NMDAR inhibition-independent antidepressant actions of ketamine metabolites

Journal name:
Nature
Volume:
533,
Pages:
481–486
Date published:
DOI:
doi:10.1038/nature17998
Received
Accepted
Published online
Corrected online

Abstract

Major depressive disorder affects around 16 per cent of the world population at some point in their lives. Despite the availability of numerous monoaminergic-based antidepressants, most patients require several weeks, if not months, to respond to these treatments, and many patients never attain sustained remission of their symptoms. The non-competitive, glutamatergic NMDAR (N-methyl-d-aspartate receptor) antagonist (R,S)-ketamine exerts rapid and sustained antidepressant effects after a single dose in patients with depression, but its use is associated with undesirable side effects. Here we show that the metabolism of (R,S)-ketamine to (2S,6S;2R,6R)-hydroxynorketamine (HNK) is essential for its antidepressant effects, and that the (2R,6R)-HNK enantiomer exerts behavioural, electroencephalographic, electrophysiological and cellular antidepressant-related actions in mice. These antidepressant actions are independent of NMDAR inhibition but involve early and sustained activation of AMPARs (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors). We also establish that (2R,6R)-HNK lacks ketamine-related side effects. Our data implicate a novel mechanism underlying the antidepressant properties of (R,S)-ketamine and have relevance for the development of next-generation, rapid-acting antidepressants.

At a glance

Figures

  1. NMDAR inhibition is not sufficient for the antidepressant actions of ketamine.
    Figure 1: NMDAR inhibition is not sufficient for the antidepressant actions of ketamine.

    a, Antidepressant-like responses of (R,S)-ketamine (KET) and desipramine (DSP) in the forced-swim test (FST) 1- and 24-h after treatment. SAL, saline. bd, Compared to (S)-KET, (R)-KET showed greater and longer-lasting antidepressant-like effects in the FST (b), novelty-suppressed feeding (NSF) test (c) and learned helplessness test (d). eg, The alternative NMDAR antagonist MK-801 did not elicit 24-h antidepressant actions in the FST (e), and did not reverse social avoidance induced by chronic social defeat stress (f, g), where purple lines represent the video-tracked movements of mice (f). Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 (see Supplementary Table 1 for statistical analyses and n numbers).

  2. Metabolism of ketamine to (2R,6R)-HNK is necessary and sufficient to exert antidepressant actions.
    Figure 2: Metabolism of ketamine to (2R,6R)-HNK is necessary and sufficient to exert antidepressant actions.

    a, Simplified diagram of (R,S)-KET metabolism. be, Greater antidepressant-like actions of ketamine in female mice compared to males in the FST (b) are associated with higher brain levels of (2S,6S;2R,6R)-HNK (e), but not KET (c) or norketamine (norKET) (d). fh, Brain levels of KET (f), norKET (g) and (2S,6S;2R,6R)-HNK (h) after administration of (R,S)-KET and 6,6-dideuteroketamine ((R,S)-d2-KET). i, j, Effects of (R,S)-KET and (R,S)-d2-KET in the 1-h and 24-h FST (i) and the learned helplessness test (j). k, l, Compared to (2S,6S)-HNK, (2R,6R)-HNK manifested greater potency and longer-lasting antidepressant-like effects in the FST (k) and learned helplessness test (l). m, (2R,6R)-HNK reversed chronic social defeat-induced social interaction deficits. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 (see Supplementary Table 1 for statistical analyses and n numbers).

  3. Role of NMDA and AMPA glutamate receptors in the acute antidepressant effects of (2R,6R)-HNK.
    Figure 3: Role of NMDA and AMPA glutamate receptors in the acute antidepressant effects of (2R,6R)-HNK.

    a, (2R,6R)-HNK does not displace [3H]MK-801 binding. b, c, (R,S)-KET inhibited, but (2S,6S)-HNK and (2R,6R)-HNK did not inhibit currents evoked by application of NMDA to stratum radiatum interneurons in rat hippocampal slices (b), quantified as percentage inhibition (INMDA; c). Arrows indicate 30-s agonist pulse. ACSF, artificial cerebrospinal fluid. d, e, Normalized fEPSP slope (d) and amplitude (e) from stimulation of the Schaffer collateral pathway in rat hippocampal slices. f, Representative field-potential traces in the same hippocampal slice before (baseline) and 60 min after application of SAL or (2R,6R)-HNK. g, h, Pre-treatment with the AMPAR inhibitor NBQX 10 min before (R,S)-KET or (2R,6R)-HNK prevented their antidepressant-like actions in the 1-h (g) or 24-h (h) FST. i, Representative qEEG spectrograms for 10-min before (baseline) and 1-h after administration of (R,S)-ketamine or (2R,6R)-HNK (indicated by a dashed line). j, Normalized gamma power changes after administration of (R,S)-KET, (2R,6R)-HNK or vehicle (SAL). Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001; in j, * denotes (R,S)-KET, # denotes (2R,6R)-HNK (see Supplementary Table 1 for statistical analyses and n numbers).

  4. Role of AMPARs in the sustained antidepressant effects of (2R,6R)-HNK.
    Figure 4: Role of AMPARs in the sustained antidepressant effects of (2R,6R)-HNK.

    ah, Protein and protein phosphorylation levels from hippocampal synaptoneurosomes. a, b, A single administration of (R,S)-KET or (2R,6R)-HNK decreased phosphorylation of eEF2 (p-eEF2), 1 h (a) and 24 h (b) after injection. c, d, Although administration of (2R,6R)-HNK or (R,S)-KET did not alter the levels of proBDNF or mature BDNF (mBDNF) 1 h after injection (c), it increased mBDNF levels 24 h after treatment (d). e, f, (R,S)-KET and (2R,6R)-HNK did not change GluA1 and GluA2 levels at 1 h after treatment (e), but did increase levels 24 h after injection (f). g, h, Administration of the AMPAR inhibitor NBQX 30 min before the 24-h FST prevented the antidepressant effects of both (R,S)-KET and (2R,6R)-HNK administered 23.5 h before NBQX. Data are mean ± s.e.m. Images cropped; see Supplementary Fig. 1 for complete blot images. GAPDH was used as a loading control. *P < 0.05, **P < 0.01, ***P < 0.001 (see Supplementary Table 1 for statistical analyses and n numbers).

  5. (2R,6R)-HNK lacks side effects of ketamine.
    Figure 5: (2R,6R)-HNK lacks side effects of ketamine.

    a, b, After recording baseline activity for 1 h, mice received drug (dashed line) and locomotor activity was monitored for 1 h. Administration of (2S,6S) HNK dose-dependently changed locomotor activity (a), whereas administration of (2R,6R)-HNK did not (b). c, d, (2S,6S)-HNK (c) but not (2R,6R)-HNK (d) induced motor in-coordination in the rotarod. eh, Unlike (R,S)-KET, (2R,6R)-HNK administration did not induce pre-pulse inhibition deficits (e), (R,S)-KET-associated discriminative stimulus (f, g), or self-administration (h). Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 (see Supplementary Table 1 for statistical analyses and n numbers).

  6. The metabolic transformations of ketamine in vivo.
    Extended Data Fig. 1: The metabolic transformations of ketamine in vivo.

    Ketamine is metabolised in vivo via P450 enzymatic transformations. i, (R,S)-KET is selectively demethylated to give (R,S)-norketamine (norKET). ii, NorKET can be then dehydrogenated to give (R,S)-dehydronorketamine (DHNK). iii, Alternatively, norKET can be hydroxylated to give the hydroxynorketamines (HNKs). iv, (R,S)-KET can also be hydroxylated at the 6 position to give either the E-6-hydroxyketamine ((2S,6R;2R,6S)-HK) or Z-6-hydroxyketamine ((2S,6S;2R,6R)-HK). v, Demethylation of (2S,6R;2R,6S)-HK yields the production of (2S,6R;2R,6S)-HNK. vi, Demethylation of (2S,6S;2R,6R)-HK further gives (2S,6S;2R,6R)-HNK.

  7. Circulating levels of ketamine and its metabolites following i.p. administration in mice.
    Extended Data Fig. 2: Circulating levels of ketamine and its metabolites following i.p. administration in mice.

    a, b, Plasma (a) and brain (b) levels of ketamine and its metabolites after administration of (R,S)-KET (10 mg kg−1) in mice. c, d, Brain levels of KET (c), norKET (d) and HNK (e) following administration of (S)- and (R)-KET. f, g, Chemical structure of (R,S)-6,6-dideuteroketamine ((R,S)-d2-KET) (f), which displaces [3H]MK-801 binding with a similar affinity to (R,S)-KET ((R,S)-KET: Ki = 799 nM; (R,S)-d2-KET: Ki = 883 nM) (g). See Supplementary Table 1 for statistical analyses and n numbers.

  8. Additional social defeat stress data.
    Extended Data Fig. 3: Additional social defeat stress data.

    a, Chronic social defeat stress and social interaction/avoidance test timeline. b, c, 24 h after administration, neither (R,S)-KET nor MK-801 affected locomotor activity (b) or total number of compartmental crosses in the social interaction apparatus (c). Data are mean ± s.e.m. ***P < 0.001. See Supplementary Table 1 for statistical analyses and n numbers.

  9. Locomotor effects of (R,S)-KET and (R,S)-d2-KET.
    Extended Data Fig. 4: Locomotor effects of (R,S)-KET and (R,S)-d2-KET.

    After recording baseline activity for 60 min, mice received drug (marked by a vertical dashed line) and locomotor activity was monitored for another 1 h. a, b, Administration of (R,S)-KET (10 mg kg−1), induced hyperlocomotor responses equally in both male and female mice. c, d, (R,S)-KET and (R,S)-d2-KET were equally potent in inducing a hyperlocomotor response at the dose of 10 mg kg−1. Data are mean ± s.e.m. *P < 0.05, **P < 0.01 (see Supplementary Table 1 for statistical analyses and n numbers).

  10. Acute and sustained antidepressant and anti-anhedonic effects of (2R,6R)- and (2S,6S)-HNK.
    Extended Data Fig. 5: Acute and sustained antidepressant and anti-anhedonic effects of (2R,6R)- and (2S,6S)-HNK.

    a, A single injection of (2R,6R)-HNK resulted in dose-dependent antidepressant-like responses in the learned helplessness test at the doses of 5–75 mg kg−1. b, A single injection of (2S,6S)- HNK induced antidepressant-like effects in the learned helplessness test at the dose of 75 mg kg−1. c, Administration of (2R,6R)-HNK induced dose-dependent antidepressant effects in the 1- and 24-h FST. d, Administration of (2S,6S)-HNK at the dose of 25 mg kg−1 induced antidepressant effects in the 1- and 24-h FST. e, Despite the greater antidepressant efficacy of (2R,6R)-HNK, administration of (2S,6S)-HNK (HNK) results in higher brain hydroxynorketamine levels compared to (2R,6R)-HNK. f, (2R,6R)-HNK manifested dose-dependent antidepressant-like effects in the NSF test. g, Similar to (R,S)-KET, the antidepressant-like effects of (2R,6R)-HNK in the FST persisted for at least 3 days after treatment. h, A single administration of (2R,6R)-HNK reversed chronic corticosterone-induced decreases in sucrose preference. i, A single administration of (2R,6R)-HNK reversed chronic corticosterone-induced decrease in female urine sniffing preference, specifically in mice that developed an anhedonic phenotype. Administration of (2R,6R)-HNK was not associated with changes in locomotor activity (j) or total compartmental crosses in the social interaction test after chronic social defeat stress (k). Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 (see Supplementary Table 1 for statistical analyses and n numbers).

  11. (2R,6R)-HNK rapidly increases the frequency and amplitude of AMPAR spontaneous excitatory postsynaptic currents in the hippocampus.
    Extended Data Fig. 6: (2R,6R)-HNK rapidly increases the frequency and amplitude of AMPAR spontaneous excitatory postsynaptic currents in the hippocampus.

    a, Representative traces of spontaneous excitatory postsynaptic currents (sEPSCs) mediated via AMPARs during baseline (before) and 20 min after drug administration. b, Example CA1 stratum radiatum interneuron recorded from a rat hippocampal slice. cj, Twenty-minute exposure of (2R,6R)-HNK (e, i), but not (R,S)-(KET) (c, g) or (2S,6S)-HNK (d, h), increased AMPA sEPSCs frequency and amplitude compared to baseline. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 (see Supplementary Table 1 for statistical analyses and n numbers). SLM, stratum lacunosum-moleculare; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum.

  12. Administration of the AMPAR antagonist, NBQX, prevents (2R,6R)-HNK-induced increases in gamma oscillations in vivo.
    Extended Data Fig. 7: Administration of the AMPAR antagonist, NBQX, prevents (2R,6R)-HNK-induced increases in gamma oscillations in vivo.

    a, Administration of (R,S)-KET, but not (2R,6R)- HNK, increased locomotor home-cage activity of mice. be, Neither (R,S)-KET nor (2R,6R)-HNK altered cortical alpha (b), beta (c), delta (d) or theta (e) oscillations in vivo. fk, Pre-treatment with the AMPAR antagonist, NBQX, did not change the locomotor activity (f), alpha (g), beta (h), delta (j) or theta (k) oscillations, but it prevented (2R,6R)-HNK-induced increases of gamma oscillations in vivo (i). Data are mean ± s.e.m. *P < 0.05, **P < 0.01 (see Supplementary Table 1 for statistical analyses and n numbers).

  13. Effects of (2R,6R)-hydroxynorketamine on protein and protein phosphorylation levels in synaptoneurosomes.
    Extended Data Fig. 8: Effects of (2R,6R)-hydroxynorketamine on protein and protein phosphorylation levels in synaptoneurosomes.

    a, b, A single administration of (R,S)-KET (10 mg kg−1) or (2R,6R)- HNK (10 mg kg−1), did not alter levels of mTOR or phosphorylated mTOR in the hippocampus 1 h (a) or 24 h (b) after injection. cj, Administration of (R,S)-KET or (2R,6R)-HNK did not alter levels of mTOR/phosphorylated mTOR (c, d), eEF2/phosphorylated eEF2 (e, f), proBDNF/mBDNF (g, h), or GluA1/GluA2 (i, j), in the prefrontal cortex of mice. The values for the phosphorylated forms of proteins were normalized to phosphorylation-independent levels of the same protein. Phosphorylation-independent levels of proteins were normalized to GAPDH. Data are mean ± s.e.m, and were normalized to the saline-treated control group for each protein. Images are cropped; see Supplementary Fig. 1 for complete blot images. *P < 0.05 (see Supplementary Table 1 for statistical analyses and n numbers).

  14. (2R,6R)-HNK administration does not alter startle amplitude, drug discrimination rate or self-administration drug intake.
    Extended Data Fig. 9: (2R,6R)-HNK administration does not alter startle amplitude, drug discrimination rate or self-administration drug intake.

    a, Startle amplitude as measured in the pre-pulse inhibition task was not affected by administration of (R,S)-KET or (2R,6R)-HNK. b, c, Response rate of overall lever pressing per sec in the drug discrimination model was not changed by administration of (R,S)-KET, (2R,6R)-HNK (b) or PCP (c). d, Unlike ketamine, (2R,6R)-HNK did not alter drug intake in the self-administration task in mice. Data are mean ± s.e.m. *P < 0.05 (see Supplementary Table 1 for statistical analyses and n numbers).

Change history

Corrected online 13 May 2016
The competing financial interests statement did not display correctly online when this paper was first published; this has been corrected and the statement is now available.

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Author information

  1. Present address: Mitchell Woods Pharmaceuticals, Shelton, Connecticut 06484, USA.

    • Irving W. Wainer

Affiliations

  1. Department of Psychiatry, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA

    • Panos Zanos,
    • Polymnia Georgiou,
    • Greg I. Elmer,
    • Heather J. Pribut,
    • Scott M. Thompson &
    • Todd D. Gould
  2. Biomedical Research Center, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224, USA

    • Ruin Moaddel,
    • Nagendra S. Singh,
    • Katina S. S. Dossou &
    • Irving W. Wainer
  3. Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland 20850, USA

    • Patrick J. Morris,
    • Yuhong Fang &
    • Craig J. Thomas
  4. Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA

    • Jonathan Fischell &
    • Scott M. Thompson
  5. Department of Pharmacology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA

    • Greg I. Elmer,
    • Edson X. Albuquerque &
    • Todd D. Gould
  6. Maryland Psychiatric Research Center, University of Maryland School of Medicine, Baltimore, Maryland 21228, USA

    • Greg I. Elmer &
    • Cheryl L. Mayo
  7. Department of Epidemiology and Public Health, Division of Translational Toxicology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA

    • Manickavasagom Alkondon &
    • Edson X. Albuquerque
  8. Experimental Therapeutics and Pathophysiology Branch, Intramural Research Program, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892, USA

    • Peixiong Yuan &
    • Carlos A. Zarate Jr
  9. NIMH Psychoactive Drug Screening Program, Department of Pharmacology and Division of Chemical Biology and Medicinal Chemistry, University of North Carolina Chapel Hill Medical School, Chapel Hill, North Carolina 27516, USA

    • Xi-Ping Huang
  10. Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA

    • Edson X. Albuquerque
  11. Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA

    • Todd D. Gould

Contributions

P.Z., R.M., P.J.M., I.W.W., C.J.T., C.A.Z. and T.D.G. were responsible for the overall experimental design. P.J.M., Y.F. and C.J.T. synthesised the ketamine metabolites and deuterated ketamine derivatives, and provided mass spectrometer confirmations. Bioanalytical quantitation of ketamine and metabolites were performed by R.M., N.S.S. and K.S.S.D.. P.Z., P.G. and H.J.P. conducted and analysed the results of the behavioural and qEEG experiments. X.-P.H. supervised and analysed the results of the binding experiments. P.Y. performed the western blot experiments. E.X.A., M.A., J.F. and S.M.T. helped design and analyse the electrophysiology experiments, which were conducted by M.A., J.F. and S.M.T. G.I.E. and C.L.M. conducted and analysed the results of the i.v. self-administration. P.Z. and T.D.G. outlined and wrote the paper, which was reviewed by all authors.

Competing financial interests

The authors declare competing financial interests: I.W.W., R.M., and C.A.Z. are listed as co-inventors on a patent for the use of (2R,6R)-hydroxynorketamine, (S)-dehydronorketamine and other stereoisomeric dehydro and hydroxylated metabolites of (R,S)-ketamine metabolites in the treatment of depression and neuropathic pain. They have assigned their rights in the patent to the U.S. government but will share a percentage of any royalties that may be received by the government. I.W.W., C.A.Z., R.M., T.G., P.Z., C.T., and P.M. are listed as co-inventors on a patent application for the use of (2R, 6R)-hydroxynorketamine and (2S, 6S)-hydroxynorketamine in the treatment of depression, anxiety, anhedonia, suicidal ideation and post-traumatic stress disorders. I.W.W., C.A.Z., R.M., C.T., and P.M. have assigned their rights in this patent to the U.S. government but will share a percentage of any royalties that may be received by the government. T.G. and P.Z. have assigned their rights in this patent to the University of Maryland but will share a percentage of any royalties that may be received by the University of Maryland.

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Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: The metabolic transformations of ketamine in vivo. (130 KB)

    Ketamine is metabolised in vivo via P450 enzymatic transformations. i, (R,S)-KET is selectively demethylated to give (R,S)-norketamine (norKET). ii, NorKET can be then dehydrogenated to give (R,S)-dehydronorketamine (DHNK). iii, Alternatively, norKET can be hydroxylated to give the hydroxynorketamines (HNKs). iv, (R,S)-KET can also be hydroxylated at the 6 position to give either the E-6-hydroxyketamine ((2S,6R;2R,6S)-HK) or Z-6-hydroxyketamine ((2S,6S;2R,6R)-HK). v, Demethylation of (2S,6R;2R,6S)-HK yields the production of (2S,6R;2R,6S)-HNK. vi, Demethylation of (2S,6S;2R,6R)-HK further gives (2S,6S;2R,6R)-HNK.

  2. Extended Data Figure 2: Circulating levels of ketamine and its metabolites following i.p. administration in mice. (339 KB)

    a, b, Plasma (a) and brain (b) levels of ketamine and its metabolites after administration of (R,S)-KET (10 mg kg−1) in mice. c, d, Brain levels of KET (c), norKET (d) and HNK (e) following administration of (S)- and (R)-KET. f, g, Chemical structure of (R,S)-6,6-dideuteroketamine ((R,S)-d2-KET) (f), which displaces [3H]MK-801 binding with a similar affinity to (R,S)-KET ((R,S)-KET: Ki = 799 nM; (R,S)-d2-KET: Ki = 883 nM) (g). See Supplementary Table 1 for statistical analyses and n numbers.

  3. Extended Data Figure 3: Additional social defeat stress data. (247 KB)

    a, Chronic social defeat stress and social interaction/avoidance test timeline. b, c, 24 h after administration, neither (R,S)-KET nor MK-801 affected locomotor activity (b) or total number of compartmental crosses in the social interaction apparatus (c). Data are mean ± s.e.m. ***P < 0.001. See Supplementary Table 1 for statistical analyses and n numbers.

  4. Extended Data Figure 4: Locomotor effects of (R,S)-KET and (R,S)-d2-KET. (252 KB)

    After recording baseline activity for 60 min, mice received drug (marked by a vertical dashed line) and locomotor activity was monitored for another 1 h. a, b, Administration of (R,S)-KET (10 mg kg−1), induced hyperlocomotor responses equally in both male and female mice. c, d, (R,S)-KET and (R,S)-d2-KET were equally potent in inducing a hyperlocomotor response at the dose of 10 mg kg−1. Data are mean ± s.e.m. *P < 0.05, **P < 0.01 (see Supplementary Table 1 for statistical analyses and n numbers).

  5. Extended Data Figure 5: Acute and sustained antidepressant and anti-anhedonic effects of (2R,6R)- and (2S,6S)-HNK. (287 KB)

    a, A single injection of (2R,6R)-HNK resulted in dose-dependent antidepressant-like responses in the learned helplessness test at the doses of 5–75 mg kg−1. b, A single injection of (2S,6S)- HNK induced antidepressant-like effects in the learned helplessness test at the dose of 75 mg kg−1. c, Administration of (2R,6R)-HNK induced dose-dependent antidepressant effects in the 1- and 24-h FST. d, Administration of (2S,6S)-HNK at the dose of 25 mg kg−1 induced antidepressant effects in the 1- and 24-h FST. e, Despite the greater antidepressant efficacy of (2R,6R)-HNK, administration of (2S,6S)-HNK (HNK) results in higher brain hydroxynorketamine levels compared to (2R,6R)-HNK. f, (2R,6R)-HNK manifested dose-dependent antidepressant-like effects in the NSF test. g, Similar to (R,S)-KET, the antidepressant-like effects of (2R,6R)-HNK in the FST persisted for at least 3 days after treatment. h, A single administration of (2R,6R)-HNK reversed chronic corticosterone-induced decreases in sucrose preference. i, A single administration of (2R,6R)-HNK reversed chronic corticosterone-induced decrease in female urine sniffing preference, specifically in mice that developed an anhedonic phenotype. Administration of (2R,6R)-HNK was not associated with changes in locomotor activity (j) or total compartmental crosses in the social interaction test after chronic social defeat stress (k). Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 (see Supplementary Table 1 for statistical analyses and n numbers).

  6. Extended Data Figure 6: (2R,6R)-HNK rapidly increases the frequency and amplitude of AMPAR spontaneous excitatory postsynaptic currents in the hippocampus. (308 KB)

    a, Representative traces of spontaneous excitatory postsynaptic currents (sEPSCs) mediated via AMPARs during baseline (before) and 20 min after drug administration. b, Example CA1 stratum radiatum interneuron recorded from a rat hippocampal slice. cj, Twenty-minute exposure of (2R,6R)-HNK (e, i), but not (R,S)-(KET) (c, g) or (2S,6S)-HNK (d, h), increased AMPA sEPSCs frequency and amplitude compared to baseline. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 (see Supplementary Table 1 for statistical analyses and n numbers). SLM, stratum lacunosum-moleculare; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum.

  7. Extended Data Figure 7: Administration of the AMPAR antagonist, NBQX, prevents (2R,6R)-HNK-induced increases in gamma oscillations in vivo. (319 KB)

    a, Administration of (R,S)-KET, but not (2R,6R)- HNK, increased locomotor home-cage activity of mice. be, Neither (R,S)-KET nor (2R,6R)-HNK altered cortical alpha (b), beta (c), delta (d) or theta (e) oscillations in vivo. fk, Pre-treatment with the AMPAR antagonist, NBQX, did not change the locomotor activity (f), alpha (g), beta (h), delta (j) or theta (k) oscillations, but it prevented (2R,6R)-HNK-induced increases of gamma oscillations in vivo (i). Data are mean ± s.e.m. *P < 0.05, **P < 0.01 (see Supplementary Table 1 for statistical analyses and n numbers).

  8. Extended Data Figure 8: Effects of (2R,6R)-hydroxynorketamine on protein and protein phosphorylation levels in synaptoneurosomes. (240 KB)

    a, b, A single administration of (R,S)-KET (10 mg kg−1) or (2R,6R)- HNK (10 mg kg−1), did not alter levels of mTOR or phosphorylated mTOR in the hippocampus 1 h (a) or 24 h (b) after injection. cj, Administration of (R,S)-KET or (2R,6R)-HNK did not alter levels of mTOR/phosphorylated mTOR (c, d), eEF2/phosphorylated eEF2 (e, f), proBDNF/mBDNF (g, h), or GluA1/GluA2 (i, j), in the prefrontal cortex of mice. The values for the phosphorylated forms of proteins were normalized to phosphorylation-independent levels of the same protein. Phosphorylation-independent levels of proteins were normalized to GAPDH. Data are mean ± s.e.m, and were normalized to the saline-treated control group for each protein. Images are cropped; see Supplementary Fig. 1 for complete blot images. *P < 0.05 (see Supplementary Table 1 for statistical analyses and n numbers).

  9. Extended Data Figure 9: (2R,6R)-HNK administration does not alter startle amplitude, drug discrimination rate or self-administration drug intake. (200 KB)

    a, Startle amplitude as measured in the pre-pulse inhibition task was not affected by administration of (R,S)-KET or (2R,6R)-HNK. b, c, Response rate of overall lever pressing per sec in the drug discrimination model was not changed by administration of (R,S)-KET, (2R,6R)-HNK (b) or PCP (c). d, Unlike ketamine, (2R,6R)-HNK did not alter drug intake in the self-administration task in mice. Data are mean ± s.e.m. *P < 0.05 (see Supplementary Table 1 for statistical analyses and n numbers).

Supplementary information

PDF files

  1. Supplementary Information (6.9 MB)

    This file contains (1) Supplementary Table 1, which includes the details for the number of animals and the results of the statistical analyses in each experiment; (2) Supplementary Figure 1, with the complete blot images for Figure 4 and Extended Data Figure 8; (3) the Supplementary Methods for the synthesis of (2R,6R)-HNK, (2S,6S)-HNK, and D-Ket, in addition to the analytical data that supports the synthetic steps and the X-ray crystallographic data that confirms the absolute and relative conformation for (2S,6S)-HNK and (2R,6R)-HNK.

Additional data