Botulinum Neurotoxin a Blocks Synaptic Vesicle Exocytosis but Not Endocytosis at the Nerve Terminal

Cultures

Timed pregnant C57Bl/6NCR mice were obtained from the Frederick Cancer Research and Development Center. Neuronal cell cultures were prepared from mouse fetuses, 13 d in gestation as described previously (Ransom et al. 1977; Fitzgerald 1989). Dissociated spinal cord cells were plated at 1 × 10⁶ cells per 35-mm plastic culture dish coated with Vitrogen-100^® (Collagen Corp.). For immunohistochemistry and FM1-43 uptake, spinal cord cells were plated at 2.5 × 10⁵ cells per dish onto a confluent glial feeder layer (Neale et al. 1991). Cultures were maintained at 35°C in a humidified atmosphere containing 10% CO₂, fed twice weekly by partial medium replacement, and were used between the third and fifth week after plating.

TeNT (10–100 ng/ml) or BoNT A (10–300 ng/ml) was added to the cultures in serum-free medium for 16–26 h before experiments (unless noted otherwise). Immunohistochemistry for synapsin, VAMP, and SNAP-25 was performed as described in detail (Williamson et al. 1996), using antisynapsin at 1:1,000, anti-VAMP at 1:400, and anti–SNAP-25 at 1:400.

Neurotransmitter Release

K⁺-evoked, Ca²⁺-dependent release of glycine was assayed as described previously (Williamson et al. 1996). In brief, cultures were labeled with [³H]glycine, rinsed, and incubated in Ca²⁺-free solutions containing 3 mM K⁺ with 0.5 mM EGTA, followed by 56 mM K⁺ (no EGTA), and finally in depolarizing medium containing 2 mM Ca²⁺ and 56 mM K⁺. For this study, 20 μl of incubation medium was withdrawn either at 1-min or 20-s intervals as noted (see Fig. 2) for scintillation spectrometry.

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Figure 2

Time course of release of [³H]glycine in response to K⁺ depolarization. (top) Control and toxin-treated spinal cord cultures were incubated with [³H]glycine, rinsed as described in Materials and Methods, and changed into HBSS containing 56 mM K⁺ and no calcium. Samples were withdrawn at 1-min intervals and counted. Levels of radioactivity released from control and toxin-blocked cultures are essentially identical. (middle) The medium was changed to one containing 56 mM K⁺ and 2 mm Ca²⁺ and samples were withdrawn every 20 s. Radioactivity released from control cultures in the presence of calcium is increased, whereas that released from toxin-treated cultures is virtually unchanged from the zero-Ca²⁺ samples. (bottom) Difference in radioactivity released in the presence and absence of Ca²⁺. Values for TeNT and BoNT A are very similar and show no Ca²⁺ dependence. Experiments were performed on cultures from three dissections, assayed between 23 and 30 d after plating. Each data point is the mean ± SD. Triangles, controls, n = 7; circles, TeNT (100 ng/ml for 24–26 h), n = 6; and squares, BoNT A (200 ng/ml for 24–26 h), n = 6.

Electrophysiology

Nystatin-perforated whole cell patch clamp experiments were performed on control and toxin-treated neurons. Patch electrodes were fire-polished; electrode resistance was ∼2 MΩ. Resting membrane potentials were measured and spontaneous postsynaptic currents (PSCs) were continually recorded with an Axopatch-1B amplifier, and analyzed with an ITC-16 computer interface, a Macintosh computer, and Synapse software developed by Instrutech Corporation and Synergistic Research Systems. For recording, the cultures were bathed in 1 ml of a Hepes-buffered salt solution (HBSS) containing 136 mM NaCl, 3 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM Hepes, 10 mM glucose, and 0.1% BSA at pH 7.25 with the osmolality adjusted with sucrose to 325 ± 5 mmol/kg (Westbrook and Brenneman 1984). The pipette medium contained 145 mM KCl, 5 mM NaCl, 2 mM MgCl₂, 5 mM Hepes, 1 mM EGTA, and 100 nM free Ca²⁺, pH 7.3. After baseline recordings were obtained, 1 ml of HBSS containing 56–90 mM KCl (with NaCl adjusted) was applied over the course of 8–18 s to the surface of the patched neuron under direct microscopic observation. With addition of the high K⁺ solution, resting membrane potentials were depolarized (average −60.7 ± 4.1 mV to −16.7 ± 1.7 mV; n = 9). PSC amplitude and duration, beginning within 1 s of the full application of K⁺, were integrated as PSC area with Synapse software.

Uptake of FM1-43 and of HRP

For FM1-43 loading, control and toxin-exposed cultures were rinsed in HBSS and depolarized for 5 min at 35°C with 56 mM KCl in isosmotic HBSS containing 2 mM CaCl₂ and 2 μM FM1-43 (Betz and Bewick 1992). Cultures were rinsed several times over 20–30 min in HBSS either containing 1 μM tetrodotoxin, or without Ca²⁺ and containing 0.5 mM EGTA, to clear surface membranes of the dye while preventing spontaneous network activity and consequent loss of FM1-43 from within labeled terminals. Living cultures were photographed in this rinse solution; some cultures underwent a second round of depolarization (in the absence of FM1-43) to destain synaptic terminals. FM1-43 labeled cultures were photographed using a Zeiss Photomicroscope II with a 40× plan-neofluar lens and T-MAX 3200 film. For uptake of HRP, incubations were identical to the above except that the stimulation medium contained 8–10 mg/ml HRP and the cultures were depolarized for 2–5 min. HRP was visualized after glutaraldehyde fixation (see below) by a 15-min incubation in 3,3′-diaminobenzidine tetrahydrochloride (0.75 mg/ml) in 0.05 M Tris-HCl, pH 7.6, containing 0.01% hydrogen peroxide.

Electron Microscopy

Cultures were prepared for electron microscopy as described in detail in Neale et al. 1978. In brief, fixation in 2.5% glutaraldehyde in 0.15 M sodium cacodylate buffer, pH 7.3, was followed by postfixation in 1% osmium tetroxide, en bloc staining with uranyl acetate, rapid dehydration in an ethanol series and embedding in the culture dish. After curing, the Epon disc was separated from the plastic dish and fields of interest were selected, removed, and mounted on Epon blanks for ultrathin sectioning. Stained ultrathin sections were examined in a JEM-1010 (JEOL USA Inc.) electron microscope fitted with a goniometer stage. Synaptic terminals were located and the specimens were tilted if necessary to obtain a cross-sectional view of the apposing synaptic membranes. Terminals were photographed at 40,000×.

Toxin Effects on Vesicle Recycling

To demonstrate further the toxin-induced electrical quiescence, we used the styryl dye FM1-43 for the optical detection of synaptic activity (Betz and Bewick 1992). Depolarization of control cultures in the presence of FM1-43 results in a pattern of fluorescence consonant with the labeling of synaptic terminals (Fig. 6 a). Uptake of FM1-43 is not observed with depolarization in the absence of Ca²⁺ (Fig. 6 b) and is markedly reduced without depolarization (Fig. 6 c). As expected, cultures treated for 22 h with 10 ng/ml TeNT (Fig. 6 d), 10–100 ng/ml BoNT C (Fig. 6 e) (Williamson et al. 1996), or BoNTs B or D (10 ng/ml; not shown), fail to release neurotransmitter with K⁺ depolarization, and also fail to take up FM1-43. In marked contrast, cultures blocked with 10 ng/ml of BoNT A, which exhibit no K⁺-evoked release of neurotransmitter (Williamson et al. 1996) show intense K⁺-stimulated uptake of FM1-43. Increasing the concentration of BoNT A to 200 ng/ml (Fig. 6 f) or 300 ng/ml (not shown) failed to abolish FM1-43 uptake. As in controls, FM1-43 loading of BoNT A–treated cultures requires both extracellular Ca²⁺ (Fig. 6 g) and elevated K⁺ (Fig. 6 h). These results indicate that BoNT A blockade of neurotransmitter release does not prevent vesicle membrane reuptake by endocytosis, and that membrane retrieval is dependent on Ca²⁺.

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Figure 6

FM1-43 staining (a–h) and destaining (i–k) with K⁺ depolarization. (a–c) Control cultures. (a) 56 mM KCl; 2 mM CaCl₂. The fluorescent image is strikingly similar to Fig. 1b and Fig. 4a. The labeled structures are individual active synaptic terminals containing a number of synaptic vesicles whose membranes are labeled with the fluorescent dye as a result of vesicle membrane recycling during the stimulation interval. (b) 56 mM KCl, 0 mM CaCl₂, and 0.5 mM EGTA. Uptake of FM1-43 is not detectable. (c) 3 mM KCl and 2 mM CaCl₂. Some uptake of FM1-43 occurs as a result of spontaneous network activity. (d) TeNT (10 ng/ml for 22 h), 56 mM KCl, and 2 mM CaCl₂. (e) BoNT C (100 ng/ml for 22 h), 56 mM KCl, and 2 mM CaCl_2. When synaptic vesicle exocytosis is blocked by TeNT or BoNT C, uptake of FM1-43 is decreased substantially. (f–h) BoNT A (200 ng/ml; i.e., >20 times that required to block neurotransmitter release). (f) 56 mM KCl and 2 mM CaCl₂. Although intensity is reduced from controls, FM1-43 labeling is unequivocal in cultures blocked by BoNT A. (g) 56 mM KCl, 0 mM CaCl₂, and 0.5 mM EGTA. As in control cultures, uptake of FM1-43 requires Ca²⁺. (h) 3 mM KCl and 2 mM CaCl₂. There is essentially no labeling of toxin-blocked cultures without K⁺ depolarization. (i and j) Control cultures loaded with FM1-43 in 56 mM KCl and 2 mM CaCl₂, as in a. (i) Loaded culture subsequently depolarized with 56 mM KCl and 2 mM CaCl₂. There is substantial loss of FM1-43 from synaptic terminals as a result of exocytosis of labeled vesicles (compare with a). (j) Loaded culture subsequently depolarized with 56 mM KCl, 0 mM CaCl₂, and 0.5 mM EGTA. The absence of Ca²⁺ prevents synaptic vesicle exocytosis and the destaining of labeled terminals. (k) BoNT A (200 ng/ml for 22 h)–blocked culture loaded with FM1-43 in 56 mM KCl and 2 mM CaCl₂, as in f, and subsequently incubated with 56 mM KCl and 2 mM CaCl₂ in an attempt to destain. In contrast to control cultures, it is not possible to destain BoNT A–blocked cultures, providing further evidence that vesicle exocytosis cannot be stimulated. Bar, 25 μm.

Evidence that FM1-43 labeling is associated with recycled synaptic vesicles is provided by the ability to destain a labeled preparation by further stimulation in the absence of dye. Indeed, K⁺ stimulation of control cultures preloaded with FM1-43 results in dye loss (Fig. 6 i). When dye-loaded cultures are stimulated in the absence of Ca²⁺, exocytosis of labeled vesicles is prevented and fluorescence is retained (Fig. 6 j). However, in BoNT A–blocked cultures that have been loaded with FM1-43, subsequent depolarization (in the presence of Ca²⁺) does not induce destaining (Fig. 6 k). This provides further evidence that synaptic vesicles in BoNT A–blocked cultures are unable to undergo exocytosis.

To strengthen the hypothesis that FM1-43 staining of BoNT A–blocked terminals is related to vesicle membrane endocytosis, we examined the fine structure of synaptic terminals. In spontaneously active control cultures (Fig. 7 a), clathrin-coated pits and vesicles are seen only occasionally, although empty clathrin baskets (arrowheads; Kanaseki and Kadota 1969) are not uncommon. When control cultures are stimulated (56 mM K⁺ and 2 mM Ca²⁺ for 5 min; Fig. 7 b), small coated pits and vesicles (arrows) are observed with increased frequency and empty clathrin baskets with decreased frequency. In cultures blocked with TeNT (Fig. 7 c) or with BoNT A (Fig. 7 e), synaptic vesicles accumulate at the presynaptic membrane, empty clathrin baskets are typical, and coated pits and vesicles are extremely rare. When TeNT-blocked cultures are stimulated with K⁺ (Fig. 7 d), these synaptic features remain unchanged, suggesting a lack of membrane movement. When BoNT A–blocked cultures are depolarized (Fig. 7 f), the number of docked synaptic vesicles remains elevated, although clathrin-coated pits are found with increased frequency and empty clathrin baskets have disappeared. Thus, stimulated BoNT A cultures take on those features which suggest active retrieval of synaptic vesicle membrane.

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Figure 7

Clathrin and synaptic vesicle recycling. (a) Resting control culture. Clathrin-coated pits and vesicles are seen only occasionally, whereas empty clathrin baskets (arrowheads) are more numerous. (b) K⁺-depolarized control culture. Clathrin-coated pits and vesicles (arrows) are seen commonly; empty clathrin baskets are rare. Note the electron lucent matrix of mitochondria (asterisks) in cultures fixed during stimulation. (c) Resting TeNT-blocked culture. Clathrin-coated pits and vesicles are almost never seen although empty clathrin baskets are found easily. (d) K⁺-depolarized TeNT-blocked culture. K⁺ stimulation causes a change in the density of the mitochondrial matrix, but no noteworthy change in the distribution of synaptic vesicles at the active zone or frequency of clathrin baskets or clathrin-coated membranes. (e) Resting BoNT A–blocked culture. This culture appears similar to the TeNT-blocked culture in the absence of clathrin-coated membranes and the persistence of empty clathrin baskets. (f) K⁺-depolarized BoNT A–blocked culture. This culture appears more similar to the stimulated control than to the stimulated TeNT-blocked culture. Empty clathrin baskets have all but disappeared and clathrin-coated membranes can be found. Toxin concentrations as in Fig. 5. Bar, 0.5 μm.

To confirm that synaptic terminals incapable of vesicle exocytosis can be stimulated to retrieve vesicle membrane, the vesicle-loading experiments were repeated with HRP substituted for FM1-43 in the stimulation medium. Control and toxin-blocked cultures were depolarized by K⁺ with Ca²⁺ for 5 min in the presence of 10 mg/ml HRP, rinsed for 30 min in medium containing TTX, and then fixed. A large proportion of synaptic vesicles in control cultures (Fig. 8 a) contain HRP reaction product. As expected, HRP-labeled vesicles are rare in TeNT-blocked cultures (Fig. 8 b). Depolarization of BoNT A–blocked cultures, however, results in the labeling of a much greater proportion of small vesicles (Fig. 8 c). When BoNT A–blocked cultures are fixed almost immediately after depolarization (i.e., without an extended rinse), clathrin-coated vesicles of the appropriate size are labeled with HRP (insets). These experiments lend further credence to the proposal that, in BoNT A–blocked neurons, synaptic vesicles can be endocytosed in the absence of temporally associated vesicle exocytosis, and indicate that the recycling process involves the clathrin-coated membrane organelles observed in control preparations.

Vesicle Exocytosis Requires Ca²⁺

To confirm that HRP-labeled vesicles in control cultures are competent for exocytosis, we loaded terminals by stimulating for 2 min in the presence of 8 mg/ml HRP. HRP-loaded terminals were subjected to a second interval of K⁺ depolarization and the change in percentage of labeled vesicles was determined (Fig. 9 and Table ). After HRP-loading of the control cultures, ∼10% of total synaptic vesicles contain HRP (Fig. 9 a). In cultures stimulated after loading, labeled vesicles decrease to 2.5% of total vesicles (Fig. 9 c). Without Ca²⁺ in the stimulation medium, the percentage of labeled vesicles remains high (10%; not shown). Thus, the loss of HRP is consistent with synaptic vesicle exocytosis.

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Figure 9

Exocytosis of HRP stimulated by K⁺ depolarization. (a and c) Control; and (b and d) BoNT A (200 ng/ml for 18 h). Synaptic terminals are loaded with horseradish peroxidase (a, b). After subsequent stimulation in the absence of HRP, the percent labeled vesicles is lower in control (c) but unchanged in toxin-blocked (d) cultures. These micrographs are analogous to Fig. 6, a and i, and Fig. 6f and Fig. k, providing further demonstration of the block in exocytosis induced by BoNT A. Note coated pits and coated vesicles implicated in synaptic vesicle membrane retrieval (arrows) in b and c and conventional coated pits (double arrowheads) in a and d. Bar, 0.5 μm.

Vesicle Endocytosis Requires Ca²⁺

In BoNT A–blocked cultures, HRP loading (as above) labels ∼5% of the synaptic vesicles (Fig. 9 b). This percentage is not decreased by subsequent depolarization (Fig. 9 d and Table ), confirming that exocytosis is not stimulated in the presence of BoNT A. When loading of either BoNT A–blocked or control cultures is attempted in the absence of extracellular Ca²⁺, after a rinse in Ca²⁺-free buffer, there is no HRP in synaptic vesicles (Table ), and there are no HRP-labeled cisternae. In control cultures, failure to load can be taken as a consequence of the zero Ca²⁺-induced block in exocytosis. The absence of vesicle recycling in BoNT A–blocked terminals under these conditions is not due to a failure of vesicle exocytosis since the endocytosis of HRP in blocked cultures occurs in the absence of measurable exocytosis. Therefore, it must be concluded that the endocytic retrieval of synaptic vesicle membrane requires Ca²⁺.

Toxin Substrates and Vesicle Docking

Atypical accumulation of docked synaptic vesicles is associated with tetanus intoxication (Hunt et al. 1994; Mellanby et al. 1988; Neale et al. 1989; Broadie et al. 1995) and with disruption of the function of the N-ethylmaleimide–sensitive factor (Kawasaki et al. 1998; Schweizer et al. 1998). This morphology suggests an inability of synaptic vesicles to fuse with the presynaptic membrane and is entirely compatible with the demonstration that TeNT is a zinc endopeptidase that cleaves VAMP (Schiavo et al. 1992), a homologue of a protein critical for transport vesicle docking and fusion at the yeast (Protopopov et al. 1993) and mammalian (McMahon et al. 1993) Golgi apparatus. TeNT and BoNTs A and C, each acting on a different protein of the ternary core complex (Montecucco and Schiavo 1995), induce blockade of neurotransmitter release, which correlates with toxin cleavage of its specific substrate (Williamson et al. 1996). We report here that BoNTs A and B also cause an accumulation of synaptic vesicles at the active zone at a time when neurotransmitter release is arrested. These observations support the requirement of the ternary complex (VAMP, syntaxin, and SNAP-25) for vesicle fusion but not for vesicle docking. The occurrence of docked vesicles in toxin-treated preparations has been explained by the ability of synaptotagmin, a synaptic vesicle integral membrane protein, to bind to SNAP-25 on the presynaptic membrane (Schiavo et al. 1997). This binding is not dependent on Ca²⁺ or on the presence of syntaxin or VAMP, and occurs even with BoNT A- or E-cleaved SNAP-25. Functional docking (i.e., leading to vesicle fusion), however, requires that the proteins of the core complex be intact (Schiavo et al. 1997). This is consistent with our observation that K⁺-induced depolarization of control cultures reduces the number of vesicles at the active zone, whereas depolarization of toxin-blocked cultures is not similarly effective. Thus, the clostridial neurotoxins block neurotransmitter release by preventing synaptic vesicle fusion.

Failure to Demonstrate Exocytosis

The synaptic blockade induced by BoNT A in this study is at variance with several reports of the failure of BoNT A to produce a complete block in transmitter release. The latter studies introduced toxin into normally nonreceptive PC12 or chromaffin cells by mechanical cracking (Banerjee et al. 1996) or digitonin permeabilization (Lawrence et al. 1997) of the cells, or intracellular injection of recombinant BoNT A light chain (Xu et al. 1998). It remains unclear whether this difference in the extent of blockade reflects the underlying biology of toxin internalization into intact neurons or differences in assay conditions.

It might be argued that, under the experimental conditions described here, BoNT A blocks the release of glycine and glutamate (Williamson et al. 1996), but fails to block the release of other transmitters. If so, we would expect to see some nerve terminals with, and others without, evidence of vesicle recycling. In fact, HRP-labeled vesicles are seen in every synaptic terminal. Alternatively, if BoNT A had caused the loss of stored transmitter from synaptic vesicles, depolarization would induce the exocytosis of empty vesicles. However, there is considerable evidence that BoNT A does not interfere with the synthesis and/or storage of acetylcholine (for review see Gundersen 1980) nor TeNT with that of GABA (Collingridge and Davies 1980).

Finally, vesicle recycling in BoNT A–blocked cultures may have occurred as a result of rapid or low level K⁺-evoked exocytosis that was below the sensitivity or time resolution of our assays. Both glycine release and synaptic currents are maintained in high K⁺ for several minutes in control cultures, and about half the number of vesicles are recycled in BoNT A–blocked cultures as in untreated controls. It seems unlikely that both the neurotransmitter release assays and whole cell patch clamp analysis would fail to detect exocytic responses of this magnitude. While it may not be possible to prove that absolutely no exocytosis occurs with BoNT A, there remains a striking disproportion between the level of exocytosis that could be measured and what might be expected to account for the endocytosis that is observed.

Ca²⁺ Requirement for Endocytosis

We have used the dissociation of synaptic vesicle exo- from endocytosis to show that vesicle endocytosis requires Ca²⁺. Our results contrast with those of Ryan et al. 1993(1996) who reported endocytosis of synaptic vesicle membrane, in cultures of hippocampal neurons, in the absence of extracellular Ca²⁺. The time course of the decrease in intraterminal Ca²⁺ (Ryan et al. 1993), elevated by the initial membrane depolarization, suggests that superfusion with FM1-43 was begun before the free Ca²⁺ concentration had returned to the baseline. This residual elevation may have been sufficient, even in the absence of either extracellular Ca²⁺ or simultaneous depolarization, to initiate membrane retrieval.

Ca²⁺ is required to initiate vesicle endocytosis after vesicle depletion induced by α-latrotoxin at the frog neuromuscular junction (NMJ) (Ceccarelli and Hurlbut 1980) or by prolonged high frequency action potential stimulation in lamprey giant reticulospinal synapses (Gad, H., O. Shupliakov, V.A. Pieribone, and L. Brodin. Soc. Neurosci. 1995. 138.8; Shupliakov et al. 1997). In the latter preparation, an extracellular Ca²⁺ concentration of 11 μM was sufficient to induce clathrin-mediated synaptic vesicle endocytosis (Gad et al. 1998). Synaptic vesicle recycling studied in synaptosomes also supports a Ca²⁺ requirement for endocytosis at a concentration lower than that required for maximal exocytosis (Marks and McMahon 1998). In that study, Ca²⁺ entry acts as the trigger by activating calcineurin which dephosphorylates a number of proteins, including amphiphysin and dynamin, implicated in clathrin-mediated endocytosis (for reviews see De Camilli and Takei 1996; Bauerfeind et al. 1998). Low extracellular Ca²⁺ allowed near maximal endocytosis after very submaximal exocytosis, leading to the speculation that the synaptic terminal membrane might always contain a pool of recoverable membrane with “exocytosis adding to this pool and endocytosis drawing from it” (Marks and McMahon 1998; see below). Experiments at the NMJ of Drosophila shibire ^ts1 mutants suggest that any Ca²⁺-requiring step in endocytosis occurs early, i.e., before the accumulation of endocytic membrane invaginations (Ramaswami et al. 1994). Secretory vesicle membrane retrieval by rapid endocytosis in adrenal chromaffin cells and melanotrophs also appears to be Ca²⁺-dependent although it is not mediated by clathrin-coated vesicles (Thomas et al. 1994; Artalejo et al. 1995; Mansvelder and Kits 1998).

In this study, endocytosed FM1-43 and HRP in control cultures are associated with recycled synaptic vesicles that are exocytosis-competent. Although tracer exocytosis is not possible in BoNT A–blocked terminals, HRP-labeled vesicles are observed docked (Fig. 8 c and 9 d). Further studies are required to confirm that the recycled vesicle membrane contains the appropriate complement of proteins and that the vesicles themselves contain neurotransmitter. Nonetheless, the present work stands as a direct demonstration of a Ca²⁺ requirement for endocytosis.

Membrane Source for Vesicle Recycling

In neuroendocrine cells, endocytosis does not always strictly follow exocytosis and, under certain circumstances, exocytosed membrane may reside in the plasma membrane until specific conditions drive retrieval (Thomas et al. 1990; Engisch, K.L., and M.C. Nowycky. Soc. Neurosci. 1996. 494.5; Hsu and Jackson 1996; Smith and Neher 1997). However, immunofluorescence fails to detect synaptic vesicle proteins on the terminal plasmalemma of the NMJ under resting conditions (von Wedel et al. 1981; Valtorta et al. 1988; Torri-Tarelli et al. 1990). Similar studies in rat hippocampal cell cultures, using antibodies directed against the lumenal NH₂ terminus of synaptotagmin I (Syt_lum-Abs) (Matteoli et al. 1992; Kraszewski et al. 1995) also demonstrated a lack of surface membrane signal, even after depolarization for several minutes (Mundigl et al. 1995). However, radioimmunoassays using [¹²⁵I]Syt_lum-Abs provide quantitative data demonstrating a significant pool of synaptotagmin I at the cell surface even at steady state (Mundigl et al. 1995). This pool comprises about half the synaptotagmin measured after K⁺ depolarization for 3 min. Given that antibody binding reflects the presence of synaptic vesicle membrane, it could be inferred that a sizable membrane reserve exists in the surface membrane of the synaptic terminal. This membrane reserve, then, could serve as the source of membrane internalized in BoNT A–treated neurons.

The present results suggest that TeNT may disrupt a component of the reuptake mechanism that is unaffected by BoNT A. VAMP-2 is recognized by AP3 for the formation of synaptic vesicles from endosomes of PC12 cells. Tetanus toxin inhibited the formation of synaptic vesicles and their coating with AP3 in vitro, whereas BoNTs C and E had no effect (Salem et al. 1998). If VAMPs were similarly a sorting signal for synaptic vesicle endocytosis in the nerve terminal, TeNT might prevent the retrieval of vesicle membrane, whereas BoNT A would not.

The authors wish to thank: Mss. Sandra Fitzgerald and Veronica Dunlap for providing spinal cord cell cultures; Drs. William Habig and Jane Halpern (both from the Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD) for gifts of tetanus toxin and valuable suggestions over many years; Dr. J. Edward Brown (US Army Medical Research Institute of Infectious Diseases, Ft. Detrick, MD) for gifts of botulinum neurotoxins; Dr. Phillip G. Nelson for advice on electrophysiology; Dr. Cesare Montecucco for antibodies against the toxin substrates; Mss. Sara Behar and Barbara Piesiecki for assistance with morphometry, and Dr. Andrew Parfitt for a critical reading of this manuscript.