Essential roles of AMPA receptor GluA1 phosphorylation and presynaptic HCN channels in fast-acting antidepressant responses of ketamine

Abstract

Although the molecular mechanism is not clear, the clinically tested drug ketamine has rapid antidepressant action that does not require the multiple weeks of treatment needed for other antidepressant drugs to have an effect. We showed that ketamine potentiated Schaffer collateral-CA1 cell excitatory synaptic transmission in hippocampal slice preparations from rodents and enhanced the phosphorylation of the GluA1 subunit on Ser⁸⁴⁵ of the AMPA-type glutamate receptor in the hippocampal area CA1. These effects persisted when γ-aminobutyric acid (GABA) receptors were pharmacologically blocked. Ketamine reduced behavioral despair in wild-type mice but had no effect in GluA1 S845A knock-in mutant mice. Presynaptic (CA3 pyramidal cell), but not postsynaptic (CA1 pyramidal cell), deletion of N-methyl-d-aspartate (NMDA)-type glutamate receptors eliminated the ketamine-induced enhancement of excitatory synaptic transmission in hippocampal slices and the antidepressant actions of ketamine in mice. The synaptic and behavioral actions of ketamine were completely occluded by inhibition or deletion of the hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1). Our results implicate presynaptic NMDA receptor inhibition followed by reduced activity of presynaptic HCN1 channels, which would result in an increase in glutamate release and postsynaptic glutamate receptor activity, as a mechanism of ketamine action. These data provide a mechanism for changes in synaptic activity that could explain the fast-acting antidepressant effects of this drug.

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.

Fig. 2. Ket increases the abundance of GluA1 at the cell surface in PKA-dependent manner

(A) Effect of ket on the abundance of GluA1 and phosphorylated GluA1 Ser⁸⁴⁵ (p-S845) at the cell surface. Acutely prepared rat hippocampal slices were incubated with ACSF, ket (20 µM), or H89 (10 µM) and ket (20 µM) (H89 + ket) for 2 hours before biotinylation assays were performed. Top: Representative blots. Bottom: Quantified data from six independent membrane protein biotinylation experiments (surface GluA1: 189 ± 33.3% of control for ket; 92 ± 13.6% for H89 plus ket; F_2,18 = 6.68, P = 0.0068, ANOVA; P = 0.012, Bonferroni post hoc test between the two groups; total GluA1: 166 ± 15.4% for ket; 159 ± 10.8% for H89 plus ket; F_2,15 = 9.59, P = 0.0021, ANOVA; P > 0.99, Bonferroni post hoc test, H89 plus ket versus control). (B) Effect of a single ket injection on animal behavior in the forced swim. Rats were subjected to the FST 30 min and 2, 8, and 24 hours after saline (control) or ket injection [10 mg/kg, intraperitoneally (i.p.)]. n = 5 animals in each group; *P < 0.05; **P < 0.01, control versus ket, t test. (C) Effect of a single ket injection on GluA1 Ser⁸⁴⁵ phosphorylation and total GluA1 abundance in CA1 area tissue wedges from rat hippocampal slices. Top: Representative Western blots. Bottom: Quantified data from n = 8 rats for each time point. *P < 0.05; **P < 0.01, compared to saline injection group.

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; F_2,23=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.

Fig. 4. Phosphorylation of GluA1 Ser⁸⁴⁵ is required for the effect of ket in the FST model of antidepressant action

(A) Effect of ket on SC-CA1 fEPSPs in hippocampal slices from wildtype and GluA1 S845A knock-in mice. Acutely prepared mice hippocampal slices were bath-applied with ket (20 µM). Ket enhanced SC-CA1 fEPSPs in wildtype mice but not in GluA1 S845A mutant mice (wildtype: 175 ± 11.4% of baseline, n = 8 slices; P < 0.001, paired t test; GluA1 S845A mice: 108 ± 4.3% of baseline, n = 6 slices; P = 0.20, paired t test). (B) Effect of ket on the abundance of GluA1 at the cell surface in wildtype and GluA1 S845A knock-in mice. Surface proteins were extracted by biotinylation assay, then GluA1 and Ser⁸⁴⁵ phosphorylated GluA1 (p-S845) were detected by Western blotting. Top: Representative blots. Bottom: Surface GluA1 and GluA1 Ser⁸⁴⁵ phosphorylation quantified from six independent experiments. *P < 0.05 compared with control, paired t test. (C and D) Effect of a single ket injection on animal behavior in the FST for wildtype mice (C) and GluA1 S845A knock-in mice (D). Mice were tested at the indicated times up to 7 days after injection of ket (10 mg/kg, i.p.) (wildtype mice: n = 6; GluA1 S845A mice: n = 7. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to corresponding time point before ket, paired t test.

Fig. 5. Presynaptic (CA3) but not the postsynaptic (CA1) NMDARs are required for the antidepressant actions of ket

(A) Effect of ket on SC-CA1 fEPSPs in hippocampal slices from control (control floxed-NR1) and CA1-NR1 KO mice. Ket potentiated SC-CA1 fEPSPs in slices from control floxed-NR1 mice and CA1-NR1 KO mice (control floxed-NR1 mice: 139.1 ± 5.1%, n = 7 slices from five mice; CA1-NR1 KO mice: 143.3 ± 4.3%, n = 7 slices from six mice; P = 0.42, t test). (B) Effect of ket on SC-CA1 fEPSPs in hippocampal slices from control and CA3-NR1 KO mice. Ket failed to enhance SC-CA1 fEPSPs in slices from CA3-NR1 KO mice (control floxed-NR1 mice: 159.1 ± 12.2%, n = 8 slices from four mice; CA3-NR1 KO mice: 102 ± 4.9%, n = 7 slices from four mice; P = 0.005, t test). (C) Effect of ket on behavior in novelty-suppressed feeding test of control floxed-NR1 mice and CA1-NR1 KO mice. Five mice of each genotype were tested. (D) Effect of ket on behavior in novelty-suppressed feeding test of control floxed-NR1 mice and CA3-NR1 KO mice. Five mice of each genotype were tested. (E) Effect of ket on behavior in the FST of control floxed-NR1 mice and CA1-NR1 KO mice. Five mice of each genotype were tested. (F) Effect of ket on behavior in the FST of control floxed-NR1 mice and CA3-NR1 KO mice. Five mice of each genotype were tested. For (B), (C), (E), and (F), *P < 0.05, **P < 0.01, and ***P < 0.001 compared to saline-treated group, unpaired t test.

Fig. 6. The synaptic and behavioral actions of ket are mimicked and occluded by HCN channel inhibition or deletion

(A) Effect of HCN inhibition by ZD on the peak amplitude of SC-CA1 EPSCs and the ability of ket-induced potentiation. Left: Data were presented as the hippocampal slices were pretreated by picrotoxin (100 µM) and CGP (4 µM) for 30 min and then first bath-applied with ZD (15 µM) followed by combined ket (20 µM) treatment (ZD: 155.6 ± 9.5% of baseline, n = 7 cells, P < 0.05, paired t test). Right: Representative traces. (1) Baseline treated with picrotoxin and CGP, (2) after ZD application, and (3), after ket plus ZD application; merged traces of 1, 2, and 3. Scale bar, 50 ms/50 pA. SC-CA1 EPSCs were recorded in the presence of picrotoxin and CGP to prevent any effects of GABAergic input. (B) Effect of HCN inhibition by ZD on PPR of SC-CA1 EPSCs. Rat hippocampal slices were recorded in the presence of picrotoxin (100 µM) and CGP (4 µM) to prevent any effects of GABAergic input. Left: Representative data from a single-cell recording are presented as peak amplitude or PPR of SC-CA1 EPSCs normalized to the baseline before ZD (15 µM) application. Middle: Representative traces before and after ZD application as well as merged and normalized (to before ZD) traces. Scale bar, 50 ms/50 pA. Right: Quantified data from seven cells show that ket significantly reduced PPR of SC-CA1 EPSCs (75.3 ± 5.9% of baseline at 50 to 60 min after ZD; ***P < 0.001, paired t test). (C) Effect of HCN inhibition by zatebradine (10 µM) on SC-CA1 EPSCs. EPSCs were recorded, and data are presented as in (A) (zatebradine: 576.9 ± 87.2% of baseline, n = 3 cells; P < 0.05, paired t test). Scale bar, 50 ms/50 pA. (D) Effect of ZD on GluA1 Ser⁸⁴⁵ phosphorylation (pGluR1) and abundance in the presence and absence of ket. Representative blots (left) and quantified data (right) of five independent Western blot experiment show that ZD occluded ket-induced enhancement of GluA1 phosphorylation and abundance. *P < 0.05 compared with control, Tukey’s post hoc tests. (E) Effect of ket on SC-CA1 fEPSPs in HCN1-KO mice. Ket failed to enhance SC-CA1 fEPSPs in hippocampal slices from HCN1-KO mice (104.1 ± 5.5% of baseline, n = 10 slices from five mice; P = 0.3611, paired t test) but significantly enhanced SC-CA1 fEPSPs in slices from wild-type mice (148 ± 8.7% of baseline, n = 12 slices from five mice; P = 0.001, paired t test). Left: Time course of SC-CA1 fEPSPs slop. Right: Representative traces. Scale bar, 10 ms/0.1 mV. (F) Effect of ket in three behavioral tests in wild-type and HCN1-KO mice. Animals were assessed using the sucrose preference test (top), the novelty-suppressed feeding test (middle), and the FST (bottom). *P < 0.05; n = 6 mice in each saline-treated and ket-treated group. (G) Effect of HCN inhibition by ZD dialyzed into CA1 cells on SC-CA1 EPSCs and I_h. ZD (15 µM) was dialyzed through the patch pipette into the CA1 neurons, and SC-CA1 EPSCs were recorded. Amplitude and decay times are shown normalized to the values at the beginning when the cell was impaled by the pipette. Left: Time courses of peak amplitude, decay time of SC-CA1 EPSCs, and peak amplitude of h current during ZD dialysis. Middle: Representative traces of SC-CA1 EPSCs 0, 20, and 50 min after implement. Scale bar, 50 ms/100 pA. Right: Representative h currents measured by comparing the difference between the instantaneous (peak) tail current amplitude and the steady-state current amplitude. Scale bar, 500 ms/300 pA. (H) Effect of ket on SC-CA1 EPSCs after HCN inhibition by ZD dialysis into CA1 cells. CA1 neurons were patched with pipette containing with ZD (15 µM) for 1 hour, then ket (20 µM) was applied to the bath solution, and SC-EPSCs were recorded. Ket significantly increased the peak amplitude of SC-CA1 EPSCs (156.1 ± 17.2% of baseline 50 to 60 min after ket, n = 6 slices; P < 0.01, paired t test).