Polymnia Georgiou

NMDAR inhibition-independent antidepressant actions of ketamine metabolites

Major depressive disorder affects around 16 per cent of the world population at some point in their lives. Despite the availability of numerous monoaminergic-based antidepressants, most patients require several weeks, if not months, to respond to these treatments, and many patients never attain sustained remission of their symptoms. The non-competitive, glutamatergic NMDAR (N-methyl-d-aspartate receptor) antagonist (R,S)-ketamine exerts rapid and sustained antidepressant effects after a single dose in patients with depression, but its use is associated with undesirable side effects. Here we show that the metabolism of (R,S)-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-related actions in mice. These antidepressant actions are independent of NMDAR inhibition but involve early and sustained activation of AMPARs (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors). We also establish that (2R,6R)-HNK lacks ketamine-related side effects. Our data implicate a novel mechanism underlying the antidepressant properties of (R,S)-ketamine and have relevance for the development of next-generation, rapid-acting antidepressants.

The oxytocin analogue carbetocin prevents emotional impairment and stress-induced reinstatement of opioid-seeking in morphine-abstinent mice.

The main challenge in treating opioid addicts is to maintain abstinence due to the affective consequences associated with withdrawal which may trigger relapse. Emerging evidence suggests a role of the neurohypophysial peptide oxytocin (OT) in the modulation of mood disorders as well as drug addiction. However, its involvement in the emotional consequences of drug abstinence remains unclear. We investigated the effect of 7-day opioid abstinence on the oxytocinergic system and assessed the effect of the OT analogue carbetocin (CBT) on the emotional consequences of opioid abstinence, as well as relapse. Male C57BL/6J mice were treated with a chronic escalating-dose morphine regimen (20–100 mg/kg/day, i.p.). Seven days withdrawal from this administration paradigm induced a decrease of hypothalamic OT levels and a concomitant increase of oxytocin receptor (OTR) binding in the lateral septum and amygdala. Although no physical withdrawal symptoms or alterations in the plasma corticosterone levels were observed after 7 days of abstinence, mice exhibited increased anxiety-like and depressive-like behaviors and impaired sociability. CBT (6.4 mg/kg, i.p.) attenuated the observed negative emotional consequences of opioid withdrawal. Furthermore, in the conditioned place preference paradigm with 10 mg/kg morphine conditioning, CBT (6.4 mg/kg, i.p.) was able to prevent the stress-induced reinstatement to morphine-seeking following extinction. Overall, our results suggest that alterations of the oxytocinergic system contribute to the mechanisms underlying anxiety, depression, and social deficits observed during opioid abstinence. This study also highlights the oxytocinergic system as a target for developing pharmacotherapy for the treatment of emotional impairment associated with abstinence and thereby prevention of relapse.

Chronic methamphetamine treatment induces oxytocin receptor up-regulation in the amygdala and hypothalamus via an adenosine A2A receptor-independent mechanism.

There is mounting evidence that the neuropeptide oxytocin is a possible candidate for the treatment of drug addiction. Oxytocin was shown to reduce methamphetamine self-administration, conditioned place-preference, hyperactivity and reinstatement in rodents, highlighting its potential for the management of methamphetamine addiction. Thus, we hypothesised that the central endogenous oxytocinergic system is dysregulated following chronic methamphetamine administration. We tested this hypothesis by examining the effect of chronic methamphetamine administration on oxytocin receptor density in mice brains with the use of quantitative receptor autoradiographic binding. Saline (4ml/kg/day, i.p.) or methamphetamine (1mg/kg/day, i.p.) was administered daily for 10 days to male, CD1 mice. Quantitative autoradiographic mapping of oxytocin receptors was carried out with the use of [(125)I]-vasotocin in brain sections of these animals. Chronic methamphetamine administration induced a region specific upregulation of oxytocin receptor density in the amygdala and hypothalamus, but not in the nucleus accumbens and caudate putamen. As there is evidence suggesting an involvement of central adenosine A2A receptors on central endogenous oxytocinergic function, we investigated whether these methamphetamine-induced oxytocinergic neuroadaptations are mediated via an A2A receptor-dependent mechanism. To test this hypothesis, autoradiographic oxytocin receptor binding was carried out in brain sections of male CD1 mice lacking A2A receptors which were chronically treated with methamphetamine (1mg/kg/day, i.p. for 10 days) or saline. Similar to wild-type animals, chronic methamphetamine administration induced a region-specific upregulation of oxytocin receptor binding in the amygdala and hypothalamus of A2A receptor knockout mice and no genotype effect was observed. These results indicate that chronic methamphetamine use can induce profound neuroadaptations of the oxytocinergic receptor system in brain regions associated with stress, emotionality and social bonding and that these neuroadaptations are independent on the presence of A2A receptors. These results may at least partly explain some of the behavioural consequences of chronic methamphetamine use.

Differential regulation of mGlu5 R and ΜOPr by priming- and cue-induced reinstatement of cocaine-seeking behaviour in mice.

The key problem for the treatment of drug addiction is relapse to drug use after abstinence that can be triggered by drug‐associated cues, re‐exposure to the drug itself and stress. Understanding the neurobiological mechanisms underlying relapse is essential in order to develop effective pharmacotherapies for its prevention. Given the evidence implicating the metabotropic glutamate receptor 5 (mGlu5R), μ‐opioid receptor (MOPr), κ‐opioid receptor (ΚOPr) and oxytocin receptor (OTR) systems in cocaine addiction and relapse, our aim was to assess the modulation of these receptors using a mouse model of cue‐ and priming‐induced reinstatement of cocaine seeking. Male mice were trained to self‐administer cocaine (1 mg/kg/infusion, i.v.) and were randomized into different groups: (1) cocaine self‐administration; (2) cocaine extinction; (3) cocaine‐primed (10 mg/kg i.p.); or (4) cue‐induced reinstatement of cocaine seeking. Mice undergoing the same protocols but receiving saline instead of cocaine were used as controls. Quantitative autoradiography of mGlu5R, MOPr, KOPr and OTR showed a persistent cocaine‐induced upregulation of the mGlu5R and OTR in the lateral septum and central amygdala, respectively. Moreover, a downregulation of mGlu5R and MOPr was observed in the basolateral amygdala and striatum, respectively. Further, we showed that priming‐ but not cue‐induced reinstatement upregulates mGlu5R and MOPr binding in the nucleus accumbens core and basolateral amygdala, respectively, while cue‐ but not priming‐induced reinstatement downregulates MOPr binding in caudate putamen and nucleus accumbens core. This is the first study to provide direct evidence of reinstatement‐induced receptor alterations that are likely to contribute to the neurobiological mechanisms underpinning relapse to cocaine seeking.

The oxytocin analogue carbetocin prevents priming-induced reinstatement of morphine-seeking: Involvement of dopaminergic, noradrenergic and MOPr systems.

Relapse to illicit drug-seeking following abstinence is a major challenge for the treatment of addiction as no effective pharmacotherapy is available. We have recently shown that activating the central oxytocinergic system prevents emotional impairment and stress-induced reinstatement associated with opioid withdrawal. Here, we investigated whether the oxytocin analogue carbetocin (CBT) is able to reverse morphine-primed reinstatement of conditioned-place preference (CPP) in mice. The mechanism underlining the behavioural effect of CBT was investigated by assessing the involvement of the striatal noradrenergic and dopaminergic systems in CBT reversal of priming- and stress-induced reinstatement of opioid CPP. In addition, given recent evidence suggesting the presence of oxytocin receptor (OTR)-μ-opioid receptor (MOPr) interactions in the brain, we further explored these interactions by carrying out OTR autoradiographic binding in brain of mice lacking MOPr. CBT administration prevented priming-induced reinstatement of morphine CPP. While an acute effect of CBT in enhancing dopamine turnover was observed following stress- and priming-induced reinstatement, CBT significantly decreased striatal noradrenaline turnover only following priming-induced reinstatement. Moreover, a significant brain region- specific increase in OTR binding was observed in MOPr knockout mice, indicating the presence of a possible OTR-MOPr interaction, which may be involved in the modulation of relapse. These results support the oxytocinergic system as a promising target for the prevention of relapse to opioid use and highlight the differential involvement of monoaminergic systems on the effects of OTR stimulation in preventing stress- and priming-induced reinstatement of opioid CPP behaviour.

Region-specific up-regulation of oxytocin receptor binding in the brain of mice following chronic nicotine administration

Nicotine addiction is considered to be the main preventable cause of death worldwide. While growing evidence indicates that the neurohypophysial peptide oxytocin can modulate the addictive properties of several abused drugs, the regulation of the oxytocinergic system following nicotine administration has so far received little attention. Here, we examined the effects of long-term nicotine or saline administration on the central oxytocinergic system using [(125)I]OVTA autoradiographic binding in mouse brain. Male, 7-week old C57BL6J mice were treated with either nicotine (7.8 mg/kg daily; rate of 0.5 μl per hour) or saline for a period of 14-days via osmotic minipumps. Chronic nicotine administration induced a marked region-specific upregulation of the oxytocin receptor binding in the amygdala, a brain region involved in stress and emotional regulation. These results provide direct evidence for nicotine-induced neuroadaptations in the oxytocinergic system, which may be involved in the modulation of nicotine-seeking as well as emotional consequence of chronic drug use.

A critical role of striatal A2AR–mGlu5R interactions in modulating the psychomotor and drug-seeking effects of methamphetamine

Addiction to psychostimulants is a major public health problem with no available treatment. Adenosine A2A receptors (A2AR) co‐localize with metabotropic glutamate 5 receptors (mGlu5R) in the striatum and functionally interact to modulate behaviours induced by addictive substances, such as alcohol. Using genetic and pharmacological antagonism of A2AR in mice, we investigated whether A2AR–mGlu5R interaction can regulate the locomotor, stereotypic and drug‐seeking effect of methamphetamine and cocaine, two drugs that exhibit distinct mechanism of action. Genetic deletion of A2AR, as well as combined administration of sub‐threshold doses of the selective A2AR antagonist (SCH 58261, 0.01 mg/kg, i.p.) with the mGlu5R antagonist, 3‐((2‐methyl‐4‐thiazolyl)ethynyl)pyridine (0.01 mg/kg, i.p.), prevented methamphetamine‐ but not cocaine‐induced hyperactivity and stereotypic rearing behaviour. This drug combination also prevented methamphetamine‐rewarding effects in a conditioned‐place preference paradigm. Moreover, mGlu5R binding was reduced in the nucleus accumbens core of A2AR knockout (KO) mice supporting an interaction between these receptors in a brain region crucial in mediating addiction processes. Chronic methamphetamine, but not cocaine administration, resulted in a significant increase in striatal mGlu5R binding in wild‐type mice, which was absent in the A2AR KO mice. These data are in support of a critical role of striatal A2AR–mGlu5R functional interaction in mediating the ambulatory, stereotypic and reinforcing effects of methamphetamine but not cocaine‐induced hyperlocomotion or stereotypy. The present study highlights a distinct and selective mechanistic role for this receptor interaction in regulating methamphetamine‐induced behaviours and suggests that combined antagonism of A2AR and mGlu5R may represent a novel therapy for methamphetamine addiction.

Zanos et al . reply

Clinical data have demonstrated rapid and sustained antidepressant effects of ketamine, a noncompetitive NMDAR (N-methyl-daspartate receptor) antagonist1. Recently, Zanos et al.2 claimed that the ketamine metabolite (2R,6R)-hydroxynorketamine (HNK) is essential for the antidepressant effects of ketamine in mice in an NMDAR-independent manner, although no alternative mechanism was proposed, beyond unspecific activation of AMPAR (α -amino-3hydroxy-5-methyl-4-isoxazole propionic acid receptor)2. Here we report that (2R,6R)-HNK blocks synaptic NMDARs in a simi lar manner to its parent compound, and we show that the effects of (2R,6R)-HNK on intracellular signalling are coupled to NMDAR inhibition. These data demonstrate that (2R,6R)-HNK inhibits synaptic NMDARs and subsequently elicits the same signal transduction pathway previously associated with NMDAR inhibition by ketamine. There is a Reply to this Comment by Zanos, P. et al. Nature 546, http://dx.doi.org/10.1038/nature22085 (2017). In previous work3, we showed that ketamine exerts its antidepressant effects by blocking NMDARs at rest, which deactivates eukaryotic elongation factor 2 kinase (eEF2K), thereby dephosphorylating eukaryotic elongation factor 2 (eEF2) and resulting in a subsequent desuppression of brain-derived neurotrophic factor (BDNF) protein translation. This signalling pathway then potentiates synaptic AMPAR responses in the hippocampus through insertion of GluA1 and GluA2 subunits3–5. Notably, Zanos et al. show that (2R,6R)-HNK triggers the same intracellular pathway and downstream effects that we demonstrated for ketamine, namely inhibition of eEF2K, increased expression of BDNF, GluA1 and GluA2, and a form of synaptic potentiation in the hippocampus that is sensitive to AMPAR blockers3–5. The similarity between the molecular findings of ketamine and (2R,6R)-HNK led us to re-examine the potential involvement of (2R,6R)-HNK in NMDAR function. We assessed the effects of (2R,6R)-HNK in NMDAR-mediated miniature excitatory postsynaptic currents (NMDAR-mEPSCs) in cultured hippocampal neurons and compared its properties to the NMDAR antagonists 2R-amino-5-phosphonopentanoate (AP5) and ketamine. NMDA-mEPSCs were isolated in the presence of

Ketamine and Ketamine Metabolite Pharmacology: Insights into Therapeutic Mechanisms

Ketamine, a racemic mixture consisting of (S)- and (R)-ketamine, has been in clinical use since 1970. Although best characterized for its dissociative anesthetic properties, ketamine also exerts analgesic, anti-inflammatory, and antidepressant actions. We provide a comprehensive review of these therapeutic uses, emphasizing drug dose, route of administration, and the time course of these effects. Dissociative, psychotomimetic, cognitive, and peripheral side effects associated with short-term or prolonged exposure, as well as recreational ketamine use, are also discussed. We further describe ketamine’s pharmacokinetics, including its rapid and extensive metabolism to norketamine, dehydronorketamine, hydroxyketamine, and hydroxynorketamine (HNK) metabolites. Whereas the anesthetic and analgesic properties of ketamine are generally attributed to direct ketamine-induced inhibition of N-methyl-D-aspartate receptors, other putative lower-affinity pharmacological targets of ketamine include, but are not limited to, γ-amynobutyric acid (GABA), dopamine, serotonin, sigma, opioid, and cholinergic receptors, as well as voltage-gated sodium and hyperpolarization-activated cyclic nucleotide-gated channels. We examine the evidence supporting the relevance of these targets of ketamine and its metabolites to the clinical effects of the drug. Ketamine metabolites may have broader clinical relevance than was previously considered, given that HNK metabolites have antidepressant efficacy in preclinical studies. Overall, pharmacological target deconvolution of ketamine and its metabolites will provide insight critical to the development of new pharmacotherapies that possess the desirable clinical effects of ketamine, but limit undesirable side effects.

Dopamine and Stress System Modulation of Sex Differences in Decision Making

Maladaptive decision making is associated with several neuropsychiatric disorders, including problem gambling and suicidal behavior. The prevalence of these disorders is higher in men vs women, suggesting gender-dependent regulation of their pathophysiology underpinnings. We assessed sex differences in decision making using the rat version of the Iowa gambling task. Female rats identified the most optimal choice from session 1, whereas male rats from session 5. Male, but not female rats, progressively improved their advantageous option responding and surpassed females. Estrus cycle phase did not affect decision making. To test whether pharmacological manipulations targeting the dopaminergic and stress systems affect decision making in a sex-dependent manner, male and female rats received injections of a dopamine D2 receptor (D2R) antagonist (eticlopride), D2R agonist (quinpirole), corticotropin-releasing factor 1 (CRF1) antagonist (antalarmin), and α2-adrenergic receptor antagonist (yohimbine; used as a pharmacological stressor). Alterations in mRNA levels of D2R and CRF1 were also assessed. Eticlopride decreased advantageous responding in male, but not female rats, whereas quinpirole decreased advantageous responding specifically in females. Yohimbine dose-dependently decreased advantageous responding in female rats, whereas decreased advantageous responding was only observed at higher doses in males. Antalarmin increased optimal choice responding only in female rats. Higher Drd2 and Crhr1 expression in the amygdala were observed in female vs male rats. Higher amygdalar Crhr1 expression was negatively correlated with advantageous responding specifically in females. This study demonstrates the relevance of dopaminergic- and stress-dependent sex differences to maladaptive decision making.