Figure 2.

Tetanus toxin (TnTx) ablates activity‐dependent SV exocytosis. Cultures were preincubated either with or without 2 nM TnTx for 24 h. (a) Representative immunoblots of cerebellar granule neuron lysates probed with synaptobrevin II (SybII) antibodies. The same blot was reprobed with synaptophysin (Syp) antibodies. (b) Quantification of sybII levels after normalisation against Syp ± SEM. SybII levels were zero in each experiment. ***p = 0.0005, Student's t‐test, n = 3 independent experiments per condition. (c and d) Cultures were transfected with the genetic reporter synaptophysin‐pHluourin (sypHy) and stimulated with a train of 800 action potentials (80 Hz). They were subsequently exposed to alkaline buffer to reveal the total sypHy fluorescence. (c) Graph displays the mean ΔF/F₀ time course for sypHy ± SEM in either untreated (blue circles) or treated (green circles) neurons normalised to the total sypHy response in alkaline buffer. Bar indicates period of stimulation. (d) Bar graph displays the maximal evoked ∆F of sypHy for untreated (blue bars) or treated (green bars) neurons normalized to the total SV pool (revealed by the alkaline buffer) ± SEM. ***p = 0.0006, Student's t‐test, n = 3 independent experiments per condition.