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Departments of 1Neuroscience and 2Physiology, University of Texas Southwestern Medical Center, Dallas, Texas
Submitted 26 March 2008; accepted in final form 14 August 2008
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ABSTRACT |
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-latrotoxin have been extremely informative in studying neurotransmission. Lanthanum and lanthanides can also trigger neurotransmitter release through an unknown mechanism. Here, we studied the effect of lanthanides on neurotransmission in hippocampal cultures. Application of 2 mM La3+ caused rapid and robust neurotransmitter release within seconds. In addition, transient application of La3+ uncovered a sustained facilitation of miniature neurotransmission. The response to La3+ was detectable at 2 µM and increased in a concentration-dependent manner 
2 mM. Rapid effect of La3+ was independent of extracellular and intracellular Ca2+ and did not require La3+ entry into cells or activation of phospholipaseCβ. Synapses deficient in synaptobrevin-2, the major synaptic vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein in the brain, did not display any rapid release in response to La3+, whereas the slow facilitation of release detected after La3+ removal remained intact. In contrast, preincubation with intracellular Ca2+ chelators selectively attenuated the delayed release triggered by La3+. Moreover, synapses deficient in synaptotagmin-1 maintained a rapid response to La3+, suggesting that La3+-triggered neurotransmitter release does not require synaptotagmin-1 as a sensor. Therefore La3+ has two separate effects on synaptic transmission. For its rapid action, La3+ interacts with a target on the surface membrane, and unlike other forms of release, it triggers strictly synaptobrevin-2–dependent fusion, implying that in central synapses synaptobrevin-2 function is secretagogue specific. For the delayed action, La3+ may act intracellularly after its entry or through intracellular Ca2+ via a mechanism that does not require synaptobrevin-2. |
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INTRODUCTION |
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-latrotoxin have been valuable tools in studying the mechanisms underlying neurotransmission. Hypertonicity is widely used to estimate the size of readily releasable pool (Rosenmund and Stevens 1996
) and trigger neurotransmitter release independent of Ca2+ to assess selective defects in Ca2+ regulation of neurotransmission (Geppert et al. 1994). -latrotoxin, on the other hand, helps to analyze the properties of unitary release events (Auger and Marty 1997; Bevan and Wendon 1984; Fesce et al. 1986) and has also been an extremely informative molecular bait to uncover several key components of synaptic junctions (Ushkaryov et al. 1992). Lanthanum (La3+) and other rare earth metals, collectively referred to as lanthanides, can also trigger neurotransmitter release but the mechanisms underlying their action is unknown. Previous studies showed that La3+ blocks the action potential evoked end plate potentials (Heuser and Miledi 1971; Miledi 1971) while increasing the frequency of miniature end plate potentials at the neuromuscular synapses of frog and goldfish (Bowen 1972; Curtis et al. 1986; Dekhuijzen et al. 1989; Heuser and Miledi 1971). Because these two consequences of La3+ treatment seemed inconsistent, La3+ and other lanthanides were often called to have dual action. Inhibition of evoked neurotransmission by La3+ is attributed to its potent ability to block voltage-gated Ca2+ channels (VGCCs) (Lansman 1990; Lansman et al. 1986; Reichling and MacDermott 1991). In addition, La3+ can also inhibit Ca2+ uptake by mitochondria (Mela 1969a,b), as well as by the plasma membrane Ca2+-ATPase (PMCA) (Herrington et al. 1996). However, it remains unclear how La3+ increases spontaneous neurotransmission in synapses. Moreover, the impact of La3+ treatment on synaptic transmission in central synapses remains to be characterized. Increasing use of LaCl3 as a therapeutic agent such as in treatment for hyperphosphatemia further necessitates a better understanding of its impact on neurotransmission (Finn 2006). Therefore here we evaluated La3+'s potential as a tool to pinpoint unconventional signaling pathways regulating central synapses, which are inaccessible to canonical secretagogues.
In these experiments, La3+ application onto dissociated hippocampal cultures caused immediate neurotransmitter release (rapid effect) in a concentration-dependent manner. Interestingly, on La3+ washout, the frequency of spontaneous neurotransmission was increased, and this delayed release (delayed effect) was only slowly reversible. Other lanthanides such as praseodymium (Pr3+), gadolinium (Gd3+), erbium (Er3+), and yttrium (Y3+) could mimic La3+ effect in central synapses, implying these two actions (rapid effect vs. delayed effect) are common features of all lanthanides. Rapid effect of La3+ was independent of both extracellular and intracellular Ca2+ as well as activation of phospholipase C (PLC)β. In addition, the rapid action of La3+ was insensitive to heavy metal chelators, thus it does not require La3+ entry into the cell. In hippocampal cultures obtained from mice deficient in the synaptic vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein synaptobrevin-2, the rapid effect of La3+ was abolished, whereas the delayed effect was still detectable. In contrast, lowering internal [Ca2+] using Ca2+ chelators significantly attenuated the delayed effect while leaving the rapid La3+-triggered neurotransmission intact. Therefore La3+ has two separate effects on synaptic transmission. For its rapid action, La3+ seems to interact with a target on the surface membrane, triggering a SNARE-dependent fusion while La3+ and/or [Ca2+]i is required for the delayed action. These multiple effects of La3+ on neurotransmitter release in central synapses might mediate neurotoxic consequences of chronic exposure to La3+ (Feng et al. 2006a,b).
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METHODS |
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Dissociated hippocampal cultures were prepared from Sprague-Dawley rats or synaptotagmin-1–deficient mice pups (gift of Dr. T. C. Sudhof) as previously described (Kavalali et al. 1999). Synaptobrevin-2–deficient mice cultures were prepared at embryonic day 18, as previously described (Deak et al. 2004). Rats and mice were rapidly killed by decapitation after sedation by chilling on an ice-cold metal plate. All experiments with hippocampal cultures were performed during 14–21 days in vitro (DIV), the time period already known as the time that synapses become fully mature and represent the mature connections as in vivo (Mozhayeva et al. 2002).
Electrophysiology
Pyramidal neurons were voltage clamped to –70 mV using whole cell patch-clamp technique, using an Axopatch 200B amplifier and Clampex 8.0 software (Molecular Devices, Sunnyvale, CA), filtered at 2 kHz, and sampled at 5 kHz. The pipette solution contained the following (in mM): 115 Cs-MeSO3, 10 CsCl, 5 NaCl, 10 HEPES, 0.6 EGTA, 20 tetraethylammonium chloride, 4 Mg-ATP, 0.3 Na2GTP, and 10 QX-314 (lidocaine N-ethyl bromide), pH 7.35, 300 mOsm (Sigma, St. Louis, MO). A modified Tyrode solution was used as the extracellular solution with 2 mM Ca2+ and 1 µM TTX. It contained the following (in mM): 150 NaCl, 4 KCl, 2 MgCl2, 10 glucose, 10 HEPES, and 2 CaCl2, pH 7.4, 310 mOsm; 0 mM Ca2+ solution had the same ionic composition except Ca2+ concentration and contained 1 mM EGTA. To record and isolate miniature excitatory postsynaptic currents (mEPSCs), picrotoxin (PTX; 50 µM; Sigma) and TTX were added to the bath solution. In the experiments at which miniature inhibitory postsynaptic currents (IPSCs) aimed to be isolated and recorded, ionotropic glutamate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 µM; Sigma) and aminophosphonopentanoic acid (AP-5; 50 µM; Sigma) were added to the bath solution. To chelate intracellular Ca2+, neurons were pretreated with either 2 µM BAPTA-AM (Molecular Probes, Eugene, OR) for 30 min or 100 µM EGTA-AM (Molecular Probes) for 10 min. The exchange between extracellular solutions such as challenge with a 2 mM La3+-containing solution was achieved by direct perfusion of solutions onto the field of interest by gravity. A two-tailed unpaired t-test was used for statistical comparisons, and values are given as means ± SE.
Lentivirus production
A 12 amino acid–inserted synaptobrevin 2 mutant (12-ins syb 2) construct was generated as described before (Deák et al. 2006a). HEK 293 cells were transfected with the Fugene 6 transfection system (Roche Molecular Biochemicals) with the expression plasmid and two helper plasmids, which are delta 8.9 and vesicular stomatitis virus G protein (3 µg of each DNA per 75-cm2 flask of HEK cells). After incubation of HEK cells at 37°C for 48 h, lentivirus-containing culture medium was harvested and filtered at a 0.45-µm pore size before use for infection. Synaptobrevin 2–deficient mice cultures were infected with 12-ins syb 2 at 4 DIV by adding 400 µl of viral suspension to each well. Patch-clamp recording was carried out at 14–21 DIV.
Ca2+ imaging
Ten microliters of 4% pluronic F-127 (low UV absorbance, Molecular Probes) and 3 µM Fura-2-AM (Invitrogen, Eugene, OR) were added to growth media of 18–19 DIV hippocampal cultures, and cells were incubated for 45 min at 37°C. Cells were transferred to the chamber and washed with Tyrode's solution, containing nominal Ca2+ and 1 mM EGTA, for a couple of minutes. Images were obtained using DeltaRAM illuminator (Photon Technology International, Birmingham, NJ) and an IC-300 camera (Photon Technology International) at a frequency of 0.5 Hz. Baseline was recorded for 1 min before stimulation. We used 2 and 20 mM of Ca2+ or La3+ for stimulation. Data analysis was performed using ImageMaster Pro software (Photon Technology International).
Statistical analysis
ANOVA was used for statistical analysis of all multiple comparison experiments. The Student's t-test (2-tailed) was used for pairwise comparisons.
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RESULTS |
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To study the effect of La3+ on neurotransmitter release from central synapses, we used a whole cell patch-clamp technique in rat dissociated hippocampal cultures and examined the basic properties of synaptic transmission. Bath application of 2 mM La3+ caused rapid neurotransmitter release (Fig. 1, A and B). This rapid effect of La3+ has not been detected earlier, because in previous studies of the neuromuscular junction preparations, the La3+ effect was monitored and analyzed not in real time but after incubation for a certain period. In these earlier experiments, the incubation times ranged between 1 and 4 min, and in some cases, synapses were incubated with La3+ for 45 min up to hours (Curtis et al. 1986; Heuser and Miledi 1971). In contrast, here, we monitored the change of synaptic responses while bath-applying La3+. Thus we could observe prompt neurotransmitter release, which we refer to as the rapid effect, both in excitatory and inhibitory synapses (Fig. 2). With sustained La3+ application, the neurotransmitter release was decreased, presumably because of depletion of available vesicles akin to synaptic responses seen after hypertonic sucrose application (Moulder and Mennerick 2005; Rosenmund and Stevens 1996).
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The following sets of experiments were aimed to test the origin of vesicles mobilized by La3+. To study whether La3+ mobilizes the same readily releasable pool (RRP) of synaptic vesicles that fuse in response to hypertonic sucrose application, we applied 2 mM La3+ 20 s after initial application of hypertonic sucrose in the continued presence of hypertonic solution. If La3+ induced the fusion of RRP vesicles, we expected to observe no additional release. However, if La3+ mobilizes a separate set of synaptic vesicles, La3+ application on top of hypertonicity would be expected to trigger a comparable amount of neurotransmitter release regardless of hypertonic sucrose application. In these experiments, prior exposure to +500 mOsm hypertonic sucrose solution resulted in a 70% reduction in La3+-induced neurotransmitter release compared with cells that were challenged with 2 mM La3+ alone (compare gray bars in Fig. 1, D and F; P < 0.001). Perfusion of 2 mM La3+ on top of +500 mOsm hypertonic sucrose solution was able to augment release by an additional 17% (n = 6), whereas 2 mM La3+ typically triggered 53% of release induced by hypertonicity alone (n = 20; Fig. 1G). Taken together, these findings suggest that the pools of vesicles mobilized by La3+ and hypertonic sucrose are largely overlapping but not completely identical.
As briefly mentioned earlier, the rapid effect of La3+ was observed both in excitatory and inhibitory synapses (Fig. 2, A and B). When we varied La3+ concentrations from 2 µM to 2 mM, the rapid effect of La3+ was detectable at 2 µM and showed a concentration-dependent increase in rate and magnitude (Fig. 2). The half rise time of release (t1/2) at 2 µM was 11.59 ± 2.43 nC/s and reached a maximum of 1.92 ± 0.4 nC/s at 2 mM La3+ (Fig. 2D).
La3+ has been shown to activate TRPC4 and 5 channels specifically through an unknown mechanism (Jung et al. 2003), and these transient receptor potential (TRP) channels are expressed in hippocampal neurons (Chung et al. 2006). To verify that La3+ acts by releasing neurotransmitters presynaptically and does not have a direct postsynaptic effect, we applied La3+ in the presence of TTX, PTX, and CNQX to block voltage-gated Na+ channels, GABA receptors, and postsynaptic AMPA receptors, respectively. If La3+ response was caused by direct activation of postsynaptic TRP channels, we would expect a similar pattern of activity in the absence of neurotransmitter receptor activation. However, we could completely eliminate La3+ response with TTX, PTX, and CNQX and thus rule out the possibility that postsynaptic TRP channels were responsible for the neurotransmitter release induced by La2+ (Fig. 2E).
La3+ application does not alter the properties of unitary synaptic responses
To further evaluate the specificity of La3+ action on neurotransmitter release, we probed the properties of unitary transmission triggering by low concentrations of La3+. Here, we applied 2 µM La3+ because higher concentrations of La3+ caused several overlapping quantal events, which were hard to assess individually. Unitary release events activating a specific type of receptor were recorded with the aid of pharmacological blockers. In this way, we measured a large number of AMPA-mEPSCs, N-methyl-D-aspartate (NMDA)-mEPSCs, and mIPSCs, before and during La3+ application in isolation. This analysis showed no significant changes in the 10–90% rise times and amplitudes of individual events before and after La3+ application (Fig. 3). These results indicate that the neurotransmitter release triggered by La3+ is quantal in nature and not likely caused by a nonspecific disruption of presynaptic terminals or postsynaptic responsiveness and may share the same neurotransmitter release mechanism with physiological forms of synaptic transmission.
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Other earth rare metals such as Pr3+, Gd3+, ytterbium (Yb3+), Er3+, and Y3+ have been reported to show La3+-like dual action on synaptic transmission at the neuromuscular junction (Alnaes and Rahamimoff 1974; Bowen 1972; Metral et al. 1978; Molgo et al. 1991). To test whether the stimulatory action of La3+ on neurotransmitter release from hippocampal synapses is shared by other lanthanides, we applied 2 mM of europium (Eu3+), Pr3+, Gd3+, and Yb3+ for 30 s and examined resulting neurotransmission. In these experiments, all lanthanides tested triggered a similar effect on neurotransmission (Fig. 4A), although the amount of release was typically smaller than that of La3+ (Fig. 4B). The average charge transfer triggered by 2 mM Gd3+, Eu3+, Yb3+, and Pr3+ for 10 s was 0.33 ± 0.13, 0.37 ± 0.09, 0.43 ± 0.09, and 0.60 ± 0.12 nC, respectively (n = 4 each). Among these lanthanides, Pr3+ was the most potent, reaching 80% of the La3+ effect. These observations suggest that the ability to trigger neurotransmitter release is shared by all lanthanides.
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After La3+ washout, the frequency of mEPSCs or mIPSCs was highly increased, which we refer as the "delayed effect," and the increased level of mPSCs was maintained for 
10 min (Fig. 5). The ability of La3+ to cause a sustained increase in the frequency of mPSCs has been already documented in neuromuscular junction preparations (Curtis et al. 1986; Heuser and Miledi 1971). We could also consistently observe this effect in central synapses as a delayed consequence on withdrawal of La3+, distinct from the rapid effect we observed during La3+ application. Delayed effect was also concentration dependent in both mEPSCs and mIPSCs (Fig. 5, A–C). Removal of 2 µM La3+solution caused a 1.74 ± 0.16-fold increase in mEPSC frequency compared with rest, whereas at 2 mM, this increase was 9.91 ± 1.53-fold (n = 5–9). Interestingly, the fold increase in the frequency of mEPSCs was greater than the change in mIPSCs (Fig. 5C). The fold increase in mIPSC frequencies after 2 µM La3+ was 1.11 ± 0.2 and reached a maximum of 4.48 ± 0.17-fold at 200 µM (n = 5–9). In contrast, we observed no significant difference in amplitudes of mEPSCs and mIPSCs (Fig. 5D).
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What is the underlying mechanism of lanthanide-evoked neurotransmission at central synapses? Our data suggest that La3+ can stimulate a releasable pool of vesicles presumably shared by the hypertonic sucrose stimulation, and La3+ application has no detectable direct postsynaptic effect (e.g., activation of TRP channels; Fig. 1H). In the next set of experiments, we pursued how La3+ may mobilize the RRP at central synapses. La3+ may trigger neurotransmitter release by one of three possible scenarios. First, La3+ might trigger Ca2+ entry presynaptically (e.g., via presynaptic TRP channels) or cause intracellular Ca2+ release and augment Ca2+-dependent neurotransmission. In this case, La3+ would require Ca2+ as a mediator for its action. Second, La3+ might enter into a cell and cause neurotransmitter release by substituting Ca2+. This scenario predicts that the action of La3+ does not require Ca2+ but requires its entry to a cell to surrogate Ca2+. La3+ has a roughly similar ionic radius (3.1 Å) to Ca2+ (2.8 Å) (Lettvin et al. 1964); thus VGCCs can be a possible entryway of La3+ into a cell (Lansman 1990; Lansman et al. 1986). In addition, several lines of evidence including electron micrographs (Pecot-Dechavassine 1983) showed that La3+ could also enter a cell via the Na+/Ca2+ exchanger (Powis et al. 1994; Reeves and Condrescu 2003; Shimizu et al. 1997). Finally, if La3+ neither stimulates Ca2+ signaling nor enters into a cell for its rapid action, La3+ might act at the surface of membrane and interact with surface receptors, which can eventually evoke neurotransmission. An earlier study suggested that La3+ might act at superficial side of sartorius muscle (Weiss 1970). More recently, another study in synaptosomes also proposed that La3+ may act extracellularly (Lopatina et al. 2005), possibly by modifying membrane lipid packing (Verstraeten et al. 1997).
To elucidate the underlying mechanism of prompt La3+-evoked neurotransmission, we tested the first scenario that La3+ may act by increasing Ca2+-evoked neurotransmission. We carried out experiments similar to above with extracellular solution containing 1 mM EGTA and nominal Ca2+, which is expected to remove extracellular Ca2+ that can enter a cell on La3+ stimulation. In addition, to test whether increase in intracellular Ca2+ mediates rapid action of La3+ either by release from internal stores or by attenuated uptake by mitochondria, cells were treated with 100 µM EGTA-AM for 10 min or 2 µM BAPTA-AM for 30 min before La3+ stimulation. We examined the effect of 2 mM La3+ in the presence of 1 mM EGTA and nominal extracellular Ca2+. Chelating extracellular Ca2+ as well as buffering intracellular Ca2+ could not antagonize the rapid action of La3+ (Fig. 6A). La3+ could still cause a similar amount of release independent of intracellular or extracellular Ca2+. In the presence of EGTA, the cumulative charge transfer by 2 mM La3+ was 1.34 ± 0.39 nC, which is comparable to the control experiments shown in Fig. 1. The release induced by La3+ after incubation with EGTA-AM or BAPTA-AM was 0.94 ± 0.14 and 1.18 ± 0.22 nC, respectively. These findings suggest that the neurotransmitter release by La3+ is not likely caused by its potential effects on Ca2+ signaling pathways. In agreement with these observations, depletion of Ca2+ from internal Ca2+ stores by for 30-min treatment of neurons with 1 µM thapsigargin did not impair release induced by subsequent application of 2 mM La3+ (Supplementary Fig. S1).1
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), implying that pretreatment with EGTA-AM or BAPTA-AM could significantly eliminate intracellular La3+ and block its action. For this reason, although we applied 2 mM La3+ under the conditions shown in Fig. 6A, we estimate the actual concentration of La3+ in the solution would be <2 mM. Previous studies showed that La3+ could enter into a cell through the Na+/Ca2+ exchanger albeit with a slow time course around 30 s (Powis et al. 1994; Reeves and Condrescu 2003). Therefore the Na+/Ca2+ exchanger is not a likely mediator of La3+ entry for its rapid action because 30 s is longer than the typical response time to La3+ application (<5 s). Nevertheless, to examine the second possibility more directly, we incubated cells with 200 µM Cd2+, a potent pore blocker of voltage-gated Ca2+ channels for 10 min before application of 2 mM La3+ and hypertonic solution to examine whether La3+ entry through VGCCs is responsible for rapid action of La3+. If the rapid action of La3+ requires its entry through Ca2+ channels, preincubation with 200 µM Cd2+ would be expected to impede the rapid effect of La3+. However, 200 µM Cd2+ did not interrupt the rapid action of La3+ because La3+ could trigger substantial release (1.35 ± 0.19 nC) in its presence (Fig. 6B). This finding indicates that the possible entry of La3+ VGCCs is not required for its rapid effect.
In addition to Ca2+ influx through VGCCs, presynaptic Ca2+ can be regulated by several mechanisms such as the Na+/Ca2+ exchanger, Ca2+ uptake and release from mitochondria, and PMCA. La3+ is also known as a strong inhibitor of Ca2+ uptake by mitochondria (Mela 1969a,b) and as an inhibitor of PMCA (Herrington et al. 1996), indicating that La3+ might interfere with Ca2+ homeostasis in presynaptic terminals in multiple ways, leading to augmentation of neurotransmitter release. Besides the activation of channels or their permeation into cells, lanthanides can also bind to Ca2+ receptors, which are found in central nerve terminals (Smith et al. 2004), and these receptors are typically coupled to the activation of the PLCβ pathway (Breitwieser et al. 2004). To test the possibility that binding of La3+ to Ca2+ receptors and consequent activation of PLCβ pathway is responsible for its rapid action, we treated hippocampal neurons with U73122
[GenBank]
, a PLCβ inhibitor, for 10 min before 2 mM La3+ stimulation. After U73122
[GenBank]
treatment, 2 mM La3+ still caused neurotransmitter release (1.34 ± 0.28 nC) comparable to its effect on untreated cells, indicating that U73122
[GenBank]
treatment failed to disrupt the rapid effect of La3+ (Fig. 6B). Figure 6C summarizes the results of these experiments using 2 mM La3+ under different conditions as a fraction of La3+ responses in untreated cells. La3+-evoked responses with nominal Ca2+ and 1 mM EGTA containing extracellular solution, after EGTA-AM and BAPTA-AM treatment, were 1.19 ± 0.35 (n = 5), 0.84 ± 0.13 (n = 4), and 1.06 ± 0.20 nC (n = 6), respectively. Average postsynaptic charge transfer values after 200 µM Cd2+ treatment or U73122
[GenBank]
application were similar to controls [Cd2+: 1.21 ± 0.17 nC (n = 7); U73122
[GenBank]
: 1.19 ± 0.25 nC (n = 6)].
In the next set of experiments, we evaluated the effectiveness of EGTA-AM treatment to inhibit rapid action potential–evoked release in response to 10-Hz stimulation (Fig. 6D). For this purpose, we evoked IPSCs (eIPSCs) for 1 s in 8 mM Ca2+ before and after treatment with 100 µM EGTA-AM (Fig. 6E). These experiments showed that brief treatment with EGTA-AM is sufficient to lower the concentration of interacellular Ca2+ and inhibit release (Fig. 6 F and G). This finding further supports the premise that, if La3+-evoked release was caused by a rise in intracellular Ca2+, it should have been susceptible to EGTA-AM treatment.
Taken together, we ruled out two possibilities. First, the action of La3+ does not seem to require rises in intracellular Ca2+. Second, La3+ entry through VGCCs or activation of PLCβ is unlikely to mediate a rapid effect of La3+.
La3+ entry proceeds with a slow time course and cannot account for the rapid release induced by La3+
La3+ might be able to enter into a cell via several poorly characterized pathways. Therefore in the next set of experiments, we aimed to monitor La3+ entry optically using Fura-2-AM, a membrane-permeable derivative of the ratiometric Ca2+-selective fluorescent dye Fura-2. Fura-2's affinity to La3+ is a 1,000-fold higher than its affinity to Ca2+(Reeves and Condrescu 2003). In addition, in response to La3+ binding, Fura-2 shows a spectral shift (increase in fluorescence emission ratio in response to excitation at 340- vs. 380-nm wavelengths), similar to the shift seen after its Ca2+ binding (Reeves and Condrescu 2003).
In these experiments, Fura-2-AM was added in growth media for 45 min at 37°C (final concentration = 3 µM), and cells were transferred to the recording chamber and briefly washed with Tyrode's solution containing nominal Ca2+ and 1 mM EGTA. We measured the fluorescence baseline in Tyrode's solution in the presence of 1 mM EGTA for 1 min and applied 2 mM La3+ or Ca2+ to monitor the entry of each ion for 30 s. Because we detected a very small increase in the 340/380 ratio with 2 mM La3+ application, cells were challenged again with higher concentrations of La3+ or Ca2+ (20 mM) as a positive control. In this setting, we observed only a small increase in the 340/380 ratio when 2 mM La3+ was applied, in contrast to the rapid and robust increase in the 340/380 ratio by 2 mM Ca2+ (Fig. 7). This suggests that there is small, if any, entry of La3+ into a cell on its bath application. Second, the rise in the 340/380 ratio after La3+ application started slower than after Ca2+. The initiation of rise in the 340/380 ratio by La3+ is indicated by the black dotted line in Fig. 7B. Compared with the gray dotted line, which indicates the rise in the 340/380 ratio by Ca2+, there is a delay between the two lines, which is 
15 s (Fig. 7B). We did not detect a significant difference between the baseline fluorescence ratios in cells treated with La3+ or Ca2+ (0.4458 for La3+, n = 55 from 6 coverslips and 0.434 for Ca2+, n = 30 from 3 coverslips). Thus it takes longer for La3+ to enter into a cell, and the entry occurs slower compared with the entry of Ca2+ (Fig. 7). This result strongly argues against the possibility that the rapid effect of La3+ is mediated by intracellular action of La3+.
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). This finding suggests that La3+ is not readily extruded from cells, and it is buffered rather ineffectively once it is introduced. This observation may explain the hardly reversible nature of the delayed effect of La3+on neurotransmitter release. Rapid effect of La3+ is strictly dependent on the vesicular SNARE protein synaptobrevin-2
Our results thus far indicated that La3+ triggers rapid neurotransmitter release independent of its entry into cells. Thus it is likely that La3+ acts at an extracellular site, presumably by binding to a putative receptor or by direct modification of plasma membrane lipids to trigger neurotransmitter release (Andjus et al. 1997; Cheng et al. 1999). To test whether the form of release triggered by La3+ shares the same molecular machinery as the physiological action potential–driven release, we took advantage of mice deficient in the synaptic vesicle protein synaptobrevin-2. Synaptobrevin-2 (also called VAMP-2) is a SNARE protein, which interacts with the plasma membrane–bound syntaxin and SNAP-25 to trigger neurotransmitter release (Sollner et al. 1993). Together, these proteins form a four-helix bundle (Otto et al. 1997; Sutton et al. 1998). Analysis of cultured hippocampal neurons from synaptobrevin-2 (syb2) knockout mice showed less severe impairment of spontaneous and hypertonic sucrose-induced release compared with evoked neurotransmitter release (Schoch et al. 2001). In addition, loss of synaptobrevin leads to a facilitation of release during high-frequency stimulation and a defect in fast endocytosis (Deak et al. 2004).
To test whether SNARE complex–mediated fusion mechanism involving synaptobrevin-2 (syb2) mediates the rapid action of La3+, we applied 2 mM La3+ onto cortical or hippocampal cultures obtained from syb2 knockout mice. Synapses in cultures obtained from wildtype mice showed the same sensitivity to La3+ as responses from wildtype rat cultures. Interestingly, the rapid effect of La3+ was completely abolished in syb2-deficient cultures (Fig. 8B, middle), supporting the premise that rapid action of La3+ requires functional SNARE proteins. The average amount of neurotransmitter release triggered by 2 mM La3+ in syb2-deficient cultures was 0.05 ± 0.01 nC (n = 14), which was indistinguishable from the baseline level of activity (0.03 ± 0.01 nC; n = 4). In contrast, parallel control experiments in wildtype cultures showed normal release in response to La3+ (0.86 ± 0.11 nC, n = 6; Fig. 8, B and C). In the same set of experiments, application of 2 mM Gd3+ was also ineffective in triggering rapid neurotransmitter release in syb2-deficient cultures. In a separate set of experiments, we tested whether the loss of La3+ response in syb2-deficient synapses was indeed caused by the absence of syb2 by reintroducing syb2. In syb2-deficient cultures infected with syb2, the charge transfer during 30 s of 250 µM La3+ was 1.11 ± 0.11 nC (n = 3), nearly 60% of neurotransmitter release compared with wildtype controls. This indicates that the defect shown in syb2-deficient culture was a specific consequence of the loss of syb2.
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). Mutants with insertion of 12 and 24 amino acids between the SNARE motif and the transmembrane domain (TMD) of synaptobrevin-2 showed that the physical distance between the two regions of synaptobrevin is indeed critical for the rescue of evoked fusion. Surprisingly, in contrast to the mutant with insertion of 24 amino acids, the 12 amino acids insertion mutant syb2 (12-ins syb2; Fig. 8A) completely rescued spontaneous release, suggesting that constraints on SNARE function during spontaneous fusion are more flexible than for evoked fusion. Interestingly, the La3+ effect on neurotransmission was also strictly dependent on the distance between the SNARE motif and the TMD domain of synaptobrevin-2, because La3+ application on synaptobrevin-2–deficient cultures infected with the lentivirus expressing 12-ins syb2 could not evoke any release (Fig. 8B, bottom). This result strongly suggests that La3+-triggered transmission shares the same strict molecular constraints as rapid action potential-evoked fusion. This is in striking contrast to spontaneous fusion or hypertonic sucrose evoked fusion, which both persist at a reduced but readily detectable level in synaptobrevin-2–deficient synapses (Deák et al. 2004; Schoch et al. 2001). The delayed action of La3+ was still detectable in cultures from synaptobrevin-2–deficient mice where the baseline rate of spontaneous release was typically 10-fold lower than in wildtype cultures (Fig. 8, D–G). The delayed effect could also be elicited by Gd3+, which also manifested a rapid synaptobrevin-2–dependent stimulation of neurotransmitter release. However, rapid application and washout of 2 mM La3+ or Gd3+ caused significant increase in the frequency of mPSCs in knockout and wild-type cultures, suggesting that lanthanides have two distinct effects on central synapses. The frequencies of mPSCs after 2 mM La3+ application showed more than fivefold increase (before La3+: 0.34 ± 0.05 Hz; after La3+: 1.97 ± 0.32 Hz; n = 14, P < 0.01) and a twofold increase after 2 mM Gd3+ application (before Gd3+: 0.46 ± 0.08 Hz; after Gd3+: 1.01 ± 0.14 Hz; n = 8, P < 0.02; Fig. 8F). The change in the amplitude of mPSCs was not statistically significant under all conditions (Fig. 8G). Thus in contrast to their rapid effects on neurotransmission, the delayed effect of La3+ and Gd3+ is only partially dependent on SNARE interactions.
To probe the molecular machinery underlying the rapid effect of La3+ further, we next took advantage of synaptotagmin-1 (syt-1)-deficient mice to examine whether La3+ may use the same pathway as rapid Ca2+-dependent synchronous release. We first stimulated cortical cultures obtained from syt-1–/– mice to confirm the genotype electrophysiologically and applied 2 mM La3+ for 30 s (Fig. 9A). Neurons obtained from Syt-1+/– were used as controls. The absence of syt-1 failed to block the rapid effect of La3+ (Fig. 9B). The cumulative charge transfer triggered during the first 10 s of La3+ application was 1.87 ± 0.49 nC in syt-1+/– (n = 4) and 1.94 ± 0.22 nC in syt-1–/– (n = 6) cultures (Fig. 9). This result suggests that, although the La3+-triggered release is strictly dependent on synaptobrevin-2, it does not require the function of synaptotagmin-1 as a sensor. This finding also argues against the possibility that the rapid effect of La3+ requires La3+ entry because intracellular La3+ could readily interact with synaptotagmin-1 and trigger release.
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To further characterize the delayed effect of La3+, we quantified the mPSC frequency before and after application of 2 mM La3+ after treatment of cultures with Ca2+ chelators such as EGTA-AM or BAPTA-AM (Fig. 10, A and B). These treatments substantially attenuated the delayed release while leaving the rapid effect intact (cf. Fig. 6A). Following incubation with 100 µM EGTA-AM for 10 min, the frequency of spontaneous release was largely unchanged compared with baseline after La3+ washout (before La3+: 3.84 ± 0.73 Hz; after La3+: 4.64 ± 1.01 Hz; n = 4). Incubation with 2 µM BAPTA-AM for 30 min also significantly diminished the delayed effect of La3+ (before La3+: 1.94 ± 0.61 Hz; after La3+: 3.58 ± 0.81 Hz; n = 6). In the presence of 1 mM EGTA and nominal Ca2+ in the extracellular environment, La3+ application caused an 11-fold increase in the frequency of mPSCs (from 2.96 ± 0.49 to 34.28 ± 1.36 Hz; n = 5, P < 0.001; Fig. 10C). This finding suggests that either intracellular Ca2+ or delayed entry of La3+ (e.g., Fig. 7) but not extracellular Ca2+ is responsible for the delayed release seen after removal of La3+ (Fig. 10D). In agreement with this premise, Cd2+ was ineffective in blocking the delayed effect, which suggests that Ca2+/La3+ entry through VGCCs was not required (Fig. 10D). The delayed effect was also insensitive to a specific inhibitor of PLCβ (U73122
[GenBank]
; Fig. 10D), which suggests that Ca2+ mobilization from internal stores that could be triggered by La3+ binding to the Ca2+ receptor is an unlikely source for this effect (Smith et al. 2004). Accordingly, depletion of Ca2+ from internal Ca2+ stores by for 30-min treatment of neurons with 1 µM thapsigargin did not impair the delayed release induced by subsequent application of 2 mM La3+ (Supplementary Fig. 1).
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DISCUSSION |
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In this study, we examined the effect of La3+ and other lanthanides, in particular Gd3+, on neurotransmitter release in dissociated hippocampal cultures and found that they triggered release in a rapid phase, which occurred within seconds, followed by a slow phase of release detectable after washout, which was hardly reversible. Our data showed that the rapid effect of La3+ is not mediated by augmenting existing Ca2+-dependent release, by increasing internal Ca2+, or by La3+ entry through various putative pathways. Instead, our findings suggest that La3+ acts extracellularly and mediates rapid neurotransmitter release, which requires the SNARE-dependent fusion machinery. The most salient feature of rapid La3+-triggered release is its strict dependence on synaptobrevin-2. We could only abolish the rapid effect of La3+ in the absence of synaptobrevin-2 but not synaptotagmin-1. This is surprising because other forms of Ca2+-dependent and -independent release including release driven by hypertonic sucrose are not fully eliminated in synaptobrevin-2–deficient synapses. Synaptobrevin-2 mutant with 12 amino acid insertion was also ineffective to rescue the rapid effect of La3+, suggesting that La3+-triggered fusion events are mechanistically closely related to fast evoked release. In addition, the properties of unitary synaptic responses during La3+ application were not different from events detected during normal spontaneous neurotransmission, providing further support that La3+ triggers neurotransmitter release by activating the conventional fusion machinery.
We could also detect a delayed effect of La3+, which is characterized as an increase in the frequency of mPSCs seen after removal of La3+ as previously shown in neuromuscular junction studies (Heuser and Miledi 1971). Delayed effect of La3+ is hardly reversible and persistent even in the absence of La3+, leading us to conclude that the entry of La3+ might mediate this delayed effect. Our data showed that pretreatment with well-known Ca2+ chelators, such as EGTA-AM or BAPTA-AM, significantly attenuated the delayed effect of La3+. As mentioned earlier, EGTA and BAPTA are effective chelators of La3+ as well as Ca2+. The absolute stability constants of EGTA:Ca2+ and EGTA:La3+ complexes indicate that EGTA can bind to La3+ with 10,000-fold higher affinity than Ca2+ (Sillren and Martell 1971). In cells pretreated with Ca2+ chelators, we assume that internal Ca2+ is effectively chelated before application of La3+. However, once we applied La3+, EGTA inside of a cell is expected to dissociate from internal Ca2+ and bind to La3+ because of its higher affinity to La3+. Thus for its delayed effect, La3+ entry through sources other than VGCCs might be required, and internalized La3+ itself might mediate neurotransmitter release. Alternatively, La3+ might use Ca2+ as a mediator for its delayed action. In this case, La3+ contributes indirectly to delayed effect by increasing internal Ca2+ concentration, presumably through its ability to inhibit Ca2+ uptake by mitochondria (Mela 1969a,b) or the function of PMCA (Herrington et al. 1996). Therefore both internal Ca2+ and La3+ uptake can be responsible for the delayed effect. In the absence of specific tools to clearly distinguish between Ca2+ and La3+, it is difficult to conclusively pinpoint whether the delayed release triggered by La3+ is a direct and indirect effect of La3+. However, these data strongly bolster our hypothesis that the rapid effect of La3+ does not require intracellular Ca2+ or La3+ because it is not susceptible to the same intracellular chelators.
La3+ as a specific tool to study synaptobrevin-2–dependent neurotransmitter release
As indicated above, rapid neurotransmitter release triggered by La3+ strictly required synaptobrevin-2. This argues for a strong specificity of La3+'s mechanism of action in contrast to other means to trigger neurotransmission. Most forms of release can persist in the absence of synaptobrevin-2, albeit at severely reduced levels (Deitcher et al. 1998; Schoch et al. 2001). This remaining release is thought to be triggered by an alternative vesicular SNARE(s) (Borisovska et al. 2005; Deak et al. 2006b). However, the results we present here indicate that, in the case of La3+-mediated release, these noncognate SNAREs may not be able to substitute synaptobrevin-2 functionally. This finding raises two possibilities. First, different vesicular SNAREs may manifest high functional specificity for different secretagogues. Alternatively, these noncognate vesicular SNAREs are located in a distinct population of vesicles that are not mobilized in response to La3+. The strong specificity of La3+-evoked fusion, compared with other means to trigger release, makes it a powerful tool to probe synaptobrevin-2 function in central synapses. Another advantage of La3+ as a tool to probe release machinery stems from the fact that most other secretagogues can cause morphological distortion of cell membranes either by inducing shrinkage (e.g., hypertonicity) or forming Ca2+ permeable channels (-latrotoxin). In contrast, in our hands, the concentrations of La3+ used to trigger release did not have any negative impact on membrane integrity. Another key feature of La3+-triggered rapid release is its persistence in the absence of synaptotagmin-1, which is consistent with the observation that rapid release triggered by La3+ is independent of Ca2+ or La3+ entry and may provide a useful probe to examine release independent of the fast Ca2+ sensing machinery.
What is the transduction mechanism that links La3+ to SNARE-mediated fusion machinery? As indicated above, our results suggest that La3+ seems to act at an extracellular site to trigger rapid SNARE-mediated neurotransmitter release, whereas the delayed release seems either to be a consequence of La3+'s slow entry into the cell or an indirect effect of Ca2+ signaling initiated by La3+ binding to an extracellular receptor (or both). There are several earlier studies that suggest that neurotransmitters or neuromodulators acting on presynaptic G protein–coupled seven transmembrane domain receptors may directly regulate neurotransmission in a SNARE-dependent manner. For instance, in lamprey central synapses, interaction of Gβ
subunits with SNAP-25 during serotonergic stimulation results in kiss-and-run–type fusion events (Gerachshenko et al. 2005; Photowala et al. 2006) and restricts glutamate release, suggesting a direct interaction between a G protein–coupled receptor and the fusion machinery. In addition, recent studies propose that the time-course of acetylcholine release is regulated by the voltage-sensitive muscarinic autoreceptors (Parnas and Parnas 2007). These findings suggest that muscarinic acetylcholine receptors tonically inhibit the release machinery, and this inhibition is relieved by membrane depolarization. Thus acting through a voltage-driven conformational change in muscarinic receptors, membrane voltage can exert tight control on the timing of neurotransmitter release (Slutsky et al. 2003). Furthermore, several G protein–coupled receptors such as the Ca2+ receptor (Smith et al. 2004) and group I metabotropic glutamate receptors (Abe et al. 2003) are present in central nerve terminals and possess binding sites for lanthanides. Therefore La3+ and other lanthanides may act on these presynaptic receptors that directly impact the release machinery.
Taken together, our results suggest that lanthanides act as powerful secretagogues to induce neurotransmitter release in a Ca2+-independent manner, taking advantage of the conventional SNARE-mediated release machinery. Further study of the mechanism underlying this process will not only help us better understand Ca2+-independent mechanisms that mediate neurotransmission but also provide insight into how extracellular heavy metals impact synaptic transmission under physiological and pathological circumstances.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 The online version of this article contains supplemental data. 
Address for reprint requests and other correspondence: E. T. Kavalali, Dept. of Neuroscience, Univ. of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9111 (E-mail: Ege.Kavalali{at}UTSouthwestern.edu)
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ABSTRACT
INTRODUCTION
