Fig. 1. Ketamine increases phosphorylation of GluA1 at hippocampal CA1 and enhances SC-CA1 synaptic transmission through a PKA-dependent mechanism.

(A) Effect of ketamine (ket) on SC-CA1 fEPSPs, recorded in the stratum radiatum of CA1 in acutely prepared rat hippocampal slices. Ket (20 µM) was applied to the bath in the presence or absence of H89 (10 µM, PKA inhibitor), which was preapplied for 1 hour and maintained throughout the experiment. Left: Data are presented as the time course of SC-CA1 fEPSPs slope before and after ket application (blue shading) in control artificial cerebrospinal fluid (ACSF) or in the presence of H89 (ket in control ACSF: 175 ± 7.2% of baseline at 51 to 60 min after ket application; n = 13 from 10 animals, P = 0.00012, paired t test; H89: 105 ± 5.4% of baseline; n = 6 from four animals, P = 0.69, paired t test). Right: Representative fEPSP averages before and after ket application. Drug responses were measured at 51 to 60 min after applied for all electrophysiological experiments in this paper. (B) Dose-response relationship of ket and the slope of SC-CA1 fEPSPs plotted with a best-fit sigmoidal function. Concentrations on the abscissa are log₁₀ coordinates. The value of n presents the number of slices recorded. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to control. (C) Effect of ket on GluA1 Ser⁸⁴⁵ phosphorylation and GluA1 abundance. Representative Western blots and data summary of six independent experiments showing that phosphorylation of GluA1 Ser⁸⁴⁵ and expression of total GluA1 were both significantly increased after ket bath application. (D) Effect of PKA inhibition on ket-induced increase in GluA1 Ser⁸⁴⁵ phosphorylation and GluA1 abundance. Rat hippocampal slices were exposed to saline (Ctrl) and ket (20 µM) in the presence or absence of H89 (10 µM). Top: Representative Western blots. Bottom: Data quantified from six independent Western blot experiments. (E) Effect of ket on GluA1 abundance and GluA1 Ser⁸⁴⁵ phosphorylation in the presence of a protein synthesis inhibitor. Rat hippocampal slices were exposed to saline and ket (20 µM) in the presence or absence of anisomycin (20 µM). Top: Representative Western blots. Bottom: Data quantified from four independent Western blot experiments [GluA1 Ser⁸⁴⁵ phosphorylation: F_2,9 = 39.52, P < 0.0001, analysis of variance (ANOVA); P = 0.245, Bonferroni post hoc test between ket group and anisomycin plus ket group; total GluA1: F_2,9 = 7.635, P = 0.0115, ANOVA; P = 0.021 for Bonferroni post hoc test between ket group and anisomycin plus ket group]. *P < 0.05 and ***P < 0.001 compared to control, and ^#P < 0.05 compared to ket alone, Bonferroni post hoc test after ANOVA. (F) Effect of ket on SC-CA1 fEPSPs in the presence of a protein synthesis inhibitor. Rat hippocampal slices were preexposed to anisomycin (20 µM) for 30 min and then ket (20 µM, blue shading). Graph shows SC-CA1 fEPSP slope, and inset shows representative traces before and after ket application. n = 6 slices from four rats; P < 0.05, paired t test. Scale bar, 5 ms/0.2 mV. (G) Effect of Trk, PKA, and CaMKII inhibition on the ket-induced increase in GluA1 abundance. Acutely prepared rat hippocampal slices were incubated with ACSF (Ctrl) or ket (20 µM), or ket and K252a (0.1 µM, Trk inhibitor), H89 (10 µM, PKA inhibitor), or KN62 (5 µM, CaMKII inhibitor). Left: Representative Western blots. Right: Data quantified from five independent Western blot experiments. F_4,16 = 6.587, P = 0.0025, ANOVA. *P < 0.05 and **P < 0.01 compared to control, and ^###P < 0.001 compared to ket alone, Tukey’s post hoc test after ANOVA.