(2R, 6R)-hydroxynorketamine ameliorates PTSD-like behaviors during the reconsolidation phase of fear memory in rats by modulating the VGF/BDNF/GluA1 signaling pathway in the hippocampus.

RATIONALE: Fear memory, a fundamental symptom of post-traumatic stress disorder (PTSD), is improved by (2R, 6R)-hydroxynorketamine ((2R, 6R)-HNK) administration. However, the phase of fear memory in which the injected drug is the most effective at mitigating PTSD-like effects remains unknown. OBJECTIVE: This study aimed to explore the effects of (2 R, 6 R)-HNK administration during three phases [acquisition (AP), reconsolidation (RP), and extinction (EP)] on PTSD-like behaviors in single prolonged stress (SPS) and contextual fear conditioning (CFC) rat models. The effects of VGF-inducible type of nerve growth factor (VGF), brain-derived neurotrophic factor (BDNF), and GluA1 on hippocampus (HIP) expression were also explored. METHODS: SPS and CFC (SPSC) were used to establish a PTSD rat model. After lateral ventricle injection of 5 µL (2 R, 6 R)-HNK (0.5 nmol). Anxiety-depression-like behaviors were assessed in rats by the open field test (OFT) and elevated plus maze test (EPMT). Situational fear responses were evaluated in rodents by freezing behavior test (FBT) test. In addition, GluA1, VGF, and BDNF were assessed in the hippocampus by Western blot assay (WB) and Immunohistochemistry assay (IF). RESULTS: SPSC procedure induced PTSD-like behaviors. The SPSC group had decreased spontaneous exploratory behavior and increased fear response. The (2R, 6R)-HNK group showed improved SPSC-induced reduction in GluA1, VGF, and BDNF levels in the HIP. During RP, anxiety and fear avoidance behaviors were alleviated, and the protein levels of GluA1, VGF, and BDNF in the HIP were restored. In contrast, no significant improvement was noted during AP and EP. CONCLUSIONS: (2R,6R)-HNK modulates the VGF/BDNF/GluA1 signaling pathway in the hippocampus and improves PTSD-like behaviors during the reconsolidation phase of fear memory in rats, which may provide a new target for the clinical treatment and prevention of fear-related disorders such as PTSD.

Ketamine and hydroxynorketamine as novel pharmacotherapies for the treatment of Opioid-Use Disorders.

Opioid use disorder (OUD) has reached epidemic proportions, with many countries facing high opioid use and related fatalities. Although currently-prescribed medications for OUD (MOUD) are considered life-saving, they inadequately address negative affect and cognitive impairment, resulting in high relapse rates to non-medical opioid use, even years after drug cessation (protracted abstinence). Evidence supports the notion that ketamine, an anesthetic and rapid-acting antidepressant drug, holds promise as a candidate for OUD treatment, including the management of acute withdrawal somatic symptoms, negative affect during protracted opioid abstinence and prevention of re-taking non-medical opioids. In this review, we comprehensively discuss preclinical and clinical research evaluating ketamine and its metabolites as potential novel therapeutic strategies for treating OUDs. We further examine evidence supporting the relevance of the molecular targets of ketamine and its metabolites in relation to their potential effects and therapeutic outcomes in OUDs. Overall, existing evidence demonstrates that ketamine and its metabolites can effectively modulate pathophysiological processes affected in OUD, suggesting their promising therapeutic role in the treatment of OUD and the prevention of return to opioid use during abstinence.

Study on the antidepressant activity of (2R,6R; 2S,6S)-Hydroxynorketamine (HNK) and its derivatives.

OBJECTIVE: This study mainly explores (2R,6R; 2S,6S)-HNK and its compounds whether there are antidepressant effects. METHODS: Four HNK compounds were obtained from 2-(Chlorophenyl) Cyclopentylmethanone. Forced swimming test, locomotor sensitization test, and conditioned location preference test were used to screen the antidepressant activity of the synthesized target compounds. RESULTS: In the case of 10 mg HNK treatment, compared with saline, the immobile time of mice in the HNK group, I5 group and I6 group at 1 h and 7 days had statistical significance. In the case of 10 mg HNK treatment, compared with saline, the immobile time of compound C and D groups in the glass cylinder area was significantly different. In the locomotor sensitization test, the movement distance of compound C and D groups on day 15 and day 7 mice increased significantly compared with the first day. In the conditioned place preference experiment, compound C and compound D induced conditioned place preference in mice compared with the Veh group. CONCLUSION: The results of the forced swimming test, locomotor sensitization test, and conditioned location preference test showed that compounds C and D may have certain anti-depressant activity. However, HNK exerts a rapid and significant antidepressant effect within 1 week, but the duration is short.

Ketamine metabolism via hepatic CYP450 isoforms contributes to its sustained antidepressant actions.

(R,S)-ketamine (ketamine) has rapid and sustained antidepressant (AD) efficacy at sub-anesthetic doses in depressed patients. A metabolite of ketamine, including (2R,6R)-hydroxynorketamine ((6)-HNKs) has been reported to exert antidepressant actions in rodent model of anxiety/depression. To further understand the specific role of ketamine's metabolism in the AD actions of the drug, we evaluated the effects of inhibiting hepatic cytochrome P450 enzymes on AD responses. We assessed whether pre-treatment with fluconazole (10 and 20 mg/kg, i. p.) 1 h prior to ketamine or HNKs (10 mg/kg, i. p.) administration would alter behavioral and neurochemical actions of the drugs in male BALB/cJ mice with a highly anxious phenotype. Extracellular microdialysate levels of glutamate and GABA (Gluext, GABAext) were also measured in the medial prefrontal cortex (mPFC). Pre-treatment with fluconazole altered the pharmacokinetic profile of ketamine, by increasing both plasma and brain levels of ketamine and (R,S)-norketamine, while robustly reducing those of (6)-HNKs. At 24 h post-injection (t24 h), fluconazole prevented the sustained AD-like response of ketamine responses in the forced swim test and splash test, as well as the enhanced cortical GABA levels produced by ketamine. A single (2R,6R)-HNK administration resulted in prevention of the effects of fluconazole on the antidepressant-like activity of ketamine in mice. Overall, these findings are consistent with an essential contribution of (6)-HNK to the sustained antidepressant-like effects of ketamine and suggest potential interactions between pharmacological CYPIs and ketamine during antidepressant treatment in patients.

The ketamine metabolite (2R,6R)-hydroxynorketamine rescues hippocampal mRNA translation, synaptic plasticity and memory in mouse models of Alzheimer's disease.

INTRODUCTION: Impaired brain protein synthesis, synaptic plasticity, and memory are major hallmarks of Alzheimer's disease (AD). The ketamine metabolite (2R,6R)-hydroxynorketamine (HNK) has been shown to modulate protein synthesis, but its effects on memory in AD models remain elusive. METHODS: We investigated the effects of HNK on hippocampal protein synthesis, long-term potentiation (LTP), and memory in AD mouse models. RESULTS: HNK activated extracellular signal-regulated kinase 1/2 (ERK1/2), mechanistic target of rapamycin (mTOR), and p70S6 kinase 1 (S6K1)/ribosomal protein S6 signaling pathways. Treatment with HNK rescued hippocampal LTP and memory deficits in amyloid-ß oligomers (AßO)-infused mice in an ERK1/2-dependent manner. Treatment with HNK further corrected aberrant transcription, LTP and memory in aged APP/PS1 mice. DISCUSSION: Our findings demonstrate that HNK induces signaling and transcriptional responses that correct synaptic and memory deficits in AD mice. These results raise the prospect that HNK could serve as a therapeutic approach in AD. HIGHLIGHTS: The ketamine metabolite HNK activates hippocampal ERK/mTOR/S6 signaling pathways. HNK corrects hippocampal synaptic and memory defects in two mouse models of AD. Rescue of synaptic and memory impairments by HNK depends on ERK signaling. HNK corrects aberrant transcriptional signatures in APP/PS1 mice.

Loss of the sustained antidepressant-like effect of (2R,6R)-hydroxynorketamine in NMDA receptor GluN2D subunit knockout mice.

Ketamine, an N-methyl-d-aspartate (NMDA) receptor antagonist, has attracted attention for its acute and sustained antidepressant effects in patients with depression. Hydroxynorketamine (HNK), a metabolite of ketamine, exerts antidepressant effects without exerting ketamine's side effects and has attracted much attention in recent years. However, the detailed pharmacological mechanism of action of HNK remains unclear. We previously showed that the GluN2D NMDA receptor subunit is important for sustained antidepressant-like effects of (R)-ketamine. Therefore, we investigated whether the GluN2D subunit is involved in antidepressant-like effects of (2R,6R)-HNK and (2S,6S)-HNK. Treatment with (2R,6R)-HNK but not (2S,6S)-HNK exerted acute and sustained antidepressant-like effects in the tail-suspension test in wildtype mice. Interestingly, sustained antidepressant-like effects of (2R,6R)-HNK were abolished in GluN2D-knockout mice, whereas acute antidepressant-like effects were maintained in GluN2D-knockout mice. When expression levels of GluN2A and GluN2B subunits were evaluated, a decrease in GluN2B protein expression in the nucleus accumbens was found in stressed wildtype mice but not in stressed GluN2D-knockout mice. These results suggest that the GluN2D subunit and possibly the GluN2B subunit are involved in the sustained antidepressant-like effect of (2R,6R)-HNK.

Study on the antidepressant activity of (2R,6R; 2S,6S)-Hydroxynorketamine (HNK) and its derivatives

Abstract Objective: This study mainly explores (2R,6R; 2S,6S)-HNK and its compounds whether there are antidepressant effects. Methods: Four HNK compounds were obtained from 2-(Chlorophenyl) Cyclopentylmethanone. Forced swimming test, locomotor sensitization test, and conditioned location preference test were used to screen the antidepressant activity of the synthesized target compounds. Results: In the case of 10 mg HNK treatment, compared with saline, the immobile time of mice in the HNK group, I5 group and I6 group at 1 h and 7 days had statistical significance. In the case of 10 mg HNK treatment, compared with saline, the immobile time of compound C and D groups in the glass cylinder area was significantly different. In the locomotor sensitization test, the movement distance of compound C and D groups on day 15 and day 7 mice increased significantly compared with the first day. In the conditioned place preference experiment, compound C and compound D induced conditioned place preference in mice compared with the Veh group. Conclusion: The results of the forced swimming test, locomotor sensitization test, and conditioned location preference test showed that compounds C and D may have certain anti-depressant activity. However, HNK exerts a rapid and significant antidepressant effect within 1 week, but the duration is short.

The effects of (2R,6R)-hydroxynorketamine on oxycodone withdrawal and reinstatement.

Despite the thousands of lives lost during the ongoing opioid crisis, a scarcity of new and effective clinical treatments for opioid use disorder (OUD) remains. To address this unmet need, some researchers have turned to dissociative and psychedelic drugs to treat multiple psychiatric conditions. In particular, low doses of ketamine have been shown to attenuate opioid withdrawal and drug use in clinical and preclinical studies. However, ketamine has misuse liability and dissociative side effects that may limit its widespread application as a treatment for OUD. More recently, (2R,6R)-hydroxynorketamine (HNK), a ketamine metabolite that lacks misuse potential, has gained attention for its effectiveness in depression and stress models. To uncover its role in OUD, we tested the time-dependent effects of (2R,6R)-HNK on oxycodone withdrawal and reinstatement of oxycodone conditioned place preference (CPP). In male and female oxycodone-dependent mice, we found that 24h pretreatment with (2R,6R)-HNK (10 or 30mg/kg, s.c.) reduced the frequency of withdrawal-like behaviors and global withdrawal scores during naloxone-precipitated withdrawal, whereas 1h pretreatment with (2R,6R)-HNK only reduced paw tremors and the sum of global withdrawal scores but not GWS Z-scores. In other experiments, both 1h and 24h pretreatment with (2R,6R)-HNK (30mg/kg, s.c.) blocked drug-induced reinstatement of oxycodone CPP. Finally, we found (2R,6R)-HNK (30mg/kg, sc) had no effect on locomotor activity and thigmotaxis. Together, these results indicate that acute (2R,6R)-HNK has efficacy in some preclinical models of OUD without producing locomotor or anxiety-like side effects.

Ketamine metabolite alleviates morphine withdrawal-induced anxiety via modulating nucleus accumbens parvalbumin neurons in male mice.

Opioid withdrawal generates extremely unpleasant physical symptoms and negative affective states. A rapid relief of opioid withdrawal-induced anxiety has obvious clinical relevance but has been rarely reported. We have shown that injection of ketamine metabolite (2R,6R)-hydroxynorketamine (HNK) leads to a rapid alleviation of anxiety-like behaviors in male mice undergoing chronic morphine withdrawal. Here we investigated the contribution of nucleus accumbens shell (sNAc) parvalbumin (PV)-neurons to this process. Chronic morphine withdrawal was associated with higher intrinsic excitability of sNAc PV-neurons via reduced voltage-dependent potassium currents. Chemogenetic inhibition of sNAc PV-neurons reversed the enhanced excitability of PV-neurons and anxiety-like behaviors in these morphine withdrawal male mice, while activation of sNAc PV-neurons induced anxiety-like behaviors in naive male mice. (2R,6R)-HNK reversed the altered potassium currents and intrinsic excitability of sNAc PV-neurons. Our findings demonstrate an important contribution of sNAc PV-neurons to modulating morphine withdrawal-induced anxiety-like behaviors and rapid relief of anxiety-like behaviors by (2R,6R)-HNK, this newly identified target may have therapeutic potentials in treating opioid addiction and anxiety disorders.

(2R,6R)-hydroxynorketamine improves PTSD-associated behaviors and structural plasticity via modulating BDNF-mTOR signaling in the nucleus accumbens.

BACKGROUND: Post-traumatic stress disorder (PTSD) is a mental illness caused by either experiencing or observing a traumatic event that is perceived to pose a serious risk to one's life. (2R,6R)-HNK has an alleviating effect on negative emotions, nevertheless, the mechanism of (2R,6R)-HNK action is unclear. METHODS: In this study, the single prolonged stress and electric foot shock (SPS&S) method was used to establish a rat model of PTSD. After determining the validity of the model, (2R,6R)-HNK was administered to the NAc by microinjection using a concentration gradient of 10, 50, and 100 µM, and the effects of the drug in the SPS&S rat model were evaluated. Moreover, our study measured changes in related proteins in the NAc (BDNF, p-mTOR/mTOR, and PSD95) and synaptic ultrastructure. RESULTS: In the SPS&S group, the protein expression of brain-derived neurotrophic factor (BDNF), mammalian target of rapamycin (mTOR), and PSD95 was reduced and synaptic morphology was damaged in the NAc. In contrast, after the administration of 50 µM (2R,6R)-HNK, SPS&S-treated rats improved their exploration and depression-linked behavior, while protein levels and synaptic ultrastructure were also restored in the NAc. With the administration of 100 µM (2R,6R)-HNK, locomotor behavior, and social interaction improved in the PTSD model. LIMITATIONS: The mechanism of BDNF-mTOR signaling after (2R,6R)-HNK administration was not explored. CONCLUSION: (2R,6R)-HNK may ameliorate negative mood and social avoidance symptoms in PTSD rats by regulating BDNF/mTOR-mediated synaptic structural plasticity in the NAc, providing new targets for the development of anti-PTSD drugs.

(2R,6R)-Hydroxynorketamine Treatment of Rats Exposed to Repetitive Low-Level Blast Injury.

Many military veterans who experienced blast-related traumatic brain injuries (TBIs) in the conflicts in Iraq and Afghanistan suffer from chronic cognitive and mental health problems, including post-traumatic stress disorder (PTSD). Male rats subjected to repetitive low-level blast exposure develop chronic cognitive and PTSD-related traits that develop in a delayed manner. Ketamine has received attention as a treatment for refractory depression and PTSD. (2R,6R)-hydroxynorketamine [(2R,6R)-HNK] is a ketamine metabolite that exerts rapid antidepressant actions. (2R,6R)-HNK has become of clinical interest because of its favorable side-effect profile, low abuse potential, and oral route of administration. We treated three cohorts of blast-exposed rats with (2R,6R)-HNK, beginning 7-11 months after blast exposure, a time when the behavioral phenotype is established. Each cohort consisted of groups (n = 10-13/group) as follows: 1) Sham-exposed treated with saline, 2) blast-exposed treated with saline, and 3) blast-exposed treated with a single dose of 20 mg/kg of (2R,6R)-HNK. (2R,6R)-HNK rescued blast-induced deficits in novel object recognition (NOR) and anxiety-related features in the elevated zero maze (EZM) in all three cohorts. Exaggerated acoustic startle was reversed in cohort 1, but not in cohort 3. (2R,6R)-HNK effects were still present in the EZM 12 days after administration in cohort 1 and 27 days after administration in NOR testing of cohorts 2 and 3. (2R,6R)-HNK may be beneficial for the neurobehavioral syndromes that follow blast exposure in military veterans. Additional studies will be needed to determine whether higher doses or more extended treatment regimens may be more effective.

NMDA Receptor Activation-Dependent Antidepressant-Relevant Behavioral and Synaptic Actions of Ketamine.

Ketamine is a well-characterized NMDA receptor (NMDAR) antagonist, although the relevance of this pharmacology to its rapid (within hours of administration) antidepressant actions, which depend on mechanisms convergent with strengthening of excitatory synapses, is unclear. Activation of synaptic NMDARs is necessary for the induction of canonical long-term potentiation (LTP) leading to a sustained expression of increased synaptic strength. We tested the hypothesis that induction of rapid antidepressant effects requires NMDAR activation, by using behavioral pharmacology, western blot quantification of hippocampal synaptoneurosomal protein levels, and ex vivo hippocampal slice electrophysiology in male mice. We found that ketamine exerts an inverted U-shaped dose-response in antidepressant-sensitive behavioral tests, suggesting that an excessive NMDAR inhibition can prevent ketamine's antidepressant effects. Ketamine's actions to induce antidepressant-like behavioral effects, up-regulation of hippocampal AMPAR subunits GluA1 and GluA2, as well as metaplasticity measured ex vivo using electrically-stimulated LTP, were abolished by pretreatment with other non-antidepressant NMDAR antagonists, including MK-801 and CPP. Similarly, the antidepressant-like actions of other putative rapid-acting antidepressant drugs (2R,6R)-hydroxynorketamine (ketamine metabolite), MRK-016 (GABAAα5 negative allosteric modulator), and LY341495 (mGlu2/3 receptor antagonist) were blocked by NMDAR inhibition. Ketamine acted synergistically with an NMDAR positive allosteric modulator to exert antidepressant-like behavioral effects and activation of the NMDAR subunit GluN2A was necessary and sufficient for such relevant effects. We conclude rapid-acting antidepressant compounds share a common downstream NMDAR-activation dependent effector mechanism, despite variation in initial pharmacological targets. Promoting NMDAR signaling or other approaches that enhance NMDAR-dependent LTP-like synaptic potentiation may be an effective antidepressant strategy.SIGNIFICANCE STATEMENT The anesthetic and antidepressant drug ketamine is well-characterized as an NMDA receptor (NMDAR) antagonist; though, the relevance and full impact of this pharmacology to its antidepressant actions is unclear. We found that NMDAR activation, which occurs downstream of their initial actions, is necessary for the beneficial effects of ketamine and several other putative antidepressant compounds. As such, promoting NMDAR signaling, or other approaches that enhance NMDAR-dependent long-term potentiation (LTP)-like synaptic potentiation in vivo may be an effective antidepressant strategy directly, or acting synergistically with other drug or interventional treatments.

Chiral LC-MS/MS method for the simultaneous determination of (R,S)-ketamine, (R,S)-norketamine, and (2R,6R;2S,6S)-hydroxynorketamine in mouse plasma and brain.

A convenient LC-MS/MS assay method to simultaneously and sensitively determine (R,S)-ketamine (Ket), (R,S)-norketamine (NK), and (2R,6R;2S,6S)-hydroxynorketamine (HNK) enantiomers in plasma and brain from mice was developed. This method enables the chiral separations of these six enantiomers in one analysis by constructing a column-switching system composed of one achiral column and two chiral columns with a relatively short analysis time (17 min). The chromatography involves the separation of (2R,6R;2S,6S)-HNK from (R,S)-Ket and (R,S)-NK on an octadecyl-silica column, followed by chiral separations on a CHIRALPAK AY-RH column for (2R,6R;2S,6S)-HNK or on a CHIRALPAK AS-RH column for the other analytes. The calibration curves for plasma and brain showed a good linearity in the range of 3-1000 ng/mL and 1.5-500 ng/g, respectively. The accuracy ranged from 90.0% to 104.0% in within-run and between-run. This validated method was applicable to determine the stereoselective pharmacokinetic profiles of (R,S)-Ket, (R,S)-NK, and (2R,6R;2S,6S)-HNK in plasma and brain collected from individual mice after a single intraperitoneal dosing of racemic Ket at an antidepressant dose. It is hoped that this assay will greatly help for understanding the relationship between the antidepressant actions of (R,S)-Ket enantiomers or their metabolites and their pharmacokinetics.

Intranasal (2R, 6R)-hydroxynorketamine for acute pain: Behavioural and neurophysiological safety analysis in mice.

Ketamine is known for its antinociceptive effect and is also used for treatment-resistant depression. However, the efficacy and safety of (2R, 6R)-hydroxynorketamine (HNK), a ketamine metabolite has been sparingly investigated for acute pain management. The current study aims at investigating the antinociceptive effect of intranasal (2R, 6R)-HNK using pre-clinical models of acute pain. Additionally, the behavioural and neurophysiological safety analyses were carried out for the effective time window. Antinociceptive efficacy of (2R, 6R)-HNK was evaluated using the hot plate test and Hargreaves' plantar test. The formalin test was carried out in both the acute and tonic phases. The neurophysiological and behavioural safety analyses were carried out separately for the haemodynamic function, cortical electroencephalography (EEG), and spontaneous behavioural functions. Analgesic effect of (2R, 6R)-HNK was evident by a significant increase in paw-withdrawal latency in both Hargreaves' and hot plate tests. Additionally, the (2R, 6R)-HNK showed a significant ameliorative effect on pain-related behaviour in the second phase of the formalin test. (2R, 6R)-HNK exhibited an anxiolytic effect without causing any significant changes in locomotor activity and haemodynamic parameters. Power spectral density (PSD) analysis of electroencephalogram revealed no significant changes except a comparative increase in the gamma band range. Both the locomotor functions in the open field test and the PSD value of delta wave indicated no sedative effect at the given dose of (2R, 6R)-HNK. The results demonstrated the pain-alleviating effect of (2R, 6R)-HNK without compromising the neurophysiological and behavioural function. Therefore, intranasal (2R, 6R)-HNK is suggested as a safe candidate for further clinical study in the management of acute pain.

Enantioselective CE-MS analysis of ketamine metabolites in urine.

The chiral drug ketamine has long-lasting antidepressant effects with a fast onset and is also suitable to treat patients with therapy-resistant depression. The metabolite hydroxynorketamine (HNK) plays an important role in the antidepressant mechanism of action. Hydroxylation at the cyclohexanone ring occurs at positions 4, 5, and 6 and produces a total of 12 stereoisomers. Among those, the four 6HNK stereoisomers have the strongest antidepressant effects. Capillary electrophoresis with highly sulfated γ-cyclodextrin (CD) as a chiral selector in combination with mass spectrometry (MS) was used to develop a method for the enantioselective analysis of HNK stereoisomers with a special focus on the 6HNK stereoisomers. The partial filling approach was applied in order to avoid contamination of the MS with the chiral selector. Concentration of the chiral selector and the length of the separation zone were optimized. With 5% highly sulfated γ-CD in 20 mM ammonium formate with 10% formic acid and a 75% filling the four 6HNK stereoisomers could be separated with a resolution between 0.79 and 3.17. The method was applied to analyze fractionated equine urine collected after a ketamine infusion and to screen the fractions as well as unfractionated urine for the parent drug ketamine and other metabolites, including norketamine and dehydronorketamine.

Ketamine and its metabolites: Potential as novel treatments for depression.

Depression is a well-known serious mental illness, and the onset of treatment using traditional antidepressants is frequently delayed by several weeks. Moreover, numerous patients with depression fail to respond to therapy. One major breakthrough in antidepressant therapy is that subanesthetic ketamine doses can rapidly alleviate depressive symptoms within hours of administering a single dose, even in treatment-resistant patients. However, specific mechanisms through which ketamine exerts its antidepressant effects remain elusive, leading to concerns regarding its rapid and long-lasting antidepressant effects. N-methyl-d-aspartate receptor (NMDAR) antagonists like ketamine are reportedly associated with serious side effects, such as dissociative symptoms, cognitive impairment, and abuse potential, limiting the large-scale clinical use of ketamine as an antidepressant. Herein, we reviewed the pharmacological properties of ketamine and the mechanisms of action underlying the rapid antidepressant efficacy, including the disinhibition hypothesis and synaptogenesis, along with common downstream effector pathways such as enhanced brain-derived neurotrophic factor and tropomyosin-related kinase B signaling, activation of the mechanistic target of rapamycin complex 1 and transforming growth factor ß1. We focused on evidence supporting the relevance of these potential mechanisms of ketamine and its metabolites in mediating the clinical efficacy of the drug. Given its reported antidepressant efficacy in preclinical studies and limited undesirable adverse effects, (R)-ketamine may be a safer, more controllable, rapid antidepressant. Overall, understanding the potential mechanisms of action of ketamine and its metabolites in combination with pharmacology may help develop a new generation of rapid antidepressants that maximize antidepressant effects while avoiding unfavorable adverse effects. This article is part of the Special Issue on 'Ketamine and its Metabolites'.

(2R,6R)-hydroxynorketamine targeting the basolateral amygdala regulates fear memory.

(2R,6R)-Hydroxynorketamine (HNK), a ketamine metabolite, has been proposed as an ideal next-generation antidepressant due to its rapid-acting and long-lasting antidepression-relevant actions. Interestingly, recent studies have shown that (2R,6R)-HNK may have diverse impacts on memory formation. However, its effect on fear memory extinction is still unknown. In the present study, we assessed the effects of (2R,6R)-HNK on synaptic transmission and plasticity in the basolateral amygdala (BLA) and explored its actions on auditory fear memory extinction. Adult male C57BL/6J mice were used in this study. The extracellular electrophysiological recording was conducted to assay synaptic transmission and plasticity. The auditory fear conditioning paradigm was performed to test fear extinction. The results showed that (2R,6R)-HNK at 30 mg/kg increased the number of c-fos-positive cells in the BLA. Moreover, (2R,6R)-HNK enhanced the induction and maintenance of long-term potentiation (LTP) in the BLA in a dose-dependent manner (at 1, 10, and 30 mg/kg). In addition, (2R,6R)-HNK at 30 mg/kg and directly slice perfusion of (2R,6R)-HNK enhanced BLA synaptic transmission. Furthermore, intra-BLA application and systemic administration of (2R,6R)-HNK reduced the retrieval of recent fear memory and decreased the retrieval of remote fear memory. Both local and systemic (2R,6R)-HNK also inhibited the spontaneous recovery of remote fear memory. Taken together, these results indicated that (2R,6R)-HNK could regulate BLA synaptic transmission and plasticity and act through the BLA to modulate fear memory. The results revealed that (2R,6R)-HNK may be a potential drug to treat posttraumatic stress disorder (PTSD) patients.

(2R,6R)-hydroxynorketamine acts through GluA1-induced synaptic plasticity to alleviate PTSD-like effects in rat models.

Post-traumatic stress disorder (PTSD) is a debilitating mental disorder with high morbidity and great social and economic relevance. However, extant pharmacotherapies of PTSD require long-term use to maintain effectiveness and have enormous side effects. The glutamatergic system, especially the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), is an important target of current research on the mechanism of PTSD. Postsynaptic AMPAR function and expression are known to be increased by (2R, 6R)-hydronorketamine (HNK), the primary metabolite of ketamine. However, whether (2R,6R)-HNK alleviates PTSD-like effects via AMPAR upregulation is yet to be known. In the present study, rats were exposed to single prolonged stress and electric foot shock (SPS&S). Afterwards, gradient concentrations of (2R,6R)-HNK (20, 50, and 100 µM) were administered by intracerebroventricular (i.c.v.) injection. Open field, elevated plus maze, freezing behavior, and forced swimming tests were used to examine PTSD-like symptoms. In addition, the protein levels of GluA1, BDNF and PSD-95 were analyzed using western blotting and immunofluorescence, and the synaptic ultrastructure of the prefrontal cortex (PFC) was observed by transmission electron microscopy. We found that (2R,6R)-HNK changed SPS&S-induced behavioral expression, such as increasing autonomous activity and residence time in the open arm and decreasing immobility time. Likewise, (2R,6R)-HNK (50 µM) increased GluA1, BDNF, and PSD-95 protein expression in the PFC. Changes in synaptic ultrastructure induced by SPS&S were reversed by administration of (2R,6R)-HNK. Overall, we find that (2R,6R)-HNK can ameliorate SPS&S-induced fear avoidance in rats, as well as rat cognates of anxiety and depression. This may be related to GluA1-mediated synaptic plasticity in the PFC.

(2R,6R)-hydroxynorketamine (HNK) reverses mechanical hypersensitivity in a model of localized inflammatory pain.

The ketamine metabolite (2R,6R)-hydroxynorketamine, or (2R,6R)-HNK, was recently reported to evoke antinociception in response to a noxious thermal stimulus in healthy mice and reverse mechanical hypersensitivity in a murine model of neuropathic pain. This study reports the behavioral effects of (2R,6R)-HNK in male and female C57BL/6J mice exposed to a localized inflammatory pain condition and the broad pharmacological mechanism underlying this effect. Hind paw intraplantar injection of λ-carrageenan (CARR) caused inflammation and mechanical hypersensitivity in mice within 2 h, lasting at least 48 h. Administration of (2R,6R)-HNK (10-30 mg/kg i.p.) 2 h following CARR injection significantly reversed mechanical hypersensitivity within 1 h in male and female mice, and the effect persisted for 24 h following a single dose. The magnitude and timing of the analgesic effect of (2R,6R)-HNK were comparable to the non-steroidal anti-inflammatory drug carprofen. The reversal of hypersensitivity by (2R,6R)-HNK was blocked at 4 and 24 h after administration by pretreatment with the AMPA receptor antagonist NBQX and was not accompanied by changes in locomotor activity. These findings reinforce the growing evidence supporting (2R,6R)-HNK as a novel analgesic in multiple preclinical pain models and further support an AMPAR-dependent mechanism of action. SIGNIFICANCE: The ketamine metabolite (2R,6R)-HNK reversed mechanical hypersensitivity associated with localized inflammation with onset less than 1 h and duration greater than 24 h, an effect comparable to the NSAID carprofen. Reversal of mechanical hypersensitivity by (2R,6R)-HNK is AMPAR-dependent.

(2R,6R)-hydroxynorketamine rapidly potentiates optically-evoked Schaffer collateral synaptic activity.

(2R,6R)-hydroxynorketamine (HNK) is a metabolite of ketamine that exerts rapid and sustained antidepressant-like effects in preclinical studies. We hypothesize that the rapid antidepressant actions of (2R,6R)-HNK involve an acute increase in glutamate release at Schaffer collateral synapses. Here, we used an optogenetic approach to assess whether (2R,6R)-HNK promotes glutamate release at CA1-projecting Schaffer collateral terminals in response to select optical excitation of CA3 afferents. The red-shifted channelrhodopsin, ChrimsonR, was expressed in dorsal CA3 neurons of adult male Sprague Dawley rats. Transverse slices were collected four weeks later to determine ChrimsonR expression and to assess the acute synaptic effects of an antidepressant-relevant concentration of (2R,6R)-HNK (10 µM). (2R,6R)-HNK led to a rapid potentiation of CA1 field excitatory postsynaptic potentials evoked by recurrent optical stimulation of ChrimsonR-expressing CA3 afferents. This potentiation is mediated in part by an increase in glutamate release probability, as (2R,6R)-HNK suppressed paired-pulse facilitation at CA3 projections, an effect that correlated with the magnitude of the (2R,6R)-HNK-induced potentiation of CA1 activity. These results demonstrate that (2R,6R)-HNK increases the probability of glutamate release at CA1-projecting Schaffer collateral afferents, which may be involved in the antidepressant-relevant behavioral adaptations conferred by (2R,6R)-HNK in vivo. The current study also establishes proof-of-principle that genetically-encoded light-sensitive proteins can be used to investigate the synaptic plasticity induced by novel antidepressant compounds in neuronal subcircuits.