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Clinical data have demonstrated rapid and sustained antidepressant effects of ketamine, a noncompetitive NMDAR (N-methyl-daspartate receptor) antagonist1. 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-3hydroxy-5-methyl-4-isoxazole propionic acid receptor)2. Here we report that (2R,6R)-HNK blocks synaptic NMDARs in a simi lar 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. There is a Reply to this Comment by Zanos, P. et al. Nature 546, http://dx.doi.org/10.1038/nature22085 (2017). In previous work3, we showed that ketamine exerts its antidepressant effects by blocking NMDARs at rest, which deactivates eukaryotic elongation factor 2 kinase (eEF2K), thereby dephosphorylating eukaryotic elongation factor 2 (eEF2) and resulting in a subsequent desuppression of brain-derived neurotrophic factor (BDNF) protein translation. This signalling pathway then potentiates synaptic AMPAR responses in the hippocampus through insertion of GluA1 and GluA2 subunits3–5. Notably, Zanos et al. show that (2R,6R)-HNK triggers the same intracellular pathway and downstream effects that we demonstrated for ketamine, namely inhibition of eEF2K, increased expression of BDNF, GluA1 and GluA2, and a form of synaptic potentiation in the hippocampus that is sensitive to AMPAR blockers3–5. The similarity between the molecular findings of ketamine and (2R,6R)-HNK led us to re-examine the potential involvement of (2R,6R)-HNK in NMDAR function. We assessed the effects of (2R,6R)-HNK in NMDAR-mediated miniature excitatory postsynaptic currents (NMDAR-mEPSCs) in cultured hippocampal neurons and compared its properties to the NMDAR antagonists 2R-amino-5-phosphonopentanoate (AP5) and ketamine. NMDA-mEPSCs were isolated in the presence of