAUC and G. BAUX Laboratoire de Neurobiologie Cellulaire et Mole´culaire, C.N.R.S., 91198 Gif sur Yvette cedex, France Abstract––2,5-Diterbutyl-1,4-benzohydroquinone, a specific blocker of Ca2+-ATPase pumps, increased acetylcholine release from an identified synapse of Aplysia, as well as from Torpedo and mouse caudate nucleus synaptosomes. Because 2,5-diterbutyl-1,4-benzohydroquinone does not change the presynaptic Ca2+ influx, the enhancement of acetylcholine release could be due to an accumulation of Ca2+ in the terminal. This possibility was further checked by studying the effects of 2,5-diterbutyl-1,4benzohydroquinone on twin pulse facilitation, classically attributed to residual Ca2+. While preventing the fast sequestration of Ca2+ by presynaptic organelles, 2,5-diterbutyl-1,4-benzohydroquinone magnified both twin pulse facilitation observed under low extracellular Ca2+ concentration and twin pulse dysfacilitation observed under high extracellular Ca2+ concentration. Thus, it is concluded that 2,5-diterbutyl-1,4-benzohydroquinone, by preventing Ca2+ buffering near transmitter release sites, modulates acetylcholine release. As 2,5-diterbutyl-1,4-benzohydroquinone was also shown to decrease by 50% the uptake of 45Ca2+ by isolated synaptic vesicles, we propose that synaptic vesicles can control the presynaptic Ca2+ concentration triggering the release of neurotransmitter. 1998 IBRO. Published by Elsevier Science Ltd. Key words: acetylcholine release, synaptic vesicles, Ca2+, facilitation, Ca2+-ATPase pumps.
vesicles.4,5,16,28,33 Third, they were shown to have an ATP-dependent Ca2+ uptake system, with a low Km for Ca2+, which allows them to be effective in the normal physiological control of the concentration of Ca2+ in the terminal.18,22,27 The aim of the present work was to check whether a modulation of synaptic transmission could be achieved by a presynaptic Ca2+ store located in the vicinity of transmitter release sites. For this purpose, we have suppressed the ability of these stores to accumulate Ca2+ by using 2,5-diterbutyl-1,4benzohydroquinone (tBuBHQ), a specific blocker of Ca2+-ATPase pumps,23,29,31 and have checked whether this treatment induces changes in transmitter release at different cholinergic synapses. We indeed observed an increase in acetylcholine (ACh) release from various identified cholinergic synapses of Aplysia, as well as from Torpedo and mouse caudate nucleus synaptosomes. The next step was to identify the presynaptic structures which may be involved. As synaptic vesicles are present at the terminals and are known to accumulate Ca2+, we have investigated the action of tBuBHQ on the uptake of 45Ca2+ by isolated synaptic vesicles.
Calcium transients due to the opening of voltagegated Ca2+ channels are well known to play a primary role in triggering transmitter release. In addition, Ca2+ storage organelles could also play a crucial role in controlling Ca2+ concentration, because they can perform as sinks as well as sources for free calcium.38 The endoplasmic reticulum and its specializations are present in almost all parts of the neuron, including synaptic terminals. These Ca2+ storage organelles can act as Ca2+ buffering systems through their Ca2+-ATPases, which enable rapid sequestration of cytoplasmic calcium,32,34 and they are able to release Ca2+ in the cytosol via the activation of inositol trisphosphate receptors or ryanodine receptors.15,34,38,42 However, little is known on the nature of the intracellular structures participating in the fast buffering of Ca2+ ions in the immediate vicinity of release sites. Synaptic vesicles are good candidates for participating in Ca2+ buffering for three reasons. First, they accumulate at the nerve terminals and occupy strategic positions at the active zone.8 Second, ultrastructural studies have demonstrated the accumulation of Ca2+ within *To whom correspondence should be addressed. Abbreviations: ACh, acetylcholine; ASW, artificial seawater; EGTA, ethyleneglycolbis(aminoethyl ether)tetra-acetate; EPSC, excitatory postsynaptic current; IPSC, inhibitory postsynaptic current; tBuBHQ, 2,5diterbutyl-1,4-benzohydroquinone.
EXPERIMENTAL PROCEDURES
Materials . Aplysia californica were purchased from Marinus (Long Beach, CA, U.S.A.) and Torpedo marmorata from the 85
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Marine Stations of Arcachon and Roscoff (France). Concentrated solutions of tBuBHQ (Aldrich) and thapsigargin (Sigma) were made in dimethylsulfoxide and diluted as necessary. The highest concentration of dimethylsulfoxide attained in the incubation medium was 1‰; and, although it had no effect on synaptic transmission in Aplysia or ACh release from synaptosomes, it was added to all controls. 45 CaCl2 (5–50 mCi/mg Ca2+) was from Amersham. All other chemicals were of the highest purity from common commercial sources. Electrophysiological methods Inhibitory postsynaptic currents (IPSCs) were recorded in the postsynaptic neuron (B3 or B6) following the stimulation of the cholinergic presynaptic neuron (B4 or B5) in the buccal ganglion of Aplysia. Excitatory postsynaptic currents (EPSCs) were elicited in the cell R15 (abdominal ganglion) by stimulation of the presynaptic axon located in the right pleuro-abdominal connective. This axon forms a cholinergic synapse on R15. The protective tissue sheath covering the neurons was removed with fine forceps and the preparation was pinned to the bottom of an experimental chamber. During the experiment, the ganglion was continuously perfused with artificial seawater (ASW; 460 mM NaCl, 10 mM KCl, 11 mM CaCl2, 25 mM MgCl2, 28 mM MgSO4, 10 mM Tris–HCl buffer, pH 7.8) at 0.5–1 ml/min flow rate. Neurons were each impaled with two microelectrodes filled with 3 M KCl. The presynaptic neuron was polarized at 50 mV with a manual current device and the action potential was evoked by a constant depolarizing step. The consecutive Cl-dependent IPSC was recorded in the postsynaptic neuron clamped at 80 mV. The use of lowresistance, KCl-filled electrodes led to a leakage of Cl ions from the electrode, resulting in the loading of the postsynaptic neuron with Cl ions and thus in a shift in the Cl equilibrium potential that would increase the size of the postsynaptic response. To take into account this problem, the reversal potential of the response was regularly reassessed throughout the experiments and the amplitude of the response was expressed as a conductance. Facilitation of the postsynaptic response was measured in paired-pulse experiments with a 15-ms time interval between the two stimuli. Facilitation was quantified by measuring the ratio a2/a1, where a1 is the amplitude of the postsynaptic response evoked by the first action potential and a2 the amplitude of the second postsynaptic response. a2 was measured 15 ms after the peak of the first response by extrapolating the shape of the first response beyond the beginning of the second response according to its monoexponential decay.12 Values are expressed either as meanS.D. from at least 20 values normalized to 100% taken from at least four independent experiments or as means with their 95% confidence intervals calculated from at least 20 individual values, using Prism Software. Biochemical methods Measurement of evoked acetylcholine release by synaptosomes. Synaptosomes were purified from the electric organ of Torpedo, according to the procedure of Israe¨l et al.21 and Morel et al.30 A crude synaptosomal fraction from mouse caudate nucleus was prepared as follows. Slices of the caudate nucleus from one to two mouse brains were washed in a large volume (200 ml) of mammalian Krebs for about 60 min at room temperature. They were then homogenized in 200–300 µl of Krebs medium by repetitive sucking through a blue Eppendorf tip. After a 10-fold dilution with Krebs, this homogenate was filtered through a 50-µm nylon gaze and centrifuged at 4342g for 20 min at 4C. The
resulting pellet (crude P2 synaptosomal fraction) was then resuspended in 500 µl of Krebs medium. ACh release from synaptosomes was monitored by continuous recording, using the choline oxidase chemiluminescent procedure described by Israe¨l and Lesbats.19,20 Release was triggered by addition of 50 mM KCl and 5 mM Ca2+. 45 Ca uptake by synaptic vesicles. Synaptic vesicles from the electric organ of Torpedo were purified on a sucrose sedimentation gradient as described by Israe¨l et al.22 Vesicles were preincubated in the presence of tBuBHQ or vehicle for 5 min at room temperature before initiating 45Ca uptake. The ATP-dependent uptake reaction was performed as described previously by Israe¨l et al.22 in the presence of 1 mM ATP, 2 mM MgCl2, 80 µM CaCl2 and 45Ca (40 d.p.m./pmol, final specific activity). Incubation was stopped after 5 or 10 min by vacuum filtration on GF/C glass fiber filters (Whatman), followed by a 3-ml wash (2 mM EGTA in 400 mM KCl, 10 mM Tris buffer, pH 7.1). Radioactivity bound to the filters was measured by scintillation counting in 3 ml Ready Protein (Beckman). Nonspecific uptake measured in the absence of ATP was subtracted from total uptake.
RESULTS
Effect of 2,5-diterbutyl-1,4-benzohydroquinone on evoked postsynaptic responses in Aplysia ganglia Bath application of tBuBHQ (5 µM) increased the amplitude of the IPSC evoked by a presynaptic action potential in the buccal ganglion. The effect of tBuBHQ appeared after a 20-min delay and was maximal after 45 min of continuous application. At the plateau, the amplitude of the IPSC was increased by 25.27.3% (Fig. 1A) compared to the control value. This potentiating effect remained at the same level for at least 90 min (i.e. the duration of the experiment) and persisted when the preparation was washed out with normal ASW. Identical studies were carried out on an excitatory cholinergic synapse in the abdominal ganglion (Fig. 1B). The application of 5 µM tBuBHQ resulted in an increase of the EPSC amplitude by 28.26.6% with respect to controls.
Effect of 2,5-diterbutyl-1,4-benzohydroquinone on acetylcholine release from isolated nerve endings Presynaptic action of tBuBHQ was further studied on vertebrate isolated cholinergic nerve endings, using synaptosomes purified from the Torpedo electric organ and from the mouse caudate nucleus. Figure 2 shows that, in both preparations, tBuBHQ had a stimulatory effect on the release of ACh evoked by KCl depolarization. This effect was dose dependent and reached a maximum at 10 µM tBuBHQ. ACh release was then increased about four-fold in Torpedo synaptosomes and three-fold in mouse synaptosomes, with respect to control preparations. The stimulating action of tBuBHQ on ACh release by synaptosomes was reduced or absent when ACh release was evoked by a Ca2+ ionophore, A23187
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Fig. 1. tBuBHQ increases the amplitude of evoked postsynaptic responses. (A) Typical potentiation of the IPSC recorded in the buccal ganglion as a function of time of exposure to 5 µM tBuBHQ. Insets: IPSC (upper trace) evoked by a presynaptic action potential (lower trace) in control conditions (a) and after the complete effect of tBuBHQ (b). Calibrations: horizontal bar=20 ms; vertical bars=200 nS (upper trace) and 20 mV (lower trace). (B) Typical potentiation of the EPSC recorded in the abdominal ganglion. Insets show EPSC recordings in control conditions (a) and after application of the drug (b). Calibrations: horizontal bar=20 ms; vertical bar=100 nS.
(data not shown), which induced a large and non-localized entry of Ca2+ into the synaptosomes. Calcium dependency of the effect of 2,5-diterbutyl-1,4benzohydroquinone In order to assess the dependency of the synaptic effects of tBuBHQ on the Ca2+ concentration within the terminal, three series of experiments were conducted on the inhibitory synaptic couple of Aplysia. The influx of Ca2+ into the terminal during the action potential was varied by manipulating the Ca2+ and Mg2+ concentrations in the seawater bath. The effect of tBuBHQ on the amplitude of the IPSC was recorded in the presence of normal seawater (ASW), high Mg2+ seawater (MgCl2 was increased from 25 to 50 mM; 2Mg2+ ASW) and high Ca2+ seawater (33 mM Ca2+; 3Ca2+ ASW). The percentage increases of IPSCs induced by 5 µM tBuBHQ were 37.59.2% in 2Mg2+ ASW, 25.21.2% in ASW and 7.91.8% in 3Ca2+ ASW (95% confidence interval).
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Fig. 2. Dose-dependent increase of ACh release from Torpedo synaptosomes (A) and mouse caudate nucleus synaptosomes (B) by tBuBHQ. ACh release elicited by addition of 50 mM KCl and 5 mM Ca2+ was measured by the chemiluminescent method of Israe¨l and Lesbats.19,20 The slope of the rising phase of the signal was used to estimate the ACh release. Calibration was obtained by injecting ACh standards at the end of release. In A, two independent experiments are plotted. In B, each point is the mean of three determinations from one experiment. (C) The effect of tBuBHQ on ACh release monitored from caudate nucleus synaptosomes. The left trace is a control release. The right trace shows the increased release after drug action.
Effect of 2,5-diterbutyl-1,4-benzohydroquinone on twin pulse facilitation Facilitation of transmitter release observed during paired-pulse stimulation is believed to be due to residual Ca2+ in the terminal.39,45 Using the inhibitory cholinergic synapse in the buccal ganglion of Aplysia, we have shown previously that tBuBHQ potentiates quantal ACh release by increasing the intraterminal concentration of calcium,13 and we report above that the intensity of the tBuBHQ effect is related to the Ca2+ concentration in the terminal. These observations prompted us to investigate the effects of tBuBHQ on facilitation at various
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Fig. 4. Specificity of tBuBHQ action. tBuBHQ (5 µM) had no additional facilitatory effect on IPSC amplitude after the increase induced by thapsigargin (2.5 µM), another inhibitor of the reticulum ATP-dependent Ca2+ pump. Fig. 3. Effects of 5 µM tBuBHQ on the amplitude of IPSCs evoked by paired pulses at a 15-ms time interval. IPSCs are shown in the control condition and after a 45-min application of tBuBHQ under 2Mg2+ ASW or 3Ca2+ ASW. Table 1. Facilitation or dysfacilitation of the second inhibitory postsynaptic current with respect to the first, calculated by the ratio a2/a1, as shown in Fig. 3
a2/a1 2Mg2+ ASW a2/a1 3Ca2+ ASW
Control
tBuBHQ
2.000.16*
2.430.16*
0.380.01*
0.3420.006*
*95% confidence interval. In 2Mg2+ ASW, this ratio was increased, whereas it was decreased in 3Ca2+ ASW.
extracellular Ca2+ concentrations. Figure 3 shows that facilitation in a 15-ms paired pulse was dependent on the influx of Ca2+. It was important in conditions where the Ca2+ influx was reduced (2Mg2+ ASW), but it was no longer observed when the presynaptic Ca2+ influx was potentiated in 3Ca2+ ASW (Table 1). Indeed, in the latter condition, the amplitude of the second IPSC was depressed with respect to the first. In the presence of 5 µM tBuBHQ, facilitation in 2Mg2+ ASW and depression in 3Ca2+ ASW were both enhanced (Table 1). Specificity of the blockade of the ATPdependent calcium pumps by 2,5-diterbutyl-1,4benzohydroquinone To relate the calcium-dependent potentiating effect of tBuBHQ on ACh release with a specific action on ATP-dependent Ca2+ pumps, two types of experiment were carried out. First, the well-known inhibitor of these Ca2+ pumps, thapsigargin,43 had a similar effect as tBuBHQ on IPSC amplitude. Thapsigargin (2.5 µM) increased the IPSC amplitude by 28.88.1% (95% confidence interval), and sub-
sequent addition of 5 µM tBuBHQ in the bath did not produce any additional increase of the IPSC amplitude (Fig. 4). Second, in the presence of 5 mM NaCN, which inhibits ATP synthesis and hence the accumulation of Ca2+ by ATP-dependent Ca2+ pumps, the amplitude of the IPSC increased to 118.64.3% (95% confidence interval), and the subsequent application of tBuBHQ did not induce any additional effect (117.24.2%; 95% confidence interval). Effect of 2,5-diterbutyl-1,4-benzohydroquinone on 45 Ca2+ uptake by isolated cholinergic synaptic vesicles Cholinergic synaptic vesicles were shown previously to take up Ca2+ in the presence of ATP18,22,27 and, more recently, the presence of a Ca2+-ATPase has been reported.14 In order to determine whether vesicles are involved in regulating the Ca2+ concentration triggering ACh release, we investigated the effects of tBuBHQ on the ATP-dependent Ca2+ uptake by pure fractions of synaptic vesicles isolated from the Torpedo electric organ. Figure 5 shows that tBuBHQ inhibits the ATP-dependent calcium uptake by vesicles in a concentration range similar to that effective on ACh release from synaptosomes (see Fig. 2). Fifty percent inhibition was reached at about 5 µM tBuBHQ. DISCUSSION
The present experiments aimed towards the further understanding of the relationship between transmitter release and the intraterminal Ca2+ concentration. Inhibition of ATP-dependent Ca2+ pumps by thapsigargin or tBuBHQ results in an increase of cytosolic free Ca2+ concentration.17,35,41 We have introduced tBuBHQ in the field of cholinergic synaptic transmission13 in order to see whether it can interfere with Ca2+-dependent ACh release. Using
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cannot be discarded. Nevertheless, it does not seem likely, because in the Aplysia synaptic preparations we have shown that, in the presence of EGTA, the inactivation of the nifedipine-resistant Ca2+ channels triggering ACh release does not depend on intracellular Ca2+ concentration.44 Several possibilities can then be envisaged:
Fig. 5. Inhibition of ATP-dependent 45Ca uptake by Torpedo cholinergic synaptic vesicles by tBuBHQ. Synaptic vesicles were pretreated for 5 min with the indicated concentrations of the drug before initiating the Ca2+ uptake reaction. Calcium uptake was stopped after 5 or 10 min, as described in Experimental Procedures. The ATP-dependent 45 Ca uptake was determined after subtracting the nonspecific uptake measured in the absence of ATP. Each point (duplicate or triplicate determinations) is the mean (S.E.M.) from duplicate or triplicate determinations on three different preparations. Data are expressed as a percentage of control.
the inhibitory cholinergic neurons of the buccal ganglion of Aplysia, we showed that tBuBHQ induces an increase of the evoked Ca2+-dependent ACh release from the presynaptic neuron as a result of an increased cytoplasmic Ca2+ concentration without changing the presynaptic Ca2+ influx.13 We report here that tBuBHQ also enhanced ACh release from vertebrate isolated nerve endings. tBuBHQ (5 µM) and thapsigargin (2.5 µM) have a similar potentiating effect on ACh release in Aplysia. As the effects of these two drugs were not additive, this suggests that they act on a common target site. However, 2 µM thapsigargin has also been reported to block high-voltage-activated Ca2+ channels.6,36 The present studies have thus focused on tBuBHQ, which exerts no action on Ca2+ channels, as a pharmacological probe to identify the subcellular compartment participating in discrete Ca2+ buffering related to the modulation of transmitter release. Increasing the external Ca2+ concentration affects both the magnitude of the Ca2+ influx during depolarization and the level of internal resting Ca2+ in such a way that an increase of transmitter release can be expected. The present experiments demonstrated that the potentiating effects of tBuBHQ on ACh release are more efficient when the calcium entry is small. Facilitation experiments using twin pulses in the absence or in the presence of tBuBHQ showed that a transient increase in Ca2+ concentration in the terminal can facilitate the release of transmitter elicited by the second pulse, up to a maximum level beyond which any additional increase in Ca2+ concentration will lead to a reduced transmitter release. To explain the observed dysfacilitation of the second response in twin pulse protocols applied in the presence of high external Ca2+ concentration, the desensitization of the presynaptic Ca2+ channels
(1) Our results on twin pulse facilitation in control conditions, as well as in the presence of tBuBHQ, are in agreement with the model proposed by Bertram et al.3 on the basis of former observations in which a fourth power relationship between transmitter release and external Ca2+ concentration1,11 or internal Ca2+ concentration24 has been demonstrated. In this model, transmitter release is proposed to be activated by the binding of Ca2+ to four acceptors of low and high affinities, and facilitation is due to residual Ca2+ still bound to one or more of four sites of the mechanism triggering ACh release after the first pulse. However, in contrast to total release, the amplitude of the second response evoked by a twin pulse is a decreasing function of Ca2+ concentration, and a dysfacilitation is observed while the release elicited by the first pulse is increased. One possible explanation is the saturation of the Ca2+ acceptors by high Ca2+ entering during the first pulse, thus reducing the release elicited by the second pulse. Indeed, increasing the presynaptic Ca2+ influx increases the amplitude of the IPSC, but leads to a dysfacilitation of the second response, a phenomenon which was dramatized by application of tBuBHQ, which increased the intracellular Ca2+ concentration. Interestingly, although the identity of the Ca2+ binding protein(s) which trigger(s) ACh release is still under investigation, synaptotagmin, a protein of the vesicle membrane which is very often presented as a ‘‘negative clamp’’ of synaptic transmission, can bind four calcium ions.9 In view of this, the model of Bertram et al.,3 which is in agreement with our observations on twin pulse facilitation, could fit with two low-affinity binding sites for Ca2+ of this protein associated with two highaffinity binding sites of another, still unknown protein. (2) An alternative model in which the number of releasing sites plays a major role can also explain our present results on facilitation. In this model, at low external Ca2+ concentration, a single stimulus would recruit a limited number of releasing sites, while a second stimulus causes other releasing sites to be activated. In the presence of a Ca2+ uptake inhibitor such as tBuBHQ, the cytoplasmic Ca2+ concentration reaches a higher level and both pulses become more efficient because many releasing sites are still available. At high extracellular Ca2+
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concentration, it can be expected that a large number of releasing sites will already be recruited by the first pulse, and the second stimulus can only activate a limited number of sites which have recovered during the time interval after the first one. The observations of Llinas et al.26 showed that the high increase in cytoplasmic Ca2+ concentration (of the order of several hundred micromoles) generated by the opening of presynaptic voltage-gated Ca2+ channels,25 and which triggers the transmitter release, occurs in microdomains and falls off steeply away from the cytoplasmic mouth of the channel.7,37,47 This implies that tBuBHQ, which facilitates ACh release, should act on Ca2+ stores located close to the release sites, allowing the unsequestered Ca2+ to participate in the building up of the Ca2+ concentration triggering ACh release in the active zone. Due to the short latency between Ca2+ influx and transmitter release (200 µs) and the restricted diffusion of Ca2+, Llinas et al.25 proposed a distance within 100 nm between Ca2+ channels and Ca2+ binding sites triggering transmitter release, an estimate which has been largely substantiated.2,46 It is obvious that the only organelles situated in such a strategic location and having the required mechanisms are the synaptic vesicles, or at least a specialized fraction of them. The finding in chick ciliary ganglia that Ca2+ influx through a single opened channel may be sufficient to
gate a transmitter release mechanism supports a highly structured model in which the synaptic vesicle release mechanism is closely tethered to one or more presynaptic Ca2+ channels.40 The accumulation of Ca2+ by synaptic vesicles via an ATP-dependent Ca2+ pump has been shown biochemically22,27 and histochemically.10,28 Our observation that tBuBHQ inhibits Ca2+ uptake by a purified preparation of synaptic vesicles from Torpedo electric organ, and is effective in the same concentration range as on ACh release, supports the view that synaptic vesicles play a role as Ca2+ buffers in the nerve terminal. A possible contamination by the endoplasmic reticulum can be discarded because of the absence of sarcoplasmic/ endoplasmic reticulum ATPase in isolated vesicles fractions from Torpedo.14
CONCLUSION
We present evidence here that synaptic vesicles can control the presynaptic Ca2+ concentration triggering ACh release and in this way modulate the release of neurotransmitter.
Acknowledgements—This work was partly supported by grants from Association Franc¸aise contre les Myopathies (Myasthe´nie no. 4664 to P.F. and no. 4297 to G.B.) and from Direction des Recherches Etudes et Techniques (95/ 141) to P.F. and L.T.
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