Tetanus toxin blocks the exocytosis of synaptic vesicles clustered at synapses but not of synaptic vesicles in isolated axons

Abstract

Recycling synaptic vesicles are already present in isolated axons of developing neurons (Matteoli et al., Zakharenko et al., 1999). This vesicle recycling is distinct from the vesicular traffic implicated in axon outgrowth. Formation of synaptic contacts coincides with a clustering of synaptic vesicles at the contact site and with a downregulation of their basal rate of exo-endocytosis (Kraszewski et al, 1995; Coco et al., 1998) We report here that tetanus toxin-mediated cleavage of synaptobrevin/vesicle-associated membrane protein (VAMP2), previously shown not to affect axon outgrowth, also does not inhibit synaptic vesicle exocytosis in isolated axons, despite its potent blocking effect on their exocytosis at synapses. This differential effect of tetanus toxin could be seen even on different branches of a same neuron. In contrast, botulinum toxins A and E [which cleave synaptosome-associated protein of 25 kDa. (SNAP-25)] and F (which cleaves synaptobrevin/VAMP1 and 2) blocked synaptic vesicle exocytosis both in isolated axons and at synapses, strongly suggesting that this process is dependent on "classical" synaptic SNAP receptor (SNARE) complexes both before and after synaptogenesis. A tetanus toxin-resistant form of synaptic vesicle recycling, which proceeds in the absence of external stimuli and is sensitive to botulinum toxin F, E, and A, persists at mature synapses. These data suggest the involvement of a tetanus toxin-resistant, but botulinum F-sensitive, isoform of synaptobrevin/VAMP in synaptic vesicle exocytosis before synapse formation and the partial persistence of this form of exocytosis at mature synaptic contacts.

Fig. 1.

Synaptic protein expression during neuronal development. Western blot analysis of cell extracts from neuronal cultures maintained in vitro for 2, 7, and 15 d (DIV) and from rat brain at different developmental stages (E18, P1, and adult). SV (synaptophysin, synaptotagmin, synaptobrevin/VAMP2, and rab3a) and plasma membrane (SNAP-25 and syntaxin) proteins are expressed since early developmental stages and undergo a progressive increase in their expression in parallel with neuronal maturation. The same amount of material (30 μg) has been loaded in each lane.

Fig. 2.

Tetanus toxin treatment inhibits SV exocytosis at mature synapses but not in developing neurons.A, B, Fifteen-day-old neurons were incubated for 5 min in the presence of Syt-ecto Abs in 55 mm external KCl before (A, B) or after (C, D) treatment with 10 nm TeNT. After this incubation, neurons were washed, fixed, detergent-permeabilized, reacted with rhodamine-conjugated goat anti-rabbit IgGs (B, D), and counterstained with antibodies against total synaptotagmin (syt), followed by FITC-conjugated goat anti-mouse IgGs (A, C). Puncta of immunoreactivity represent presynaptic nerve terminals, which outline perikarya and dendrites. Syt-ecto Abs are internalized at synaptic contacts when applied in control conditions (B) but not after treatment with TeNT (D). E–H, Exocytosis-dependent uptake of Syt-ecto Abs (applied for 5 min in the presence of 55 mm KCl in the external medium) in living neurons before synaptogenesis, in control conditions (F), or after treatment with 10 nmTeNT (H). E,G, Double immunofluorescence of total synaptotagmin (syt) of the same neurons as in F andH. Note that an efficient internalization of Syt-ecto Abs takes place in axons, even after treatment with TeNT (H). Scale bar: A–D, 20 μm; E–H, 28 μm. I, Quantitative analysis of Syt-ecto internalization in neurons before and after synaptogenesis, both in control conditions or after treatment with 10 nm TeNT.

Fig. 3.

TeNT treatment cleaves synaptobrevin/VAMP2 without blocking SV recycling in neurons before synaptogenesis. A, B, Immature control neuron exposed to Syt-ecto Abs (for 5 min in the presence of 55 mm KCl in the external medium) (B) and double labeled for synaptobrevin/VAMP2 (A). C,D, Immature neuron exposed to Syt-ecto Abs (D) and double labeled for synaptobrevin/VAMP2 (C) after treatment with 10 nm TeNT. Note that TeNT treatment cleaves synaptobrevin/VAMP2 without impairing SV recycling. Scale bar, 11.25 μm. E, Quantitative analysis of Syt-ecto Ab internalization (●) and synaptobrevin/VAMP2 cleavage (■) in developing neurons at different times after culture intoxication. Note that TeNT treatment dramatically reduces synaptobrevin/VAMP2 immunoreactivity over time without significantly reducing SV recycling. F, Quantitative analysis of Syt-ecto Ab internalization (●) and synaptobrevin/VAMP2 cleavage (■) in developing neurons exposed to increasing doses of BoNT/F. Note the existence of a strict correlation between synaptobrevin/VAMP2 cleavage and inhibition of SV recycling. Values were expressed as a ratio between the signals produced by Syt-ecto Abs or by synaptobrevin/VAMP2 antibodies and those produced by antibodies directed against total synaptotagmin I or synaptophysin.

Fig. 4.

BoNT/A and E block SV recycling in neurons before synaptogenesis. A, B, Exocytosis-dependent uptake of Syt-ecto Abs (applied for 5 min in the presence of 55 mm KCl in the external medium) in immature neurons after treatment with 20 nm BoNT/A. Virtually no internalization takes place in neuronal processes (B), visualized by double labeling with antibodies against total synaptotagmin (syt,A). Scale bar, 12.8 μm. C, Quantitative analysis of Syt-ecto internalization in cultures treated with 20 nm BoNT/A and 80 nm BoNT/E. Note that BoNTs of both serotypes strongly reduce SV recycling.

Fig. 5.

SV recycling is TeNT-insensitive in isolated axons of mature cultures. An efficient internalization of Syt-ecto Abs, applied for 5 min in 55 mm KCl, takes place in the isolated axons present in fully differentiated cultures after exposure to 10 nm TeNT (B). No labeling is detectable at synaptic contacts (arrowheads).A, Double labeling of the same culture as inB with antibodies against total synaptotagmin(syt). Insets a, b, After TeNT intoxication, synaptobrevin/VAMP2 immunoreactivity is no longer visible in an isolated axon (b) double labeled with the SV marker synaptophysin (a).C–H, SV exocytosis is differentially affected by TeNT in distinct compartments of a same neuron grown in microisland and forming autaptic contacts. Exposure to 10 nm TeNT completely prevents Syt-ecto Ab internalization at autapses, the sites where the axon gets in touch with dendrites (small arrowheads), whereas an efficient internalization of Syt-ecto antibodies takes place in the isolated axon (large arrowhead) (D). F,H, High magnification details of Syt-ecto Ab internalization at autaptic contacts (F) and in the isolated axon (H). C,E, G, Double labeling of the same neuron with antibodies against total synaptotagmin. Scale bar:A, B, 11 μm; C,D, 25 μm; G, H, 10 μm.

Fig. 6.

Persistence of spontaneous release at the synapse after TeNT but not BoNT treatment. A, B, Spontaneous Syt-ecto internalization at synaptic sites in TeNT-poisoned neurons (B) double labeled with antibodies against total synaptotagmin (syt, A). C, Quantitative analysis of Syt-ecto Ab internalization in mature neurons intoxicated with TeNT or with BoNTs (20 nm BoNT/A, 80 nm BoNT/E, and 80 nm BoNT/F). Note that BoNTs of all serotypes prevent similar spontaneous and evoked Syt-ecto Ab internalization at the majority of synaptic contacts, whereas TeNT inhibits evoked Syt-ecto internalization without substantially impairing spontaneous uptake. Incubation with Syt-ecto antibodies is performed for 5 min in the presence of 55 mm KCl in the external medium or for 1 hr in low KCl (5 mm) and 1 μm TTX.