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.