Significance Despite available medications for depression, currently approved antidepressants take months to exert therapeutic effects, and ∼30% of patients remain treatment resistant. In contrast, a single subanesthetic dose of ketamine exerts rapid (within hours) and sustained antidepressant actions. Preclinical studies indicate that the ketamine metabolite (2R,6R)-hydroxynorketamine [(2R,6R)-HNK] is a rapid-acting antidepressant candidate with limited adverse effects compared with ketamine. Using behavioral, genetic, and pharmacological approaches and EEG measurements, we determined that the mechanism underlying antidepressant-relevant actions of (2R,6R)-HNK converges with metabotropic glutamate receptor subtype 2 (mGlu 2 ) receptor signaling and identified high-frequency EEG oscillations as a marker associated with rapid antidepressant responses. Our data support the use of individually subtherapeutic doses of mGlu 2 receptor inhibitors with ketamine or (2R,6R)-HNK in clinical trials for the treatment of depression.

Abstract Currently approved antidepressant drugs often take months to take full effect, and ∼30% of depressed patients remain treatment resistant. In contrast, ketamine, when administered as a single subanesthetic dose, exerts rapid and sustained antidepressant actions. Preclinical studies indicate that the ketamine metabolite (2R,6R)-hydroxynorketamine [(2R,6R)-HNK] is a rapid-acting antidepressant drug candidate with limited dissociation properties and abuse potential. We assessed the role of group II metabotropic glutamate receptor subtypes 2 (mGlu 2 ) and 3 (mGlu 3 ) in the antidepressant-relevant actions of (2R,6R)-HNK using behavioral, genetic, and pharmacological approaches as well as cortical quantitative EEG (qEEG) measurements in mice. Both ketamine and (2R,6R)-HNK prevented mGlu 2/3 receptor agonist (LY379268)-induced body temperature increases in mice lacking the Grm3, but not Grm2, gene. This action was not replicated by NMDA receptor antagonists or a chemical variant of ketamine that limits metabolism to (2R,6R)-HNK. The antidepressant-relevant behavioral effects and 30- to 80-Hz qEEG oscillation (gamma-range) increases resultant from (2R,6R)-HNK administration were prevented by pretreatment with an mGlu 2/3 receptor agonist and absent in mice lacking the Grm2, but not Grm3−/−, gene. Combined subeffective doses of the mGlu 2/3 receptor antagonist LY341495 and (2R,6R)-HNK exerted synergistic increases on gamma oscillations and antidepressant-relevant behavioral actions. These findings highlight that (2R,6R)-HNK exerts antidepressant-relevant actions via a mechanism converging with mGlu 2 receptor signaling and suggest enhanced cortical gamma oscillations as a marker of target engagement relevant to antidepressant efficacy. Moreover, these results support the use of (2R,6R)-HNK and inhibitors of mGlu 2 receptor function in clinical trials for treatment-resistant depression either alone or in combination.

Although monoamine-targeting pharmacotherapies to treat depression, such as selective serotonin-reuptake inhibitors, are commonly used, more than 30% of severely depressed individuals remain treatment resistant (1). Moreover, even when effective, existing antidepressants often take months to exert their full therapeutic effects (2). Recent efforts to identify more effective antidepressant medications have focused on agents modulating glutamatergic neurotransmission (3). The relevance of targeting glutamatergic synapses is supported by numerous placebo-controlled trials that have provided strong evidence for the rapid (within 2 h) and sustained (∼7 d) antidepressant effects of subanesthetic doses of (R,S)-ketamine (ketamine) in depressed (4) and treatment-resistant depressed patients (5⇓⇓⇓–9). Ketamine has been proposed to exert antidepressant actions via inhibition of glutamate NMDA receptor (NMDAR) function, which is also the mechanism attributed to its dissociative/anesthetic actions (10⇓⇓–13). However, no human clinical studies have replicated the full spectrum of robust, rapid, and sustained antidepressant actions observed with ketamine using alternative drugs that directly inhibit NMDAR function (14). Also, preclinical findings indicate NMDAR inhibition-independent mechanisms for the antidepressant-relevant actions of ketamine (15, 16). Thus, ketamine’s complete mechanism of action as a rapid-acting antidepressant remains controversial (10⇓⇓–13, 17).

In vivo, ketamine is rapidly metabolized to norketamine and thereafter, is hydroxylated at multiple locations to produce the hydroxynorketamines (HNKs). (2S,6S;2R,6R)-HNKs, which are produced by hydroxylation of the cyclohexyl ring at the C6 position, are the major HNK metabolites found in the plasma of humans, as well as plasma and brain of rodents after ketamine administration (18). Metabolism of ketamine to (2S,6S;2R,6R)-HNK was shown to be involved in ketamine’s sustained antidepressant-relevant actions in mice (15). The (2R,6R)-HNK stereoisomer has been identified as a potent, putative rapid-acting antidepressant drug in multiple animal behavioral tests (15, 19⇓⇓–22) and is reported to share ketamine’s antidepressant-relevant downstream signaling mechanisms (15, 16, 19, 22⇓⇓–25). Importantly, (2R,6R)-HNK does not share ketamine’s robust sensory dissociation or abuse potential properties in rodents (15, 20) and also, does not seem to inhibit NMDAR function at antidepressant-relevant concentrations (15, 18, 26⇓⇓⇓–30). Thus, mechanistic studies with (2R,6R)-HNK enable the study of rapid and sustained antidepressant actions after a single administration that are independent of ketamine’s effect to inhibit NMDAR function.

Antidepressant-relevant actions of ketamine and (2R,6R)-HNK converge on a glutamate-associated enhancement of neuronal activity in mood-regulating synapses, which are proposed to both initiate and sustain their effects (13, 31, 32). Both ketamine (33⇓⇓–36) and (2R,6R)-HNK (15) enhance glutamatergic excitatory synaptic transmission in rodent brain slices. Such glutamate neurotransmission is regulated by the actions of group II metabotropic glutamate receptors subtypes 2 and 3 (mGlu 2/3 ) (37). mGlu 2 receptors are mainly expressed perisynaptically in close proximity to the presynaptic terminals, where they act as autoreceptors (38, 39) to decrease synaptic glutamate release when activated (40, 41). Indeed, activation of mGlu 2/3 receptors reduces glutamate-dependent excitatory neurotransmission in rodent brain slices (42). In contrast, mGlu 3 receptors are mainly expressed postsynaptically on neurons and glia (43⇓–45).

Inhibitors of group II mGlu receptors have gained interest for their actions to exert rapid antidepressant-relevant effects, comparable with those observed with ketamine, in rodents (46⇓⇓⇓⇓⇓⇓⇓–54). In addition to their similar antidepressant-relevant behavioral actions, downstream signaling pathways considered necessary for the antidepressant actions of ketamine are similarly involved in the actions of mGlu 2/3 receptor antagonists (47, 48, 53, 55⇓⇓–58). Some evidence exists to suggest convergence in the mechanism of action of ketamine and mGlu 2/3 receptor modulation. In particular, ketamine-induced enhancement of cortical extracellular glutamate levels was abolished by pretreatment with an mGlu 2/3 receptor agonist in rats (59). Prevention of glutamate-dependent neurotransmission in the prefrontal cortex is expected to prevent hyperactivation of this brain region. Indeed, ketamine-induced cortical activation [as measured by [14C]2-deoxyglucose autoradiography, blood oxygenation-level dependence (BOLD) pharmacological magnetic resonance imaging, or [18F]fluorodeoxyglucose μ-positron emission tomography] was prevented by pretreatment with mGlu 2/3 receptor agonists (60, 61) or selective mGlu 2 receptor-positive allosteric modulators (62) in rodents. A similar effect was observed in humans using two different mGlu 2/3 receptor agonist prodrugs while assessing BOLD pharmacological magnetic resonance imaging (63). In addition, pretreatment with mGlu 2/3 agonists (64⇓⇓–67) or mGlu 2 receptor-selective positive allosteric modulators (68, 69) prevented ketamine-induced enhancement of cortical quantitative EEG (qEEG) gamma oscillations, a marker of neuronal activation. These convergent processes have been hypothesized to involve ketamine-induced NMDAR inhibition, since mGlu 2/3 activation also prevents cortical activation induced by other noncompetitive NMDAR open channel blockers, including MK-801 (68, 69), memantine (60, 62), and phencyclidine (70). However, whether there are convergent actions between mGlu 2/3 receptor inhibition and (2R,6R)-HNK and if such effects are dependent on NMDAR inhibition are not known.

Here, we investigated the role of the mGlu 2 and mGlu 3 receptors in the antidepressant-relevant actions of (2R,6R)-HNK using pharmacological manipulations as well as mice in which mGlu 2 (Grm2−/−) or mGlu 3 (Grm3−/−) receptors are constitutively deleted. We also assessed the role of mGlu 2 and mGlu 3 receptors in the effects of (2R,6R)-HNK on cortical qEEG power. The results of our experiments reveal that (2R,6R)-HNK’s antidepressant-relevant actions converge with mGlu 2 , but not mGlu 3 , receptor signaling and highlight an NMDAR inhibition-independent mechanism underlying these effects. Cortical qEEG measurements implicate increases in high-frequency synchronized gamma oscillations as a putative mechanism contributing to rapid antidepressant efficacy and provide a target engagement marker with translational utility for human clinical trials.

Discussion Preclinical studies indicate that the ketamine metabolite (2R,6R)-HNK is a putative fast-acting antidepressant (15, 16, 19⇓⇓⇓⇓⇓–25) devoid of ketamine’s adverse effects (15, 20). In particular, this metabolite was shown to be effective in rodent behavioral tests predictive of rapid antidepressant efficacy (15, 19⇓⇓–22) and to exert a long-lasting (at least 21-d) effect in rescuing chronic stress-induced behavioral despair and anhedonia in rats (19). In this study, using an in vivo measure predictive of mGlu 2/3 receptor antagonist activity, we demonstrated that (2R,6R)-HNK, similar to the mGlu 2/3 receptor antagonist LY341495, reverses mGlu 2/3 receptor agonist-induced hyperthermia. Our experiments revealed this effect to be mGlu 2 , but not mGlu 3 , receptor mediated. We also show that subeffective doses of an mGlu 2/3 receptor antagonist combined with (2R,6R)-HNK exert synergistic antidepressant-relevant behavioral actions and enhancement of cortical gamma qEEG power in mice. In addition, administration of the mGlu 2/3 receptor agonist LY379268 before (2R,6R)-HNK prevented both the acute and sustained antidepressant-relevant actions of (2R,6R)-HNK. Importantly, the antidepressant-relevant behavioral effects of (2R,6R)-HNK were absent in mice lacking the Grm2, but not Grm3, gene. (2R,6R)-HNK’s action to increase gamma qEEG power was absent in mice lacking the Grm2, but not Grm3, gene, and it was abolished by pretreatment with an mGlu 2/3 receptor agonist. We note that our findings do not reveal a direct interaction of (2R,6R)-HNK with the mGlu 2 receptor, and we, therefore, do not exclude the possibility that this metabolite acts either upstream or downstream of mGlu 2 receptors to exert convergent effects. It was previously shown that peripheral administration of ketamine enhances extracellular glutamate levels in the prefrontal cortex of rats (59, 83) and that pretreatment with an mGlu 2/3 receptor agonist blocked this effect (59), while the agonist itself had no effect on glutamate levels, suggesting that ketamine may act similarly to an mGlu 2/3 receptor antagonist in vivo. Indeed, using the mGlu 2/3 receptor agonist-induced hyperthermia assay, we show that both ketamine and (2R,6R)-HNK act similarly to mGlu 2/3 receptor antagonists in vivo. This finding supports the hypothesis of a convergent mechanism of action between mGlu 2/3 receptor inhibition and the effects of ketamine and (2R,6R)-HNK. In line with this hypothesis, in vitro experiments have previously shown that ketamine (33⇓⇓–36), (2R,6R)-HNK (15), and the mGlu 2/3 receptor antagonist LY341495 (84, 85) similarly enhance excitatory glutamatergic synaptic transmission in rodent brain slices. Glutamate-dependent amplification of neuronal activity via activation of synaptic AMPARs has been broadly hypothesized to underlie the antidepressant actions of ketamine and other putative rapid-acting antidepressants, including (2R,6R)-HNK and mGlu 2/3 receptor antagonists (52). An in vivo marker of neuronal excitation, which is dependent on AMPAR throughput (15, 86), is the enhancement of high-frequency (gamma) qEEG oscillations (87). Results from human studies reveal that an antidepressant dose of ketamine induces an acute enhancement of the power (amplitude) of qEEG oscillations within the 30- to 80-Hz gamma range in the parietal (88), cingulate (88), and cerebral (89) as well as general cortical brain areas (90). Increases in cortical gamma power have been ascribed to a mechanism related to the psychotomimetic actions of ketamine (91⇓–93), putatively via NMDAR inhibition-mediated modulation of interneuron activity (94). Similar to ketamine, (2R,6R)-HNK administration increases high-frequency cortical gamma qEEG oscillations in mice (15), but unlike ketamine, this metabolite does not exert behavioral changes in the prepulse inhibition task at doses up to 375 mg/kg, indicative of a lack of psychosis potential (15) and relevant NMDAR inhibition (26⇓⇓⇓–30). Although ketamine’s overall effect on gamma power is likely influenced by its actions to inhibit the NMDAR expressed on GABAergic interneurons (83, 94, 95), the results here suggest an explanation whereby (2R,6R)-HNK exerts its effects on gamma oscillations via a mechanism that does not involve NMDAR inhibition. High-frequency oscillations in vivo have parallels to many forms of activity-dependent plasticity, such as long-term potentiation, in which high-frequency stimuli induce sustained strengthening of excitatory synapses via an enhanced synchrony between limbic-connected brain regions, likely necessary for antidepressant behavioral actions (31, 96). The finding that (2R,6R)-HNK enhances cortical gamma qEEG oscillations indicates that this metabolite may engage endogenous processes that promote synaptic strengthening. Therefore, it is possible that enhancement of gamma qEEG oscillations is directly involved in the rapid antidepressant-relevant behavioral actions of (2R,6R)-HNK, especially considering our previous findings that in vivo pharmacological inhibition of AMPARs blocks both the behavioral actions and the increases in gamma power induced by (2R,6R)-HNK (15). These data highlight increases in gamma qEEG activity as a putative translational measure of antidepressant response. In agreement, ketamine-induced increases in gamma power have been recently associated with better antidepressant responses in patients treated for depression (97). In this study, we demonstrated that administration of either (2R,6R)-HNK or an mGlu 2/3 receptor antagonist at an antidepressant-relevant dose enhances cortical gamma qEEG oscillations and that these effects are mGlu 2 , but not mGlu 3 , receptor dependent. We also show that the antidepressant-relevant behavioral actions of (2R,6R)-HNK, similar to the effects previously shown for mGlu 2/3 receptor antagonists (71), are also mGlu 2 , but not mGlu 3 , receptor dependent. These findings indicate that both mGlu 2/3 receptor antagonists and (2R,6R)-HNK act in an mGlu 2 receptor-dependent manner to enhance high-frequency neuronal activity and to exert antidepressant-relevant actions. In line with our findings, a previous study also showed increases in the gamma power range (30–49 Hz) 2–4 h (but not 1 h) after functional inhibition of the mGlu 2/3 receptor in rats (98); however, that study restricted qEEG analyses to frequencies between 1 and 49 Hz, thus excluding gamma frequencies between 50 and 80 Hz. Moreover, the 10-mg/kg dose used for LY341495 to enhance gamma EEG oscillations in the aforementioned study is higher than the identified antidepressant-relevant dose (3 mg/kg) used in this study, where we observed increases of gamma power within 10 min after administration. Important for understanding the mechanism of (2R,6R)-HNK action as an antidepressant is our finding that combined subeffective doses of an mGlu 2/3 receptor antagonist and (2R,6R)-HNK synergistically exerted antidepressant-relevant actions and increased gamma qEEG oscillations. This finding suggests that a convergent mechanism in both the behavioral and physiological actions of these compounds exists. A similar synergistic antidepressant-relevant effect between ketamine and mGlu 2/3 inhibition was previously demonstrated in the FST when rats were tested both acutely and 24 h after administration (99). This synergistic effect was reported to require AMPAR activity since pretreatment with an AMPAR antagonist prevented the effect (100). Our study extends these findings to (2R,6R)-HNK and implicates a non-NMDAR inhibition-mediated synergistic effect. It was previously shown that activation of mGlu 2/3 (64⇓⇓–67) or selective positive allosteric modulation of mGlu 2 receptors (68, 69) can prevent ketamine-induced enhancement of gamma qEEG oscillations. This effect was thought to be due to the antipsychotic actions of mGlu 2/3 receptor activation, since this pharmacological manipulation can prevent excessive glutamate levels in the prefrontal cortex of rodents after administration of NMDAR antagonists, which causes schizophrenia endophenotypes (59, 101, 102). Nevertheless, activation of mGlu 2/3 receptors using the same doses as the ones used to prevent ketamine-induced gamma qEEG oscillations did not prevent ketamine-associated disruption in sensorimotor gating assessed via the prepulse inhibition task in rodents (66, 103, 104). These findings highlight that the actions of mGlu 2/3 activation to reduce ketamine-induced increase in gamma power are unlikely to represent prevention of NMDAR inhibition-mediated psychotomimetic actions. Here, we show that gamma qEEG oscillation enhancement induced by (2R,6R)-HNK is also prevented by pretreatment with an mGlu 2/3 receptor agonist and is absent in mice lacking the mGlu 2 receptor. Therefore, mGlu 2 receptor-dependent modulation of gamma frequency changes induced by (2R,6R)-HNK does not represent a mechanism whereby mGlu 2/3 activation prevents NMDAR inhibition-induced excitation of cortical pyramidal neurons and thus, psychotomimetic effects, but rather, this could be important for (2R,6R)-HNK’s antidepressant mechanism of action. Indeed, pretreatment with an mGlu 2/3 receptor agonist [similar to what was reported for ketamine (54)] or lack of mGlu 2 , but not mGlu 3 , receptors also prevented the antidepressant-relevant actions of (2R,6R)-HNK. This finding supports the hypothesis of a critical convergent mechanism between mGlu 2 receptor signaling and (2R,6R)-HNK effects and suggests mGlu 2 receptor inhibition as a promising target for rapid antidepressant effects. Although a clinical trial (n = 310) assessing the effects of an mGlu 2/3 receptor-negative allosteric modulator (decoglurant) in patients suffering from depression failed to induce antidepressant actions compared with placebo (105), there was no measure of target engagement (such as gamma power) to ensure sufficient drug brain exposure. Taken together, our findings highlight the presence of a convergent mechanism underlying the antidepressant-relevant actions of (2R,6R)-HNK and mGlu 2/3 receptor antagonists and indicate that (2R,6R)-HNK acts in an mGlu 2 receptor-dependent manner to exert these actions. Our data also support high-frequency gamma qEEG power as a marker relevant to the mechanism underlying rapid antidepressant efficacy. Moreover, our data support the use of drugs with mGlu 2 receptor antagonist activity in experimental therapeutic trials either alone or in combination with low doses of (2R,6R)-HNK for treatment-resistant depression.

Materials and Methods Detailed methods are described in SI Appendix. Animals. Male and female CD-1 mice (8–11 wk old at the start of testing; Charles River Laboratories) were housed in groups of four to five per cage with a 12-h light/dark cycle (lights on at 0700 h). Food and water were available ad libitum. For social defeat experiments, 8- to 9-wk-old male C57BL/6J mice (University of Maryland, Baltimore veterinary resources breeding colony) and retired male CD-1 breeders (Charles River Laboratories) were used. Grm2−/− and Grm3−/− mice (bred on a CD-1 background) as previously described by Linden et al. (106) and WT littermate controls were provided from an Eli Lilly Pharmaceuticals colony maintained at Taconic Biosciences. All experimental procedures were approved by the University of Maryland, Baltimore Animal Care and Use Committee and were conducted in full accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (107). Behavioral Assays. Mice were tested in the mGlu 2/3 receptor agonist-induced hyperthermia assay as an in vivo measure of mGlu 2/3 receptor antagonist activity (71). In addition, mice were assessed for behavioral despair in the FST 1 and/or 24 h postinjection (15), for escape deficits after inescapable shock (108), and for sucrose preference deficits after chronic social defeat stress (15). Details are in SI Appendix. Cortical qEEG. Surgeries, recordings, and data analysis for the qEEG studies were performed as previously described (15), with minor modifications. Tissue Distribution and Clearance Measurements of (2R,6R)-HNK. The concentrations of (2R,6R)-HNK in brain tissue were determined by achiral liquid chromatography–tandem mass spectrometry as previously described (15). Statistical Analysis. Statistical analyses were performed using GraphPad Prism software version 6. Holm–Šídák post hoc comparison was applied where ANOVAs reached statistical significance (i.e., P ≤ 0.05). The sample sizes, the specific statistical tests used, and the main effects of our statistical analyses for each experiment are reported in SI Appendix, Table S1. All post hoc comparison results are indicated in the figures. Raw data are provided in Dataset S1.

Acknowledgments This work was supported by Brain & Behavior Research Foundation (NARSAD) Young Investigator Grant 26826 (to P.Z.), NIH Grant MH107615 (to T.D.G.), Veterans Affairs Merit Award 1I01BX004062 (to T.D.G.), and a Harrington Discovery Institute Scholar–Innovator grant (to T.D.G.). The laboratories of C.J.T., R.M., and C.A.Z. are supported by the NIH Intramural Research Program. The contents do not represent the views of the US Department of Veterans Affairs or the US Government.

Footnotes Author contributions: P.Z., P.J.M., C.J.T., C.A.Z., and T.D.G. designed research; P.Z., J.N.H., B.W.S., P.G., C.E.J., J.L., and R.M. performed research; P.J.M. and C.J.T. contributed new reagents/analytic tools; P.Z., J.N.H., B.W.S., P.G., and R.M. analyzed data; and P.Z., J.N.H., P.G., C.A.Z., and T.D.G. wrote the paper.

Conflict of interest statement: P.Z., P.J.M., C.J.T., R.M., C.A.Z., and T.D.G. are listed as coauthors in patent applications related to the pharmacology and use of (2R,6R)-HNK in the treatment of depression, anxiety, anhedonia, suicidal ideation, and posttraumatic stress disorders. R.M. and C.A.Z. are listed as coinventors on a patent for the use of ketamine in major depression and suicidal ideation. T.D.G. has received research funding from Janssen, Allergan, and Roche Pharmaceuticals and was a consultant for FSV7 LLC during the preceding 3 years. All of the other authors report no conflict of interest.

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