NMDAR inhibition-independent antidepressant actions of ketamine metabolites
This review (2106) explores findings from rodent studies that examined whether a ketamine-metabolite (HNK) with fewer side effects is sufficient to induce antidepressant effects using a range of measurement techniques. Results indicated that the metabolite could exert antidepressant effects through early activation of glutaminergic AMPA receptors, independent of NMDA receptor inhibition typically induced by ketamine.
Abstract
Introduction: Major depressive disorder afflicts ~16 per cent of the world population at some point in their lives. Despite a number of available monoaminergic-based antidepressants, most patients require many weeks, if not months, to respond to these treatments, and many patients never attain sustained remission of their symptoms. The non-competitive glutamatergic N-methyl-D-aspartate receptor (NMDAR) antagonist, (R,S)-ketamine (ketamine), exerts rapid and sustained antidepressant effects following a single dose in depressed patients.Methods: Here we show that the metabolism of 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 actions in vivo.Results: Notably, we demonstrate that these antidepressant actions are NMDAR inhibition-independent but they involve early and sustained α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor activation. We also establish that (2R,6R)-HNK lacks ketamine-related side effects. Our results indicate a novel mechanism underlying ketamine’s unique antidepressant properties, which involves the required activity of a distinct metabolite and is independent of NMDAR inhibition.Discussion: These findings have relevance for the development of next-generation, rapid-acting antidepressants.
Research Summary of 'NMDAR inhibition-independent antidepressant actions of ketamine metabolites'
Introduction
Major depressive disorder is common and existing monoaminergic antidepressants typically require weeks to produce clinical improvement, leaving an unmet need for faster-acting therapies. Sub-anaesthetic doses of (R,S)-ketamine produce rapid and sustained antidepressant effects in treatment-resistant unipolar and bipolar depression, but its clinical use is limited by dissociative side effects and abuse liability. The conventional mechanistic explanation holds that ketamine's antidepressant actions arise from direct inhibition of N-methyl-d-aspartate receptors (NMDARs), yet other selective NMDAR antagonists have failed to reproduce ketamine's robust and enduring clinical profile, prompting uncertainty about the underlying mechanism. Zanos and colleagues set out to test whether metabolism of ketamine to downstream hydroxynorketamine (HNK) metabolites is necessary for its antidepressant-like effects and to characterise the pharmacology of the HNK enantiomers. The study examines behavioural antidepressant readouts in mice, compares stereoisomers and metabolites, probes receptor-level actions (NMDAR versus AMPAR), measures electrophysiological and electroencephalographic biomarkers, and evaluates side-effect and abuse-related outcomes. Their central aim was to determine whether a specific ketamine metabolite can produce antidepressant actions independently of NMDAR inhibition and with an improved side-effect profile.
Methods
This paper reports a preclinical, mechanistic study using mice (and some rat tissue for electrophysiology) combining behavioural pharmacology, pharmacokinetics, receptor binding, electrophysiology, molecular assays and abuse-liability paradigms. Behavioural assays included the forced-swim test (FST) assessed at 1 h (acute) and 24 h (sustained) after treatment, novelty-suppressed feeding (NSF), learned helplessness, chronic social defeat stress with social interaction/avoidance testing, and two models of anhedonia induced by chronic corticosterone (sucrose preference and female urine sniffing). Motor coordination (rotarod), open-field locomotion, pre-pulse inhibition, drug discrimination and intravenous self-administration were used to evaluate side effects and abuse liability. Pharmacological interventions comprised (R,S)-ketamine, its isolated (R)- and (S)-enantiomers, the NMDAR antagonist MK-801, the AMPAR antagonist NBQX, and the HNK metabolites (2R,6R)-HNK and (2S,6S)-HNK. To test the requirement for metabolism, the group synthesised 6,6-dideuteroketamine ((R,S)-d2-KET) expected to slow conversion to HNK without altering parent ketamine pharmacology. Doses reported in the extracted text include ketamine at 10 mg kg-1 for many assays and HNK doses ranging up to 375 mg kg-1 for safety testing; experimental timelines generally assessed behaviour 1 h and 24 h after a single administration. Pharmacokinetic measurements of ketamine and metabolites in plasma and brain were performed by achiral liquid chromatography–tandem mass spectrometry at multiple time points. NMDAR binding used [3H]MK-801 displacement assays on rat brain membranes. Electrophysiological studies included extracellular field recordings of AMPAR-mediated fEPSPs in hippocampal CA1 slices (Schaffer collateral stimulation), and whole-cell patch-clamp recordings from CA1 stratum radiatum interneurons to measure NMDA-evoked currents and spontaneous AMPAR-mediated EPSCs. Quantitative electroencephalography (qEEG) was recorded with frontal electrodes referenced to the cerebellum to measure gamma-band power. Molecular assays on hippocampal and prefrontal cortex synaptoneurosomes included western blots for phospho- and total eEF2, phospho- and total mTOR, BDNF, and AMPAR subunits GluA1 and GluA2. Drug-discrimination procedures trained mice to discriminate ketamine from saline; intravenous self-administration assessed reinforcement of ketamine versus (2R,6R)-HNK using jugular catheters and fixed-ratio schedules. Experimental design elements reported include random assignment and blinded assessment for most assays (exceptions specified for whole-cell patch-clamp and self-administration). Statistical analysis used two-tailed tests with P < 0.05 as significance threshold, ANOVAs with appropriate post-hoc tests when assumptions were met, and non-parametric tests when they were not. For NSF latency analyses, Kaplan–Meier survival analysis was applied.
Results
Behavioural pharmacology: A single administration of (R,S)-ketamine produced a dose-dependent reduction in immobility in the FST at 1 h and 24 h, indicating both acute and sustained antidepressant-like effects, whereas the tricyclic desipramine showed only an acute effect at 1 h. Contrary to what would be expected if NMDAR inhibition were the primary mechanism, (R)-ketamine exhibited greater antidepressant potency than (S)-ketamine across the FST, NSF and learned helplessness tests despite (S)-ketamine being the more potent NMDAR inhibitor. The selective NMDAR antagonist MK-801 did not produce sustained (24 h) antidepressant-like effects in the FST nor did it reverse social avoidance after chronic social defeat. Metabolism and sex differences: Ketamine is metabolised stereoselectively into multiple metabolites including norketamine and several hydroxynorketamines (HNKs). After ketamine administration, the (2S,6S;2R,6R)-HNK pool was the predominant HNK species measured in mouse plasma and brain. Female mice showed greater antidepressant potency of ketamine in the FST and approximately three-fold higher brain levels of the summed (2S,6S;2R,6R)-HNK species compared to males, while brain levels of parent ketamine and norketamine were equivalent between sexes. Role of HNKs and metabolism: Deuteration at C6 (6,6-dideuteroketamine ((R,S)-d2-KET)) substantially hindered metabolism to (2S,6S;2R,6R)-HNK without altering ketamine brain levels or NMDAR binding/hyperlocomotion, and, importantly, (R,S)-d2-KET failed to produce sustained antidepressant actions in FST and learned helplessness tests at 24 h. Direct administration of the isolated HNK enantiomers showed that (2R,6R)-HNK produced more potent and longer-lasting antidepressant-like effects than (2S,6S)-HNK across the FST, learned helplessness and NSF tests. A single (2R,6R)-HNK dose produced effects that persisted at least 3 days in the FST, reversed chronic corticosterone-induced anhedonia (sucrose preference and female urine sniffing) and rescued social interaction deficits after chronic social defeat stress. Receptor pharmacology and electrophysiology: (2R,6R)-HNK did not displace [3H]MK-801 binding in vitro and did not functionally inhibit NMDARs on hippocampal interneurons, indicating a lack of direct NMDAR antagonism. Instead, (2R,6R)-HNK robustly increased AMPAR-mediated synaptic transmission: it enhanced AMPAR-mediated field EPSPs in CA1 slices after Schaffer collateral stimulation and elevated the frequency and amplitude of AMPAR-mediated spontaneous EPSCs in CA1 interneurons. Acute blockade of AMPARs with NBQX administered 10 min before treatment prevented both the 1-h and 24-h antidepressant-like effects of ketamine and (2R,6R)-HNK in the FST. Likewise, administering NBQX 30 min prior to the 24-h FST (that is, after antidepressant treatment) also abolished sustained antidepressant effects, implicating AMPAR activation in both induction and maintenance phases. qEEG and molecular correlates: Surface qEEG recordings showed that (2R,6R)-HNK, like ketamine, acutely increased gamma-band power without affecting other frequency bands, and NBQX pre-treatment prevented this gamma increase. At the molecular level, ketamine decreased phosphorylation of eEF2 in the hippocampus at 1 h and 24 h and increased hippocampal BDNF at 24 h; (2R,6R)-HNK reproduced these hippocampal changes. No consistent changes in mTOR phosphorylation were observed in hippocampus or prefrontal cortex. Both ketamine and (2R,6R)-HNK increased synaptic GluA1 and GluA2 levels in hippocampal synaptoneurosomes at 24 h but not at 1 h, consistent with a delayed upregulation of synaptic AMPARs corresponding to sustained behavioural effects. Safety and abuse liability: (2R,6R)-HNK lacked several ketamine-associated side effects. Unlike ketamine and (2S,6S)-HNK, (2R,6R)-HNK did not increase locomotor activity or impair motor coordination on the rotarod. High doses of (2R,6R)-HNK did not alter sensory gating as measured by pre-pulse inhibition or startle amplitude. In ketamine-trained drug-discrimination assays, (2R,6R)-HNK failed to substitute for ketamine while PCP did. In intravenous self-administration paradigms, mice readily self-administered ketamine but did not self-administer pharmacologically relevant doses of (2R,6R)-HNK, indicating low reinforcement under these conditions. Overall, (2R,6R)-HNK showed antidepressant efficacy in mice with an attenuated side-effect and abuse profile compared to ketamine.
Discussion
Zanos and colleagues interpret their findings to indicate that ketamine's unique antidepressant properties depend on its metabolic conversion to a specific hydroxynorketamine metabolite, most notably the (2R,6R)-HNK enantiomer. They argue that the antidepressant-like actions of (2R,6R)-HNK occur independently of direct NMDAR inhibition, because the metabolite neither displaced MK-801 binding nor inhibited NMDAR-mediated currents, and because a prototypical NMDAR antagonist (MK-801) failed to produce sustained behavioural effects. Instead, the data support a model in which administration of (2R,6R)-HNK causes an acute enhancement of glutamatergic signalling through AMPARs, evidenced by increased AMPAR-mediated EPSPs/EPSCs and elevated gamma power on qEEG, followed by a longer-term adaptation characterised by increased synaptic AMPAR subunit levels and changes in hippocampal eEF2 phosphorylation and BDNF that may underpin sustained antidepressant effects. The investigators emphasise that pharmacological blockade of AMPARs with NBQX prevented both the acute and the sustained antidepressant-like effects, strengthening the claim that AMPAR activation is necessary for both induction and maintenance phases. They also highlight the favourable safety profile of (2R,6R)-HNK relative to ketamine — absence of locomotor activation, sensory-dissociative interoceptive effects and reinforcing self-administration — and propose that these properties make HNKs promising leads for next-generation rapid-acting antidepressants. The authors note that (2R,6R)-HNK produced persistent behavioural effects even at time points when brain concentrations were below detectable levels, and they suggest that synaptic plasticity changes involving AMPARs may account for such long-lasting actions. While the paper focuses on preclinical data in rodents, the authors reference human data showing a positive correlation between plasma (2S,6S;2R,6R)-HNK levels and antidepressant response to ketamine, implying translational relevance. Online Materials and Extended Data are cited for methodological and supplemental detail. The extracted text does not present a distinct limitations paragraph; therefore, specific caveats explicitly acknowledged by the authors beyond those implicit in translational extrapolation are not available in the provided extract.
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demonstrated that the NMDAR antagonist MK-801, which binds at the same receptor site as ketamine, does not exert sustained (24 h) antidepressant-like effects in the FST (Fig.; see also refs 15, 16), or reverse social interaction deficits induced by chronic social defeat stress (Fig.and Extended Data Fig.). These findings indicate a probable NMDAR inhibition-independent mechanism underlying the antidepressant responses of ketamine.
ANTIDEPRESSANT ACTIONS OF KETAMINE METABOLITES
Ketamine is stereoselectively metabolised into a broad array of metabolites, including norketamine, hydroxyketamines, dehydronorketamine and the HNKs(Fig.and Extended Data Fig.). After ketamine administration, (2S,6S;2R,6R)-HNK is the major HNK metabolite found in the plasma and brain of mice (Extended Data Fig.), and plasma of humans. Similar to previous evidence revealing enhanced ketamine antidepressant responses in female rodents compared to males, we observed greater antidepressant potency of ketamine in female mice in the FST (Fig.), which was not associated with sex differences in ketamine-induced hyperlocomotion (probably mediated by NMDAR inhibition; Extended Data Fig.). To investigate whether these sex-dependent antidepressant differences are explained by a different pharmacokinetic profile of ketamine in males versus females, we measured the levels of ketamine and its metabolites in the brains of mice after ketamine administration. While equivalent levels of ketamine and norketamine were found, (2S,6S;2R,6R)-HNK was approximately three-fold higher in the brains of female mice compared to males (Fig.), suggesting a role of (2S,6S;2R,6R)-HNK in the antidepressant effects of ketamine. To directly determine e-g, 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 Tablefor statistical analyses and n numbers). b-e, 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). f-h, 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)-d 2 -KET). i, j, Effects of (R,S)-KET and (R,S)-d 2 -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 Tablefor statistical analyses and n numbers).
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whether metabolism of ketamine to (2S,6S;2R,6R)-HNK is required for its antidepressant actions, we deuterated ketamine at the C6 position (6,6-dideuteroketamine; (R,S)-d 2 -KET, Extended Data Fig.). This alteration would not change the pharmacological properties of unmetabolized ketamine, but may change the relative rate of metabolism. Indeed, 6,6-dideuteroketamine did not change NMDAR binding affinity (Extended Data Fig.), or NMDAR-mediated hyperlocomotion (Extended Data Fig.), but robustly hindered its metabolism to (2S,6S;2R,6R)-HNK, without changing the ketamine levels in the brain (Fig.). Unlike ketamine, administration of 6,6-dideuteroketamine did not induce antidepressant actions in the FST (Fig.) or learned helplessness test (Fig.) 24 h after administration, indicating a role of (2S,6S;2R,6R)-HNK in the sustained antidepressant effects. Notably, published human data reveal a positive correlation between the antidepressant responses of ketamine and plasma (2S,6S;2R,6R)-HNK metabolite levels. To determine whether (2S,6S)-HNK or (2R,6R)-HNK exert antidepressant effects independently of ketamine administration, we compared their behavioural effects in the 24-h (sustained) FST and learned helplessness test. We observed more potent antidepressant effects after administration of the (2R,6R)-HNK metabolite (Fig.), which is exclusively derived from (R)-ketamine, and thus consistent with the greater antidepressant actions of (R)-ketamine relative to (S)-ketamine (Fig.). Moreover, (2R,6R)-HNK resulted in a dosedependent antidepressant action in the learned helplessness test, FST and NSF test (Extended Data Fig.). We note that (2S,6S)-HNK also exerts antidepressant actions at higher doses (Extended Data Fig.). The greater antidepressant effects of (2R,6R)-HNK do not result from higher brain levels of the drug compared to (2S,6S)-HNK (Extended Data Fig.). Similar to ketamine, a single (2R,6R)-HNK administration induced persistent antidepressant effects in the FST, lasting for at least 3 days (Extended Data Fig.). A single (2R,6R)-HNK administration also reversed chronic corticosterone-induced anhedonia assessed with the sucrose preference and female urine sniffing behavioural tasks (Extended Data Fig.), as well as social avoidance induced by chronic social defeat stress (Fig.; Extended Data Fig.).
(2R,6R)-HNK EFFECTS ON GLUTAMATE RECEPTORS
A prominent hypothesis for the mechanism of action of ketamine is that it acts via direct inhibition of NMDARs localized to interneurons. This is suggested to lead to disinhibition of glutamatergic neurons, which receive input from interneurons, and a resultant rapid increase in glutamate synaptic transmission in mood-relevant brain regions. However, in contrast to ketamine, (2R,6R)-HNK does not displace [ 3 H]MK-801 binding to the NMDAR in vitro (Fig.; also see ref. 12) and does not functionally inhibit NMDARs localized to stratum radiatum interneurons in hippocampal slices (Fig.). Instead, (2R,6R)-HNK induced a robust increase in AMPAR-mediated excitatory post-synaptic potentials (EPSPs) recorded from the CA1 region of hippocampal slices after stimulation of Schaffer collateral axons, which was sustained after washout of the drug (Fig.). (2R,6R)-HNK also increased the frequency and amplitude of AMPAR-mediated excitatory postsynaptic currents (EPSCs) recorded from CA1 stratum radiatum interneurons (Extended Data Fig.), which receive glutamatergic inputs from the Schaffer collaterals. To test the extent to which the antidepressant effect of (2R,6R)-HNK depends on AMPAR activation in vivo, mice were pre-treated with the AMPAR antagonist 2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2, 3-dione (NBQX) 10 min before treatment with ketamine or (2R,6R)-HNK. Mice were then assessed in the FST 1 or 24 h after the treatment. 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 Tablefor statistical analyses and n numbers).
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Similar NBQX treatment has previously been shown to prevent the antidepressant actions of ketamine, without affecting other behaviours in rodents. Treatment with NBQX, prior to (2R,6R)-HNK, prevented both the 1-h and 24-h antidepressant effects of (2R,6R)-HNK (Fig.), indicating that its antidepressant actions require the acute activation of AMPARs. A non-invasive method used to assess ketamine-activated circuitry in both humans and rodents is the quantitative electroencephalography (qEEG) measurement of gamma-band power, which is dependent on activation of fast ionotropic excitatory receptors, including AMPARs. We show that, similar to ketamine, (2R,6R)-HNK administration acutely increases gamma power measured via surface electrodes in vivo (Fig.), independent of locomotor activity changes, and without altering alpha, beta, delta or theta oscillations (Extended Data Fig.). Importantly, pre-treatment with NBQX prevented (2R,6R)-HNK-induced increases in gamma power, thus further implicating AMPARs in the (2R,6R)-HNK mechanism of action (Extended Data Fig.), and validating a potential human translational biomarker of the central nervous system response to (2R,6R)-HNK. Evidence indicates that mammalian target of rapamycin (mTOR) signalling, protein synthesis through eukaryotic translation elongation factor 2 (eEF2) dephosphorylation, as well as brain-derived neurotrophic factor (BDNF) increases, underlie the antidepressant responses of ketamine. We examined whether administration of (2R,6R)-HNK affects phosphorylation of mTOR (Ser2448) and eEF2 (Thr56), or BDNF levels in synaptoneurosome fractions of the hippocampus and prefrontal cortex. No differences were observed in mTOR phosphorylation after administration of ketamine or (2R,6R)-HNK in the hippocampus or prefrontal cortex of mice (Extended Data Fig.). However, ketamine induced a decrease in eEF2 phosphorylation in the hippocampus 1 h and 24 h after injection, and increased hippo campal BDNF at 24 h (Fig.). These changes did not occur in the prefrontal cortex (Extended Data Fig.), but were recapitulated by (2R,6R)-HNK administration (Fig.), and may be partially responsible for its sustained antidepressant actions. It is noteworthy that (2R,6R)-HNK resulted in antidepressant actions (Fig.; Extended Data Fig.) at time points (for example, 24 h) past when its brain concentrations are below detectable levels (for example, 2 h; Extended Data Fig.). Synaptic plasticity changes involving AMPARs are thought to underlie such long-term antidepressant actions of ketamine. Here we show that while neither ketamine nor (2R,6R)-HNK administration altered the levels of AMPAR subunits GluA1 and GluA2 in hippocampal synaptoneurosomes 1 h after treatment (Fig.), they both increased GluA1 and GluA2 levels 24 h after treatment in mouse hippocampal (Fig.), but not prefrontal cortex synaptoneurosomes (Extended Data Fig.). Consistent with an increase in synaptic AMPARs being involved in the sustained, 24-h, antidepressant actions, administration of NBQX 30 min prior to the 24-h FST (23.5 h after antidepressant treatment; see timeline Fig.) prevented the antidepressant actions of both ketamine and (2R,6R)-HNK (Fig.). These findings implicate an AMPAR-mediated maintenance of synaptic potentiation to underlie the sustained antidepressant effects of (2R,6R)-HNK.
(2R,6R)-HNK LACKS KETAMINE-RELATED SIDE EFFECTS
Ketamine has abuse potential, as well as sensory-dissociation properties and other side effects, which limit its potential widespread use for the treatment of depression 9 . While administration of ketamine (Extended Data Fig.) and (2S,6S)-HNK (Fig.) were associated with increased locomotor activity and motor incoordination (Fig.), (2R,6R)-HNK did not induce any significant changes in locomotion, and did not affect coordination as measured by the accelerating rotarod test (Fig.). We show that unlike ketamine, (2R,6R)-HNK
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administration, even at high doses (375 mg kg -1 ), did not affect sensory gating as assessed with pre-pulse inhibition (Fig.) or startle amplitude (Extended Data Fig.). Non-competitive NMDAR antagonists, including ketamine and phencyclidine, produce discriminative stimulus effects in drug discrimination protocols and manifest crossdrug substitution profiles at an antidepressant-relevant dose range. In ketamine-trained mice, (2R,6R)-HNK administration did not produce ketamine-related discrimination responses, whereas phencyclidine (PCP) did (Fig.), without either of these drugs changing overall lever pressing response rates (Extended Data Fig.). These findings further support a non-NMDAR mechanism for (2R,6R)-HNK action including interoceptive effects, unlike the abused drugs ketamine and PCP. Since drug discrimination does not independently predict abuse potential per se, we further assessed the effects of ketamine and (2R,6R)-HNK in an intravenous drug self-administration model, classically used for the evaluation of abuse/addiction liability. Intravenous ketamine was readily self-administered and resulted in a significant increase in drug intake (Fig.; Extended Data Fig.). By contrast, mice did not self-administer pharmacologically relevant doses of (2R,6R)-HNK under the same conditions (Fig.; Extended Data Fig.). Overall, (2R,6R)-HNK administration revealed an innocuous side-effect profile compared to ketamine.
DISCUSSION
Our data provide new evidence explaining the unique antidepressant effects of ketamine and implicate an NMDAR inhibition-independent mechanism. These findings reveal that production of a distinct metabolite of ketamine is necessary and sufficient to produce the ketamine antidepressant actions. Overall, our data indicate that administration of (2R,6R)-HNK induces an acute increase in glutamatergic signalling (as supported by our EPSP, EPSC and qEEG measurements), followed by a long-term adaptation involving the upregulation of synaptic AMPARs, as evidenced by an increase in GluA1 and GluA2 in hippocampal synapses. This is supported by the finding that NBQX reverses both the acute (delivered before (2R,6R)-HNK) (Fig.) and sustained (delivered after (2R,6R)-HNK; Fig.) antidepressant actions of (2R,6R)-HNK. Considering the lack of side effects, and the favourable physiochemical properties of HNKs 33 , these findings have relevance for the development of next-generation, rapid-acting antidepressants. Online Content Methods, along with any additional Extended Data display items and Source Data, are available in the online version of the paper; references unique to these sections appear only in the online paper. All drugs were dissolved in 0.9% saline, and administered intraperitoneally (i.p.) in a volume of 7.5 ml kg -1 of body mass by a male experimenter for the behavioural studies. Corticosterone (4-pregnen-11β, 21-diol-3, 20-dione 21-hemisuccinate; Steraloids) was dissolved in tap water. For the electrophysiology recordings, test drugs were diluted in ACSF. FST. Mice were tested in the FST 1 h and/or 24 h after injection. During the test, mice were subjected to a 6-min swim session in clear Plexiglass cylinders (30 cm height × 20 cm diameter) filled with 15 cm of water (23 ± 1 °C). The test was performed in normal light conditions (800 Lx). Sessions were recorded using a digital video camera. Immobility time, defined as passive floating with no additional activity other than that necessary to keep the animal's head above the water, was scored for the last 4 min of the 6-min test by a trained observer. We conducted three different FST experiments where we used the AMPAR antagonist NBQX. In the first two experiments, we administered NBQX 10 min before ketamine, (2R,6R)-hydroxynorketamine, or vehicle and then tested the mice 1 h and 24 h later to assessed whether AMPAR activity is necessary for the acute actions of these drugs, leading to both acute and sustained antidepressant effects. In the third experiment we first administered ketamine, (2R,6R)-HNK, or vehicle and 23.5 h later mice received either NBQX or vehicle. Thirty minutes later we tested these mice in the FST, to assess effects of AMPAR activity on sustained antidepressant actions. Open-field test. Mice were placed into individual open-field arenas (50 × 50 × 38 cm (length × width × height); San Diego Instruments) for a 60-min habituation period. Mice then received an injection of the respective drug and assessed for locomotor activity for another 60 min. Distance travelled was analysed using TopScan v2.0 (CleverSys, Inc.). NSF test. Mice were singly housed and food-deprived for 24 h in freshly made home-cages. Two normal chow diet pellets were placed on an inverted weighingboat platform (10 × 10 × 1.5 cm) in the centre of an open-field arena (40 × 40 cm). Thirty or sixty minutes (see figure legends) after drug administration, mice were introduced into a corner of the arena. The time needed for the mice to take a bite of food was recorded over a 10-min period by a trained observer. After the test, the mice were returned to their home cage containing pre-weighed food pellets, and latency to start biting the pellet, as well as consumption was recorded for a period of 10 min. There was no significant change in home cage latency or consumption in any of our experiments (data not shown). Learned helplessness test. The learned helplessness model consisted of three different phases: inescapable shock training, learned helplessness screening, and the test. For the inescapable shock portion of the test (day 1), the animals were placed in one side of two-chambered shuttle boxes (34 × 37 × 18 cm (height × width × depth); Coulbourn Instruments), with the door between the chambers closed. After a 5-min adaptation period, 120 inescapable foot-shocks (0.45 mA, 15 s duration, randomized average inter-shock interval of 45 s) were delivered through the floor. During the screening session (day 2), the mice were placed in one of the two chambers of the apparatus for 5 min. A shock (0.45 mA) was then delivered, and the door between the two chambers was raised simultaneously. Crossing over into the second chamber terminated the shock. If the animal did not cross over, the shock terminated after 3 s. A total of 30 screening trials of escapable shocks were presented to each mouse with an average of 30-s delay between each trial. Mice that developed helplessness behaviour (>5 escape failures during the last 10 screening shocks) received the assigned drug 24 h after screening (day 3). During the learned helplessness test phase (day 4), the animals were placed in the shuttle boxes and, after a 5-min adaptation period, a 0.45-mA shock was delivered concomitantly with door opening for the first five trials, followed by a 2-s delay for the next 40 trials. Crossing over to the second chamber terminated the shock. If the animal did not cross over to the other chamber, the shock was terminated after 24 s. A total of 45 trials of escapable shocks were presented to each mouse with 30-s inter-trial intervals. The number of escape failures was recorded for each mouse by automated computer software (Graphic State v3.1; Coulbourn Instruments). Chronic social defeat stress and social interaction. The timeline for the social defeat experiments is presented in Extended Data Fig.. Male C57BL/6J mice underwent a 10-day chronic social defeat stress model, as described elsewhere, with some modifications. In brief, experimental mice were introduced to the home cage (43 × 11 × 20 cm (length × width × height)) of a resident aggressive retired CD-1 breeder, pre-screened for aggressive behaviours, for 10 min. After this physical attack phase, mice were transferred and housed in the opposite side of the resident's cage divided by a Plexiglas perforated divider, to maintain continuous sensory contact. This process was repeated for 10 days. Experimental mice were introduced to a novel aggressive CD-1 mouse each day. On day 11, test mice were screened for susceptibility in a social interaction/avoidance choice test. The social interaction apparatus consisted of a rectangular three-chambered box (mouse conditioned-place preference chamber; Stoelting Co., see Extended Data Fig.) containing two equal sized end-chambers and a smaller central chamber. The social interaction/avoidance choice test consisted of two 5-min phases. During the habituation phase, mice explored the empty apparatus. During the test phase, two small wire cages (Galaxy Cup, Spectrum Diversified Designs, Inc.), one containing a 'stranger' CD-1 mouse and the other one empty, were placed in the far corners of each chamber. The time spent interacting (nose within close proximity of the cage) with the stranger mouse versus the empty cage was analysed using TopScan video tracking software (CleverSys). Locomotor activity (total distance moved over 5 min) and number of total crosses into and out of the central chamber were also measured. The social interaction ratio was calculated by dividing the time spent interacting with the stranger by the time spent with the empty cage. Mice having a social interaction ratio higher than 1.0 were considered resilient, and mice with a social interaction ratio lower than 1.0 were considered susceptible. On day 13 resilient and susceptible mice received an i.p. injection of saline, (R,S)-KET (20 mg kg -1 ; chosen based on dose previously effective in C57BL/6J mice), MK-801 (0.1 mg kg -1 ) or (2R,6R)-HNK (20 mg kg -1 ). Mice were re-tested for social interaction/avoidance on day 15 (24 h after treatment). Chronic corticosterone-induced anhedonia. Sucrose preference test. For assessing the baseline sucrose preference, mice were singly housed for 24 h and presented with two identical bottles containing either tap water or 1% sucrose solution. After baseline sucrose measurement, mice were re-group housed (5 mice per cage) and treated for 4 weeks with corticosterone (25 μg ml -1 equivalent) given in water bottles. Before initiation of any behavioural measurements, animals were weaned off corticosterone treatment; 3 days corticosterone 12.5 μg ml -1 and 3 days corticosterone 6.25 μg ml -1 , followed by 1 week of complete withdrawal. Mice were subsequently singly housed in freshly made home cages and provided with two bottles containing either tap water or 1% sucrose solution. Twenty-four hours later, mice that developed the anhedonia phenotype (<70% sucrose preference) were treated with saline or (2R,6R)-HNK (10 mg kg -1 ) and sucrose preference was measured after an additional 24 h. Female urine sniffing test. A separate cohort of mice was treated with the same chronic corticosterone administration model as described above but assessed for female urine sniffing preference as a measure of hedonic behaviour. Mice were singly housed in freshly made home cages for a habituation period of 10 min. Subsequently, one plain cotton tip was secured on the centre of the cage wall and mice were allowed to sniff and habituate to the tip for a period of 30 min. Then, the plain cotton tip was removed and replaced by two cotton tip applicators, one infused with fresh female mouse oestrus urine and the other with fresh male mouse urine. These applicators were presented and secured at the two corners of the cage wall simultaneously. Sniffing time for both female and male urine was scored by a trained observer for a period of 3 min. Twenty-four hours later, mice that developed the anhedonia phenotype (<65% female urine preference; susceptible phenotype), as well as mice that did not develop the anhedonia phenotype (>75% female urine preference; resilient phenotype) were treated with either saline or (2R,6R)-HNK (10 mg kg -1 ). Mice were re-tested for female urine preference 24 h later. Pre-pulse inhibition. Mice were individually tested in acoustic startle boxes (SR-LAB, San Diego Instruments). After drug administration, mice were placed in the startle chamber for a 30-min habituation period. The experiment started
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with a further 5-min adaptation period during which the mice were exposed to a constant background noise (67 dB), followed by five initial startle stimuli (120 dB, 40 ms duration each). Subsequently, animals were exposed to four different trial types: pulse alone trials (120 dB, 40 ms duration), three pre-pulse trials of 76 and 81 of white noise bursts (20 ms duration) preceding a 120 dB pulse by 100 ms, and background (67 dB) no-stimuli trials. Each of these trials was randomly presented five times. The dose of ketamine (30 mg kg -1 ) was selected based on a dose-response experiment we performed in a previous study. The percentage pre-pulse inhibition was calculated using the following formula: ((magnitude on pulse-alone trialmagnitude on pre-pulse + pulse trial)/magnitude on pulsealone trial) × 100. Drug discrimination. Mice were food-restricted until they reached 85% of their initial body weight and were maintained at 85% throughout the duration of the experiment. Animals were trained to lever press for food (20 mg sucrose pellets; TestDiet) in standard two-lever operant conditioning chambers (Coulbourn Instruments), under a fixed-ratio 5 schedule of reinforcement (FR5) in daily 30-min sessions. After stable responding was maintained over three consecutive sessions, mice were trained to discriminate ketamine (10 mg kg -1 ) from saline under a double alternation schedule (that is, ketamine, ketamine, saline, saline), which required on average 40 training sessions. Mice received either ketamine (10 mg kg -1 ; i.p.) or saline 15 min before the start of the 30-min session. Responding to the correct lever resulted in the delivery of a reward, while incorrect responding reset the FR for correct lever-responding. Drug discrimination test sessions were conducted when mice reached the following criteria: (1) first FR5 completed on the correct lever, and (2) ≥85% correct lever responding over the entire session. Fifteen minutes prior to the 30-min test sessions mice received either saline, ketamine (10 mg kg -1 ), PCP (3 mg kg -1 ) or (2R,6R)-HNK (10 and 50 mg kg -1 ). At this stage, completion of the FR5 schedule on either lever resulted in the delivery of food reward. Lever response and pellet delivery were monitored and controlled by an automated computer system (Graphic State v3.1; Coulbourn Instruments). Intravenous drug self-administration. Apparatus. Each operant chamber was equipped with one lever, a dipper liquid delivery system, a 22-gauge liquid swivel and a syringe pump (located outside the chamber). The lever was a balanced rocker arm that broke an infrared photo beam when 0.5 g of force was applied. Two stimulus lights were used; one was positioned to illuminate the translucent lever and the other was positioned above the liquid delivery recess. The lever light was illuminated during periods of water or drug availability; the second light was illuminated during water or drug delivery. The system was controlled by an integrated Coulbourn environmental control system and Med Associates interface. Water training. Mice were first trained to complete an operant response for water reinforcement. Completion of the response requirements on the lever illuminated stimulus lights above a spout and delivered a small amount of water. Initially, the response requirement was one lever press (FR1); after completion of each 50 reinforcements the fixed ratio requirement was increased by one (FRX + 1). Mice were trained for 24 h per day for 4 days with free access to food. Surgery. After completion of the water training, mice were surgically prepared with a catheter implanted in the jugular vein. Surgical procedures were performed under ketamine-(90 mg kg -1 , i.p.) and xylazine-(16 mg kg -1 , i.p.) induced anaesthesia. Silastic tubing (0.012 inch (0.30 mm) inner diameter) was implanted in the right jugular vein to the level of the atrium, passed subcutaneously and exited in the midscapular region. The catheter was connected to a tether/swivel system that was mounted to the skull of the mouse with dental cement. Intravenous drug self-administration. Seven days after surgery, mice were placed in the operant chamber and given access (FR4) to 0.32, 1.0, 3.2 or 0 (saline) mg kg -1 drug per infusion for 5 days at each dose. Completion of each FR resulted in the illumination of the overhead house light and the stimulus light above the spout. Infusions of 5-8 μl (based on body weight) were given over a period of 15 s. A 30-s time-out period, during which house and stimulus lights were out, followed the completion of each infusion. Each mouse had access to drug for 6 h per day and free access to food and water 24 h per day. A 12-h light/dark cycle was maintained (lights on/off at 07:00/19:00). Each animal remained in its operant chamber for the duration of the experiment. A stimulus light illuminating the lever signalled drug availability. Only those animals with patent catheters at the end of the experiment were included in the analysis. The average number of reinforcements and drug intake during the last 3 days at each dose were used as dependent measures. Rotarod. The rotarod test was conducted to compare the effects of ketamine, (2S,6S)-HNK and (2R,6R)-HNK on motor coordination. The experiment consisted of two phases: training phase (4 days) and a test phase (1 day). On each of the training days five trials (trial time: 3 min) were conducted with an inter-trial interval of two min. Mice were individually placed on the rotarod apparatus (IITC Life Science) and the rotor (3.75 inch diameter) accelerated from 5 to 20 r.p.m. over a period of three minutes. Latency to fall was recorded for each trial. Animals with an average of <100 s of latency to fall during the last training day were excluded from the experiment. On the test day (day 5), mice received (i.p.) injections of saline, (R,S)-KET (10 mg kg -1 ), (2S,6S)-HNK (25 or 125 mg kg -1 ) or (2R,6R)-HNK (25 or 125 mg kg -1 ) and were tested in the rotating rod 5, 10, 15, 20, 30 and 60 min after injection using the same procedure described for the training days.
TISSUE DISTRIBUTION AND CLEARANCE MEASUREMENTS OF KETAMINE AND METABOLITES.
At 10, 30, 60, or 240 min after drug administration mice were exposed to 3% isoflurane and subsequently decapitated. Trunk blood was collected in EDTA-containing tubes and centrifuged at 5,938g for 6 min (4 °C). Plasma was collected and stored at -80 °C until analysis. Whole brains were simultaneously collected, rinsed with PBS, immediately frozen in dry ice and stored at -80 °C until analysis. The concentrations of (R,S)-ketamine and 6,6-dideuteroketamine and their respective metabolites in plasma and brain tissue were determined by achiral liquid chromatography-tandem mass spectrometry following a previously described method, with slight modifications. The analysis was accomplished using an Eclipse XDB-C18 guard column (4.6 × 12.5 mm) and a Varian Pursuit XRs 5 C18 analytical column (250 × 4.0 mm ID, 5 μm; Varian). The mobile phase consisted of ammonium acetate (5 mM, pH 7.6) as component A and acetonitrile as component B. A linear gradient was run as follows: 0 min 20% B; 5 min 20% B; 15 min 80% B; 20 min 20% B at a flow rate of 0.4 ml min -1 . The total run time was 30 min per sample. For plasma and brain samples, the calibration standards ranged from 10,000 ng ml -1 to 19.5 ng ml -1 for (R,S)-ketamine, (R,S)-norketamine, (2R,6R;2S,6S)-HNK, (R,S)-dehydronorketamine, (R,S)-d 2 -ketamine, (R,S)-d 2norketamine and d-(2S,6S;2R,6R)-HNK. The quantification of (R,S)-ketamine, (R,S)-d 2 -ketamine and their respective metabolites was accomplished by calculating area ratios using d 4 -ketamine (10 μl of 10 μg ml -1 solution) as the internal standard. The MS/MS analysis was performed using a triple quadrupole mass spectrometer model API 4000 system from Applied Biosystems/MDS Sciex equipped with Turbo Ion Spray (TIS) (Applied Biosystems). The data was acquired and analysed using Analyst version 1.4.2 (Applied Biosystems). Positive electrospray ionization data were acquired using multiple reaction monitoring (MRM) using MK-801 displacement binding. NMDAR binding assays were performed according to ref. 37, with minor modifications. Rat brains were homogenized and membrane fractions were collected. Aliquoted membranes were stored at -80 °C until use. Membrane pellets were washed five times with an ice-cold buffer (20 mM HEPES, 1 mM EDTA, pH 7.0) before use. The binding assays were set up in 96-well plates using 5 nM [ 3 H]MK-801 and rat brain membranes (100 μg per well) in a final volume of 125 μl per well in the NMDAR binding buffer (20 mM HEPES, 1 mM EDTA, 100 μM glutamate, 100 μM glycine, pH 7.0). Test compounds were first distributed in 96-well plates (25 μl per well at 5× final concentrations ranging from 0.1 nM to 10 μM, 11 points) in triplicate. The radioligand, [ 3 H]MK-801, was added (50 μl per well at 2.5× of final concentration of 5 nM) to all wells. Reactions started with the addition of 50 μl rat brain membrane and were incubated for 1 h in the dark at room temperature. The reactions were harvested via rapid filtration onto Whatman GF/B glass fibre filters pre-soaked with 0.3% polyethyleneimine using a 96-well Brandel harvester, followed by three quick washes each with 500 μl chilled wash buffer (50 mM Tris HCl, pH 7.4). Filters were microwave-dried and scintillation cocktail was then melted onto the filter mates on a hot plate. The radioactivity retained on the filters was counted in a MicroBeta scintillation counter. All assays were performed in triplicates. Western blots. To purify synaptoneurosomes, mouse prefrontal cortex and hippocampi were dissected and homogenized in Syn-PER Reagent (ThermoFisher Scientific; 87793) with 1× protease and phosphatase inhibitor cocktail (ThermoFisher Scientific; 78440). The homogenate was centrifuged for 10 min at 1,200g at 4 °C. The supernatant was centrifuged at 15,000g for 20 min at 4 °C. After centrifugation, the pellet (synaptosomal fraction) was re-suspended and sonicated in N-PER Neuronal Protein Extraction Reagent (ThermoFisher Scientific; 87792). Protein concentration was determined via the BCA protein assay kit (ThermoFisher Scientific; 23227). Equal amount of proteins (10-40 μg as optimal for each antibody) for each sample was loaded into NuPage 4-12% Bis-Tris gel for electrophoresis. Gel transfer was performed with the TransBlot Turbo Transfer System (Bio-Rad) Nitrocellulose membranes with transferred proteins were blocked with 5% milk in TBST (TBS plus 0.1% Tween-20) for 1 h and kept with primary antibodies overnight at 4 °C. The following primary antibodies were used: phospho-eEF2 (at Thr56; Cell Signaling Technology; 2331), total eEF2 (Cell Signaling Technology; 2332), phospho-mTOR (at Ser2448; Cell Signaling Technology; 2971), total mTOR (Cell Signaling Technology; 2983), GluA1 (Cell Signaling Technology; 2983), GluA2 (Cell Signaling Technology; 13607), BDNF (Santa Cruz Biotechnology; sc-546), and GAPDH (Abcam; ab8245). The next day, blots were washed three times in TBST and incubated with horseradish peroxidase conjugated anti-mouse or anti-rabbit secondary antibody (1:5,000 to 1:10,000) for 1 h. After three final washes with TBST, bands were detected using enhanced chemiluminescence (ECL) with the Syngene Imaging System (G:Box ChemiXX9). After imaging, the blots were incubated in the stripping buffer (ThermoFisher Scientific; 46430) for 10-15 min at room temperature followed by three washes with TBST. The stripped blots were incubated in blocking solution for 1 h and incubated with the primary antibody directed against total levels of the respective protein or GAPDH for loading control. Densitometric analysis of phospho-and total immunoreactive bands for each protein was conducted using Syngene's GenTools software. The values for the phosphorylated forms of proteins were normalized to phosphorylationindependent levels of the same protein. Phosphorylation-independent levels of proteins were normalized to GAPDH. Fold change was calculated by normalization to saline-treated control group for each protein or phosphoprotein. qEEG. Surgery. qEEG experiments were performed according to ref. 38, with minor modifications. Mice were anaesthetized with isoflurane (3.5%) and maintained under anaesthesia (2-2.5%) throughout the surgery. Mice received analgesia (carprofen, 5 mg kg -1 , i.p.) before the start of surgery. An F20-EET radio-telemetric transmitter (Data Sciences International) was placed subcutaneously and its leads implanted over the dura above the frontal cortex (1.7 mm anterior to bregma) and the cerebellum (6.4 mm posterior to bregma). Animals recovered from surgery for 7 days before recordings. qEEG recordings. For comparisons between saline, ketamine, and (2R,6R)-HNK, mice were singly housed and acclimated to the behavioural room for 24 h before qEEG recordings. qEEGs were recorded using the Dataquest A.R.T. acquisition system (Data Sciences International) with frontal qEEG recordings referenced to the cerebellum. Baseline qEEG (30 min) recordings were followed by an i.p. injection of saline, ketamine (10 mg kg -1 ) or (2R,6R)-HNK (10 mg kg -1 ) and a further 60 min of post-injection recordings. To assess effects of NBQX on (2R,6R)-HNK-induced changes in qEEG oscillations, mice were acclimated to the behavioural room 1.5-2 h before recordings. Baseline (30 min) recordings were followed by an i.p. injection of either saline or NBQX (10 mg kg -1 ) and 30 min later mice received an injection (i.p.) of (2R,6R)-HNK (10 mg kg -1 ) and recordings continued for 60 min after injection. In vivo data analysis. qEEGs were analysed using custom-written MATLAB scripts (Version 2012a, Mathworks) and the mtspecgramc routine in the Chronux Toolbox (). Oscillation power in each bandwidth (delta = 1-3 Hz; theta = 4-7 Hz; alpha = 8-12 Hz; beta = 13-29 Hz; gamma = 30-80 Hz) was computed in 10-min bins from spectrograms for each animal. Field recordings. Removal of the rat brains, as well as dissection of the hippocampi were performed in ice-cold ACSF bubbled with 95% O 2 , 5% CO 2 . The ACSF contained (in mM): 124 NaCl, 3 KCl, 1.25 NaH 2 PO 4 , 1.5 MgCl 2 , 2.5 CaCl 2 , 26 NaHCO 3 and 10 glucose. Hippocampal slices were cut at 400 μm using a vibratome and kept in a holding chamber at the interface of ACSF and humidified 95% O 2 , 5% CO 2 for at least 1 h. For the fEPSPs, slices were transferred to a submersion-type recording chamber and perfused with ACSF (1-2 ml min -1 ; room temperature). Picrotoxin (0.1 mM), CGP52432 (2 μM) and APV (80 μM) were added to block GABA A , GABA B and NMDA receptors respectively. Concentric bipolar tungsten electrodes were placed in stratum radiatum to stimulate the Schaffer collateral afferents. Extracellular recording pipettes were filled with ACSF (3-5 MΩ) and placed in stratum radiatum of area CA1. Field potentials were evoked by monophasic stimulation (100 μs duration) at 0.1 Hz. The stimulus intensity was set at 150% of threshold intensity, resulting in fEPSPs amplitude of 0.1-0.3 mV. A stable baseline was recorded for at least 10 min. Vehicle and (2R,6R)-HNK were applied by perfusion over a period of 1 h followed by washout using ACSF. For AMPARmediated responses, peak fEPSP amplitudes and slopes, measured over a window of 1-4 ms following the rising phase of the response, are reported as percentage change from baseline. DNQX (50 μM) was bath-applied to ensure AMPA-mediated responses. Experiments were performed and analysed blind to treatment groups, using pCLAMP software (Molecular Devices). Whole-cell patch-clamp recordings. Rats were euthanized by CO 2 asphyxiation followed by decapitation. Removal of the brains, as well as dissection and slicing of the hippocampi were performed in an ice-cold solution consisting of a mixture of equal parts of regular ACSF and sucrose-containing ACSF. ACSF was composed of (in mM): 125 NaCl, 26 NaHCO 3 , 2.5 KCl, 1.25 NaH 2 PO 4 , 2 CaCl 2 , 1 MgCl 2 , and 25 glucose. Sucrose-containing ACSF was composed of (in mM): 230 sucrose, 2.5 KCl, 1.25 NaH 2 PO 4 , 26 NaHCO 3 , 0.5 CaCl 2 , 10 MgSO 4 , and 10 glucose. Hippocampal slices of 300-μm thickness were cut using a vibratome (Leica VT1000S; Leica Microsystems Inc.) and transferred to an immersion chamber containing regular ACSF that was continuously bubbled with 95% O 2 , 5% CO 2 and maintained in a water bath at 30 °C. At the time of recordings, hippocampal slices were transferred to a 1-ml recording chamber, where they were superfused at 2 ml min -1 with ACSF that was continuously bubbled with 95% O 2 , 5% CO 2 . In all experiments, ACSF used to superfuse the slices contained the muscarinic antagonist atropine (0.5 μM) and the GABA A receptor antagonist picrotoxin (50 μM). Whole-cell patch-clamp recordings were obtained from the soma of CA1 stratum radiatum interneurons in hippocampal slices according to standard patch-clamp techniques using an EPC9 amplifier (HEKA Elektronik). The signals were filtered at 3 kHz and analysed using pCLAMP 10.3 or WinEDR v3.2.6 (University of Strathclyde, UK). The patch-clamp pipettes were pulled from a borosilicate glass capillary (1.2-mm OD) and had resistances between 3 and 5 MΩ when filled with internal solution. The internal pipette solution contained (in mM): 10 ethylene-glycol-bis(β-aminoethylether)-N-N′-tetraacetic acid, 10 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 130 Cs-methane sulfonate, 10 CsCl, 2 MgCl 2 , 5 lidocaine N-ethyl bromide, and 0.5% biocytin (pH adjusted to 7.3 with 340 mOsm CsOH). A specially adapted U-tube developed in the Albuquerque laboratory was used to apply NMDA (50 μM) to the neurons for the NMDA-evoked current experiments. NMDA-evoked currents and spontaneous AMPA-mediated excitatory postsynaptic currents (sEPSCs) were recorded at -40 mV and -60 mV respectively. A single neuron was studied in each slice. All experiments were carried out at room temperature (20-22 °C). The peak amplitude of NMDA-evoked currents was analysed using the pCLAMP v10.3 software, with baseline determined as the mean value obtained before drug application and the final (16 min) washout time point. Frequency and peak amplitude of AMPA sEPSCs were analysed using WinEDR v3.2.6. Statistical analyses. Required sample sizes were estimated based on our past experience performing similar experiments. Experimentation and analysis were performed in a manner blinded to treatment assignments in all experiments with the exception of the whole-cell patch-clamp recordings and intravenous drug self-administration. For all blinded experiments, mice were randomly assigned to treatment groups. Statistical analyses were performed using GraphPad Prism software v6. By pre-established criteria, values greater than ±2 s.d. from individual group means were excluded from the analyses. All statistical tests were two-tailed, and significance was assigned at P < 0.05. Normality and equal variances between group samples were assessed using the Kolmogorov-Smirnov and Brown-Forsythe tests respectively. When normality and equal variance between sample groups was achieved, ANOVAs were followed by a Holm-Šídák post-hoc comparison when significance was reached, and significant results are indicated with asterisks in the figures. As a secondary analysis, pairwise comparisons at each equivalent dose were performed followed by multiple comparison corrections, where appropriate, and are reported in Supplementary Information Table. Where normality or equal variance of samples failed, non-parametric one-way ANOVAs (Kruskal-Wallis one-way ANOVA on ranks or Friedman repeated-measures one-way ANOVA on ranks) were performed, followed by Dunn's correction. For assessment of the NSF test results, Kaplan-Meier survival analysis was used followed by the Mantel-Cox log-rank test. The sample sizes (biological replicates), specific statistical tests used, and the main effects of our statistical analyses for each experiment are reported in Supplementary Information Table. Extended Data Figure| 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-6hydroxyketamine ((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.
SOCIAL INTERACTION
(test for social avoidance reversal) a Extended Data Figure| 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 Tablefor statistical analyses and n numbers.
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Extended Data Figure| Acute and sustained antidepressant and antianhedonic 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 1and 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 Tablefor statistical analyses and n numbers).
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