Figure 1

3,4-DAP restores evoked synaptic release in neuronal cultures intoxicated by BoNT/A but not BoNT/D or BoNT/E. (A) Representative whole-cell recordings of evoked neurotransmission in the presence of vehicle, TTX (5 µM), CNQX (20 µM), or 3,4-DAP (50 µM) and quantitation of normalized EPSC amplitudes versus vehicle (n = 6 each for TTX and CNQX; n = 16 for 3,4-DAP). (B) Representative Western blots of SNAP-25, STX1 and SYB2 from lysates harvested from primary neuron cultures treated with vehicle, BoNT/A, BoNT/D, or BoNT/E treatment and schematic of SNAP-25 and SYB structure with BoNT/A, /D and /E cleavage sites. Each panel representes a complete image from blots stained for STX1 and SNAP-25 (top) or SYB2 (bottom). Note that while proteolytic cleavage fragments of SNAP-25 can be immunologically detected, cleavage of SYB2 results in the rapid degradation of the N-terminal fragment, and therefore loss of reactivity is considered evidence of intoxication¹⁹. (C–E): Representative whole-cell recordings and of evoked neurotransmission and mean EPSC amplitudes in cultures intoxicated by BoNT/A, BoNT/D, or BoNT/E and treated with vehicle (n = 12), 3,4-DAP (50 µM; n = 12 for BoNT/A and n = 9 for BoNT/D or E) or 3,4-DAP plus VGCC antagonists (n = 8). Mean EPSC amplitudes are normalized to recordings from age-matched, non-intoxicated control cultures. Arrows represent stimulation times. Scale bars represent 400 ms (x-axis) and 100 pA (y-axis). All data presented as mean ± SEM. *Indicates p < 0.05, **Indicates p < 0.01, ns = not significant.