Fig. 6.

Pharmacological blockade of sphingolipid signaling occluded the effect of BDNF on EPSC amplitude. A: cumulative percent histogram plot showing that the neutral sphingomyelinase inhibitor 25 μM sphingolactone-24 blocked the BDNF enhancement of EPSC amplitude (n = 6 cells; 606 and 306 EPSCs for sphingolactone-24 and sphingolactone-24/BDNF, respectively). This is also seen in a mean amplitude bar plot (inset; −128.6 ± 21.3 pA vs. −120.3 ± 23.6 pA for sphingolactone-24 and sphingolactone-24/BDNF, respectively; n = 6). B: cumulative percent histogram plot showing that postsynaptically applied fumonisin B1 (10 μM), a blocker of endogenous ceramide production, blocked the ability of BDNF to increase EPSC amplitude (n = 8 cells; 384 and 336 EPSCs for fumonisin and fumonisin/BDNF, respectively). This is also reflected in a bar plot of mean amplitudes before and after a 10- to 15-min 100 ng/ml BDNF application in the presence of postsynaptic fumonisin B1 (inset; −43 ± 4 pA vs. −45.8 ± 6.5 pA for control and BDNF, respectively, n = 8). C: plot of spike output vs. stimulus amplitude (normalized to threshold) showing that cytosolic fumonisin B1 application (10 μM) caused an increase in spike discharge in response to square-wave current injections and blocked the previously reported BDNF-dependent decrease in spike output (n = 24 for saline control, n = 5 for internal fumonisin B1 alone, and n = 11 for internal fumonisin B1 and bath-applied 100 ng/ml BDNF; P < 0.001, 2-way ANOVA, saline was different from both other groups). Inset: current-clamp trace showing large overshooting action potentials generated in response to a 400-pA current injection (500% threshold for this cell) in a cell 40 min after establishing the whole cell recording with fumonisin B1 in the recording electrode, suggesting that cells remained healthy during cytosolic fumonisin perfusion.