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First published online 29 June 2004
doi: 10.1242/jcs.01218

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Snake presynaptic neurotoxins with phospholipase A2 activity induce punctate swellings of neurites and exocytosis of synaptic vesicles

¹ Department of Biomedical Sciences and CNR Institute of Neuroscience, University of Padova, Viale G. Colombo 3, 35121 Padova, Italy
² Molecular NeuroPathoBiology Laboratory, Cancer Research UK, London Research Institute, 61 Lincoln's Inn Fields, London, WC2A 3PX, UK
³ Department of Neuroscience, S. Raffaele Scientific Institute and `Vita-Salute' University, Via Olgettina 58, 20132 Milan, Italy

View larger version (16K): [in a new window]   Fig. 1. Time-dependent induction of cell death of CGNs (A) or cortical neurons (B) by ß-BTx, notexin, taipoxin and textilotoxin. Neurons were incubated for the indicated times with 5 nM of each toxin, washed extensively and further incubated for 4 hours in the presence of resazurin to quantify the mitochondrial activity of viable cells. Values are averages of three independent experiments with triplicate measurements; error bars indicate s.d.

View larger version (110K): [in a new window]   Fig. 2. Morphological changes induced by ß-bungarotoxin in intoxicated primary neuronal cultures. CGNs (A,B) and cortical (C,D) neurons at 6 days in vitro or hippocampal neurons (E,F) at 15 days in vitro were examined by Nomarski optics after incubation for 1 hour in either control medium (A,C,E) or medium with 5 nM ß-BTx. Notice the presence of many bulges in the neuronal extensions of the toxin-treated samples. Scale bars, 50 µm (A-D), 10 µm (E,F). Insets are higher magnifications of the areas indicated by the square.

View larger version (35K): [in a new window]   Fig. 3. FESEM analysis of control and intoxicated CGNs. Control neurons (A) and neurons treated for 1 hour with 5 nM taipoxin (B,C) were processed and analysed by FESEM. (C) Higher magnification of the area indicated by the square in (B). Scale bars, 10 µm (A,B), 1 µm (C).

View larger version (17K): [in a new window]   Fig. 4. Quantification of bulge density in CGNs by FESEM analysis. (A) Bulges were counted and expressed as bulge density (bulges µm–2) in control cells or in cells treated for 1 hour with 200 nM pancreatic PLA2 or with 5 nM taipoxin or ß-BTx. (B) Dose-dependent quantification of bulges in CGNs treated for 1 hour with taipoxin concentration 0.1-5.0 nM. Bars represent the data obtained from three different cell preparations ± s.d.

View larger version (48K): [in a new window]   Fig. 5. Effect of SPANs on the distribution of cytoskeletal markers. Untreated CGNs (A-C) were compared with CGNs treated for 1 hour with ß-BTx (D-F). The distribution of F-actin (A,D) and of neuron-specific neurofilaments (NF) (B,E) changed from filamentous structures to dotted aggregates, whereas the appearance of tubulin (C,F) remained unaltered. Scale bar, 50 µm.

View larger version (75K): [in a new window]   Fig. 6. Cellular effects of SPANs are restricted to neuronal cells. Control CGNs (A) were compared with cells that were treated for 1 hour with taipoxin (B). Distribution of actin labelled with phalloidin (red) changed from filamentous structures to dots in intoxicated neurons, whereas the actin appearance of GFAP-positive astrocytes was unaltered (yellow). Scale bar, 50 µm.

View larger version (44K): [in a new window]   Fig. 7. Effect of SPANs on the distribution of synaptophysin I and VAMP2. Control CGNs (A,B) and neurons treated for 1 hour with 5 nM taipoxin (C-F) were fixed, permeabilized, counterstained for SypI (A,C,E) or for VAMP2 (B,D,F) and analysed by fluorescence confocal microscopy. The two vesicular membrane proteins changed distribution, being more intense in correspondence to the bulges of intoxicated neurons. (E,F) Higher magnifications of phase-contrast and fluorescence images of intoxicated neurons showing the intense staining of the two SV proteins in correspondence of the bulges (arrow). (G,H) Hippocampal neurons were co-transfected with expression vectors containing SypI-EYFP (G) and ECFP-VAMP2 (H), exposed to 5 nM taipoxin for 1 hour at 37°C and then analysed without fixation by epifluorescence microscopy. Scale bar, 50 µm (A-D), 10 µm (G-H).

View larger version (39K): [in a new window]   Fig. 8. SPANs induce SV exocytosis without proportionate recycling. Control CGNs (A) and cells treated for 1 hour at 37°C with notexin (B), ß-BTx (C), textilotoxin (D) or taipoxin (E) were incubated for 5 minutes at 37°C with anti-SytI-ecto antibodies before fixation and then processed for indirect immunofluorescence. (F) Higher magnification of the area indicated by the square in (E), which highlights the bright selective staining of bulges in the intoxicated sample (arrows). Scale bar, 50 µm (A-E).

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