Inhibition of SERT and NMDAR synergistically confers rapid antidepressant effects of ketamine

Abstract

While N-methyl-d-aspartate receptor (NMDAR) blockade is crucial for the rapid antidepressant effects of ketamine, the involvement of other mechanisms remains contentious, particularly regarding the role of serotonin, a key neurotransmitter in the target of traditional antidepressants. Here, we demonstrate that ketamine elevates serotonin levels by inhibiting the serotonin transporter (SERT). A cryogenic electron microscopy structure of ketamine-bound SERT in the outward-open conformation, resolved at 3.2 Å, indicates that ketamine binds to the central site of SERT. Elevated serotonin, along with NMDAR inhibition, induces ketamine-like rapid antidepressant effects. This increase in serotonin leads to the activation of vasoactive intestinal peptide (VIP)-expressing interneurons, which are essential for the rapid antidepressant effects of ketamine. Inhibition of VIP neurons blocks these effects and ketamine-like effects, highlighting a crucial cell type-specific mechanism. These findings identify a critical pathway in the rapid antidepressant actions of ketamine and offer potential pharmacological strategies for developing rapidly acting antidepressants.

Keywords: NMDAR; SERT; ketamine; rapid antidepressant effects; vasoactive intestinal peptide-expressing interneurons.

Figure 1.

Ketamine inhibits SERT to elevate 5-HT levels. (A) Schematic of LC-MS/MS after intraperitoneal (i.p.) injection of ketamine. (B) Brain concentrations of ketamine after a single i.p. injection of ketamine in mice, as measured by LC-MS/MS (four mice per group). (C) In vivo real-time microdialysis setup and experimental procedure. HPLC, high-performance liquid chromatography. (D) Concentration of mPFC 5-HT before and after drug injection (saline: six mice; ketamine: six mice). Ketamine (i.p., 10 mg/kg), Memantine (i.p., 10 mg/kg). Two-way ANOVA with Bonferroni's multiple comparisons test. (E) Workflow of the dynamic PET imaging (0–90 min) study with 4-[¹⁸F]-ADAM. (F) Representative PET images of a mouse brain after saline and ketamine injection. SUVR, standard uptake value ratio. (G) Quantification of 4-[¹⁸F]-ADAM SUVR for the mPFC relative to the cerebellum (saline: five mice; ketamine: five mice). Multiple unpaired t-tests. (H) BP_nd values after treatment with saline or ketamine in the mPFC. Two-tailed unpaired t-test. (I) Experimental procedure for the radioligand binding assay. (J) Dose-dependent inhibition of [³H]-imipramine radioligand binding to the cell membrane of human cells that express human SERT separated from the cytoplasm by ketamine in the radioligand binding assay. (K) 5-HT transporter uptake experimental procedure. Excitation wavelength: 450 nm; emission wavelength: 515 nm. (L) The dose-dependent inhibition of 5-HT fluorophore binding to SERT by ketamine in the 5-HT transporter uptake experiment. Data are shown as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (see Table S3 for statistical analyses and n numbers).

Figure 2.

Ketamine binds to the central pocket of hSERT as determined by cryo-EM. (A) Cryo-EM map of ketamine-bound hSERT. (B) Overall structure of hSERT in complex with ketamine. N-acetyl glucosamine (NAG; hSERT), N-dodecyl-β-d-maltopyranoside (DDM), cholesteryl hemisuccinate (CHS), representative lipids, sodium and chloride ions, and transmembrane helices (TM; 1–12) are labeled. (C) Chemical structure of ketamine. (D) Ketamine (pink stick model) binds to the central pocket of hSERT. (E) Cryo-EM density (mesh) of ketamine (pink stick) and the surrounding TM helices (blue ribbon). (F) Interaction of ketamine in the central pocket. The key residues are shown as sticks. Sodium and chloride ions are shown as balls. (G) LigPlot+ analysis of hSERT^ketamine.

Figure 3.

Dual inhibition of SERT and NMDAR induces ketamine-like rapid antidepressant effects. (A) Our proposed mechanism for the dual inhibition of SERT and NMDAR by ketamine. (B) Seven different treatment groups and doses of drugs. CRS, chronic restraint stress. (C–F) Mice in the indicated treatment groups were assessed for sucrose preference with the SPT (C), duration of immobility during the TST (D), total distance traveled (E) and time spent in the center zone (F) during the OFT (10 mice per treatment group). One-way ANOVA with Bonferroni's multiple comparisons test. (G) The site of cannula implantation and drug injection. (H) Three treatment groups and doses of drugs. (I–L) Mice in the indicated treatment groups were assessed for sucrose preference with the SPT (I), duration of immobility during the TST (J), total distance traveled (K) and time spent in the center zone (L) during the OFT (11 mice per treatment group). Two-way ANOVA with Bonferroni's multiple comparisons test. Data are shown as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS, not significant (see Table S3 for statistical analyses and n numbers).

Figure 4.

Ketamine activates VIP neurons in the mPFC, which requires 5-HT and requires NMDAR inhibition. (A) Experimental procedure and timeline for determining neuronal activation after ketamine administration in a VIP-Cre::Ai47 mouse. (B) Immunohistochemical staining of c-Fos-expressing neurons in the mPFC at 1.5 h after injection of saline or ketamine. Yellow arrowheads indicate the overlap among c-Fos (red), DAPI (blue) and Ai47 (green, VIP neurons) expression. Scale bar, 50 μm (left), 20 μm (right). DAPI, 4′,6-diamidino-2-phenylindole. (C) Quantitative analysis of c-Fos+ cells among VIP neurons (overlap) in the mPFC (five mice per group). Sal, saline; Ket, ketamine. Two-tailed unpaired t-test. (D) Schematic of a brain slice of a VIP-Cre::Ai47 mouse showing the site of injection of ketamine or saline and patch-clamp recording of VIP neurons in the mPFC. (E) Representative traces showing spontaneous activity of VIP neurons 1 h after injection of saline, ketamine (10 mg/kg) or p-CPA (150 mg/kg, once daily for 3 days) with ketamine (10 mg/kg) at the indicated timepoints (four mice per group). p-CPA, DL-4-chlorophenylalanine. (F) Pie charts illustrating the % abundance of silent and firing VIP neurons in (E). Chi-squared test. (G) Quantification of firing frequency (left) and neuronal RMP (right) of neurons as described in (F). RMP, resting membrane potential. Two-tailed unpaired t-test. (H) Sample traces of neurons as described in (G). (I) Spike number measurements of neurons as described in (G). Two-way ANOVA with Bonferroni's multiple comparisons test. (J) Representative traces showing spontaneous activity of silent and firing VIP neurons 1 h after AP5 (40 nmol) with saline or AP5 (40 nmol) with fluoxetine (10 mg/kg) or saline with fluoxetine (10 mg/kg) injection. (K) Pie charts illustrate the % abundance of silent and firing VIP neurons in (J). Chi-square test. (L) Quantification of firing frequency (left) and neuronal RMP (right) of neurons as described in (K). Two-tailed unpaired t-test. (M and N) Example traces (left; the sample represents 2 min before drug application, 3 min during drug application and 3 min after drug application) and statistics (right) showing the effects of 5-HT (M) and AP5 (N) on mPFC VIP neurons in brain slices from normal mice [13 neurons, 4 mice in (M); 10 neurons, 3 mice in (N)]. Two-tailed unpaired t-test. (O) Example trace (sample represents 1 min before drug application, 2 min and 3 min during drug application, and 1.5 min and 2.5 min after drug application) showing the effects of AP5 with 5-HT on mPFC VIP neurons in brain slices from normal mice. (P) Analysis of firing frequency (left) and neuronal RMP (right) of neurons as described in (O) (seven neurons, three mice). Two-tailed unpaired t-test. (Q) Example trace (sample represents 1 min before and 2 min and 3 min during drug application and 1.5 min and 2.5 min after drug application) showing the effects of AP5 with 5-HT before and after palon application on mPFC VIP neurons in brain slices from normal mice. Palon, palonosetron. (R) Analysis of firing frequency (left) of neurons as described in (Q) (nine neurons, three mice). One-way ANOVA with Bonferroni's multiple comparisons test (R). Data are shown as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS, not significant (see Table S3 for statistical analyses and n numbers).

Figure 5.

Activation of mPFC VIP neurons is necessary for the rapid antidepressant effects. (A) Experimental procedure and timeline. Mice were pre-treated with either saline (Pre-saline) or CNO (Pre-CNO) 1 h before injection with saline or ketamine. (B) Chemogenetic manipulation (left) and example site of hM4Di expression (right) in the mPFC of VIP-Cre mice. Scale bar, 200 μm. (C) A representative trace showing action potential inhibition after CNO application during cell-attached recording in mPFC VIP neurons. (D–K) hM4Di and mCherry mice pre-treated with either saline (D–G) or CNO (H–K) were then injected with either saline or ketamine and were later assessed for the duration of immobility during the TST (D and H) and the FST (E and I) and total distance traveled (F and J) and time spent in the center zone (G and K) during the OFT. Sal, saline; Ket, ketamine; fmi, forceps minor of the corpus callosum. Two-way ANOVA with Bonferroni's multiple comparisons test. (L) Three different treatment groups and doses of drugs. (M–P) hM4Di mice pre-treated with either saline or CNO were then injected with one of three treatments and were later assessed for the duration of immobility during the TST (M) and the FST (N) and the total distance traveled (O) and center zone time (P) during the OFT (10 mice per group). Two-tailed unpaired t-test. Data are shown as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS, not significant (see Table S3 for statistical analyses and n numbers).