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β-bungarotoxin-induced depletion of synaptic vesicles at the mammalian neuromuscular junction
Neuropharmacology 47 (2004) 304–314 www.elsevier.com/locate/neuropharm
b-bungarotoxin-induced depletion of synaptic vesicles at the mammalian neuromuscular junction S. Prasarnpun1, J. Walsh, J.B. Harris School of Neurology, Neurobiology and Psychiatry, Faculty of Medical Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, UK Received 23 January 2004; received in revised form 25 March 2004; accepted 22 April 2004
Abstract The neurotoxic phospholipase A2, b-bungarotoxin, caused the failure of the mechanical response of the indirectly stimulated rat diaphragm. Exposure to b-bungarotoxin had no effect on the response of the muscle to direct stimulation. Resting membrane potentials of muscle fibres exposed to the toxin were similar to control values, and the binding of FITC-labelled a-bungarotoxin to nAChR at the neuromuscular junction was unchanged. Motor nerve terminal boutons at a third of cell junctions were destroyed by exposure to b-bungarotoxin leaving only a synaptic gutter filled with Schwann cell processes and debris. At other junctions, some or all boutons survived exposure to the toxin. Synaptic vesicle density in surviving terminal boutons was reduced by 80% and synaptophysin immunoreactivity by >60% in preparations exposed to b-bungarotoxin, but syntaxin and SNAP-25 immunoreactivity was largely unchanged. Terminal bouton area was also unchanged. The depletion of synaptic vesicles was completely prevented by prior exposure to botulinum toxin C and significantly reduced by prior exposure to conotoxin x-MVIIC. The data suggest that synaptic vesicle depletion is caused primarily by a toxin-induced entry of Ca2+ into motor nerve terminals via voltage gated Ca2+ channels and an enhanced exocytosis via the formation of t- and v-SNARE complexes. # 2004 Elsevier Ltd. All rights reserved. Keywords: b-Bungarotoxin; Botulinum toxin C; Conotoxin x-MVIIC; Exocytosis
1. Introduction Envenoming bites by kraits constitute a medical emergency across SE Asia. Victims experience a severe and potentially life-threatening neuromuscular weakness that may be slow in onset but can last for several days (Warrell, 1999). Conventional treatments of neurotoxic envenomations (anticholinesterases, specific antivenoms) are ineffective and prolonged ventilinatory support and intensive care may be required. There is little doubt that post-synaptically active toxins like abungarotoxin contribute to the onset of weakness but the poor efficacy of conventional treatments suggest the involvement of additional toxic agents. There is general Corresponding author. Tel.: +44-191-222-6648; fax: 44-191-2225227. E-mail address: [email protected] (J.B. Harris). 1 Present address: Department of Biology, Naresuan University, Phitsanulok 65000, Thailand.
0028-3908/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2004.04.012
agreement that the toxins responsible for this profound treatment-resistant neuromuscular weakness are the bbungarotoxins, a family of closely related neurotoxic phospholipases A2 that constitutes the major toxic fraction of the crude venoms of all species of krait. Chang and Lee (1963) first isolated and characterised b-bungarotoxin as a pre-synaptically-active neurotoxin causing neuromuscular paralysis. Chen and Lee (1970) reported that the toxin caused synaptic vesicle depletion at the vertebrate neuromuscular junction following inoculation in vivo. Although the significance of this finding has been the subject of considerable debate, their proposal that the failure of neuromuscular transmission resulted from vesicle depletion is supported by evidence from other studies, both in vitro and in vivo (see, for example, Strong et al., 1977; Dixon and Harris, 1999). Abe et al. (1976) reported that many end-plates in vertebrate skeletal muscles exposed to b-bungarotoxin were denervated. A combined study of nerve terminal
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pathology after exposure to b-bungarotoxin in vivo and in vitro confirmed the findings of both Chen and Lee (1970) and Abe et al. (1976) and led Dixon and Harris (1999) to propose that synaptic vesicle depletion was primarily responsible for the acute onset of paralysis in victims of envenoming krait bite and that the prolonged, untreatable paralysis was due to nerve terminal degeneration. In this study, we asked how the neurotoxic phospholipases A2 causes the depletion of synaptic vesicles. We report that b-bungarotoxin causes depletion by promoting the SNARE-dependent release of transmitter from mammalian motor nerve terminals and that the release process depends on the entry of Ca2+ through functional voltage-gated Ca2+ channels. The data also suggest that the depletion of synaptic vesicles is not associated with the incorporation of synaptic vesicle membrane into the plasma membrane of the nerve terminal. We provide a simple model linking the hydrolytic activity of b-bungarotoxin to both depletion of synaptic vesicles and nerve terminal destruction.
2. Methods 2.1. Animals and muscles Female Wistar rats (Bantin and Kingman, UK) weighing 100–150 g (approximately 6 weeks of age) were used for all experiments. Prior to use, they were acclimatised in a holding room for 7 days. During this period, they had free access to food and water and were maintained in full accord with the animals (Scientific Procedures Act) of 1986 under the day to day care of a veterinarian. All experiments were made on isolated phrenic nerve/diaphragm preparations removed from animals killed by stunning and exsanguination (Bu¨lbring, 1946). The muscles were mounted in a bathing fluid of composition (mM) NaHCO3 (12), KCl (4), KH2PO4 (1), NaCl (138.8), MgCl2 (1), CaCl2 (2), glucose (11) at pH 7.2 equilibrated with 5% CO2 in O2 v and maintained at ambient temperature (19–21 C). Two innervated hemidiaphragm preparations were made from one animal. Routinely, one preparation was superfused with bathing solution and used as a control and the partner was superfused with b-bungarotoxin at a concentration of 10 lg ml1 unless otherwise stated. Muscles were stimulated either directly (0.1 Hz, 0.2 ms pulse duration, supramaximal voltage unless otherwise stated) or indirectly via the phrenic nerve (0.1 Hz, 0.02 ms pulse duration, supramaximal voltage unless otherwise stated). The mechanical responses of the muscle were recorded isometrically.
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2.2. Toxins and other reagents b-Bungarotoxin (cat. T5644) was purchased from Sigma Chemical Supplies. Botulinum toxin C (cat. 203676) was obtained from Calbiochem and conotoxin x-MVIIC (cat. 123069) was obtained from Latoxan. Fluorescein isothiocyanate (FITC)-conjugated a-bungarotoxin (cat. F1176) was obtained from Molecular Probes Inc. Murine anti-synaptophysin monoclonal antibodies (cat. S5768), anti-syntaxin monoclonal antibodies (cat. S0664) and anti-SNAP-25 monoclonal antibodies (cat. S9684) were purchased from Sigma Chemical Supplies. Primary anti-AChE antibodies raised in rabbits against rat brain AChE were a gift from Dr. A Massoulie. Rabbit anti-mouse rhodamineconjugated polyclonal antibodies (cat. R0270) and swine anti-rabbit rhodamine-conjugated polyclonal antibodies (cat. R0156) were obtained from DAKO. All secondary antibodies were incubated with rat serum and centrifuged to yield a clear supernatant before use. All other reagents were obtained from regular commercial sources and were routinely Analar grade. 2.3. Tissue sectioning Diaphragm muscles were pinned, under slight tension, to a Sylgard-lined dish. A segment of muscle, 5 5 mm, was cut out so that the band of innervation ran across the segment. The segment was rolled up, supported in a piece of calf’s liver, placed onto a piece of filter paper and frozen by immersion in liquid N2 cooled in isopentane. The tissue block was mounted on a Microm 560 cryostat and transverse sections 6 lm thick were cut. The sections were collected on chromealum/gelatine subbed slides. 2.4. Fluorescence immunocytochemistry of AChE and AChR labelling The primary anti-AChE Ab was diluted from stock 1:500 in 0.1 M phosphate buffered saline (PBS), pH 7.4, containing 0.1 M lysine and 0.3% BSA. Tissue sections were exposed to the primary antibody for 1 h at room temperature and then washed three times in PBS over 30 min. Sections were then incubated with the refined secondary antibody and FITC-conjugated abungarotoxin (1:200) in a moist chamber for 3 h. Slides were blotted dry, mounted in Vectashield and examined under the fluorescence microscope. To determine whether incubation in b-bungarotoxin blocked nAChR the ‘‘labelling index’’ was calculated. The number of end-plates recognised by the labelling of AChE (AChE) was noted. The number of those end-plates that colabelled with FITC-conjugated a-bungarotoxin (aBTx) was also counted. The calculation ðaBTx=AChEÞ 100
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was termed the labelling index. The area and intensity of labelling by FITC-conjugated a-bungarotoxin was measured using the software package Metamorph. 2.5. Fluorescence immunocytochemistry of synaptic proteins and nAChR labelling Tissue sections were permeabilised according to the primary antibody being used. For synaptophysin labelv ling, the sections were permeabilised in cold (20 C) acetone for 10 min. For syntaxin and SNAP-25 labelling, the sections were pre-treated for 30 min at room temperature in 4% paraformaldehyde. They were then permeabilised in 0.l% Triton X-100 in PBS for 10 min at room temperature. After permeabilisation, the sections to be labelled for syntaxin and SNAP-25 were rinsed in PBS three times over 15 min and the sections to be labelled for synaptophysin were air-dried. The relevant primary antibodies were diluted with 3% bovine serum albumin and 0.1 M lysine in PBS to a final strength of 1/100, and were applied to the sections v overnight in a closed moist chamber at 4 C. The following day, the sections were washed in PBS three times over 30 min, followed by incubation in secondary antibodies (1:100 rabbit anti-mouse rhodamine-conjugated immunoglobulins) for 1 h at room temperature. Routinely, counterstaining of nAChRs was done by the inclusion of 1:200 FITC-conjugated a-bungarotoxin in the secondary antibody solution. The sections were then washed three times in PBS over 30 min, and fixed in 1% paraformaldehyde for 30 min. The sections were again washed three times in PBS over 30 min before mounting in Vectashield. As each visualised image comprised fluorescein labelled nAChR and rhodamine labelled synaptic protein, it was possible to calculate and express a labelling index as the percentage of end-plates recognised by nAChR labelling that were also labelled with the synaptic protein antibody. 2.6. Confocal microscopy A dual channel Bio-Rad laser confocal system (MRC 600) mounted on an Olympus upright microscope (BH2) and equipped with an Ar–Kr laser, was used to prepare images of labelled neuromuscular
junctions on tissue sections prepared as above. The 488-nm line of the laser was used to excite the fluorescein-labelled nAChRs at the end-plates, and the 647-nm line to excite the rhodamine-labelled proteins. The emissions were collected separately into the two channels of the confocal system using 515 and 676-nm barrier filters, respectively. The optical section thickness to be scanned was set at 1 lm. The intensity threshold for control images was set at 100–250 grey. Intensity thresholds were set using control sections and those thresholds were not changed for the duration of the examination of sections of toxin-treated muscles. Typically, single sections were randomly chosen from each of between two and four muscles. Sections contained between 11 and 61 junctional profiles. All junctions seen in a single section were examined and the respective areas of label and fluorescence intensities were measured using Cosmos Version 7 software. The mean of in-muscle averages area and intensity, respectively, were then calculated. In Tables 1 and 2, these data are expressed as mean S:E:M: of n junctions/ muscles. 2.7. Transmission electron microscopy Segments of diaphragm muscles were mounted under slight tension in a Sylgard-lined dish and fixed in Karnovsky’s solution for 1.5 h (Karnovsky, 1965). After fixation the tissue was washed three times in PBS over 15 min, teased into bundles of 10–15 muscle fibres and transferred to an incubation medium containing indoxyl acetate and hexazotized paraosanilin in 0.1 M citrate buffer at pH 6.0 for 1 h (Strum and Hall-Craggs, 1982). Regions containing neuromuscular junctions could be identified by a brick red deposit. These regions were cut out to yield blocks 1 1 mm, rinsed in So¨renson’s phosphate buffer (pH 7.4) and post-fixed in OsO4 (1% w/v in So¨renson’s buffer) before being dehydrated in graded acetone and embedded in TAAB resin (TAAB Laboratory Equipment, UK). Semi-thin sections were first cut. If these contained junctional regions, ultrathin sections, 50–70 nm thick and a silver/ gold colour, were prepared, collected onto Athene New 300 grids and examined in a Philips E500 microscope. Sections were scanned blind with respect to origin.
Table 1 Characteristics of labelling of nAChR by FITC-a-bungarotoxin in nerve–muscle preparations exposed to b-bungarotoxin AChR/AChE labelling index (%)
Control (n:n) b-Bungarotoxin (n:n)
100 0% (185:4) 100 0% (134:3)
FITC-a-bungarotoxin labelling Area of label (lm2)
Intensity of label (pixels)
26 6:9ð44 : 3Þ 20 5:8 (32:3)
230 9:9 (44:3) 280 75 (32:3)
Rat innervated hemidiaphragm preparations were indirectly stimulated at 0.1 Hz for 180 min in either normal bathing medium (control) or b-bungarotoxin (10 lg ml1) before being labelled with anti-AChE Ab and FITC-a-bungarotoxin to label neuromuscular junctions. Measurements were made on n fibres in n muscles, and are expressed as means of in-muscle averages S:E:M:
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Table 2 Characteristics of labelling of synaptic proteins by indirect immunofluorescence in nerve–muscle preparations exposed to b-bungarotoxin Protein/AChR labelling index (%)
Area of label (lm2)
Intensity of label (pixels)
Synaptophysin control (n:n) b-Bungarotoxin (n:n) Difference from control
100 0% (51:4) 100 0% (87:4) NS
29 2:7 (51:4) 16 1:4 (87:4) p< 5%
492 49 (51:4) 356 27 (87:4) p< 5%
Syntaxin control (n:n) b-Bungarotoxin (n:n) Difference from control
100 0% (100:3) 89 1:4% (101:2) p< 5%
23 2:7 (42:3) 22 3:5 (33:2) NS
640 168 (42:3) 668 88 (33:2) NS
SNAP-25 control (n:n) b-Bungarotoxin (n:n) Difference from control
99 2:3% (80:3) 78 2:6% (143:3) p< 5%
n.d. n.d.
n.d. n.d.
Rat innervated hemidiaphragm preparations were indirectly stimulated at 0.1 Hz for 180 min in either normal bathing medium (control) or b-bungarotoxin (10 lg ml1) before being labelled. Measurements were made on n fibres in n muscles, and are expressed as means of in-muscle averages S:E:M:
Junctional profiles were identified by the presence of the electron dense AChE reaction product within a synaptic cleft. They were photographed without further selection for subsequent analysis provided the sections were not oblique and the images were not transected or otherwise obscured by grid bars or edges. This technique ensured that no samples were made of terminal expansions prior to their entry into the cleft. Photographic images of individual boutons were prepared and measurements of the area of the terminal were made. Manual counts of synaptic vesicles within each bouton were made and data expressed as the number of synaptic vesicles lm2 of each terminal bouton. The length of the synaptic trough within which the bouton was accommodated was measured and so too was the length of the post-junctional membrane, including in the latter the length of the synaptic folds. Nerve contact length was defined as the length of direct apposition of bouton to synaptic trough. This was clearly reduced when Schwann cell processes invaded the gap between the synaptic trough and the terminal bouton. All morphometric data were collected and analysed using the software package Metamorph. 2.8. Electrophysiology Segments of hemidiaphragm were mounted in a horizontal superfusion bath with oxygenated normal bathing fluid at room temperature. Glass microelectrodes filled with 3 M KCl, resistance 5–15 MX, were used to impale putative end-plate regions. Records were made of resting membrane potential. 2.9. Statistical evaluations Where appropriate, data were expressed as mean S:E:M: Populations of data were compared
using the unpaired Student’s t-test and differences were considered as significantly different when P < 0:05.
3. Results 3.1. b-Bungarotoxin and inhibition of evoked muscle contractions The mechanical response of control phrenic nerve/ hemidiaphragm preparations to both indirect and direct muscle stimulation was constant over a period of 240 min. In the presence of b-bungarotoxin, 3–30 lg ml1 (approximately 107–106 M), there was a lag phase of 20–60 min during which the twitch response was unchanged. Thereafter, the response began to decline and transmission failed completely between 120 and 240 min. Increasing either the concentration of the toxin or the rate of stimulation resulted in reductions in both lag phase and time to transmission failure (Fig. 1A and B), but continual indirect stimulation was not an essential requirement for toxin-induced transmission failure (Fig. 1C). Transmission failure did not occur if the exposure time to b-bungarotoxin was less than 5 min (Fig. 1D). The response to direct muscle stimulation was unchanged even when transmission had failed completely (not shown). Segments of paralysed diaphragm muscle and their relevant controls were remounted in a horizontal bath superfused with normal bathing fluid and used for electrophysiological recordings. Mean muscle fibre membrane potential of paralysed muscles (73 1:2 mV, n ¼ 57) was indistinguishable from that in control muscle (72 1:3 mV, n ¼ 52). Other segments were used for histological and electron microscopical examinations. No muscle pathology was observed. There was no difference between control and toxin-treated muscles with respect to the nAChR/AChE labelling index, area of labelling by FITC-conjugated a-bungarotoxin or
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Fig. 1. Responses of the indirectly stimulated hemidiaphragm preparation following exposure to b-bungarotoxin. In each case, time zero represents the point at which b-bungarotoxin was added to the tissue bath. Unless otherwise stated preparations were stimulated supramaximally at 0.1 Hz and b-bungarotoxin was used at a concentration of 10 lg ml1. The response is expressed as % original (pre-toxin) tension generated by a single stimulus. (A) and (B) Transmission failure is partially dependant on toxin concentration and the rate of stimulation. (C) Preparation was unstimulated during a 3 h incubation with b-bungarotoxin. Following restoration of stimulation, the control preparation responded with a normal twitch response but the preparation exposed to b-bungarotoxin was not responsive to indirect stimulation. (D) b-Bungarotoxin was added to the bath at time zero and then washed out 1–20 min later. Note that short exposure times resulted in no loss of response. Graphical representation of the results has excluded data on directly stimulated muscles for the sake of clarity. b-Bungarotoxin had no influence on the directly elicited response. In each case n ¼ 3 6.
fluorescence (pixel) intensity of nAChR labelling (Table 1). 3.2. Synaptophysin, syntaxin and SNAP-25 labelling In sections from control muscles, all end-plates labelled with FITC-a-bungarotoxin were also labelled with anti-synaptophysin Ab. The labelling index was thus 100%. The area of label was similar to that of FITC-abungarotoxin labelling. In the toxin-treated muscles, the labelling index was maintained at 100%, but the labelling was variable in intensity even at a given endplate, leading to a reduction in mean intensity of label by 30%. The area of label was also reduced by 45% (Table 2). Since synaptophysin is an integral component of the synaptic vesicle membrane the loss of both label intensity and area suggested a significant loss of synaptic vesicles from nerve terminals. The labelling indices of both syntaxin and SNAP-25 were slightly but significantly reduced in toxin-treated
muscles (Table 2; Fig. 2). Quantitative data were generated only for syntaxin: at labelled end-plates of toxintreated muscles there was no reduction in either area labelled or intensity of label (Table 2). These data suggested that at the majority of nerve terminals, proteins involved in the formation of SNARE-complexes remain in place even when muscles are paralysed by bbungarotoxin. 3.3. Nerve terminal morphology In control muscles, junctional profiles typically comprised between one and four nerve terminal boutons (mean 2:1 0:17). All terminal boutons were filled with synaptic vesicles and contained intact mitochondria. Schwann cell processes covered the exposed (non-synaptic) region of each bouton (Fig. 3A). The morphometric data are summarised in Table 3.
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Fig. 2. Representative images of concurrent fluorescent labelling of nAChR, (A) and (C), and syntaxin (B) and (D) in transverse sections of hemidiaphragm muscles maintained in either normal bathing fluid (controls) (A) and (B) or b-bungarotoxin (10 lg ml1) (C) and (D) until transmission in the preparation exposed to toxin had failed. Note the loss of syntaxin labelling at a single end-plate in the section of toxin treated diaphragm muscle (arrow).
Neuromuscular junctions in muscles exposed to bbungarotoxin were similar in general organisation to those in control muscles. The average junctional profile contained 2:0 0:14 terminal boutons. Thirty-two junctions were examined. At 12, all terminal boutons were intact. Seventeen junctions contained one bouton that was damaged and at three junctions no intact terminal boutons were identified. Synaptic vesicle density in surviving boutons was reduced to 11 vesicles lm2 (cf. 63 vesicles lm2 in control boutons) and mitochondria were swollen and flocular in appearance (Table 3, Fig. 3B). Where boutons were destroyed, the denervated synaptic cleft contained Schwann cell processes (Fig. 3E). At surviving junctions, Schwann cell processes were often observed extending into the synaptic cleft, thus reducing the length of synaptic contact between nerve terminal bouton and post-synaptic membrane (not shown). The mean area of surviving nerve terminal boutons exposed to b-bungarotoxin was not significantly different from that of nerve terminal boutons in control muscles (Table 3). Taken together, these observations suggest that boutons become detached from their anchoring points on the post-synaptic membrane before undergoing degeneration following exposure to b-bungarotoxin.
3.4. Synaptic vesicle density and botulinum toxin C Neuromuscular transmission failed approximately 120 min after exposure to botulinum C (109 M). At this point, preparations were either exposed to b-bungarotoxin (10 lg ml1) in addition to botulinum toxin C or left in contact with botulinum toxin C alone for a further 180 min. Incubation with botulinum toxin had no direct effect on synaptic vesicle density and completely blocked synaptic vesicle depletion in preparations subsequently exposed to b-bungarotoxin (Figs. 3D and 4). 3.5. Synaptic vesicle density and conotoxin x-MVIIC It was not possible to make direct measurements of the role of [Ca2+]0 on b-bungarotoxin-induced exocytosis because the neurotoxic activity of b-bungarotoxin is Ca2+-dependent. We therefore measured synaptic vesicle loss in preparations in which the voltage gated Ca2+ channels of the nerve terminal were blocked with conotoxin x-MVIIC. Incubation of phrenic nerve/ hemidiaphragm preparations with conotoxin x-MVIIC (1 lM) resulted in transmission failure in approximately 120 min. From this point, preparations were maintained for a further 180 min in the continuing presence of conotoxin x-MVIIC alone or conotoxin x-
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techniques to document synaptic vesicle depletion and nerve terminal morphometry, and established that [Ca2+]0, voltage gated Ca2+ channels and the t-SNARE proteins SNAP-25 and syntaxin are all involved in the neuropathology of b-bungarotoxin induced-neuromuscular paralysis. 4.1. Specificity of action of b-bungarotoxin b-Bungarotoxin is a member of a large class of venom-derived toxic phospholipases A2, most of which are both myotoxic and neurotoxic, and some of which also stabilise the junctional nAChR in its desensitised form (see reviews by Hawgood and Bon, 1991; Harris, 1991). b-Bungarotoxin-induced failure of neuromuscular transmission was not associated with any change in either the gross morphology of the muscle, or the postjunctional region of the neuromuscular junction. In preparations in which transmission had failed, the mechanical response to direct stimulation and the resting membrane potential of individual muscle fibres were unchanged. Exposure to the toxin had no effect on the binding of FITC-conjugated a-bungarotoxin to junctional nAChR. Chang et al. (1973) have previously shown that exposure to b-bungarotoxin does not affect either the response of nAChR to applied ACh or the characteristics of the experimentally-induced muscle fibre action potential. Thus, the acute effects of b-bungarotoxin are targeted exclusively to the nerve terminal. 4.2. Synaptic vesicle depletion and nerve terminal morphometry Fig. 3. Representative images showing the effects of b-bungarotoxin, and of pre-incubation with botulinum toxic C and x-conotoxin MVII-C on terminal boutons at the neuromuscular junction. (A) After incubation in normal bathing fluid. Note the terminal boutons sitting in the deeply folded synaptic cleft. The terminal boutons are filled with mitochondria (M) and small clear synaptic vesicles (V). Incubation in b-bungarotoxin (10 lg ml1) until transmission failed (B) resulted in the swelling of mitochondria and a loss of morphological integrity (M) and a reduction in the density of synaptic vesicles. Pre-incubation with either botulinum toxin C, 109 M (C) and (D) or x-conotoxin MVII-C, 106 M (E) reduced synaptic vesicle loss.
MVIIC plus b-bungarotoxin, 10 lg ml1. Treatment with conotoxin x-MVIIC alone had no effect on synaptic vesicle density but b-bungarotoxin induced depletion of synaptic vesicles was reduced by more than 60% (Figs. 3C and 4).
4. Discussion In this study, we have confirmed that b-bungarotoxin is pre-synaptically active. We used quantitative
Thin sections of neuromuscular junctions in both control and toxin-treated muscles contained, on average, two terminal boutons. At approximately 10% of toxin-treated junctions (i.e. 3/32) synaptic gutters contained only Schwann cells and debris. Of the remaining junctions 17 contained a mixture of destroyed and intact terminal boutons and 12 possessed entirely intact terminal boutons. The intact terminal boutons on muscles exposed to b-bungarotoxin were similar in area to those on control muscles but they contained 80% fewer synaptic vesicles, mitochondria were swollen and had lost well defined cristae, and Schwann cell processes had entered the synaptic gutter, thus reducing the area of contact between nerve terminal and muscle fibre. Cytochemical studies on frozen sections of control and toxin-treated muscle fibres showed that the average areas of nAChR and synaptophysin labelling were similar in control muscles. In toxin-treated muscles, the area of nAChR labelling and fluorescence intensity were similar to controls but the area and the fluorescence intensity of synaptophysin labelling fell—by 45% and 30%, respectively. Synaptophysin is a component of the synaptic vesicle membrane but it is difficult to
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Table 3 Effect of b-bungarotoxin on nerve terminal morphometry
Nerve terminal area (lm2) Nerve terminal perimeter (lm) Nerve terminal contact length (lm) Junctional fold length (lm) Trough length (lm) Synaptic vesicle density (vesicles lm2) Mitochondrial perimeter (lm) Damaged mitochondria (%)
Control n ¼ 32 : 65 : 65
Toxin-treated n ¼ 32 : 63 : 43
Difference from control
5:2 0:72 13:9 1:32 5:6 0:58 48 3:7 10:2 0:89 63 8 0:9 0:02 32
5:0 0:60 12:8 1:25 3:8 0:39 49 4:6 9:8 0:83 11 2 1:7 0:04 90 3
NS NS p< 5% NS NS NS p< 5% p< 5%
Morphometric data collected from surviving nerve terminals at control end-plates and nerve terminals on the end-plates of muscles exposed to bbungarotoxin (10 lg ml1). Exposure to the toxin caused the depletion of synaptic vesicles, and a reduction in nerve contact length (a result of the invagination of terminal Schwann cells). Note that nerve terminal area, junctional fold length and trough length were unchanged. Mitochondria were swollen and mitochondrial damage was severe. n is number of end-plates:number of nerve terminals:number of surviving nerve terminals used for morphometry.
relate directly the depletion of synaptic vesicles observed in the electron microscopy and the loss of immunofluorescence using light microscopy. However, a 45% reduction in an area of label combined with a 30% reduction in fluorescence intensity suggested a
total loss of immunofluorescence of >60%, which is compatible with the morphometric assessment of an 80% depletion of synaptic vesicles. 4.3. The role of [Ca2+]0 and Ca2+ voltage gated channels Physiological exocytosis in response to indirect stimulation is initiated by the entry of Ca2+ into the nerve terminal via open voltage-gated Ca2+ channels. We blocked voltage-gated Ca2+ channels in nerve terminals to test whether the b-bungarotoxin-induced depletion of synaptic vesicles was dependent on the influx of extracellular Ca2+. In mammalian motor nerve terminals, the voltage gated Ca2+ channels are mainly non-L, non-N P/Q type and they are blocked by conotoxin x-MVIIC (Hillyard et al., 1992; Wheeler et al., 1994; Randall and Tsien, 1995; Bowersox et al., 1995; Dunlap et al., 1995; Lin and Lin-Shiau, 1997). Incubation with conotoxin x-MVIIC alone caused transmission failure but had no effect on synaptic vesicle density. Pre-exposure to the conotoxin significantly reduced the depletion of synaptic vesicles from terminal boutons subsequently exposed to b-bungarotoxin. Thus, we conclude that the depletion was partially caused by the entry of Ca2+ into the terminal via opened voltage-gated Ca2+ channels.
Fig. 4. Synaptic vesicle density derived by direct measurement from images of nerve terminals at the neuromuscular junction as shown in Fig. 3. The density of synaptic vesicles in terminal boutons was not changed by incubation with botulinum toxin C (109 M) or x-conotoxin MVIIC (106 M). Incubation with b-bungarotoxin (10 lg ml1) caused a significant fall in synaptic vesicle density that was prevented by pre-incubation in either botulinum toxin C or x-conotoxin x-MVIIC. Control data and data derived from preparations exposed to b-bungarotoxin are graphic representations of data reported in Table 3. Data derived from preparations, pre-incubated with either botulinum toxin C or conotoxin x-MVIIC involved measurements of synaptic vesicle density from between 17 and 27 nerve terminals.
4.4. The role of SNAP-25 and syntaxin in synaptic vesicle depletion Botulinum toxin C hydrolyses both SNAP-25 and syntaxin (Foran et al., 1996; Williamson et al., 1996; Kalandakanond and Coffield, 2001), thus preventing the formation of a SNARE complex between synaptic vesicle and nerve terminal membranes. b-Bungarotoxin caused no depletion of synaptic vesicles in preparations previously exposed to botulinum toxin C. The simplest explanation is that the accelerated exocytosis and
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depletion of synaptic vesicles caused by b-bungarotoxin is dependent both on the entry of Ca2+ via open voltage-gated Ca2+ channels and the formation of SNARE complexes in the nerve terminal. 4.5. The fate of synaptic vesicles In most situations, the speed of endocytosis is sufficient to accommodate the 100-fold increases in the rate of exocytosis induced by many natural toxins (Ceccarelli et al., 1979; Fesce et al., 1986; Henkel and Betz, 1995). b-Bungarotoxin causes only a fivefold increase in the rate of exocytosis (Chang et al., 1973) so it is not easy to understand why synaptic vesicles are depleted. Montecucco and Rossetto (2000) have suggested that b-bungarotoxin bound to the nerve ter-
minal was internalised during endocytosis. The internalised toxin could then attack the inner leaf of the synaptic vesicle membrane, releasing free fatty acids and generating lysophosphatides. Fatty acids and lysophosphatides cause an increase in membrane fluidity, enhance membrane fusion in vitro (Rufini et al., 1990; Nishio et al., 1996) and might be expected to promote fusion between synaptic vesicle membranes and nerve terminal membranes, and to impair vesicle re-sealing and thus inhibit endocytosis. The combination of accelerated exocytosis and reduced endocytosis would be sufficient to explain the depletion of synaptic vesicles (Montecucco and Rossetto, 2000). There are, however, two major problems with this suggestion. The first is the requirement that there is the uptake of b-bungarotoxin into synaptic vesicles during endocytosis.
Fig. 5. (A) Synaptic vesicle recycling in a normal nerve terminal: a synaptic vesicle buds away from an endosome (1), is filled with transmitter (2) docks (3) and is primed for release (4). The invasion of an action potential causes the opening of a voltage gated Ca2+ channel, the influx of Ca2+, the fusion of the synaptic vesicle with the plasma membrane of the nerve terminal and the release of the transmitter (5). The now empty vesicle is recovered during endocytosis (6) loses its endocytotic clathrin coat (7) and fuses with a budding endosome. (B) b-Bungarotoxin binds to the motor nerve terminal at a site close to or part of the vesicle binding/exocytotic site (D). When the vesicle opens (5), the interior face is exposed to the toxin and the vesicle membrane is hydrolysed (6). Thus, the vesicle is destroyed. Vesicles that escape destruction may enter the full recycling process, but the inevitable consequences of the hydrolytic attack are the depletion of synaptic vesicles and failure of synaptic transmission.
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Although Herkert and his colleagues (Herkert et al., 2001; Shakhman et al., 2003) have shown that b-bungarotoxin is internalised in cultured hippocampal neurons and Nico et al. (2003) have shown that the related pre-synaptically active phospholipase taipoxin is accumulated by chromaffin cells the situation with respect to the neuromuscular junction is less clear. Both Strong et al. (1977) and Esquadera et al. (1982) have shown that the b-bungarotoxin binds primarily to the nerve terminal membrane and that the secondary uptake of b-bungarotoxin into the nerve terminal via endocytosis is very limited; others have failed to find any evidence of uptake at all (Howard and Wu, 1976; Simpson et al., 1993). The second problem is that enhanced exocytosis associated with impaired endocytosis in terminal boutons is typically associated with bouton swelling (Henkel and Betz, 1995 for example). Our data suggest that boutons do not swell. A mechanism consistent with these findings is that during exocytosis the inner face of the open synaptic vesicle comes into contact with bound toxin and is hydrolysed (Fig. 5). Noremberg and Parsons (1986) have reported that synaptic vesicles are very sensitive to hydrolysis by b-bungarotoxin. Those vesicles that escape hydrolysis could then re-enter the cycle of endocytosis/exocytosis. The combination of enhanced exocytosis and synaptic vesicle hydrolysis would be a progressive loss of synaptic vesicles available for release, the failure of neuromuscular transmission, and an empty terminal bouton of unchanged diameter. This explanation is consistent with all available experimental evidence and has many elements in common with that of Montecucco and Rossetto (2000). It differs from the latter in that our explanation, (1) depends critically on the suggestion that the toxin binding sites are closely related to the release sites, (2) does not require an internalisation step, (3) does not require the incorporation of synaptic vesicle membrane into the nerve terminal plasma membrane.
4.6. The degeneration of the nerve terminal Degeneration of nerve terminals was probably related to the hydrolytic activity of b-bungarotoxin. Hydrolysis of membrane lipids leads to an increase in membrane fluidity, loss of ion homeostasis, cellular depolarisation, entry of Ca2+ into the cell and, ultimately, disintegration of the membrane. Elevation of [Ca2+]i is enhanced by the progressive build up of free fatty acids and lysophosphatides (Sedlis et al., 1983), and results in depolarisation of mitochondria, increased mitochondrial proton permeability and mitochondrial failure (Wernicke et al., 1975; Nicholls et al., 1985; Rugulo et al., 1986; Nicholls and Budd, 2000; Jambrina et al., 2003; Granitkerich, 2003; Gulbins et al., 2003). The data suggest that the elevation of
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[Ca2+]i is the common denominator in both the enhanced exocytosis and the initiation of nerve terminal degeneration.
References Abe, T., Limbrick, A.R., Miledi, R., 1976. Acute muscle denervation induced by b-bungarotoxin. Proceedings of the Royal Society of London (Series B) 149, 545–553. Bowersox, S.S., Miljanich, G.P., Sigiura, Y., Li, C., Nadasdi, L., Hoffman, B.B., Ramachandran, J., Ko, C.P., 1995. Differential blockade of voltage sensitive calcium channels at the mouse neuromuscular junction. Journal of Pharmacology and Experimental Therapy 273, 248–256. Bu¨lbring, E., 1946. Observations on the isolated phrenic nerve diaphragm preparation of the rat. British Journal of Pharmacology 1, 38–61. Ceccarelli, B., Grohovaz, F., Hurlburt, W.P., 1979. Freeze fracture studies of frog neuromuscular junctions during intense release of transmitter. 1. Effect of black widow spider venom and Ca2+-free solutions on the structure of the active zone. Journal of Cell Biology 81, 163–177. Chang, C.C., Lee, C.Y., 1963. Isolation of toxins from the venom of Bungarus multicinctus and their modes of neuromuscular blocking action. Archives Internationale de Pharmacodynamique 144, 241– 257. Chang, C.C., Chen, T.F., Lee, C.Y., 1973. Studies of the presynaptic effect of b-bungarotoxin on neuromuscular transmission. Journal of Pharmacology and Experimental Therapy 184, 339–345. Chen, I.L., Lee, C.Y., 1970. Ultrastructural changes in the motor nerve terminals caused by b-bungarotoxin. Virchows Archiv 6, 318–325. Dixon, R.W., Harris, J.B., 1999. Nerve terminal damage by b-bungarotoxin. Its clinical significance. American Journal of Pathology 154, 447–455. Dunlap, K., Leubke, J.I., Turner, T.J., 1995. Exocytotic Ca2+ channels in mammalian central neurons. Trends in Neuroscience 18, 89–98. Esquadera, J.E., Solsona, C., Marsal, J., 1982. Binding of b-bungarotoxin to Torpedo electric organ synaptosomes. A high resolution autoradiographic study. Neuroscience 7, 751–758. Fesce, L.C., Segal, J.R., Ceccarelli, B., Hurlburt, W.P., 1986. Effects of black widow spider venom and Ca2+ on quantal secretion at frog neuromuscular junction. Journal of General Physiology 88, 59–81. Foran, P., Lawrence, G.L., Shone, C.C., Foster, K.A., Dolly, J.O., 1996. Botulinum neurotoxin C1 cleaves both syntaxin and SNAP25 in intact and permeabilised chromaffin cells; correlation with its blockade of catecholamine release. Biochemistry 35, 2630– 2636. Granitkerich, V.Y., 2003. The role of mitochondria in cytoplasmic Ca2+ recycling. Experimental Physiology 88, 91–97. Gulbins, E., Dreschers, S., Bock, J., 2003. Role of mitochondria in apoptosis. Experimental Physiology 88, 85–90. Harris, J.B., 1991. Phospholipases in snake venoms and their effects on nerve and muscle. In: Harvey, A.L. (Ed.), Snake Toxins. Pergammon Press, New York, pp. 91–129. Hawgood, B., Bon, C., 1991. Snake venom presynaptic toxins. In: Tu, A.T. (Ed.), Natural Toxins. Reptile Venoms and Toxins, vol. 5. Marcel Decker, New York, pp. 3–52. Henkel, A.W., Betz, W.J., 1995. Monitoring of black widow spider venom (BWSV) induced exo- and endocytosis in living frog nerve terminals with FMI-43. Neuropharmacology 34, 1397–1406.
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Herkert, M., Shakhman, O., Schweins, E., Becker, G.M., 2001. bBungarotoxin is a potent inducer of apoptosis in cultured rat neurons by receptor-mediated internalisation. European Journal of Neuroscience 14, 821–828. Hillyard, D.A., Monje, V.D., Mintz, I.M., Bean, B.P., Nadasdi, L., Ramachandran, J., Miljanich, G., Azimi-Zoonooz, A., McIntosh, J.M., Cruz, L.J., Imperial, J.S., Olivera, B.M., 1992. A new conus peptide ligand for mammalian presynaptic Ca2+ channels. Neuron 9, 69–77. Howard, B.D., Wu, W., 1976. Evidence that b-bungarotoxin acts at the exterior of nerve terminals. Brain Research 103, 190–192. Jambrina, E., Alonso, R., Alcalde, M., Rodriguez, M.D., Serrano, A., Martinez, A.C., Garcia-Sancho, J., Izquierdo, M., 2003. Calcium influx through receptor-operated channel induces mitochondria-triggered paraoptic cell death. Journal of Biological Chemistry 278, 14134–14145. Kalandakanond, S., Coffield, J.A., 2001. Cleavage of intracellular substrates of botulinum toxin A, C and D in a mammalian target tissue. Journal of Pharmacology and Experimental Therapy 296, 749–755. Karnovsky, M.J., 1965. A formaldehyde–glutaraldehyde fixative of high osmolarity for use in electron microscopy. Journal of Cell Biology 27, 137A. Lin, M.J., Lin-Shiau, S.Y., 1997. Multiple type of Ca2+ channels in mouse motor nerve terminals. European Journal of Neuroscience 9, 817–823. Montecucco, C., Rossetto, O., 2000. How do presynaptic PLA2 neurotoxins block nerve terminals? Trends in Biochemical Sciences 25, 266–270. Nicholls, D., Snelling, R., Dolly, J.O., 1985. Bio-energetic actions of b-bungarotoxin, dendrotoxin and bee-venom phospholipase A2 on guinea-pig synaptosomes. Biochemical Journal 229, 653–662. Nicholls, D.G., Budd, S.L., 2000. Mitochondria and neuronal survival. Physiological Reviews 80, 315–360. Nico, P., Rossetto, O., Gil, A., Montecucco, S., Gutierrez, L.M., 2003. Taipoxin induces F-actin fragmentation and enhances release of catecholamines in bovine chromaffin cells. Journal of Neurochemistry 85, 329–337. Nishio, H., Takeuchi, T., Hata, F., Yagasaki, O., 1996. Ca2+ independent fusion of synaptic vesicles with phospholipase A2 treated presynaptic membranes in vitro. Biochemical Journal 318, 981–987. Noremberg, K., Parsons, S.M., 1986. Selectivity and the regulation of phospholipase A2-mediated attack on cholinergic synaptic vesicles by b-bungarotoxin. Journal of Neurochemistry 47, 1312–1317.
Randall, A., Tsien, R.W., 1995. Pharmacological dissection of multiple types of Ca2+ channel currents in rat cerebellar granule neurons. Journal of Neuroscience 15, 2995–3012. Rufini, S., Pedersen, J.Z., Desideri, A., Luly, P., 1990. b-bungarotoxin-mediated liposome fusion: spectroscopic characterisation by fluorescence and ESR. Biochemistry 29, 9644–9651. Rugulo, M., Dolly, J.O., Nicholls, D.G., 1986. The mechanism of action of b-bungarotoxin at the presynaptic plasma membrane. Biochemical Journal 233, 519–523. Sedlis, S.P., Corr, P.B., Sobel, B.E., Ahumada, G.G., 1983. Lysophosphatidylcholine potentiates Ca2+ accumulation in rat cardiac myocytes. American Journal of Physiology 244, H32–H38. Shakhman, O., Herkert, M., Rose, C., Humeny, A., Becker, C.-M., 2003. Induction by b-bungarotoxin of apoptosis in cultured hippocampal neurons is mediated by Ca2+-dependent formation of reactive oxygen species. Journal of Neurochemistry 87, 598–698. Simpson, L.K., Lautenslager, G.T., Kaiser, I.I., Middlebrook, J.L., 1993. Identification of the site at which phospholipase A2 neurotoxins localize to produce their neuromuscular blocking effects. Toxicon 31, 13–26. Strong, P.N., Heuser, J.E., Kelly, R.O., 1977. Selective enzymatic hydrolysis of nerve terminal phospholipids by b-bungarotoxin: biological and morphological studies. In: Hall, Z., Kelly, R., Fox, C.F. (Eds.), Cellular Neurobiology. Alan Liss, New York, pp. 227–249. Strum, J.M., Hall-Craggs, E.C.B., 1982. A method demonstrating motor end-plates for light and electron microscopy. Journal of Neuroscience Methods 6, 305–309. Warrell, D.A., 1999. The clinical management of snakebites in the Southeast Asian region. Southeast Asian Journal of Tropical Medicine and Public Health 30 (Suppl. 1). Wernicke, J.F., Vanker, A.D., Howard, B.D., 1975. The mechanism of action of b-bungarotoxin. Journal of Neurochemistry 25, 483– 496. Wheeler, D.B., Randall, A.D., Tsien, R.W., 1994. Roles of N-type and A-type Ca2+ channels in supporting hippocampal synaptic transmission. Science 264, 107–111. Williamson, L.C., Halpern, J.L., Montecucco, C., Brown, J.E., Neale, E.A., 1996. Clostridial neurotoxins and substrate proteolysis in intact neurons: botulinum C acts on synaptosomal-associated protein of 24 kDa. Journal of Biological Chemistry 271, 7694–7699.
b-bungarotoxin-induced depletion of synaptic vesicles at the mammalian neuromuscular junction S. Prasarnpun1, J. Walsh, J.B. Harris School of Neurology, Neurobiology and Psychiatry, Faculty of Medical Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, UK Received 23 January 2004; received in revised form 25 March 2004; accepted 22 April 2004
Abstract The neurotoxic phospholipase A2, b-bungarotoxin, caused the failure of the mechanical response of the indirectly stimulated rat diaphragm. Exposure to b-bungarotoxin had no effect on the response of the muscle to direct stimulation. Resting membrane potentials of muscle fibres exposed to the toxin were similar to control values, and the binding of FITC-labelled a-bungarotoxin to nAChR at the neuromuscular junction was unchanged. Motor nerve terminal boutons at a third of cell junctions were destroyed by exposure to b-bungarotoxin leaving only a synaptic gutter filled with Schwann cell processes and debris. At other junctions, some or all boutons survived exposure to the toxin. Synaptic vesicle density in surviving terminal boutons was reduced by 80% and synaptophysin immunoreactivity by >60% in preparations exposed to b-bungarotoxin, but syntaxin and SNAP-25 immunoreactivity was largely unchanged. Terminal bouton area was also unchanged. The depletion of synaptic vesicles was completely prevented by prior exposure to botulinum toxin C and significantly reduced by prior exposure to conotoxin x-MVIIC. The data suggest that synaptic vesicle depletion is caused primarily by a toxin-induced entry of Ca2+ into motor nerve terminals via voltage gated Ca2+ channels and an enhanced exocytosis via the formation of t- and v-SNARE complexes. # 2004 Elsevier Ltd. All rights reserved. Keywords: b-Bungarotoxin; Botulinum toxin C; Conotoxin x-MVIIC; Exocytosis
1. Introduction Envenoming bites by kraits constitute a medical emergency across SE Asia. Victims experience a severe and potentially life-threatening neuromuscular weakness that may be slow in onset but can last for several days (Warrell, 1999). Conventional treatments of neurotoxic envenomations (anticholinesterases, specific antivenoms) are ineffective and prolonged ventilinatory support and intensive care may be required. There is little doubt that post-synaptically active toxins like abungarotoxin contribute to the onset of weakness but the poor efficacy of conventional treatments suggest the involvement of additional toxic agents. There is general Corresponding author. Tel.: +44-191-222-6648; fax: 44-191-2225227. E-mail address: [email protected] (J.B. Harris). 1 Present address: Department of Biology, Naresuan University, Phitsanulok 65000, Thailand.
0028-3908/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2004.04.012
agreement that the toxins responsible for this profound treatment-resistant neuromuscular weakness are the bbungarotoxins, a family of closely related neurotoxic phospholipases A2 that constitutes the major toxic fraction of the crude venoms of all species of krait. Chang and Lee (1963) first isolated and characterised b-bungarotoxin as a pre-synaptically-active neurotoxin causing neuromuscular paralysis. Chen and Lee (1970) reported that the toxin caused synaptic vesicle depletion at the vertebrate neuromuscular junction following inoculation in vivo. Although the significance of this finding has been the subject of considerable debate, their proposal that the failure of neuromuscular transmission resulted from vesicle depletion is supported by evidence from other studies, both in vitro and in vivo (see, for example, Strong et al., 1977; Dixon and Harris, 1999). Abe et al. (1976) reported that many end-plates in vertebrate skeletal muscles exposed to b-bungarotoxin were denervated. A combined study of nerve terminal
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pathology after exposure to b-bungarotoxin in vivo and in vitro confirmed the findings of both Chen and Lee (1970) and Abe et al. (1976) and led Dixon and Harris (1999) to propose that synaptic vesicle depletion was primarily responsible for the acute onset of paralysis in victims of envenoming krait bite and that the prolonged, untreatable paralysis was due to nerve terminal degeneration. In this study, we asked how the neurotoxic phospholipases A2 causes the depletion of synaptic vesicles. We report that b-bungarotoxin causes depletion by promoting the SNARE-dependent release of transmitter from mammalian motor nerve terminals and that the release process depends on the entry of Ca2+ through functional voltage-gated Ca2+ channels. The data also suggest that the depletion of synaptic vesicles is not associated with the incorporation of synaptic vesicle membrane into the plasma membrane of the nerve terminal. We provide a simple model linking the hydrolytic activity of b-bungarotoxin to both depletion of synaptic vesicles and nerve terminal destruction.
2. Methods 2.1. Animals and muscles Female Wistar rats (Bantin and Kingman, UK) weighing 100–150 g (approximately 6 weeks of age) were used for all experiments. Prior to use, they were acclimatised in a holding room for 7 days. During this period, they had free access to food and water and were maintained in full accord with the animals (Scientific Procedures Act) of 1986 under the day to day care of a veterinarian. All experiments were made on isolated phrenic nerve/diaphragm preparations removed from animals killed by stunning and exsanguination (Bu¨lbring, 1946). The muscles were mounted in a bathing fluid of composition (mM) NaHCO3 (12), KCl (4), KH2PO4 (1), NaCl (138.8), MgCl2 (1), CaCl2 (2), glucose (11) at pH 7.2 equilibrated with 5% CO2 in O2 v and maintained at ambient temperature (19–21 C). Two innervated hemidiaphragm preparations were made from one animal. Routinely, one preparation was superfused with bathing solution and used as a control and the partner was superfused with b-bungarotoxin at a concentration of 10 lg ml1 unless otherwise stated. Muscles were stimulated either directly (0.1 Hz, 0.2 ms pulse duration, supramaximal voltage unless otherwise stated) or indirectly via the phrenic nerve (0.1 Hz, 0.02 ms pulse duration, supramaximal voltage unless otherwise stated). The mechanical responses of the muscle were recorded isometrically.
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2.2. Toxins and other reagents b-Bungarotoxin (cat. T5644) was purchased from Sigma Chemical Supplies. Botulinum toxin C (cat. 203676) was obtained from Calbiochem and conotoxin x-MVIIC (cat. 123069) was obtained from Latoxan. Fluorescein isothiocyanate (FITC)-conjugated a-bungarotoxin (cat. F1176) was obtained from Molecular Probes Inc. Murine anti-synaptophysin monoclonal antibodies (cat. S5768), anti-syntaxin monoclonal antibodies (cat. S0664) and anti-SNAP-25 monoclonal antibodies (cat. S9684) were purchased from Sigma Chemical Supplies. Primary anti-AChE antibodies raised in rabbits against rat brain AChE were a gift from Dr. A Massoulie. Rabbit anti-mouse rhodamineconjugated polyclonal antibodies (cat. R0270) and swine anti-rabbit rhodamine-conjugated polyclonal antibodies (cat. R0156) were obtained from DAKO. All secondary antibodies were incubated with rat serum and centrifuged to yield a clear supernatant before use. All other reagents were obtained from regular commercial sources and were routinely Analar grade. 2.3. Tissue sectioning Diaphragm muscles were pinned, under slight tension, to a Sylgard-lined dish. A segment of muscle, 5 5 mm, was cut out so that the band of innervation ran across the segment. The segment was rolled up, supported in a piece of calf’s liver, placed onto a piece of filter paper and frozen by immersion in liquid N2 cooled in isopentane. The tissue block was mounted on a Microm 560 cryostat and transverse sections 6 lm thick were cut. The sections were collected on chromealum/gelatine subbed slides. 2.4. Fluorescence immunocytochemistry of AChE and AChR labelling The primary anti-AChE Ab was diluted from stock 1:500 in 0.1 M phosphate buffered saline (PBS), pH 7.4, containing 0.1 M lysine and 0.3% BSA. Tissue sections were exposed to the primary antibody for 1 h at room temperature and then washed three times in PBS over 30 min. Sections were then incubated with the refined secondary antibody and FITC-conjugated abungarotoxin (1:200) in a moist chamber for 3 h. Slides were blotted dry, mounted in Vectashield and examined under the fluorescence microscope. To determine whether incubation in b-bungarotoxin blocked nAChR the ‘‘labelling index’’ was calculated. The number of end-plates recognised by the labelling of AChE (AChE) was noted. The number of those end-plates that colabelled with FITC-conjugated a-bungarotoxin (aBTx) was also counted. The calculation ðaBTx=AChEÞ 100
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was termed the labelling index. The area and intensity of labelling by FITC-conjugated a-bungarotoxin was measured using the software package Metamorph. 2.5. Fluorescence immunocytochemistry of synaptic proteins and nAChR labelling Tissue sections were permeabilised according to the primary antibody being used. For synaptophysin labelv ling, the sections were permeabilised in cold (20 C) acetone for 10 min. For syntaxin and SNAP-25 labelling, the sections were pre-treated for 30 min at room temperature in 4% paraformaldehyde. They were then permeabilised in 0.l% Triton X-100 in PBS for 10 min at room temperature. After permeabilisation, the sections to be labelled for syntaxin and SNAP-25 were rinsed in PBS three times over 15 min and the sections to be labelled for synaptophysin were air-dried. The relevant primary antibodies were diluted with 3% bovine serum albumin and 0.1 M lysine in PBS to a final strength of 1/100, and were applied to the sections v overnight in a closed moist chamber at 4 C. The following day, the sections were washed in PBS three times over 30 min, followed by incubation in secondary antibodies (1:100 rabbit anti-mouse rhodamine-conjugated immunoglobulins) for 1 h at room temperature. Routinely, counterstaining of nAChRs was done by the inclusion of 1:200 FITC-conjugated a-bungarotoxin in the secondary antibody solution. The sections were then washed three times in PBS over 30 min, and fixed in 1% paraformaldehyde for 30 min. The sections were again washed three times in PBS over 30 min before mounting in Vectashield. As each visualised image comprised fluorescein labelled nAChR and rhodamine labelled synaptic protein, it was possible to calculate and express a labelling index as the percentage of end-plates recognised by nAChR labelling that were also labelled with the synaptic protein antibody. 2.6. Confocal microscopy A dual channel Bio-Rad laser confocal system (MRC 600) mounted on an Olympus upright microscope (BH2) and equipped with an Ar–Kr laser, was used to prepare images of labelled neuromuscular
junctions on tissue sections prepared as above. The 488-nm line of the laser was used to excite the fluorescein-labelled nAChRs at the end-plates, and the 647-nm line to excite the rhodamine-labelled proteins. The emissions were collected separately into the two channels of the confocal system using 515 and 676-nm barrier filters, respectively. The optical section thickness to be scanned was set at 1 lm. The intensity threshold for control images was set at 100–250 grey. Intensity thresholds were set using control sections and those thresholds were not changed for the duration of the examination of sections of toxin-treated muscles. Typically, single sections were randomly chosen from each of between two and four muscles. Sections contained between 11 and 61 junctional profiles. All junctions seen in a single section were examined and the respective areas of label and fluorescence intensities were measured using Cosmos Version 7 software. The mean of in-muscle averages area and intensity, respectively, were then calculated. In Tables 1 and 2, these data are expressed as mean S:E:M: of n junctions/ muscles. 2.7. Transmission electron microscopy Segments of diaphragm muscles were mounted under slight tension in a Sylgard-lined dish and fixed in Karnovsky’s solution for 1.5 h (Karnovsky, 1965). After fixation the tissue was washed three times in PBS over 15 min, teased into bundles of 10–15 muscle fibres and transferred to an incubation medium containing indoxyl acetate and hexazotized paraosanilin in 0.1 M citrate buffer at pH 6.0 for 1 h (Strum and Hall-Craggs, 1982). Regions containing neuromuscular junctions could be identified by a brick red deposit. These regions were cut out to yield blocks 1 1 mm, rinsed in So¨renson’s phosphate buffer (pH 7.4) and post-fixed in OsO4 (1% w/v in So¨renson’s buffer) before being dehydrated in graded acetone and embedded in TAAB resin (TAAB Laboratory Equipment, UK). Semi-thin sections were first cut. If these contained junctional regions, ultrathin sections, 50–70 nm thick and a silver/ gold colour, were prepared, collected onto Athene New 300 grids and examined in a Philips E500 microscope. Sections were scanned blind with respect to origin.
Table 1 Characteristics of labelling of nAChR by FITC-a-bungarotoxin in nerve–muscle preparations exposed to b-bungarotoxin AChR/AChE labelling index (%)
Control (n:n) b-Bungarotoxin (n:n)
100 0% (185:4) 100 0% (134:3)
FITC-a-bungarotoxin labelling Area of label (lm2)
Intensity of label (pixels)
26 6:9ð44 : 3Þ 20 5:8 (32:3)
230 9:9 (44:3) 280 75 (32:3)
Rat innervated hemidiaphragm preparations were indirectly stimulated at 0.1 Hz for 180 min in either normal bathing medium (control) or b-bungarotoxin (10 lg ml1) before being labelled with anti-AChE Ab and FITC-a-bungarotoxin to label neuromuscular junctions. Measurements were made on n fibres in n muscles, and are expressed as means of in-muscle averages S:E:M:
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Table 2 Characteristics of labelling of synaptic proteins by indirect immunofluorescence in nerve–muscle preparations exposed to b-bungarotoxin Protein/AChR labelling index (%)
Area of label (lm2)
Intensity of label (pixels)
Synaptophysin control (n:n) b-Bungarotoxin (n:n) Difference from control
100 0% (51:4) 100 0% (87:4) NS
29 2:7 (51:4) 16 1:4 (87:4) p< 5%
492 49 (51:4) 356 27 (87:4) p< 5%
Syntaxin control (n:n) b-Bungarotoxin (n:n) Difference from control
100 0% (100:3) 89 1:4% (101:2) p< 5%
23 2:7 (42:3) 22 3:5 (33:2) NS
640 168 (42:3) 668 88 (33:2) NS
SNAP-25 control (n:n) b-Bungarotoxin (n:n) Difference from control
99 2:3% (80:3) 78 2:6% (143:3) p< 5%
n.d. n.d.
n.d. n.d.
Rat innervated hemidiaphragm preparations were indirectly stimulated at 0.1 Hz for 180 min in either normal bathing medium (control) or b-bungarotoxin (10 lg ml1) before being labelled. Measurements were made on n fibres in n muscles, and are expressed as means of in-muscle averages S:E:M:
Junctional profiles were identified by the presence of the electron dense AChE reaction product within a synaptic cleft. They were photographed without further selection for subsequent analysis provided the sections were not oblique and the images were not transected or otherwise obscured by grid bars or edges. This technique ensured that no samples were made of terminal expansions prior to their entry into the cleft. Photographic images of individual boutons were prepared and measurements of the area of the terminal were made. Manual counts of synaptic vesicles within each bouton were made and data expressed as the number of synaptic vesicles lm2 of each terminal bouton. The length of the synaptic trough within which the bouton was accommodated was measured and so too was the length of the post-junctional membrane, including in the latter the length of the synaptic folds. Nerve contact length was defined as the length of direct apposition of bouton to synaptic trough. This was clearly reduced when Schwann cell processes invaded the gap between the synaptic trough and the terminal bouton. All morphometric data were collected and analysed using the software package Metamorph. 2.8. Electrophysiology Segments of hemidiaphragm were mounted in a horizontal superfusion bath with oxygenated normal bathing fluid at room temperature. Glass microelectrodes filled with 3 M KCl, resistance 5–15 MX, were used to impale putative end-plate regions. Records were made of resting membrane potential. 2.9. Statistical evaluations Where appropriate, data were expressed as mean S:E:M: Populations of data were compared
using the unpaired Student’s t-test and differences were considered as significantly different when P < 0:05.
3. Results 3.1. b-Bungarotoxin and inhibition of evoked muscle contractions The mechanical response of control phrenic nerve/ hemidiaphragm preparations to both indirect and direct muscle stimulation was constant over a period of 240 min. In the presence of b-bungarotoxin, 3–30 lg ml1 (approximately 107–106 M), there was a lag phase of 20–60 min during which the twitch response was unchanged. Thereafter, the response began to decline and transmission failed completely between 120 and 240 min. Increasing either the concentration of the toxin or the rate of stimulation resulted in reductions in both lag phase and time to transmission failure (Fig. 1A and B), but continual indirect stimulation was not an essential requirement for toxin-induced transmission failure (Fig. 1C). Transmission failure did not occur if the exposure time to b-bungarotoxin was less than 5 min (Fig. 1D). The response to direct muscle stimulation was unchanged even when transmission had failed completely (not shown). Segments of paralysed diaphragm muscle and their relevant controls were remounted in a horizontal bath superfused with normal bathing fluid and used for electrophysiological recordings. Mean muscle fibre membrane potential of paralysed muscles (73 1:2 mV, n ¼ 57) was indistinguishable from that in control muscle (72 1:3 mV, n ¼ 52). Other segments were used for histological and electron microscopical examinations. No muscle pathology was observed. There was no difference between control and toxin-treated muscles with respect to the nAChR/AChE labelling index, area of labelling by FITC-conjugated a-bungarotoxin or
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Fig. 1. Responses of the indirectly stimulated hemidiaphragm preparation following exposure to b-bungarotoxin. In each case, time zero represents the point at which b-bungarotoxin was added to the tissue bath. Unless otherwise stated preparations were stimulated supramaximally at 0.1 Hz and b-bungarotoxin was used at a concentration of 10 lg ml1. The response is expressed as % original (pre-toxin) tension generated by a single stimulus. (A) and (B) Transmission failure is partially dependant on toxin concentration and the rate of stimulation. (C) Preparation was unstimulated during a 3 h incubation with b-bungarotoxin. Following restoration of stimulation, the control preparation responded with a normal twitch response but the preparation exposed to b-bungarotoxin was not responsive to indirect stimulation. (D) b-Bungarotoxin was added to the bath at time zero and then washed out 1–20 min later. Note that short exposure times resulted in no loss of response. Graphical representation of the results has excluded data on directly stimulated muscles for the sake of clarity. b-Bungarotoxin had no influence on the directly elicited response. In each case n ¼ 3 6.
fluorescence (pixel) intensity of nAChR labelling (Table 1). 3.2. Synaptophysin, syntaxin and SNAP-25 labelling In sections from control muscles, all end-plates labelled with FITC-a-bungarotoxin were also labelled with anti-synaptophysin Ab. The labelling index was thus 100%. The area of label was similar to that of FITC-abungarotoxin labelling. In the toxin-treated muscles, the labelling index was maintained at 100%, but the labelling was variable in intensity even at a given endplate, leading to a reduction in mean intensity of label by 30%. The area of label was also reduced by 45% (Table 2). Since synaptophysin is an integral component of the synaptic vesicle membrane the loss of both label intensity and area suggested a significant loss of synaptic vesicles from nerve terminals. The labelling indices of both syntaxin and SNAP-25 were slightly but significantly reduced in toxin-treated
muscles (Table 2; Fig. 2). Quantitative data were generated only for syntaxin: at labelled end-plates of toxintreated muscles there was no reduction in either area labelled or intensity of label (Table 2). These data suggested that at the majority of nerve terminals, proteins involved in the formation of SNARE-complexes remain in place even when muscles are paralysed by bbungarotoxin. 3.3. Nerve terminal morphology In control muscles, junctional profiles typically comprised between one and four nerve terminal boutons (mean 2:1 0:17). All terminal boutons were filled with synaptic vesicles and contained intact mitochondria. Schwann cell processes covered the exposed (non-synaptic) region of each bouton (Fig. 3A). The morphometric data are summarised in Table 3.
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Fig. 2. Representative images of concurrent fluorescent labelling of nAChR, (A) and (C), and syntaxin (B) and (D) in transverse sections of hemidiaphragm muscles maintained in either normal bathing fluid (controls) (A) and (B) or b-bungarotoxin (10 lg ml1) (C) and (D) until transmission in the preparation exposed to toxin had failed. Note the loss of syntaxin labelling at a single end-plate in the section of toxin treated diaphragm muscle (arrow).
Neuromuscular junctions in muscles exposed to bbungarotoxin were similar in general organisation to those in control muscles. The average junctional profile contained 2:0 0:14 terminal boutons. Thirty-two junctions were examined. At 12, all terminal boutons were intact. Seventeen junctions contained one bouton that was damaged and at three junctions no intact terminal boutons were identified. Synaptic vesicle density in surviving boutons was reduced to 11 vesicles lm2 (cf. 63 vesicles lm2 in control boutons) and mitochondria were swollen and flocular in appearance (Table 3, Fig. 3B). Where boutons were destroyed, the denervated synaptic cleft contained Schwann cell processes (Fig. 3E). At surviving junctions, Schwann cell processes were often observed extending into the synaptic cleft, thus reducing the length of synaptic contact between nerve terminal bouton and post-synaptic membrane (not shown). The mean area of surviving nerve terminal boutons exposed to b-bungarotoxin was not significantly different from that of nerve terminal boutons in control muscles (Table 3). Taken together, these observations suggest that boutons become detached from their anchoring points on the post-synaptic membrane before undergoing degeneration following exposure to b-bungarotoxin.
3.4. Synaptic vesicle density and botulinum toxin C Neuromuscular transmission failed approximately 120 min after exposure to botulinum C (109 M). At this point, preparations were either exposed to b-bungarotoxin (10 lg ml1) in addition to botulinum toxin C or left in contact with botulinum toxin C alone for a further 180 min. Incubation with botulinum toxin had no direct effect on synaptic vesicle density and completely blocked synaptic vesicle depletion in preparations subsequently exposed to b-bungarotoxin (Figs. 3D and 4). 3.5. Synaptic vesicle density and conotoxin x-MVIIC It was not possible to make direct measurements of the role of [Ca2+]0 on b-bungarotoxin-induced exocytosis because the neurotoxic activity of b-bungarotoxin is Ca2+-dependent. We therefore measured synaptic vesicle loss in preparations in which the voltage gated Ca2+ channels of the nerve terminal were blocked with conotoxin x-MVIIC. Incubation of phrenic nerve/ hemidiaphragm preparations with conotoxin x-MVIIC (1 lM) resulted in transmission failure in approximately 120 min. From this point, preparations were maintained for a further 180 min in the continuing presence of conotoxin x-MVIIC alone or conotoxin x-
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techniques to document synaptic vesicle depletion and nerve terminal morphometry, and established that [Ca2+]0, voltage gated Ca2+ channels and the t-SNARE proteins SNAP-25 and syntaxin are all involved in the neuropathology of b-bungarotoxin induced-neuromuscular paralysis. 4.1. Specificity of action of b-bungarotoxin b-Bungarotoxin is a member of a large class of venom-derived toxic phospholipases A2, most of which are both myotoxic and neurotoxic, and some of which also stabilise the junctional nAChR in its desensitised form (see reviews by Hawgood and Bon, 1991; Harris, 1991). b-Bungarotoxin-induced failure of neuromuscular transmission was not associated with any change in either the gross morphology of the muscle, or the postjunctional region of the neuromuscular junction. In preparations in which transmission had failed, the mechanical response to direct stimulation and the resting membrane potential of individual muscle fibres were unchanged. Exposure to the toxin had no effect on the binding of FITC-conjugated a-bungarotoxin to junctional nAChR. Chang et al. (1973) have previously shown that exposure to b-bungarotoxin does not affect either the response of nAChR to applied ACh or the characteristics of the experimentally-induced muscle fibre action potential. Thus, the acute effects of b-bungarotoxin are targeted exclusively to the nerve terminal. 4.2. Synaptic vesicle depletion and nerve terminal morphometry Fig. 3. Representative images showing the effects of b-bungarotoxin, and of pre-incubation with botulinum toxic C and x-conotoxin MVII-C on terminal boutons at the neuromuscular junction. (A) After incubation in normal bathing fluid. Note the terminal boutons sitting in the deeply folded synaptic cleft. The terminal boutons are filled with mitochondria (M) and small clear synaptic vesicles (V). Incubation in b-bungarotoxin (10 lg ml1) until transmission failed (B) resulted in the swelling of mitochondria and a loss of morphological integrity (M) and a reduction in the density of synaptic vesicles. Pre-incubation with either botulinum toxin C, 109 M (C) and (D) or x-conotoxin MVII-C, 106 M (E) reduced synaptic vesicle loss.
MVIIC plus b-bungarotoxin, 10 lg ml1. Treatment with conotoxin x-MVIIC alone had no effect on synaptic vesicle density but b-bungarotoxin induced depletion of synaptic vesicles was reduced by more than 60% (Figs. 3C and 4).
4. Discussion In this study, we have confirmed that b-bungarotoxin is pre-synaptically active. We used quantitative
Thin sections of neuromuscular junctions in both control and toxin-treated muscles contained, on average, two terminal boutons. At approximately 10% of toxin-treated junctions (i.e. 3/32) synaptic gutters contained only Schwann cells and debris. Of the remaining junctions 17 contained a mixture of destroyed and intact terminal boutons and 12 possessed entirely intact terminal boutons. The intact terminal boutons on muscles exposed to b-bungarotoxin were similar in area to those on control muscles but they contained 80% fewer synaptic vesicles, mitochondria were swollen and had lost well defined cristae, and Schwann cell processes had entered the synaptic gutter, thus reducing the area of contact between nerve terminal and muscle fibre. Cytochemical studies on frozen sections of control and toxin-treated muscle fibres showed that the average areas of nAChR and synaptophysin labelling were similar in control muscles. In toxin-treated muscles, the area of nAChR labelling and fluorescence intensity were similar to controls but the area and the fluorescence intensity of synaptophysin labelling fell—by 45% and 30%, respectively. Synaptophysin is a component of the synaptic vesicle membrane but it is difficult to
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Table 3 Effect of b-bungarotoxin on nerve terminal morphometry
Nerve terminal area (lm2) Nerve terminal perimeter (lm) Nerve terminal contact length (lm) Junctional fold length (lm) Trough length (lm) Synaptic vesicle density (vesicles lm2) Mitochondrial perimeter (lm) Damaged mitochondria (%)
Control n ¼ 32 : 65 : 65
Toxin-treated n ¼ 32 : 63 : 43
Difference from control
5:2 0:72 13:9 1:32 5:6 0:58 48 3:7 10:2 0:89 63 8 0:9 0:02 32
5:0 0:60 12:8 1:25 3:8 0:39 49 4:6 9:8 0:83 11 2 1:7 0:04 90 3
NS NS p< 5% NS NS NS p< 5% p< 5%
Morphometric data collected from surviving nerve terminals at control end-plates and nerve terminals on the end-plates of muscles exposed to bbungarotoxin (10 lg ml1). Exposure to the toxin caused the depletion of synaptic vesicles, and a reduction in nerve contact length (a result of the invagination of terminal Schwann cells). Note that nerve terminal area, junctional fold length and trough length were unchanged. Mitochondria were swollen and mitochondrial damage was severe. n is number of end-plates:number of nerve terminals:number of surviving nerve terminals used for morphometry.
relate directly the depletion of synaptic vesicles observed in the electron microscopy and the loss of immunofluorescence using light microscopy. However, a 45% reduction in an area of label combined with a 30% reduction in fluorescence intensity suggested a
total loss of immunofluorescence of >60%, which is compatible with the morphometric assessment of an 80% depletion of synaptic vesicles. 4.3. The role of [Ca2+]0 and Ca2+ voltage gated channels Physiological exocytosis in response to indirect stimulation is initiated by the entry of Ca2+ into the nerve terminal via open voltage-gated Ca2+ channels. We blocked voltage-gated Ca2+ channels in nerve terminals to test whether the b-bungarotoxin-induced depletion of synaptic vesicles was dependent on the influx of extracellular Ca2+. In mammalian motor nerve terminals, the voltage gated Ca2+ channels are mainly non-L, non-N P/Q type and they are blocked by conotoxin x-MVIIC (Hillyard et al., 1992; Wheeler et al., 1994; Randall and Tsien, 1995; Bowersox et al., 1995; Dunlap et al., 1995; Lin and Lin-Shiau, 1997). Incubation with conotoxin x-MVIIC alone caused transmission failure but had no effect on synaptic vesicle density. Pre-exposure to the conotoxin significantly reduced the depletion of synaptic vesicles from terminal boutons subsequently exposed to b-bungarotoxin. Thus, we conclude that the depletion was partially caused by the entry of Ca2+ into the terminal via opened voltage-gated Ca2+ channels.
Fig. 4. Synaptic vesicle density derived by direct measurement from images of nerve terminals at the neuromuscular junction as shown in Fig. 3. The density of synaptic vesicles in terminal boutons was not changed by incubation with botulinum toxin C (109 M) or x-conotoxin MVIIC (106 M). Incubation with b-bungarotoxin (10 lg ml1) caused a significant fall in synaptic vesicle density that was prevented by pre-incubation in either botulinum toxin C or x-conotoxin x-MVIIC. Control data and data derived from preparations exposed to b-bungarotoxin are graphic representations of data reported in Table 3. Data derived from preparations, pre-incubated with either botulinum toxin C or conotoxin x-MVIIC involved measurements of synaptic vesicle density from between 17 and 27 nerve terminals.
4.4. The role of SNAP-25 and syntaxin in synaptic vesicle depletion Botulinum toxin C hydrolyses both SNAP-25 and syntaxin (Foran et al., 1996; Williamson et al., 1996; Kalandakanond and Coffield, 2001), thus preventing the formation of a SNARE complex between synaptic vesicle and nerve terminal membranes. b-Bungarotoxin caused no depletion of synaptic vesicles in preparations previously exposed to botulinum toxin C. The simplest explanation is that the accelerated exocytosis and
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depletion of synaptic vesicles caused by b-bungarotoxin is dependent both on the entry of Ca2+ via open voltage-gated Ca2+ channels and the formation of SNARE complexes in the nerve terminal. 4.5. The fate of synaptic vesicles In most situations, the speed of endocytosis is sufficient to accommodate the 100-fold increases in the rate of exocytosis induced by many natural toxins (Ceccarelli et al., 1979; Fesce et al., 1986; Henkel and Betz, 1995). b-Bungarotoxin causes only a fivefold increase in the rate of exocytosis (Chang et al., 1973) so it is not easy to understand why synaptic vesicles are depleted. Montecucco and Rossetto (2000) have suggested that b-bungarotoxin bound to the nerve ter-
minal was internalised during endocytosis. The internalised toxin could then attack the inner leaf of the synaptic vesicle membrane, releasing free fatty acids and generating lysophosphatides. Fatty acids and lysophosphatides cause an increase in membrane fluidity, enhance membrane fusion in vitro (Rufini et al., 1990; Nishio et al., 1996) and might be expected to promote fusion between synaptic vesicle membranes and nerve terminal membranes, and to impair vesicle re-sealing and thus inhibit endocytosis. The combination of accelerated exocytosis and reduced endocytosis would be sufficient to explain the depletion of synaptic vesicles (Montecucco and Rossetto, 2000). There are, however, two major problems with this suggestion. The first is the requirement that there is the uptake of b-bungarotoxin into synaptic vesicles during endocytosis.
Fig. 5. (A) Synaptic vesicle recycling in a normal nerve terminal: a synaptic vesicle buds away from an endosome (1), is filled with transmitter (2) docks (3) and is primed for release (4). The invasion of an action potential causes the opening of a voltage gated Ca2+ channel, the influx of Ca2+, the fusion of the synaptic vesicle with the plasma membrane of the nerve terminal and the release of the transmitter (5). The now empty vesicle is recovered during endocytosis (6) loses its endocytotic clathrin coat (7) and fuses with a budding endosome. (B) b-Bungarotoxin binds to the motor nerve terminal at a site close to or part of the vesicle binding/exocytotic site (D). When the vesicle opens (5), the interior face is exposed to the toxin and the vesicle membrane is hydrolysed (6). Thus, the vesicle is destroyed. Vesicles that escape destruction may enter the full recycling process, but the inevitable consequences of the hydrolytic attack are the depletion of synaptic vesicles and failure of synaptic transmission.
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Although Herkert and his colleagues (Herkert et al., 2001; Shakhman et al., 2003) have shown that b-bungarotoxin is internalised in cultured hippocampal neurons and Nico et al. (2003) have shown that the related pre-synaptically active phospholipase taipoxin is accumulated by chromaffin cells the situation with respect to the neuromuscular junction is less clear. Both Strong et al. (1977) and Esquadera et al. (1982) have shown that the b-bungarotoxin binds primarily to the nerve terminal membrane and that the secondary uptake of b-bungarotoxin into the nerve terminal via endocytosis is very limited; others have failed to find any evidence of uptake at all (Howard and Wu, 1976; Simpson et al., 1993). The second problem is that enhanced exocytosis associated with impaired endocytosis in terminal boutons is typically associated with bouton swelling (Henkel and Betz, 1995 for example). Our data suggest that boutons do not swell. A mechanism consistent with these findings is that during exocytosis the inner face of the open synaptic vesicle comes into contact with bound toxin and is hydrolysed (Fig. 5). Noremberg and Parsons (1986) have reported that synaptic vesicles are very sensitive to hydrolysis by b-bungarotoxin. Those vesicles that escape hydrolysis could then re-enter the cycle of endocytosis/exocytosis. The combination of enhanced exocytosis and synaptic vesicle hydrolysis would be a progressive loss of synaptic vesicles available for release, the failure of neuromuscular transmission, and an empty terminal bouton of unchanged diameter. This explanation is consistent with all available experimental evidence and has many elements in common with that of Montecucco and Rossetto (2000). It differs from the latter in that our explanation, (1) depends critically on the suggestion that the toxin binding sites are closely related to the release sites, (2) does not require an internalisation step, (3) does not require the incorporation of synaptic vesicle membrane into the nerve terminal plasma membrane.
4.6. The degeneration of the nerve terminal Degeneration of nerve terminals was probably related to the hydrolytic activity of b-bungarotoxin. Hydrolysis of membrane lipids leads to an increase in membrane fluidity, loss of ion homeostasis, cellular depolarisation, entry of Ca2+ into the cell and, ultimately, disintegration of the membrane. Elevation of [Ca2+]i is enhanced by the progressive build up of free fatty acids and lysophosphatides (Sedlis et al., 1983), and results in depolarisation of mitochondria, increased mitochondrial proton permeability and mitochondrial failure (Wernicke et al., 1975; Nicholls et al., 1985; Rugulo et al., 1986; Nicholls and Budd, 2000; Jambrina et al., 2003; Granitkerich, 2003; Gulbins et al., 2003). The data suggest that the elevation of
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[Ca2+]i is the common denominator in both the enhanced exocytosis and the initiation of nerve terminal degeneration.
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