Figure 9.

Selective suppression of spontaneous release via application of folimycin at rest is sufficient to elicit eEF2 kinase-dependent synaptic potentiation. A, Experimental design for whole-cell recordings in the presence of folimycin. B, C, Representative traces of evoked (B) and spontaneous (C) postsynaptic currents during different stages of the experiment (1, 2, 3). B, Recording evoked neuronal activity in dissociated hippocampal cultures for 4 min in Tyrode's solution (1), in presence of 10 μm TTX and 80 nm folimycin for 30 min (2), and after TTX washout (3). C, Recording spontaneous neuronal activity (mEPSC) in dissociated hippocampal cultures 10 min after application of TTX and folimycin (2), 30 min after application of TTX and folimycin (2), and after TTX washout (3). D, mEPSC frequency is significantly reduced in the presence of folimycin with or without TTX (n = 5, p < 0.001; mean ± SEM). E, Immunoblot analysis of phospho-eEF2 and total-eEF2 proteins in total protein lysates from dissociated hippocampal cultures indicates that the level of eEF2 phosphorylation in cultures treated with folimycin or ketamine is significantly decreased compared with control cultures treated with vehicle (n = 5, p < 0.02). Plotted are phospho-eEF2/total eEF2 ratios as percentage of control. F, FPs were recorded in eEF2K knock-out (KO) mice and in wild-type (WT) littermate control mice. In WT slices (filled circles, n = 5), folimycin induced synaptic strength facilitation. In contrast, no changes were observed in slices from eEF2K KO mice after application of folimycin (n = 9). Plotted are FP initial slopes (mean ± SEM) as a function of time. Inset, Representative waveforms from WT and eEF2K KO slices, recorded at different time points (1, 2). One-way ANOVA with repeated measurements, F_(39,157) = 2.85, p = 0.001; with Holm-Sidak post hoc test, p < 0.05. Scale bar, 0.2 mV/5 ms.