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GluN2A mediates ketamine-induced rapid antidepressant-like responses

Nature Neuroscience volume 26, pages 1751–1761 (2023)Cite this article

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Abstract

Ketamine was thought to induce rapid antidepressant responses by inhibiting GluN2B-containing N-methyl-d-aspartic acid (NMDA) receptors (NMDARs), which presents a promising opportunity to develop better antidepressants. However, adverse side effects limit the broader application of ketamine and GluN2B inhibitors are yet to be approved for clinical use. It is unclear whether ketamine acts solely through GluN2B-dependent mechanisms. The present study reports that the loss of another major NMDAR subunit, GluN2A, in adult mouse brains elicits robust antidepressant-like responses with limited impact on the behaviors that mimic the psychomimetic effects of ketamine. The antidepressant-like behavioral effects of broad NMDAR channel blockers, such as ketamine and MK-801 (dizocilpine), were mediated by the suppression of GluN2A, but not by the inhibition of GluN2B. Moreover, treatment with ketamine or MK-801 rapidly increased the intrinsic excitability of hippocampal principal neurons through GluN2A, but not GluN2B. Together, these findings indicate that GluN2A mediates ketamine-triggered rapid antidepressant-like responses.

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Fig. 1: MK-801-induced fast antidepressant and anxiolytic effects, not hyperlocomotion, were occluded in GluN2A−/− mice.
Fig. 2: GluN2A removal in adult mouse brains induced antidepressant-like behaviors but not anxiolytic or hyperlocomotive behaviors.
Fig. 3: KO of GluN2A in excitatory neurons, not in inhibitory neurons, led to antidepressant, anxiolytic-like and hyperlocomotive behaviors in mice.
Fig. 4: KD of hippocampal GluN2A promoted antidepressant-like behaviors.
Fig. 5: GluN2A did not regulate the excitatory and inhibitory synaptic transmission of CA1 pyramidal neurons.
Fig. 6: Suppression of GluN2A elevated intrinsic excitability of CA1 pyramidal neurons in a cell autonomous manner.
Fig. 7: Acute treatment with MK-801 or ketamine increased excitability of CA1 pyramidal neurons in WT, but not in GluN2A−/−, mice.

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Data availability

The datasets generated during and analyzed during the present study are available from the corresponding author on reasonable request. Source data are provided with this paper.

Code availability

The present study did not use any customized code or mathematical algorithm.

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Acknowledgements

We thank S. Nakanishi (Osaka Bioscience Institute and Kyoto University, Japan) for providing the Grin2a−/− mice (by the RIKEN BRC through the National Bio-Resource Project of MEXT, Japan). We thank K. He (Interdisciplinary Research Center on Biology and Chemistry, Chinese Academy of Sciences, China), J. Hu (ShanghaiTech University, China), Z. Qiu (Institute of Neuroscience, Chinese Academy of Sciences, China) and X. Xu (Institute of Neuroscience, Chinese Academy of Sciences, China) for helping us to acquire the genetically engineered tool mice. We thank the staff members of the Animal Facility at the National Facility for Protein Science in Shanghai, Shanghai Advanced Research Institute and the Chinese Academy of Sciences for providing assistance in mouse breeding and maintenance. We thank M. Sheng (Broad Institute and Massachusetts Institute of Technology, USA) and J. Yuan (Interdisciplinary Research Center on Biology and Chemistry, Chinese Academy of Sciences, China) for helping with the manuscript preparation. The present study was supported by the following agencies: Shanghai Municipal Science and Technology Major Project to Y.C. (grant no. 2019SHZDZX02) and Natural Science Foundation of Shanghai to Y.G. (grant nos. 19ZR1468600 and 201409003800).

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  1. These authors contributed equally: Tonghui Su, Yi Lu, Chaoying Fu.

Authors and Affiliations

  1. Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China

    Tonghui Su, Yi Lu, Chaoying Fu, Yang Geng & Yelin Chen

  2. University of Chinese Academy of Sciences, Beijing, China

    Tonghui Su & Yi Lu

  3. Synphatec (Shanghai) Biopharmaceutical Technology Co., Ltd, Shanghai, China

    Tonghui Su & Yang Geng

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Contributions

Y.C. and Y.G. conceived the idea and designed the study. T.S., Y.L. and C.F. performed the experiments and statistical analysis, and wrote the manuscript under the supervision of Y.C. and Y.G.

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Correspondence to Yang Geng or Yelin Chen.

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Y.C. is a visiting professor of Shanghai Jiao Tong University and a founder of Synphatec (Shanghai) Biopharmaceutical Technology Co., Ltd. Y.C., Y.G., T.S., Y.L. and C.F. are inventors on China patent application (no. 202110901806.8) held by Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences.

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Extended data

Extended Data Fig. 1 MK-801-induced fast antidepressant and anxiolytic effects, not hyperlocomotion, were occluded in GluN2A−/− mice, related to Fig. 1.

a and b, GluN2A expression was evaluated with FISH (a) and western blot (b) in cortex or hippocampus from WT and GluN2A−/− mice. Scale bar showed 200 μm. Samples of (b) derive from the same experiment and that gels/blots were processed in parallel. c–f, Measurements of GluN2A−/− mice and their WT littermates with OFT (c, p = 0.00020, d, p = 0.0040, WT n = 16, GluN2A−/− n = 14) and NSFT (e, p = 0.0031, f, WT n = 10, GluN2A−/− n = 11). g, Escape latency of WT and GluN2A−/− mice in learned helplessness test (LH) (WT-Sham n = 7, WT-IES n = 20, p = 0.0013; GluN2A−/−-Sham n = 10, GluN2A−/−-IES n = 19, p < 0.0001). h–j, Effects of MK-801 treatment on WT and GluN2A−/− mice were evaluated with OFT (WT-Sal n = 6, WT-MK-801 n = 8, GluN2A−/−-Sal n = 8, GluN2A−/−-MK-801 n = 9). h, WT-Sal v.s. WT-MK-801 p = 0.00040; WT-Sal v.s. GluN2A−/−-Sal p = 0.0080; GluN2A−/−-Sal v.s. GluN2A−/−- MK-801 p = 0.0010; i, WT-Sal v.s. WT-MK-801 p < 0.0001; WT-Sal v.s. GluN2A−/−-Sal p = 0.0080; GluN2A−/−-Sal v.s. GluN2A−/−-MK-801 p = 0.0019; j, WT-Sal v.s. WT-MK-801 p = 0.00010; WT-Sal v.s. GluN2A−/−-Sal p < 0.0001. k, Inhibition of GluN2A-containing NMDAR-mediated currents by ketamine, (2R, 6R)-HNK and LY341495. IComp/IGlu represents the normalized remaining currents after co-perfusion with each compound. Veh v.s. Ket p < 0.0001, one-way ANOVA. l, Schematic diagram showing the experimental timeline. m, Quantitation of the immobility time from WT and GluN2A−/− in TST after 7 days fluoxetine (Flx) treatment (WT-Sal n = 10, WT-Flx n = 12, p = 0.0046; GluN2A−/−-Sal n = 9, GluN2A−/−-Flx n = 12, p = 0.030; WT-Sal v.s. GluN2A−/−-Sal p = 0.0039). Error bars show SEM. Two-way ANOVA (g, h, i, j, m) and Student’s t test (two-tailed) (c–f). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Source data

Extended Data Fig. 2 Validation of adult age-specific induced GluN2A knockout in mice, related to Fig. 2.

a, Body weight of UBC-CreERT/Grin2aflox/flox mice treated with TAM (n = 22) or Veh (n = 20). b–d, GluN2A expression level was measured from hippocampus of UBC-CreERT/Grin2aflox/flox mice after treated with TAM or Veh (b, c, western blot, Veh n = 7, TAM n = 6) (d, qRT-PCR, Veh n = 5, TAM n = 5). c, p < 0.0001; d, p < 0.0001; e–i, Evaluation of Grin2aflox/flox (2A flox) and UBC-CreERT/Grin2aflox/flox (UBC-2A flox) mice with OFT (e), EPM (f, g), TST (h, p = 0.011) or FST (i, p = 0.025). The behavioral analysis was done 1 month after treatment with TAM (2A flox n = 8, UBC-2A flox n = 9). j and k, Verification of GluN2A knockout in UBC-CreERT/Grin2aflox/flox mice treated with TAM after inescapable footshocks training in LH test presented in Fig. 2i–n (Veh n = 5, TAM n = 6). k, p < 0.0001. Samples of (b)/(j) derive from the same experiment and that gels/blots were processed in parallel. Error bars show SEM. One-sample t test (c, k), two-way ANOVA (e–g) and Student’s t test (two-tailed) (d, h, i). * p < 0.05, **** p < 0.0001.

Source data

Extended Data Fig. 3 Validation of excitatory-neuron specific GluN2A conditional knockout mice (Nex-2A cKO), related to Fig. 3.

a, Greyscale images showing tdTomato signal in sagittal brain sections of Nex-Cre/Ai9 mice. C-cortex, H-hippocampus, S-striatum, T-thalamus, A-amygdala, M-midbrain. Scale bars showed 500 μm (top) and 200 μm (bottom) respectively. b and c, Representative greyscale images of Grin2a FISH in hippocampus (b) or cortex (c) of WT and Nex-2A cKO mice. Scale bar showed 50 μm in b and 100 μm in c. d and e, Western blot analysis of GluN2A in hippocampus (d) and cortex (e) of Nex-2A WT and cKO mice. Samples of (d)/(e) derive from the same experiment and that gels/blots were processed in parallel. f and g, Quantification of GluN2A expression level by western blot (f) and RT-PCR (g) in hippocampus or cortex of Nex-2A WT and cKO mice (f, Hipp: Nex-2A WT n = 4, cKO n = 5, p < 0.0001; Ctx: Nex-2A WT n = 4, cKO n = 3, p = 0.010), (g, Hipp: Nex-2A WT n = 4, cKO n = 3, p = 0.0045; Ctx: Nex-2A WT n = 4, cKO n = 3, p = 0.00020). One-sample t test (f) and Student’s t test (two-tailed) (g). Error bars show SEM. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Source data

Extended Data Fig. 4 Validation of inhibitory-neuron specific GluN2A conditional knockout mice (vGAT-2A cKO), related to Fig. 3.

a, Greyscale images showing tdTomato signal in sagittal brain sections from vGAT-Cre/Ai9 mice. C-cortex, H-hippocampus, S-striatum, T-thalamus, A-amygdala, M-midbrain. Scale bars showed 500 μm (top) and 200 μm (bottom), respectively. b and c, Western blot analysis of GluN2A expression level in hippocampus of vGAT-2A cKO or WT mice, quantified in b (vGAT-2A WT n = 3, vGAT-2A cKO n = 3). Samples of (b) derive from the same experiment and that gels/blots were processed in parallel. d, Quantification of GluN2A mRNA level in hippocampus of vGAT-2A cKO or WT mice (vGAT-2A WT n = 4, vGAT-2A cKO n = 6). e–g, Grin2a FISH and fluorescent staining of GAD1 in hippocampus of vGAT-2A cKO or WT mice (e; yellow arrows indicated the GAD1 positive cells that co-expressed with GluN2A; cyan arrowhead indicated GAD1 positive cells without GluN2A signal). f, Area of stratum radiatum (SR) and stratum lacunosum & moleculare (SLM). g, Density of GluN2A positive cells in SR and SLM (vGAT-2A WT n = 4, vGAT-2A cKO n = 4; p = 0.0024,). Scale bar showed 200 μm. h, Representative traces of evoked NMDAR-mediated EPSCs from CA1 pyramidal neurons or interneurons in WT or vGAT-2A cKO mice after incubated with Veh (black) or GluN2B selective inhibitor Ro-25 6981 (blue). i and j, Quantitation of the decay time (i) or +Ro25/-Ro25 ratio of the amplitudes of evoked NMDAR-EPSCs (j) (Pyr: pyramidal neurons, vGAT-2A WT n = 3, vGAT-2A cKO n = 5; INs: interneurons, vGAT-2A WT n = 5, vGAT-2A cKO n = 5;). i, INs: p = 0.018; j, INs: p = 0.012. Error bars show SEM. One-sample t test (c, d), two-way ANOVA (f, i, j) and Student’s t test (two-tailed) (g).* p < 0.05, ** p < 0.01.

Source data

Extended Data Fig. 5 Hippocampal interneurons of GluN2A−/− mice had normal excitability, related to Fig. 6.

a–j, Representative current-clamp recording traces after injection of a 330 pA depolarizing current (a, f), quantitation of AP number (b, g), RMP (c, h), sample traces after step hyperpolarizing current injections (d, i), current-voltage curves and RIn (e, j and inserts) from pyramidal neurons of mPFC (a–e) or visual cortex (f–j) of WT (grey) or GluN2A−/− (red) mice. (mPFC: WT n = 9, GluN2A−/− n = 16; visual cortex: WT n = 13, GluN2A−/− n = 9). b, p(130 pA) = 0.012, p(150 pA) = 0.018; e, p = 0.031, p(RIn) = 0.031. k–p, Representative current-clamp recording traces after injection of a 330 (k) or 210 (n) pA depolarizing current (k, n), quantitation of AP number after depolarizing current injections (l, o), RMP (m, p) from non-fast spiking (k–m) or fast-spiking (n–p) interneurons from CA1 of WT (grey) or GluN2A−/− (red) mice. (Non-fast, WT n = 12, GluN2A−/− n = 7; Fast, WT n = 13, GluN2A−/− n = 14). 6–8 weeks old mice were used for electrophysiological recordings. Error bars show SEM. Two-way ANOVA (b, e, g, j, l, o) or Student’s t test (two-tailed) (c, e (insert), h, j (insert), m, p). * p < 0.05.

Extended Data Fig. 6 Cell type specific removal of GluN2A did not alter excitatory or inhibitory synaptic transmission of CA1 pyramidal neurons, related to Fig. 6.

a–f, Representative traces (a, d), average individual events (b, e), quantitation of cumulative probability and average amplitude of events (c, f) of mIPSCs and sIPSCs recorded from 4–6 weeks old Nex-2A WT (black) or Nex-2A cKO (olive) mice (mIPSCs: Nex-2A WT n = 12, Nex-2A cKO n = 14; sIPSCs: Nex-2A WT n = 16, Nex-2A cKO n = 14). g–r, Representative traces, average individual events, quantitation of cumulative probability and average amplitude of events of mIPSCs (g–i), sIPSCs (j–l), mEPSCs (m–o) and sEPSCs (p–r) recorded from vGAT-2A WT (black) or vGAT-2A cKO (purple) mice. (mIPSCs: vGAT-2A WT n = 18, vGAT-2A cKO n = 21; sIPSCs: vGAT-2A WT n = 15, vGAT-2A cKO n = 13; mEPSCs: vGAT-2A WT n = 13, vGAT-2A cKO n = 10; sEPSCs: vGAT-2A WT n = 12, vGAT-2A cKO n = 16) 6–8 weeks mcie were used for electrophysiological recordings. Error bars showed SEM. Cumulative frequency was analyzed with Kolmogorov-Smirnov test. Peak amplitudes were analyzed with Student’s t test (two-tailed).

Extended Data Fig. 7 GluN2B was not responsible to the effects of MK-801 in intrinsic excitability and antidepressant-like behaviors, related to Fig. 6.

a, Representative recording traces after injection of 330 pA depolarizing current in CA1 pyramidal neurons treated with Veh or 3 μM Ro-25 6981 for 1–2 hours. b and c, Quantitation of the number of action potentials (b) after injection of depolarizing currents and RMP (c) recorded from neurons after Veh or Ro-25 6981 treatments (Veh n = 18, Ro 25-6981 n = 19). d–i, number of AP (e, p = 0.0074), RMP (f, p = 0.041), current-voltage curve (h, p = 0.00070), input resistance (RIn) (i, p = 0.00080) and sample trace recorded from CA1 pyramidal neurons after incubated with ‘Ro 25-6981 + Veh’ (n = 10) or ‘Ro 25-6981 + 10 μM MK-801’ (n = 10) for 1–2 hours from WT mice (g). j and k, Effects of MK-801 on WT mice-treated with GluN2B inhibitor Ro 25-6981 (10 mpk) were evaluated with TST (j; Ro 25-6981 + Sal n = 10; Ro 25-6981 + MK-801 n = 10, p < 0.0001) and FST (k; Ro 25-6981 + Sal n = 10; Ro 25-6981 + MK-801 n = 9, p < 0.0001). All the recordings were measured from CA1 pyramidal neurons acutely prepared from 6-8 weeks old WT mice. Error bars show SEM. Two-way ANOVA (b, e, h) or Student’s t test (two-tailed) (c, f, i, j, k). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Extended Data Fig. 8 Sag potentials were not altered in CA1 pyramidal neurons from GluN2A−/− mice.

a, Representative voltage sag were generated by hyperpolarized current of −100 pA. b, Normalized voltage sag of WT and GluN2A−/−, which were measured at the last 50-ms of 1-s stimulation (WT n = 14, GluN2A−/− n = 22). Error bars show SEM. Two-way ANOVA.

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Su, T., Lu, Y., Fu, C. et al. GluN2A mediates ketamine-induced rapid antidepressant-like responses. Nat Neurosci 26, 1751–1761 (2023). https://doi.org/10.1038/s41593-023-01436-y

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