Fig. 3. Ket alters presynaptic transmission independently of GABA inhibitory input.
(A) Effect of PKA inhibition and GABAergic input on the action of ket on SC-CA1 EPSCs recorded from CA1 neurons in hippocampal slices from rats. Hippocampal slices were pretreated for 30 min with picrotoxin (100 µM) and CGP (4 µM) to inhibit GABAergic signaling. Blue shading indicates the period of exposure to ket (20 µM) in the presence or absence of H89 (PKA inhibitor, 10 µM). All drugs were applied to the bath solution. Left: Data are presented as the EPSC amplitude normalized to the baseline before ket application (ket: 163 ± 8.3%of baseline, n=11, P < 0.001, paired t test). Right: Representative traces before and after ket in the absence (top) or presence (bottom) of H89. Scale bar, 40 ms/30 pA. (B) Effect of ket on SC-CA1 EPSCs and PPR of EPSCs in the presence of picrotoxin and CGP. Data were obtained using the same concentrations of drugs applied as in (A). Left: Representative data from a single-cell recording are presented as the ratio of the second EPSC amplitude to the first EPSC normalized to the baseline before ket application. Ket significantly decreased PPR of EPSCs: 81.3 ± 4.1% of baseline; n = 12 slices, P < 0.0001, paired t test. Right: Representative traces before and after ket application as well as merged and normalized (to before ket) traces. Scale bar, 40 ms/30 pA. (C) Effect of inhibition of NMDARs by MK-801 on SC-CA1 EPSCs and the effect to ket. Slices were pretreated by bath application for 1 hour with picrotoxin (100 µM) and CGP (CPG, 4 µM). Blue shading indicates the period of exposure to ket (20 µM). Left: Data are presented as the EPSC amplitude normalized to the baseline beforeMK-801 application (afterMK-801: 165 ± 6.0% of baseline; after MK-801 plus ket: 171 ± 20.2% of baseline; F2,23=6.87, P = 0.0046, ANOVA; P > 0.99 between the two groups, Bonferroni post hoc test). Right: Representative traces. (1) Baseline in picrotoxin and CGP, (2) after MK-801 application, and (3) after ket application in continuous MK-801 treatment. Scale bar, 40 ms/30 pA. (D) Effect of ket on GluA1 abundance and phosphorylation when GABAergic inhibition is blocked. Rat hippocampal slices were treated with drugs at the same concentrations as in (A). Representative blots are shown along with normalized quantified data from five independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control, t test.