Glycerotoxin (GLTx), a large neurotoxin isolated from the venom of the sea worm Glycera convoluta, promotes a long-lasting increase in spontaneous neurotransmitter release at the peripheral and central synapses by selective activation of Cav2.2 channels. We found that GLTx stimulates the very high frequency, long-lasting (more than 10 hours) spontaneous release of acetylcholine by promoting nerve terminal Ca2+ oscillations sensitive to the inhibitor ω-conotoxin GVIA at the amphibian neuromuscular junction. Although an estimate of the number of synaptic vesicles undergoing exocytosis largely exceeds the number of vesicles present in the motor nerve terminal, ultrastructural examination of GLTx-treated synapses revealed no significant change in the number of synaptic vesicles. However, we did detect the appearance of large pre-synaptic cisternae suggestive of bulk endocytosis. Using a combination of styryl dyes, photoconversion and horseradish peroxidase (HRP)-labeling electron microscopy, we demonstrate that GLTx upregulates presynaptic-vesicle recycling, which is likely to emanate from the limiting membrane of these large cisternae. Similar synaptic-vesicle recycling through bulk endocytosis also occurs from nerve terminals stimulated by high potassium. Our results suggest that this process might therefore contribute significantly to synaptic recycling under sustained levels of synaptic stimulation.
Our understanding of synaptic mechanisms has been greatly enhanced by the use of naturally occurring neurotoxins. Shaped by evolution, neurotoxins act on key elements of synaptic processes in a highly specific manner. Some of these neurotoxins directly affect the neurotransmitter-release machinery and have been used to decipher key steps in exocytosis by helping to characterize the role of certain proteins involved in this process (de Paiva et al., 1999; Schiavo et al., 2000). By contrast, very few neurotoxins have been found to affect synaptic-vesicle endocytosis (Ceccarelli and Hurlbut, 1980). Initially, the venom of the polychaete Glycera convoluta was studied because of its ability to stimulate spontaneous quantal acetylcholine (ACh) release at the frog neuromuscular junction (NMJ) (Morel et al., 1983). G. convoluta venom (GCV) produces a long-lasting (several hours) high-frequency discharge of miniature end-plate potentials (MEPPs). Interestingly, the number of spontaneous quantal events that occur during hours of GCV stimulation far exceeds the number of synaptic vesicles known to be present in motor nerve terminals. However, preliminary ultrastructural examination of GCV-treated motor nerve terminals did not reveal any change in the number of synaptic vesicles, which has been used historically as evidence supporting the non-vesicular hypothesis (Morel et al., 1983). Later work from Ceccarelli's group uncovered synaptic-vesicle recycling at the frog NMJ (Ceccarelli and Hurlbut, 1980; Ceccarelli et al., 1973), which follows synaptic-vesicle fusion events occurring by means of either a ‘kiss-and-run’ mechanism (Fesce et al., 1994) or full fusion with the plasma membrane (Heuser and Reese, 1973). Although evidence for a kiss- and-run type of exocytosis has been found in neurosecretory and mast cells (de Toledo et al., 1993; Fernandez et al., 1984; Neher and Marty, 1982; Xia et al., 2009), much controversy still exists regarding the relevance of such a fast retrieval mechanism at the NMJ and central synapses (Balaji and Ryan, 2007; Dickman et al., 2005; Granseth et al., 2009; Rizzoli and Betz, 2004; Rizzoli and Betz, 2005; Verstreken et al., 2002; Zhang et al., 2009).
We have purified from GCV a glycoprotein of 320 kDa named glycerotoxin (GLTx). GLTx is its most active component upon synaptic release and its molecular target was identified as N-type Ca2+ channels (Cav2.2) (Meunier et al., 2002; Schenning et al., 2006). Here, we take advantage of the sustained stimulation of neurotransmitter release, characterized by burst-like episodes reaching more than 100-200 quanta released per second, that is triggered by GLTx to explore the mechanism of fast synaptic-vesicle retrieval at the amphibian NMJ under sustained asynchronous release stimulation.
We found that GLTx promotes presynaptic Ca2+ oscillations, driving an increase in MEPP frequency lasting for more than 10 hours and therefore largely exceeding the presynaptic content of synaptic vesicles. Quantitative electron microscopy analysis demonstrated that the number of synaptic vesicles present in motor nerve terminals was not significantly affected by GLTx despite the number of quanta released. We demonstrate that GLTx drastically enhances synaptic-vesicle recycling – an effect prevented by pretreatment with the Cav2.2-specific inhibitor ω-conotoxin MVIIA. Furthermore, electron microscopy analyses upon photoconversion of the styryl dye AM1-43 or horseradish peroxidase (HRP) labeling indicated that the recycling of synaptic vesicles triggered by GLTx stimulation predominantly originates from large presynaptic endosomal structures.
Glycerotoxin elicits a long-term increase in neurotransmitter release
GLTx is capable of eliciting a dramatic increase in spontaneous quantal neurotransmitter release at the frog NMJ (Meunier et al., 2002). Simple wash by perfusion of GLTx-free Ringer's buffer completely reversed the stimulatory effect (Fig. 1A). Re-application of GLTx on the same preparation elicited an equivalent increase in MEPP frequency, demonstrating that the effect of GLTx is indeed reversible and does not seem to promote run down of ACh release from motor nerve terminals (Fig. 1A). We have previously reported that the stimulatory effect of GLTx can be prevented by pretreating the neuromuscular preparation with a Cav2.2-specific inhibitor, such as ω-conotoxin GVIA (ω-CTx-GVIA) (Meunier et al., 2002; Schenning et al., 2006), which does not compete with GLTx for the same binding site on the Ca2+ channel (Schenning et al., 2006). To test whether ω-CTx-GVIA, in addition to preventing the stimulatory action of GLTx, could also block neurotransmitter release after the activation of Cav2.2 by GLTx, we determined whether the GLTx response could be inhibited by post-treatment with ω-CTx-GVIA. ω-CTx-GVIA added 20 minutes after GLTx completely blocked the stimulatory effect within 5 minutes after its application in the continuous presence of GLTx (Fig. 1B). This result confirms that GLTx binding does not prevent the interaction of ω-CTx-GVIA with Cav2.2 and that the effect of GLTx is totally abrogated by selective Cav blockade. Similar results were obtained using ω-CTx-MVIIA, further demonstrating the essential role of Cav2.2 in the stimulatory activity of GLTx (data not shown).
Very few neurotoxins are capable of promoting neurotransmitter release at a frequency as high as 100 MEPPs/second. However, α-latrotoxin (α-LTX) from the venom of the black widow spider and trachynilysin from the stonefish Synanceia trachynis have been reported to promote such stimulatory activity (Clark et al., 1970; Colasante et al., 1996; Tsang et al., 2000). These two toxins produce a rapid depletion in synaptic vesicles, leading to a block of both evoked and spontaneous transmitter release (Clark et al., 1970; Colasante et al., 1996; Tsang et al., 2000). In the case of α-latrotoxin, this depletion only occurs under Ca2+-free conditions (Ceccarelli and Hurlbut, 1980). We therefore investigated whether GLTx could also promote long-term blockage of spontaneous release from intoxicated NMJs. Surprisingly, nerve terminals exposed to GLTx for 175 minutes still exhibited high MEPP frequency (Fig. 1C), which remained elevated even after more than 10 hours of continuous GLTx stimulation (Fig. 1C), suggesting that synaptic vesicles were being actively recycled.
Glycerotoxin promotes a long-term increase in basal intraterminal Ca2+ levels
GLTx has been previously shown to promote spontaneous rises in levels of intracellular Ca2+ in rat brain synaptosomes (Madeddu et al., 1984) and in the cell body of rat motor neurons in culture; these increases were reported to be sensitive to the blockade of Cav2.2 channels (Schenning et al., 2006). From this earlier work, it was hypothesized that GLTx stimulates neurotransmitter release by directly activating Cav2.2 and enhancing Ca2+ entry into the motor nerve terminals. To directly test this hypothesis, we imaged the spontaneous fluctuations in intracellular Ca2+ in motor nerve terminals of the frog NMJ after treatment with GLTx. As shown in Fig. 2, the morphological feature of regularly spaced active zones, each of which is about 1 μm long, can be exploited to focus the analysis of Ca2+ fluctuations in individual active zones (Fig. 2A). Using this approach, we collected Ca2+-sensitive fluorescent images within these areas of the NMJ at 0.5 Hz. We detected a GLTx-mediated increase in resting Ca2+ fluctuations (Fig. 2B,C). Strikingly, there were occasional strong Ca2+ fluctuations. After both 1 and 5 hours of incubation with GLTx (150 pM), this general increase in fluctuations and the strong Ca2+ fluctuations occurred spontaneously at similar rates within these presynaptic active zones (Fig. 2B-E).
To measure the effect of GLTx in terms of promotion of spontaneous Ca2+ entry, we calculated the frequency of those fluctuations that exceeded twice the standard deviation of average fluctuations within these active zones. Following 1 hour of incubation with GLTx, this frequency was 0.016±0.004/active zone (AZ)/second (54 active zones, 5 terminals) and, following five hours of exposure, it became 0.013±0.001/AZ/second (37 active zones, 3 terminals). There was no significant difference in frequency between 1 hour and 5 hours of GLTx incubation (p=0.56, unpaired t-test). Considering that each amphibian motor nerve terminal contains approximately 700 active zones (Soyoun Cho and S.D.M., unpublished observations), the frequency of spontaneous fluctuations in Ca2+ entry would be about 10/nerve terminal/second. In this light, it is likely that the increase in Ca2+ fluctuations leads to the reported general elevation of MEPP frequency, and that the strong Ca2+ entry events might instead trigger the large synchronous bursts in MEPP frequency observed in addition to the general increase in asynchronous MEPP frequency (Betz et al., 1992; de Paiva et al., 1999; Henkel et al., 1996; Lin et al., 2005; Meunier et al., 2002; Ribchester et al., 1994; Smith and Betz, 1996).
To confirm that Cav2.2 channels are responsible for the GLTx-evoked Ca2+ fluctuations at the frog NMJ, we examined the effects of Cav2.2 blockers in this system. Fig. 3A-C shows that these transient Ca2+ spikes disappeared after the addition of ω-CTx-GVIA (2 μM). To compare quantitatively the magnitude of Ca2+ fluctuations, we calculated the coefficient of variation (CV) of fluorescence within each active zone. One hour (n=81 active zones) of GLTx treatment significantly increased this parameter within individual active zones (P<0.01, one-way ANOVA) compared with active zones in control terminals (n=24 active zones) (Fig. 3C). Importantly, there was no significant difference in CV between control nerve terminals and nerve terminals treated with GLTx plus ω-CTx-GVIA (n=57 active zones; P>0.05, one-way ANOVA) (Fig. 3C). Furthermore, the CV of fluorescence intensity of individual active zones was still significantly increased after 5 hours of GLTx treatment compared to controls (n=37 active zones, data not shown).
Glycerotoxin promotes the long-term upregulation of endocytosis
Styryl dyes such as FM1-43 have been widely used to investigate synaptic-vesicle recycling at the NMJ (Betz et al., 1992; de Paiva et al., 1999; Henkel et al., 1996; Lin et al., 2005; Ribchester et al., 1994; Smith and Betz, 1996). FM1-43 was applied for 5 minutes in the absence of stimulation before washing the preparation with cold Ca2+-free medium. As shown in Fig. 4A, FM1-43 staining was predominantly localized on the plasma membrane of motor nerve terminals, as previously shown (Betz et al., 1992). The same procedure was then carried out 20 minutes after the initial addition of GLTx. Importantly, GLTx was capable of inducing the internalization of the styryl dye, as illustrated by the intensity profile taken across the nerve terminal (Fig. 4B) when compared to the unstimulated control (Fig. 4A). Subsequently, FM1-43-loaded motor nerve terminals were stimulated with GLTx in a Ca2+-containing Ringer's solution, rinsed and imaged 5 minutes later. Destaining of the labeled terminals was observed, with translocation of the dye from the intraterminal space to the plasma membrane (Fig. 4C). The GLTx-induced FM1-43 uptake was inhibited by pre-incubation of the preparation with ω-CTx-MVIIA (Fig. 4D). To test whether the FM1-43 taken up as a result of GLTx action could be released upon electrical stimulation, a destaining experiment was carried out. Nerve terminals were preloaded with FM1-43 for 5 minutes at the end of 30 minutes stimulation using GLTx, washed and imaged before and after stimulation at 5 Hz for 5 minutes. Clear translocation of the dye from the intraterminal space to the plasma membrane was found, suggesting that GLTx-induced recycling of vesicles can efficiently be reused for neurotransmitter release (Fig. 4E). Although most of the internal FM1-43 staining was released using electrical stimulation, few of the FM1-43-labelled internal structures did not undergo destaining, suggesting that a limited number of the endocytic compartments generated by GLTx entered a dead-end pathway. Taken together, these experiments demonstrate that GLTx triggers exocytosis of synaptic vesicles as well as promoting their active compensatory recycling.
To investigate whether the effect of GLTx on synaptic-vesicle recycling was long lasting, a short pulse of FM1-43 was applied during the last 5 minutes of GLTx treatments of variable duration. This was followed by several washes in cold Ca2+- and GLTx-free Ringer's solution prior to imaging (Fig. 5A). Interestingly, when GLTx was co-applied with FM1-43 for only 5 minutes, hotspots abutting the plasma membrane were clearly visible, indicating sites of active vesicle recycling (Fig. 5B). Preparations exposed to GLTx for longer periods (between 30 and 300 minutes) instead exhibited internal staining of the motor nerve terminals (Fig. 5B). Quantification of FM1-43 uptake shows that GLTx promotes long-lasting upregulation of synaptic-vesicle recycling (Fig. 5C). Importantly, at longer time points, the internalized FM1-43 staining was not uniform throughout nerve terminals nor did it exhibit the classical banding pattern observed following relatively mild stimulation (5 minutes high K+-induced depolarization, Fig. 6A) (Betz et al., 1992). Rather, donut-like structures displaying intense FM1-43 staining were detected (Fig. 5B and Fig. 6D). Interestingly, these structures were also induced by long-lasting depolarization with high K+ medium (Fig. 6B), suggesting that a switch in endocytosis mode occurs to allow long-term mobilization of synaptic-vesicle recycling. These structures were reminiscent of large vacuoles, which might appear following sustained stimulation of neurotransmitter release. Importantly, the high intensity of FM1-43 staining of these donut-like structures suggested that the majority of recycling vesicles could be emanating from these large endocytic structures.
Large endocytic structures make a major contribution to synaptic-vesicle recycling under sustained stimulation
We next investigated the ultrastructural changes produced by GLTx treatments of various lengths. A number of parameters were examined, including the number of synaptic vesicles, coated vesicles, large dense core vesicles and enlarged endosomes, and the area of the presynaptic nerve terminals (Table 1). Most parameters remained unchanged by GLTx treatment at all of the time points investigated. Importantly, the number of synaptic vesicles was not affected, demonstrating that the recycling process was indeed capable of maintaining an intact pool of synaptic vesicles. Another line of evidence that active recycling was taking place upon GLTx treatment was the finding that the number of large endosomes was significantly increased by GLTx treatment at longer time points (Table 1). To investigate the nature and localization of recycling vesicles, we used the photoconvertible styryl dye AM1-43 to identify AM1-43-loaded recycling vesicles in GLTx-treated preparations by electron microscopy. We next incubated preparations in the presence or absence of GLTx for 60 and 120 minutes prior to applying a pulse of AM1-43 for 5 minutes, followed by extensive washing in cold Ca2+- and GLTx-free Ringer's solution. Confocal images of the stained nerve terminal were taken (Fig. 6D) before carrying out photoconversion and processing of the preparation for electron microscopy. Unstimulated nerve terminals did not exhibit any noticeable AM1-43 precipitate (Fig. 6E). Examination of GLTx-treated nerve terminals revealed large endosomal structures (Fig. 6F,G). Importantly, the vast majority of photoconverted vesicles were either abutting or in close proximity to these large structures (Fig. 6E,F). In some instances, a number of these large structures were found to be either very close to or fused with each other (see serial sections in Fig. 6G, inserts).
The lack of detectable photoconverted AM1-43 dye inside the lumen of the large endosomal structures in GLTx-treated nerve terminals was surprising. However, this is not unprecedented because it has been previously suggested that FM dyes do not convert well inside endosomal compartments (de Lange et al., 2003). We therefore used the classical fluid-phase marker horseradish peroxidase (HRP) to probe the formation of the large endosomes under long-term GLTx stimulation. NMJ preparations, in either the presence or absence of GLTx, were exposed to HRP for 5 hours. In preparations treated with GLTx, electrophysiological recordings of MEPP frequency were performed to confirm the activity of GLTx prior to addition of HRP (data not shown). After 5 hours, samples were fixed and processed for electron microscopy. In control unstimulated preparations treated with HRP, no endosomes were detected and the HRP precipitate was confined to the synaptic cleft and around the perisynaptic Schwann cells (Fig. 7A). In contrast, in GLTx-stimulated preparations, HRP was taken up into bulk endosomes (Fig. 7B-D). HRP-positive tubular structures seemingly in contact with the plasma membrane were also detected (Fig. 7B and E). Remarkably, the electron-dense precipitate generated by HRP in our experiment did not fill the entire endosome, but was mainly found decorating its membrane (Fig. 7B-G). This is consistent with previous results obtained using the same technique (Clayton et al., 2008; Clayton et al., 2009; Meshul and Pappas, 1984). Notably, HRP-filled vesicles were detectable in close proximity to these endosomes (Fig. 7F). One example is shown in Fig. 7F (arrow): one can see clear continuity in the HRP staining between the HRP precipitate lining the endosomal membrane and an omega-shaped vesicle seemingly budding from the bulk endosome. Interestingly, we also observed C-shaped bulk cisternae and multilamellar endosomes in GLTx-treated nerve terminals (Fig. 7H and I). Endosomes with vesicles seemingly in the process of budding off were frequently observed in GLTx-treated nerve terminals (Fig. 7J-M). Occasionally, large membrane invaginations with vesicles seemingly pinching off from these intermediate structures were also observed (Fig. 7N). Altogether, our data suggest that GLTx promotes an active recycling process stemming from bulk endosomes that contributes to the maintenance of neurotransmitter release at the NMJ.
In this study, we characterized the morphological and functional changes caused by GLTx at the amphibian NMJ. We found that GLTx elicits long-term stimulation of asynchronous quantal transmitter release without noticeable changes in the number of synaptic vesicles. GLTx was therefore used as a tool to examine the modality of synaptic-vesicle recycling under such conditions of elevated neurotransmitter release. Using FM1-43, we found that GLTx greatly increased the synaptic-vesicle recycling rate, which was upregulated for over 5 hours. Photoconversion experiments carried out at various time points after GLTx application clearly show that most recycling vesicles are localized on or near large endosomal compartments.
GLTx has unique features among excitatory neurotoxins
Other excitatory neurotoxins, such as α-LTX or trachynilysin, have been shown to promote the selective depletion of small synaptic vesicles (Colasante et al., 1996; Matteoli et al., 1988). These two neurotoxins stimulate asynchronous neurotransmitter release at a similar rate to GLTx, but this rapidly results in a full blockade of neurotransmitter release caused by the depletion of synaptic vesicles (Colasante et al., 1996; Tsang et al., 2000). It therefore appears that the mechanism of GLTx action differs from that of the above-mentioned pore-forming neurotoxins (Davletov et al., 1998). Although the stimulatory effect of trachynilysin is strictly dependent on external Ca2+, the synaptic-vesicle depletion elicited by α-LTX in the absence of external Ca2+ (Ceccarelli et al., 1973) has been attributed to the mobilization of intracellular Ca2+ from mitochondria (Tsang et al., 2000). One of the major differences between the effects of α-LTX and GLTx is that the stimulatory effect of GLTx is dependent on external Ca2+ and the intracellular Ca2+ signals elicited by the two toxins are not identical in nature. Whereas α-LTX promotes a long-lasting high-amplitude rise in intracellular Ca2+, GLTx gives rise to an overall small increase in the basal Ca2+ level (low amplitude), accompanied by occasional Ca2+ spikes (Fig. 2C,E). In this light, the Ca2+ signal generated by GLTx is reminiscent of that elicited by electrical stimulation and this might explain why GLTx does not overwhelm the endocytic mechanism. Alternatively, pore-forming toxins such as trachynilysin and α-LTX might cause a run-down of the energy level of the nerve terminals, contributing to the blockade of endocytosis and vesicle recycling. GLTx targets motor nerve terminals through a direct interaction with Cav2.2, resulting in channel upregulation (Meunier et al., 2002; Benoit et al, 2002; Schenning et al., 2006). Similarly, GLTx has been shown to promote Ca2+ influx in Cav2.2-expressing HEK and bovine chromaffin cells (Meunier et al., 2002) and, more recently, in cultured motor neurons (Schenning et al., 2006). The single large Ca2+ transient detected in these cells was hard to reconcile with the long-lasting stimulatory effect of GLTx observed at the amphibian NMJ. Our results demonstrate that GLTx promotes subtle long-lasting changes in the basal level of intraterminal Ca2+ (Fig. 2), consistent with a general increase in asynchronous release. Moreover, the intermittent larger Ca2+ transient could account for the burst of MEPPs also observed in GLTx-treated preparations.
Number of vesicles released by long-term activity
The ability of neurons to sustain high levels of neurotransmitter release under high-frequency stimulation is crucial to maintaining motor and more integrative cerebral functions. The level of GLTx-induced asynchronous neurotransmitter release from motor nerve terminals is comparable to that seen in high-frequency nerve stimulation (~100 synaptic vesicles/second; Fig. 1). In 10 hours of GLTx treatment, an estimated more than 3 million synaptic vesicles underwent fusion. In our longest GLTx incubation (24 hours), the nerve terminals were still firing at 100 MEPPs/second, suggesting that, in this case, over 8 million vesicles were released. Considering that the total number of synaptic vesicles at the frog presynaptic nerve terminal has been estimated to be 300,000 (Couteaux and Pecot-Dechavassine, 1970; Heuser and Reese, 1981; Van der Kloot and Molgo, 1994), the number of released vesicles after 10 hours of GLTx action exceeded the actual total number of synaptic vesicles by more than an order of magnitude. This suggests that the synaptic-vesicle recycling mechanism is not only highly upregulated, but also robust enough to sustain high activity for many hours, with no evidence of synaptic-vesicle depletion (Table 1). The total number of readily releasable vesicles per nerve terminal at a given time point has been estimated to be ~25,000. GLTx action would therefore deplete this vesicle pool in just over 4 minutes, suggesting that, over 10 hours of stimulation, the readily releasable pool has been completely replenished over 150 times.
Other studies have used high-frequency stimulation conditions of 30 Hz for 1 minute, which corresponds to 1800 stimulations (Richards et al., 2000). Assuming that each stimulation results in 380 quanta being released (Van der Kloot and Molgo, 1994), the total number of quanta released is 684,000. Our study is perhaps more comparable to an earlier study, which describes 3 million quanta being released over 24 hours using a 2 Hz nerve-evoked simulation paradigm (Lynch, 1982). This type of stimulation would produce a higher rate of release (700-800 quanta/second), which was shown to induce significant depletion of synaptic vesicles (Lynch, 1982). To the best of our knowledge, our study establishes that the machinery for synaptic-vesicle recycling at the frog NMJ can cope with the release of over 100 but less than 700 vesicles/second.
Mode of endocytosis under GLTx stimulation
Two main hypotheses have been put forward to explain synaptic-vesicle recycling: clathrin-mediated endocytosis occurring outside the active zones; and a ‘kiss-and-run’ mechanism involving the transient fusion and immediate retrieval of synaptic vesicles in the active zone (Ceccarelli et al., 1973; Colasante et al., 1996; Colasante and Pecot-Dechavassine, 1996; Heuser and Reese, 1973). Both modes of endocytosis have been shown to play important roles in maintaining cellular communication at varying frequencies in a range of synapses (Colasante and Pecot-Dechavassine, 1996; Harata et al., 2006). At the frog NMJ, coated pits and coated vesicles were only detected far from the active zones upon electrical stimulation in the presence of the K+ channel blocker 4-aminopyridine (Colasante and Pecot-Dechavassine, 1996; Heuser and Reese, 1981), whereas during simple high K+ stimulation, neither coated pits nor synaptic-vesicle depletion were detected (Colasante and Pecot-Dechavassine, 1996); an alternative mechanism for synaptic-vesicle recycling, such as kiss-and-run, has thus been inferred.
The use of fluorescent styryl dyes has allowed the characterization of distinct vesicle pools that differ in their dynamic properties (Rizzoli and Betz, 2005). Low-frequency neurotransmission mainly relies on the readily releasable pool of synaptic vesicles, whereas high-frequency stimulation depletes the reserve pool (Gaffield et al., 2006; Richards et al., 2000). We have used GLTx as a tool to investigate which mechanism is responsible for the replenishment of synaptic vesicles under conditions of sustained high-level release of neurotransmitters at the NMJ. Our photoconversion experiment demonstrates that recycling occurs via synaptic vesicles that are likely to bud from large endosomal structures.
In the kiss-and-run mode of endocytosis, recycled vesicles are expected to localize at or very near the plasma membrane (Rizzoli and Betz, 2004). Our results show that, under GLTx-induced sustained stimulation of exocytosis, only a few recycled vesicles can be detected in this area (Fig. 6). This is in good agreement with work demonstrating that recycled vesicles were not enriched near the active zone (Rizzoli and Betz, 2004). In our experiments, most of the photoconverted vesicles were localized either abutting or in the proximity of large vacuolar structures. These compartments were not detected upon short-term high-rate stimulation; smaller oblong-shaped cisternae were mostly observed in this case (Richards et al., 2000). Similarly, low-rate chronic stimulation (24 hours at 2 Hz) has been shown to increase the number of these small cisternae at the frog NMJ (Lynch, 1982). One could speculate that the large endosomal structures highlighted in our study might result from the accumulation of plasma membrane derived from the hot-spots detected on the plasma membrane after 5 minutes exposure to GLTx (Fig. 5B). Our HRP-labeling approach confirmed the results obtained by AM1-43 photoconversion by showing that HRP was taken up by these GLTx-induced bulk endosomes (Fig. 7) and that vesicles budding off from these structures also contained some detectable precipitate (Fig. 7).
Our findings suggest that the same bulk endocytic mechanism is involved in short- and long-lasting stimulation, and that the pathway triggered by GLTx is actively involved in recycling, as demonstrated by the destaining experiment (Fig. 4E). At the frog NMJ, GLTx yields the same ultrastructural changes seen upon short-duration electrical stimulation (Richards et al., 2000), such as the appearance of C-shaped endosomes (Fig. 7H) and larger-diameter endocytic vacuoles, such as those recently described by Clayton and co-workers (Clayton et al., 2008; Clayton et al., 2009). We cannot rule out from our data that excess membrane could also be redirected to dead-end compartments, as suggested by the presence of GLTx-induced FM1-43 staining that was resistant to electrical stimulation (Fig. 4E). Consistent with this, together with bulk endosomes surrounded by recycling vesicles, a small number of multilamellar structures were also observed using HRP labeling (Fig. 7I). At present, we know neither the nature nor the precise contribution of this pathway to the overall process. However, it is tempting to hypothesize that GLTx promotes the recycling of synaptic vesicles through bulk endocytosis and that the excess membrane generated by upregulated synaptic-vesicle fusion is internalized in a dead-end pathway, as previously described (Heerssen et al., 2008; Kasprowicz et al., 2008).
In conclusion, GLTx represents a powerful research tool to investigate endocytic pathways at the nerve terminal. More detailed molecular characterization of the large vacuolar compartments will be necessary to further understand the precise function(s) of bulk endosomes and their precise contribution to synaptic-vesicle recycling.
Materials and Methods
Reagents, drugs and toxins
ω-CTx-GVIA and ω-CTx-MVIIA were purchased from Alomone (Jerusalem, Israel). FM1-43 was purchased from Molecular Probes (Carlsbad, USA) and AM1-43 was purchased from Biotium (Hayward, USA). All other reagents were from Sigma (Castle Hill, Australia).
Purification of GLTx
G. convoluta specimens were obtained from the Marine Biological Station, Roscoff (France). GLTx was prepared as previously described and tested for its ability to stimulate catecholamine release from bovine adrenal chromaffin cells (Meunier et al., 2002). Protein concentration was determined using the Bradford assay (BioRad, Hercules, USA).
Amphibian NMJ preparations were used. In these preparations, N-type (Cav2.2) Ca2+ channels are responsible for eliciting Ca2+-induced neurotransmitter release (Van der Kloot and Molgo, 1994). Electrophysiology was carried out using Rana esculenta. Ca2+ imaging was done on Rana pipiens and the FM1-43 study was carried out on Bufo marinus (School of Biomedical Sciences Teaching Facility, University of Queensland, Australia). Adult frogs (R. pipiens) were anesthetized in a 0.1% tricaine methane sulfonate solution and killed by double pithing. The cutaneous pectoris (CP) muscles with their motor nerve were dissected and bathed in normal frog Ringer's solution (NFR; in mM: 116 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES-OH, pH 7.4). To load the Ca2+-sensitive dye Calcium Green-1 (3000 Da dextran conjugate; Molecular Probes), the motor nerve was cut approximately 0.5 mm from the edge of the muscle. The cut end of the nerve was immersed in a drop of 30 mM Calcium Green-1 for 7-8 hours at room temperature. The preparation was then washed in NFR and incubated at 4°C for 2 hours before being pinned down on an elevated Sylgard platform (Dow Corning, Midland, USA) at the centre of a recording chamber. The nerve was stimulated through a suction electrode at five times the threshold intensity required to cause muscle twitching. Labeling of postsynaptic nicotinic ACh receptors with 2 μg/ml AlexaFluor594-conjugated α-bungarotoxin (α-BTX) (Molecular Probes) for 10 minutes was used to focus on the active zone and evaluate the z-axis drift over the course of data collection. Muscle contractions were blocked by adding 10 μM D-tubocurarine. Superficially positioned nerve terminals in a single focal plane were chosen for study.
Calcium imaging and analysis
The wavelengths and timing of the laser illumination (Krypton-Argon laser; Innova 70 Spectrum, Coherent, Hilton, Australia) were selected using an acousto-optic tunable filter (AOTF; ChromoDynamics, Chicago, IL, USA). The laser was fiber coupled to the illumination port of an upright fluorescence microscope (Lumplan/FL IR, Olympus). A 100× water-immersion objective (1.0 NA) was used and provided a spatial resolution of ~333 nm. Calcium Green-1 was excited with the 488 nm laser line and emitted light was collected through a 530±20 nm filter. AlexaFluor594–α-BTX was excited with the 567 nm line and light was collected through a 620±30 nm emission filter. All images were collected using a liquid-nitrogen-cooled, back-thinned CCD camera (LN1300B, Roper Scientific, Tucson, USA), which enabled the high-sensitivity detection of fluorescence signal with low noise. Using 20 Hz electrical nerve stimulation, only those terminals with a strong nerve-stimulation-evoked Ca2+ signal were chosen for further experiments. To detect spontaneous Ca2+ oscillations, 50-100 fluorescence images were collected at 0.5 Hz for each nerve terminal in the absence of nerve stimulation. For each image, the laser illuminated the calcium-sensitive dye for 1 ms. All image processing and analyses were performed using MATLab. Before analysis, images were coregistered to correct for slight movement of the preparation during data collection. To measure GLTx-induced spontaneous Ca2+ fluctuations within the active-zone regions of the adult NMJ, the average pixel intensities over the entire active zone were calculated. The region of interest for each active zone was defined by correlating the banding pattern of α-BTX labeling with the average Ca2+ image (see Fig. 2A). To assess background Ca2+ oscillations within individual active zones before GLTx treatment, a histogram of fluorescence intensity was plotted and fitted with a Gaussian distribution. The mean of the 95% range of fluorescence intensity (±2 s.d.) was taken as baseline fluorescence. Differences from baseline were determined for individual images by subtracting this baseline fluorescence. The resulting ‘differential images’ represented the relative fluorescence changes, computed as ΔF/F0=(F–F0)/F0, and were displayed in pseudo-colors. The CV in the fluorescence within each active zone was calculated as the ratio of standard deviation in F divided by mean fluorescence intensity. The magnitude of Ca2+ oscillations was determined by comparison with the mean value of CV. The frequency of spontaneous Ca2+ fluctuations following GLTx treatment was calculated by counting the number of fluorescence intensity excursions that exceeded 2×s.d. of the baseline fluorescence.
MEPPs were recorded at room temperature (22°C) with an intracellular glass capillary microelectrode filled with 3 M KCl (8–12 Ω) using conventional techniques. The motor nerve of the isolated NMJ was stimulated via a suction microelectrode. After amplification, electrical signals were digitized, displayed on an oscilloscope and simultaneously recorded on videotape with the aid of a modified digital audio processor (Sony PCM 701 ES) and a video-cassette recorder (Sony SLC 9F). Data were collected and analyzed with SCAN software kindly provided by John Dempster (University of Strathclyde, UK) running on a PC equipped with an analogue-digital converter (Model DT2821, Data Translation, Marlboro, USA). Statistical analysis was performed using the Student's t-test (two tailed, unpaired). Values are expressed as mean±s.e.m. and data were considered significant at P<0.05.
A 5 minute pulse of FM1-43 (10 μM) was applied to toad NMJ preparations in NFR solution in either the absence or presence of GLTx or high K+ (30 mM). Preparations were then washed five times with cold, Ca2+-free, EGTA Ringer's solution. NMJs were visualized and images acquired on a Zeiss 510 meta laser scanning confocal microscope. For destaining experiments, the NMJ preparations were washed with NFR solution five times prior to electrical stimulation.
Toad NMJ preparations were treated with GLTx and MEPP frequencies were recorded to confirm toxin activity. 10 mg/ml HRP (Sigma, type VI) in Ringer's solution was applied to preparations and incubated for a further 5 hours. Muscles were briefly washed in Ringer's solution and then fixed in 4% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M phosphate buffer for 2 hours at room temperature. The preparations were then washed in 0.1 M PBS three times for 20 minutes, followed by washing in acetate buffer (pH 6.0) for 5 minutes. Sections were incubated for 15 minutes in a 2% NiSO4 solution (in sodium acetate buffer) containing 2 mg/ml D-glucose, 0.4 mg/ml NH4Cl and 0.025% diaminobenzidine (DAB). Glucose oxidase (0.2 μl/ml) was added to the nickel-DAB solution, resulting in the production of H2O2 and the deposition of nickel-DAB. Sections were washed in sodium acetate buffer to stop the reaction and then washed thoroughly in PBS (three times for 15 minutes).
Our protocol was adapted from previous studies (Harata et al., 2001; Richards et al., 2003). AM1-43-labeled preparations were fixed in PBS containing 2% glutaraldehyde for 2 hours. Muscles were then quickly washed twice in PBS, quenched in PBS containing 100 mM glycine pH 7.4 for 1 hour, soaked in PBS containing 100 mM ammonium chloride for 5 minutes, and further washed in PBS for 30 minutes. Samples were treated with 1.5 mg/ml DAB diluted in PBS for 30 minutes in the dark. The preparation was then illuminated under UV light through a 10× air objective (Carl Zeiss, Oberkochen, Germany) from a 100 W mercury lamp for 45-70 minutes, depending on the rate of formation of the brown precipitate.
Muscle preparations were fixed in 2.5% glutaraldehyde, post-fixed in 1% osmium tetroxide, followed by ethanol dehydration and Epon resin embedding (ProSciTech, Thuringowa, Australia). Cross-sections (60 nm thick) were stained with uranyl acetate and Reynold's lead citrate. NMJs were examined using a JEOL 1010 transmission electron microscope.
This work was supported by a grant from the Australian Research Council (F.A.M. and T.H.N.), a fellowship to F.A.M. from the National Health and Medical Research Council of Australia, by Cancer Research UK (G.S.) and by US National Institutes of Health R01 NS043396 to S.D.M. Deposited in PMC for release after 12 months.