Abstract
Effective pharmacotherapy for major depressive disorder remains a major challenge, as more than 30% of patients are resistant to the first line of treatment (selective serotonin reuptake inhibitors)1. Sub-anaesthetic doses of ketamine, a non-competitive N-methyl-d-aspartate receptor antagonist2,3, provide rapid and long-lasting antidepressant effects in these patients4,5,6, but the molecular mechanism of these effects remains unclear7,8. Ketamine has been proposed to exert its antidepressant effects through its metabolite (2R,6R)-hydroxynorketamine ((2R,6R)-HNK)9. The antidepressant effects of ketamine and (2R,6R)-HNK in rodents require activation of the mTORC1 kinase10,11. mTORC1 controls various neuronal functions12, particularly through cap-dependent initiation of mRNA translation via the phosphorylation and inactivation of eukaryotic initiation factor 4E-binding proteins (4E-BPs)13. Here we show that 4E-BP1 and 4E-BP2 are key effectors of the antidepressant activity of ketamine and (2R,6R)-HNK, and that ketamine-induced hippocampal synaptic plasticity depends on 4E-BP2 and, to a lesser extent, 4E-BP1. It has been hypothesized that ketamine activates mTORC1–4E-BP signalling in pyramidal excitatory cells of the cortex8,14. To test this hypothesis, we studied the behavioural response to ketamine and (2R,6R)-HNK in mice lacking 4E-BPs in either excitatory or inhibitory neurons. The antidepressant activity of the drugs is mediated by 4E-BP2 in excitatory neurons, and 4E-BP1 and 4E-BP2 in inhibitory neurons. Notably, genetic deletion of 4E-BP2 in inhibitory neurons induced a reduction in baseline immobility in the forced swim test, mimicking an antidepressant effect. Deletion of 4E-BP2 specifically in inhibitory neurons also prevented the ketamine-induced increase in hippocampal excitatory neurotransmission, and this effect concurred with the inability of ketamine to induce a long-lasting decrease in inhibitory neurotransmission. Overall, our data show that 4E-BPs are central to the antidepressant activity of ketamine.
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Data availability
All data generated and analysed during this study are included in this published Article and its Supplementary Information files. Source data are provided with this paper.
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Acknowledgements
We thank A. Sylvestre, A. Lafrance, I. Harvey and T. Degenhard for technical assistance. A.A.-V. was a recipient of FRQS (Fond Recherche Québec-Santé) and CIHR (Canadian Institutes for Health Research) postdoctoral fellowships. D.D.G. is a recipient of FRQS and CIHR postdoctoral fellowships. J.-C.L. is the recipient of the Canada Research Chair in Cellular and Molecular Neurophysiology. This work was supported by a CIHR Foundation grant to N. Sonenberg (FND-148423), and a CIHR project grant to J.-C.L. (PJT-153311).
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A.A.-V. designed the study, performed experiments, supervised students, analysed data and wrote the manuscript. D.D.G. performed behavioural experiments and data analyses. E.M.-C. performed western blot experiments, supervised students and edited the manuscript. M.J.E. performed electrophysiological experiments. A.K. performed electrophysiological experiments and data analyses. A.S. performed immunohistochemistry experiments and contributed to the writing of the manuscript. M.L.-C. and A.T.-B. contributed to behavioural tests. S.B. performed data analyses. G.M.R., S.S. and N. Salmaso contributed to experimental design, data analysis and manuscript editing. G.G., J.-C.L. and N. Sonenberg supervised students, designed experiments and contributed to writing the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Expression of 4E-BP1 and 4E-BP2 in the prefrontal cortex and hippocampus.
a, Representative western blot analysis (from 3 independent experiments) of 4E-BP1 and 4E-BP2 in the prefrontal cortex and hippocampus of wild-type (Eif4ebp1/2+/+ +/+), 4E-BP1 knock out (Eif4ebp1−/−), 4E-BP2 knock out (Eif4ebp2−/−), and 4E-BP1/2 double knock out mice (Eif4ebp1/2−/−) (uncropped blots are presented in Supplementary Information). b, c, Representative confocal images of immunostaining for 4E-BP2 (green), CAMK2α (red)/GAD67 (magenta) in the hippocampus. The arrows indicate area showed in the insert, which is a higher magnification of the images. Insert scale bar: 10 μm. Brain sections were additionally counterstained with DAPI (blue). d, Representative confocal images of immunostaining for 4E-BP1 (green), GFAP (red) in the hippocampus. Brain sections were additionally counterstained with DAPI (blue). The white box indicates area shown in e. f, Representative confocal image of immunostaining for 4E-BP1 (green) and CD11b (magenta). Brain sections were additionally counterstained with DAPI (blue). All immunofluorescence experiments were repeated in 3 independent experiments.
Extended Data Fig. 2 4E-BP1 and 4E-BP2 are required for the antidepressant effect of ketamine.
a, A group of Eif4ebp1/2+/+, Eif4ebp1−/−, and Eif4ebp2−/− mice were treated with saline or ketamine (IP, 10 mg kg−1) and tested 24 h after injection in the FST (2-way ANOVA and Fisher’s LSD, n = 11, 13, 11, 12, 4, 7 from left to right). b, Wild-type (Eif4ebp1/2+/+), Eif4ebp1−/− and Eif4ebp2−/− mice were treated with saline or ketamine (IP, 10 or 30 mg kg−1) and tested in the FST 6 days after treatment (2-way ANOVA and Fisher’s LSD, n = 13, 19, 7, 11, 10, 9, 16, 14, 6 from left to right). c, A different group of mice treated as in a was tested in the open field, and general activity was measured as distance travelled (2-way ANOVA, n = 8, 8, 8, 6, 6, 5, 6, 6, 7 from left to right). d, Latency to feed in the home cage was measured immediately after the NSF task in Eif4ebp1/2+/+ +/+, Eif4ebp1−/− and Eif4ebp2−/− mice, treated with saline or Ket (corresponding to Fig. 1b; 2-way ANOVA). e, Latency to feed in the home cage was also measured after the NSF task in the three mouse strains, following saline or (2R,6R)-HNK treatment (corresponding to Fig. 1c; 2-way ANOVA). f, In the same group of mice as in e (and Fig. 1c) average distance travelled during the NSF test was measured (expressed as an average per minute to correct for differential time spent in the arena; 2-way ANOVA). g, Mice of the three genotypes were also tested for immobility in the tail suspension test, 1 h after saline or Ket treatments (2-way ANOVA and Fisher’s LSD, n = 8, 9, 9, 10, 10, 7 from left to right). h, Eif4ebp1/2+/+ +/+, Eif4ebp1−/− and Eif4ebp2−/− mice were treated with fluoxetine (Fluox, IP, 3 mg kg−1, 0.5 h) and tested in the FST (2-way ANOVA and Fisher’s LSD, n = 8, 10, 9, 8, 6, 6 from left to right). Mean ± s.e.m.; * P < 0.05; ** P < 0.01 vs. Sal-treated mice of the same genotype.
Extended Data Fig. 3 Generation of Eif4ebp1 and Eif4ebp2 floxed genes in mice.
Schematic representation of the targeting construct used for the generation of Eif4ebp1 and Eif4ebp2 knockout mice. FRT (FLPase recognition target) and loxP sequences are indicated. Positive (PGK-neo) selection markers are indicated in the schemes on the Neomycin cassette (Neo, neomycin). Numbered grey boxes represent exons in the Eif4ebp1 and Eif4ebp2 genes. The Eif4ebp1 and Eif4ebp2 neomycin alleles were produced by homologous recombination. FLPase was used to generate the Eif4ebp1 and Eif4ebp2 floxed alleles. The genotyping PCR primers are indicated for each construct, as well as the length of the PCR product.
Extended Data Fig. 4 Effects of cell-specific mutation of Eif4ebp1 and Eif4ebp2 on home cage feeding, locomotion and forced swim test.
a, Latency to feed in the home cage was measured 1 h after saline or Ket (IP, 10 mg kg−1) treatments in control, mice lacking Eif4ebp1 in CAMK2α+ (Eif4ebp1Ex) or GAD2+ neurons (Eif4ebp1In), or mice mutant for Eif4ebp2 in excitatory (Eif4ebp2Ex) or inhibitory (Eif4ebp2In) neurons (corresponding to mice in Fig. 2i; 2-way ANOVA and Dunnett’s). b, In these same mice, distance travelled during the NSF task was measured. Data were expressed as average distance travelled per minute to account for differences in latency to feed (2-way ANOVA and Dunnett’s). c, Control, Eif4ebp1Ex, Eif4ebp1In, Eif4ebp2Ex and Eif4ebpIn mice were tested 6 days (144 h) after saline or Ket treatment (IP, 10 mg kg−1) in the FST (2-way ANOVA and Fisher’s LSD, n = 17, 15, 7, 10, 12, 17, 8, 9, 5, 7 from left to right). Mean ± s.e.m.; * P < 0.05 vs. control (main effect of genotype, in a) or vs. Sal-treated mice of the same genotype.
Extended Data Fig. 5 Effects of 4E-BP2 mutation in inhibitory neurons on amplitude of sEPSC and mIPSC.
a, Cumulative distribution of sEPSC amplitude in CA1 pyramidal neurons of control and Eif4ebp2In mice treated with saline or Ket (mice recorded in Fig. 3; Kolmogorov–Smirnov test). b, Amplitude of sEPSCs recorded from pyramidal neurons in CA1 of control and Eif4ebp2In mice (mice recorded in Fig. 3; 2-way ANOVA). c, Cumulative distribution of mIPSC amplitude in CA1 pyramidal neurons from mice treated as in a (mice recorded in Fig. 4; Kolmogorov–Smirnov test). d, Amplitude of mIPSC in CA1 pyramidal neurons in mice treated as in a (mice recorded in Fig. 4; 2-way ANOVA). Mean ± s.e.m.; * P < 0.05 vs. control-Sal.
Extended Data Fig. 6 The absence of 4E-BP2 in inhibitory neurons does not affect mEPSCs in pyramidal neurons.
a, Representative whole-cell recordings of mEPSCs in CA1 pyramidal neurons of mice treated with saline or Ket 24 h before (IP, 10 mg kg−1). Control and Eif4ebp2In mice were used. b, Cumulative inter-event interval (Kolmogorov–Smirnov test, n = 7, 11, 6, 17 from left from right) and c, frequency of mEPSCs measured in a (2-way ANOVA). d, Cumulative (Kolmogorov–Smirnov test) and e, average distribution of mEPSC amplitude measured in a (2-way ANOVA). Mean ± s.e.m.; * P < 0.05 vs. Eif4ebp2In-Sal.
Supplementary information
Supplementary Information
Uncropped Western blots from Extended Data Figure 1a. Uncropped images of the films for the Western blot analyses of 4E-BP1 and 4E-BP2 in the prefrontal cortex and hippocampus of wildtype (Eif4ebp1/2+/+ +/+), 4E-BP1 knock out (Eif4ebp1-/-), 4E-BP2 knock out (Eif4ebp2-/-), and 4E-BP1/2 double knock out mice (Eif4ebp1/2-/-). Images for 4E-BP1, 4E-BP2 and GAPDH are presented.
Supplementary Table 1
Details of statistical analyses.
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Aguilar-Valles, A., De Gregorio, D., Matta-Camacho, E. et al. Antidepressant actions of ketamine engage cell-specific translation via eIF4E. Nature 590, 315–319 (2021). https://doi.org/10.1038/s41586-020-03047-0
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DOI: https://doi.org/10.1038/s41586-020-03047-0
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