Clinical data have demonstrated rapid and sustained antidepressant effects of ketamine, a noncompetitive NMDAR (N-methyl-daspartate receptor) antagonist. Recently, Zanos et al.2 claimed that the ketamine metabolite (2R,6R)-hydroxynorketamine (HNK) is essential for the antidepressant effects of ketamine in mice in an NMDAR-independent manner, although no alternative mechanism was proposed, beyond unspecific activation of AMPAR (α -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor). Here we report that (2R,6R)-HNK blocks synaptic NMDARs in a similar manner to its parent compound, and we show that the effects of (2R,6R)-HNK on intracellular signalling are coupled to NMDAR inhibition. These data demonstrate that (2R,6R)-HNK inhibits synaptic NMDARs and subsequently elicits the same signal transduction pathway previously associated with NMDAR inhibition by ketamine

Albuquerque, E.X.

Alkondon, M.

Dossou, K.S.S.

Elmer, G.I.

Fang, Y.

Fischell, J.

Georgiou, P.

Gould, T.D.

Huang, X.-P.

Mayo, C.L.

Moaddel, R.

Morris, P.J.

Pribut, H.J.

Singh, N.S.

Thomas, C.J.

Thompson, S.M.

Yuan, P.

Zanos, P.

Zarate, C.A., Jr.

Carolina Digital Repository

Zanos et al. replyreplying to K. Suzuki et al. Nature 546, http://dx.doi.org/10.1038/nature22084 (2017)In the accompanying Comment1, Suzuki et al. confirmed our previous findings2 that the ketamine metabolite (2R,6R)-hydroxynorketamine (HNK) does not functionally inhibit the NMDAR at concentrations relevant to its antidepressant actions recently reported in mice (that is, approximately 10 μM). We reported that 10 μM (2R,6R)-HNK, which equates to the maximum concentration of brain exposure (10.69 μM (ref. 2)) after intraperitoneal administration of a 10 mg kg−1 anti depressant dose of (2R,6R)-HNK in mice, robustly potentiated excitatory postsynaptic potentials in hippocampal slices without func-tionally inhibiting the NMDAR2, as Suzuki et al. now confirm. Unless NMDAR inhibition is observed at 10 μM (2R,6R)-HNK, it is probably not of major relevance to the antidepressant actions of (2R,6R)-HNK. Notably, (2R,6R)-HNK administration also resulted in significant anti-depressant actions at the dose of 5 mg kg−1 in mice, which would produce even lower concentrations of the metabolite in the brain than 10 μM (ref. 2). Furthermore, our field-potential electrophysio-logy experiments were conducted in the presence of the NMDAR antagonist AP5 (80 μM), demonstrating that the AMPAR-mediated synaptic potentiation we observed is indepen dent of any capacity of (2R,6R)-HNK to inhibit the NMDAR.We agree with Suzuki et al. that assessing higher concentrations of (2R,6R)-HNK on NMDAR-mediated miniature excitatory postsyn-aptic currents (NMDAR-mEPSCs) responses might be important in providing information for off-target effects of this metabolite, and their study indeed  provides evidence for a modest inhibition (~ 37%) at the concentration of 50 μ M. The absence of Mg2+ in the testing condi-tions may influence the apparent discrepancy between 37% inhibition of NMDAR-mEPSCs at the 50 μ M concentration (for example, as is the case with  memantine, which only inhibits NMDAR-mEPSCs in the absence of Mg2+, and does not have antidepressant properties in humans3), and the previously reported lack of displacement of [3H] MK-801 binding at less than 100 μ M (ref. 4). It will be important to follow up on this finding with a full concentration response curve in order to determine the half maximal inhibi tory concentration (IC50) of (2R,6R)-HNK on NMDAR-mEPSC responses, and to compare to the IC50 of (R,S)-ketamine, under the same conditions using physiolo-gical levels of Mg2+. Notably, we did not observe NMDAR-inhibition-associated psychostimulant, self-administration, drug discrimination and pre-pulse inhibition side effects of (2R,6R)-HNK at any dose, including up to 375 mg kg−1 (ref. 2).The authors also observed decreased phosphorylation of the eukar-yotic elongation  factor 2 (eEF2) only at the concentration of 50 μ M. Although this is the same high concentration at which (2R,6R)-HNK induced an inhibition of the NMDAR responses, these data do not provide direct evidence of a functional link between the moderate inhibition of NMDARs and the decrease in eEF2 phosphorylation. Indeed there are multiple mechanisms other than NMDAR inhibi-tion that could be responsible for the observed eEF2 dephosphoryl-ation5–8. It is also important to note that although direct application of ketamine9 or the alternative NMDAR blocker MK-801 (ref. 10) to cultured neurons or hippocampal slices was previously reported to induce NMDAR-inhibition-dependent synaptic potentiation, MK-801 has repeatedly failed to induce  ketamine-like  sustained anti-depressant effects in several animal tests2,9,11,12. It is  therefore probable  that synaptic strengthening as a consequence of NMDAR inhibition (proposed by Suzuki et al.) is not solely  responsible for the antidepres-sant actions of ketamine. Indeed, the results of clinical trials indicate that several other NMDAR antagonists lack the full antidepressant actions of ketamine in humans13.Suzuki et al. also propose that NMDAR inhibition by (R,S)-ketamine is responsible for the initiation of antidepressant responses (that is, the acute actions), and HNK metabolites mediate the long-term antidepressant effects of ketamine through continued NMDAR inhibition. This hypothesis is not supported by our pharmacokinetic measurements of ketamine and its metabolites in the brain of mice following intraperitoneal administration of (R,S)-ketamine, which show that both ketamine and its metabolites peak at similar early time points, have a similar short half-life, and are below detectable levels by four hours after treatment2. In conclusion, the data presented by Suzuki et al. further support an NMDAR-inhibition-independent mechanism of antidepressant action of the ketamine metabolite (2R,6R)-HNK at physiologically relevant concentrations. Although development of (2R,6R)-HNK as an anti depressant drug, which does not induce NMDAR-mediated side effects, can proceed independently of target identification, it remains important to identify its primary pharmacological target at anti depressant-relevant concentrations (~ 10 μ M).Author Irving W. Wainer has chosen not to be listed on this Reply.Panos Zanos1, ruin Moaddel2, Patrick J. Morris3, Polymnia Georgiou1, Jonathan Fischell4, Greg I. Elmer1,5,6, Manickavasagom Alkondon7, Peixiong Yuan8, Heather J. Pribut1, Nagendra S. Singh2, Katina S. S. Dossou2, Yuhong Fang3,  Xi-Ping Huang9, Cheryl L. Mayo6, Edson X. Albuquerque5,7,10, Scott M. Thompson1,4, Craig J. Thomas3, Carlos A. Zarate Jr8 & Todd D. Gould1,5,111Department of Psychiatry, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA. email: gouldlab@me.com2Biomedical Research Center, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224, USA.3Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland 20892, USA.4Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.5Department of Pharmacology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.6Maryland Psychiatric Research Center, University of Maryland School of Medicine, Baltimore, Maryland 21228, USA.7Department of Epidemiology and Public Health, Division of Translational Toxicology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.8Experimental Therapeutics and Pathophysiology Branch, Intramural Research Program, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20814, USA.9NIMH 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.10Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.11Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.1. Suzuki, K., Nosyreva, E., Hunt, K. W., Kavalali, E. T. & Monteggia, L. M. Effects of a ketamine metabolite on synaptic NMDAR function. Nature 546, http://dx.doi.org/10.1038/nature22084 (2017).2. Zanos, P. et al. NMDAR inhibition-independent antidepressant actions ofketamine metabolites. Nature 533, 481–486 (2016).3. Gideons, E. S., Kavalali, E. T. & Monteggia, L. M. Mechanisms underlyingdifferential effectiveness of memantine and ketamine in rapidantidepressant responses. Proc. Natl Acad. Sci. USA 111, 8649–8654 (2014)4. Moaddel, R. et al. Sub-anesthetic concentrations of (R,S)-ketaminemetabolites inhibit acetylcholine-evoked currents in α 7 nicotinic acetylcholine receptors. Eur. J. Pharmacol. 698, 228–234 (2013).5. Hizli, A. A. et al. Phosphorylation of eukaryotic elongation factor 2 (eEF2) bycyclin A-cyclin-dependent kinase 2 regulates its inhibition by eEF2 kinase. Mol. Cell. Biol. 33, 596–604 (2013).6. Knebel, A., Morrice, N. & Cohen, P. A novel method to identify protein kinasesubstrates: eEF2 kinase is phosphorylated and inhibited by SAPK4/p38δ . EMBO J. 20, 4360–4369 (2001).7. Redpath, N. T., Foulstone, E. J. & Proud, C. G. Regulation of translation elongation factor-2 by insulin via a rapamycin-sensitive signalling pathway.EMBO J. 15, 2291–2297 (1996).8. Wang, X. et al. Regulation of elongation factor 2 kinase by p90(RSK1) and p70S6 kinase. EMBO J. 20, 4370–4379 (2001).9. Autry, A. E. et al. NMDA receptor blockade at rest triggers rapid behaviouralantidepressant responses. Nature 475, 91–95 (2011).10. Nosyreva, E. et al. Acute suppression of spontaneous neurotransmission drivessynaptic potentiation. J. Neurosci. 33, 6990–7002 (2013).11. Maeng, S. et al. Cellular mechanisms underlying the antidepressant effects ofketamine: role of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acidreceptors. Biol. Psychiatry 63, 349–352 (2008).12. Yang, B., Ren, Q., Ma, M., Chen, Q. X. & Hashimoto, K. Antidepressant effects of(+ )-MK-801 and (− )-MK-801 in the social defeat stress model. Int. J.Neuropsychopharmacol. 19, pyw080 (2016).13. Newport, D. J. et al. Ketamine and other NMDA antagonists: early clinical trialsand possible mechanisms in depression. Am. J. Psychiatry 172, 950–966 (2015).doi:10.1038/nature22085

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