Ketamine: A tale of two enantiomers

1There are significant limitations to current widely prescribed antidepressant treatments. These include a significant delay in the onset of therapeutic action (weeks to months) and approximately one-third of patients with major depressive disorder (MDD) failing to demonstrate an adequate response. For individuals with depression, particularly if suffering from suicidal ideation, these time lags and resistance to standard treatments can be extremely harmful.

2Increasing evidence has revealed that the dissociative anaesthetic ketamine, an uncompetitive N-Methyl-D-Aspartate (NMDA) receptor antagonist, has the potential to overcome such limitations, demonstrating rapid antidepressant and anti-suicidal effects, even in treatment-resistant patients. It has been proposed that ketamine’s antidepressant effects are primarily mediated through NMDA receptor antagonism, resulting in disinhibition of pyramidal cells and an acute cortical glutamate surge, with downstream effects on synaptogenesis and neuroplastic pathways. However, the precise molecular and cellular processes underlying ketamine’s antidepressant effects are still not clear and evidence suggests that mechanisms other than NMDA receptor inhibition play a more crucial role in the antidepressant effects of ketamine, its component enantiomers and metabolites.

3In this review, we summarise findings on the antidepressant effects of ketamine and its enantiomers. We then discuss underlying therapeutic mechanisms, exploring the case that ketamine’s enantiomers and metabolites may produce complementary antidepressant effects via distinct mechanisms, before considering future directions of enquiry.

4Ketamine is a racemic mixture that consists of equal amounts of two enantiomers, (S)-ketamine and (R)-ketamine (or esketamine and arketamine) (Figure 1). (S)-ketamine has a three to fourfold greater binding affinity for the NMDA receptor than (R)-ketamine (Ki = 0.30 μM and Ki = 1.4 μM respectively). In humans, (S)-ketamine is more potent than (R)-ketamine both as an anaesthetic and as an analgesic, which is putatively explained by its higher affinity for the NMDA receptor. It was argued that because of its increased potency, lower doses of (S)-ketamine could be used in anaesthesia/analgesia with faster recovery times and therefore potentially some diminution in dissociative and psychotomimetic side effects. However, direct comparative studies of (S)- and (R)-ketamine have suggested otherwise. In one study, higher rates of psychotomimetic side effects were seen in an (S)-ketamine treated group, despite the dose of (S)-ketamine being lower than (R)-ketamine (0.45 mg/kg and 1.8 mg/kg respectively). Furthermore, a healthy volunteer study from found that although (S)-ketamine administration produced acute psychosis-like reactions (ego-dissolution, illusions and hallucinations, thought disorders, paranoid ideations), in the same individuals (R)-ketamine did not produce any psychotic symptoms but instead led to a state of relaxation and a feeling of wellbeing.

5(S)-ketamine and (R)-ketamine both undergo extensive metabolism by cytochrome P450 enzymes to corresponding forms of norketamine, dehydronorketamine (DHNK), hydroxyketamine (HK) and hydroxynorketamines (HNKs) (Figure 2). (S)-ketamine or (R)-ketamine is first demethylated by either CYP3A4 or CYP2B6 to (S)-norketamine or (R)-norketamine. (S)-norketamine or (R)-norketamine are subsequently metabolised to (S)-DHNK or (R)-DHNK or HNKs. Hydroxylation of (S)-norketamine or (R)-norketamine by CYP2A6 at the six position results in (2S,6S)-HNK and (2R,6R)-HNK respectively, which are the major HNK metabolites found in plasma following ketamine infusion. CYP2A6 can also directly hydroxylate (S)-ketamine or (R)-ketamine to form (2S,6S)-HK and (2R,6R)-HK, which are further transformed to (2S,6S)-HNK or (2R,6R)-HNK. Of these metabolites, (2R,6R)-HNK and (S)-norketamine have attracted particular interest as candidate antidepressants in their own right.

6(R,S)-ketamine: In the first double-blind, placebo-controlled study of racemic ketamine in MDD, it was demonstrated that a single sub-anaesthetic intravenous (IV) infusion (0.5 mg/kg over 40 mins) resulted in rapid antidepressant effects, within hours of administration. A number of subsequent studies have also demonstrated the rapid-acting antidepressant effects of ketamine in treatment-resistant unipolar and bipolar depression. Findings have been reviewed in several meta-analyses, which report robust antidepressant and anti-suicidal effects, lasting up to 1 week, in treatment-resistant MDD and bipolar depression, with acute dissociative symptoms being the most commonly reported side effect.

7Unfortunately, the antidepressant effect of a single dose of ketamine is not generally sustained beyond 1 week. In the first randomised controlled trial (RCT) of repeated ketamine administration, it was shown that twice- or thrice-weekly administration of IV ketamine (0.5 mg/kg over 40 mins) was sufficient to maintain antidepressant efficacy over 15 days in individuals with treatment-resistant depression (TRD). Similar findings have also been reported in open-label repeated infusion studies. In contrast, a recent RCT investigating the effects of six ketamine infusions (0.5 mg/kg over 45 mins) or saline placebo over 3 weeks in severe TRD with current, chronic suicidal ideation failed to demonstrate a significant difference in depression severity or suicidality at the 3 week endpoint. However, this study was limited by a small sample size (out of 26 randomised patients, n=13 per group, only 14 completed the entire study) and therefore may have been underpowered to detect a true difference between treatment groups. In addition, all patients were maintained on their medication regimes throughout the infusion phase and the impact that concomitant medications may have had on ketamine’s effects cannot be ruled out.

8Although there are specialist centres around the world and an increasing number of ketamine clinics in the United States offering ketamine infusions for depression, the use of repeated infusions may not be the most practical due to the resources required. Other routes of administration (oral, sublingual, intranasal, intramuscular or subcutaneous) could prove to be simpler and more feasible alternatives for repeated administration but few studies have evaluated these options.

9(S)-ketamine: As NMDA receptor antagonism was understood to play a key role in ketamine’s antidepressant mechanism, (S)-ketamine was investigated as a novel antidepressant candidate by Janssen Research & Development due to its higher affinity for the NMDA receptor. In a first proof-of-concept trial, IV (S)-ketamine at doses of 0.2 mg/kg and 0.4 mg/kg led to rapid and robust antidepressant effects in individuals with TRD. Side effects included headache, nausea and dissociation. It was suggested that as improvements in depressive symptoms were not significantly different between the two tested doses that a lower dose of (S)-ketamine may allow for better tolerability while maintaining efficacy.

10A fixed-dose (S)-ketamine nasal spray has subsequently been developed and tested in TRD. A number of Phase II and III trials have shown that intranasal (S)-ketamine plus an existing or newly initiated oral antidepressant outperforms placebo plus an oral antidepressant for individuals with TRD, although others failed to demonstrate positive results. In a large discontinuation study, 297 individuals with TRD who met response or remission criteria following 16 weeks of treatment with intranasal (S)-ketamine (56 mg or 84 mg twice weekly) plus an oral antidepressant were entered into a randomised withdrawal phase (to continue with (S)-ketamine or switch to placebo). Those randomised to continue treatment with intermittently administered (S)-ketamine nasal spray plus an oral antidepressant had a significantly delayed time to relapse compared to those treated with placebo nasal spray and oral antidepressants. A subsequent open-label study has examined the long-term safety of (S)-ketamine nasal spray plus a new oral antidepressant in patients with TRD. Common treatment-emergent adverse events included dizziness, dissociation, nausea and headache, which mostly occurred on dosing days, were mild to moderate in severity and resolved on the same day. Longitudinal analysis showed dissociative symptoms declined over subsequent administrations and cognitive performance was generally found to either improve or remained stable compared with baseline. Similar long-term maintenance or safety evidence of this level are not available for (R,S)- or (R)-ketamine at this time.

11Considering the available evidence, the United States Food and Drug Administration and European Medicines Agency have approved the (S)-ketamine nasal spray, SpravatoTM, for adults with TRD in combination with an oral antidepressant. However, some questions remain regarding uncertainty of efficacy, safety, potential for abuse and need for careful monitoring, which currently limit wider use.

12(R)-ketamine: Preclinical findings have suggested (R)-ketamine has the potential for more potent and longer-lasting antidepressant effects than both ketamine and (S)-ketamine and it appears to have less behavioural side-effects and abuse liability. Given initial findings from in healthy subjects, where (R)-ketamine did not produce psychosis-like symptoms as seen with (S)-ketamine, but instead feelings of relaxation and wellbeing, researchers have now begun to explore the antidepressant potential of (R)-ketamine in humans.

13In the first open-label pilot study of (R)-ketamine, seven subjects with TRD received a single IV infusion of (R)-ketamine at a dose of 0.5 mg/kg over 40 mins. Mean Montgomery-Åsberg Depression Rating Scale scores dropped significantly from 30.7 at baseline to 10.4 at day 1 after the infusion, with 71% of subjects showing an antidepressant response at day 1 and 57% at day 7. Interestingly, dissociation was nearly absent with minimal haemodynamic effects. However, it should be noted that five out of the seven patients in this study were taking antipsychotic medications (quetiapine (three), risperidone (one), aripiprazole (one)) that might have resulted in lower blood pressure or less dissociation. Naturally, the results of this small open-label study must be interpreted with caution. A clinical trial is underway by Perception Neuroscience to further investigate safety and tolerability of differing doses of (R)-ketamine in healthy volunteers before exploring its potential in depression (Universal Trial Number: U1111-1241-1005). In addition, a large trial comparing the efficacy and safety of (R)-ketamine with (S)-ketamine and (R,S)-ketamine in TRD is already underway in China (ChiCTR1800015879).

14It has been widely acknowledged that the rapid antidepressant effects of ketamine are mediated through blockade of NMDA receptors located on γ-aminobutyric acid (GABA)-ergic inhibitory interneurons. This in turn leads to a disinhibition of pyramidal cells and an acute cortical glutamate surge. Subsequent activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors appears to play a key role in the antidepressant effects as demonstrated by preclinical work that has shown pre-treatment with an AMPA receptor antagonist blocks the antidepressant effects of ketamine and its enantiomers in rodent models.

15It has been reported that the metabolism of (R,S)-ketamine to HNK, is required for ketamine’s antidepressant-like effects in rodents. Administration of the (R)-enantiomer (2R,6R)-HNK was associated with greater and longer-lasting antidepressant effects than (2S,6S)-HNK and MK-801, a more potent NMDA receptor antagonist. Importantly, (2R,6R)-HNK lacked ketamine-related side-effects and abuse potential in this model. Furthermore, the antidepressant effects were independent of any action on NMDA receptors but instead required AMPA receptor activation. Subsequent work has also suggested an important role of presynaptic group II metabotropic glutamate (mGlu2) autoreceptor inhibition in the antidepressant actions of (2R,6R)-HNK. Although findings with regards to antidepressant-like effects of (2R,6R)-HNK in rodents have been replicated by other independent laboratories, other studies from one laboratory were unable to reproduce this and instead suggest unmetabolised (R)-ketamine itself may be responsible for the antidepressant actions. Clinical trials have not yet examined the utility of (2R,6R)-HNK as a rapid-acting antidepressant. However, clinical studies investigating ketamine metabolite plasma levels as biomarkers have found that higher (2R,6R)-HNK levels were associated with less improvement in depressive symptoms, which is counterintuitive considering preclinical findings. Regardless, (2R,6R)-HNK, with its potential to modulate mGlu2 and AMPA receptor function, remains a promising candidate antidepressant and work to validate this compound for clinical use is ongoing at the United States National Institute for Mental Health.

16(S)-ketamine is primarily metabolised to (S)-norketamine and it has been shown in an animal model that although the antidepressant actions of the metabolite are similarly potent to the parent compound, (S)-norketamine appeared to lack the associated side effects. Importantly, the findings of this study suggest that AMPA receptor activation is not necessary for the antidepressant actions of (S)-nortketamine as AMPA receptor antagonists did not block its antidepressant effects, instead highlighting a role for brain-derived neurotrophic factor (BDNF), tropomyosin kinase B (TrkB) and mechanistic target of rapamycin complex (mTORC) signalling. There is, however, some recent clinical evidence that found no relationship between norketamine concentration (neither (S)-norketamine nor (R)-norketamine) and antidepressant response, following administration of (R,S)-ketamine to individuals with TRD.

17The preferential action of (R,S)-ketamine at GABAergic interneurons is supported by findings that the NMDA receptor antagonist MK-801 initially inhibits the firing of fast-spiking GABAergic interneurons and, at a delayed rate, increases the firing rate of pyramidal neurons. have since demonstrated that perfusion of hippocampal rat brain slices with (R,S)-ketamine enhances the excitability of pyramidal cells indirectly by reducing synaptic GABAergic inhibition, thus causing disinhibition. A recent study found that knockdown of a key NMDA receptor subunit, GluN2B, on GABAergic interneurons resulted in a significant increase (disinhibition) of spontaneous excitatory postsynaptic currents on layer V pyramidal cells in mouse brain slices. Moreover, knockdown of GluN2B on GABAergic interneurons but not pyramidal cells of the medial prefrontal cortex (mPFC) had antidepressant-like effects and occluded or blocked the antidepressant behavioural effects of (R,S)-ketamine. Further supporting this disinhibition hypothesis, administration of negative allosteric modulators of GABAA receptors (GABA-NAMs) exert rapid antidepressant actions similar to (R,S)-ketamine in animal models, likely through disinhibition of excitatory glutamatergic neurotransmission. Although GABAergic interneuron inhibition via NMDA receptors appears to serve as an important initial target of (R,S)-ketamine, further work is needed to determine if this mechanism is as relevant for each of ketamine’s enantiomers and metabolites.

18BDNF and its receptor TrkB have been consistently implicated in the aetiology of depression and mechanism of action of current antidepressants. BDNF serves a key a role in processes including neuronal maturation, synapse formation and synaptic plasticity. Findings from preclinical work suggest BDNF-TrkB signalling in the hippocampus and prefrontal cortex to be a critical component of antidepressant response to conventional antidepressants. The rapid antidepressant-like effects of (R,S)-ketamine have been shown in one preclinical study to depend on the rapid synthesis of BDNF. In this study, (R,S)-ketamine was shown to rapidly increase TrkB phosphorylation, an indicator of TrkB activation, in the hippocampus, suggesting BDNF-TrkB signalling in this brain region is also involved in the antidepressant response to ketamine. This is in agreement with previous work showing the acute antidepressant effects of (R,S)-ketamine administration are associated with increased BDNF protein levels in the hippocampus.

19In the study by, the ketamine-mediated suppression of resting NMDA receptor activity was also shown to deactivate eukaryotic elongation factor 2 (eEF2) kinase, resulting in reduced eEF2 phosphorylation and augmentation of BDNF synthesis. Subsequent findings confirmed the importance of this signalling pathway in the antidepressant response to ketamine as eEF2 kinase knockout mice, administered an acute low dose of (R,S)-ketamine did not show an antidepressant response or an increase in BDNF protein expression in the hippocampus. In addition to the hippocampus, BDNF in the mPFC may also be an important site of action as preclinical work has found that an infusion of a BDNF neutralising antibody into the mPFC abolishes ketamine’s antidepressant-like effects. Additional preclinical work demonstrated that (R,S)-ketamine-induced antidepressant effects are associated with upregulation of BDNF and mTORC in the hippocampus and prefrontal cortex, mediated by AMPA receptors.

20Considering the individual enantiomers, in a chronic social defeat stress and learned helplessness models of depression, a TRkB antagonist was able to block the antidepressant effects of both (S)-ketamine and (R)-ketamine. Interestingly, (R)-ketamine induced greater effects on reduced dendritic spine density, BDNF-TrkB signalling and synaptogenesis in the prefrontal cortex and hippocampus compared with (S)-ketamine and (R)-ketamine showed a greater potency and longer-lasting antidepressant effect than (S)-ketamine in this model.

21The mammalian target of rapamycin complex 1 (mTORC1) and extracellular signal-regulated kinase (ERK) are key signalling molecules in pathways that regulate protein synthesis with roles in synaptic development and plasticity. The function of mTORC1 and ERK in the antidepressant actions of ketamine and its enantiomers are not completely clear. Work in rodents initially demonstrated that (R,S)-ketamine rapidly activated the mTORC pathway, leading to increased synaptic signalling proteins and synaptic spine density. Furthermore, intracerebroventricular administration of an mTORC1 inhibitor, rapamycin, has been shown to block ketamine-induced synaptogenesis and antidepressant-like effects. Other work has shown that (R,S)-ketamine administration did not alter levels of phosphorylated mTOR in the hippocampi of control or BDNF-knockout mice, neither were the antidepressant effects of (R,S)-ketamine blocked by intraperitoneally administered rapamycin. However, this study did report reduced phosphorylation of ribosomal protein s6 kinase in brain tissues, a pharmacodynamic readout of mTORC1 inhibition, following rapamycin administration. One explanation for the failure of rapamycin to block the antidepressant effects of (R,S)-ketamine in the study by is that the peripheral route of administration may not have achieved sufficient central nervous system exposure compared to studies where intracortical rapamycin administration resulted in adequate mTORC1 inhibition to block (R,S)-ketamine’s antidepressant effects.

22Further preclinical work demonstrated that the antidepressant effects of (S)-ketamine but not (R)-ketamine were blocked by mTORC1 inhibition and that (S)-ketamine, but not (R)-ketamine, significantly attenuated decreased phosphorylation of mTOR in the prefrontal cortex of mice in a chronic social defeat stress model. This same study showed that pre-treatment with an ERK inhibitor blocked the antidepressant effects of (R)-ketamine but not (S)-ketamine and, furthermore, (R)-ketamine but not (S)-ketamine significantly attenuated the reduced phosphorylation of ERK in the prefrontal cortex and hippocampi of susceptible mice using the same model. It is interesting to note that the antidepressant effects of (S)-norketamine, (S)-ketamine’s predominant metabolite, have also been shown to be blocked by the mTORC1 inhibitor rapamycin. Taken together this suggests that mTORC1 has a role in antidepressant effects of (S)-ketamine but less so for (R)-ketamine and that ERK activation could instead mediate the antidepressant effects of (R)-ketamine (Figure 3).

23In a recent randomised double-blind cross-over study, explored whether pre-treatment with rapamycin could attenuate the rapid antidepressant effects of (R,S)-ketamine in individuals with MDD. Surprisingly, rapamycin did not alter the acute antidepressant effects of (R,S)-ketamine but instead prolonged the antidepressant effects. Two weeks after ketamine administration there were significantly higher response and remission rates following rapamycin + ketamine compared to placebo + ketamine. The authors hypothesised that the failure to block ketamine’s effects by rapamycin may have been due to the dosage used and peripheral route of administration, mirroring the findings from preclinical work that also utilised peripheral rapamycin administration. A key difference between this human work and the preclinical work, however, is follow-up time. None of the ketamine + rapamycin animal studies discussed could have discovered findings as shown in the study by as antidepressant effects were not followed up for long enough. also hypothesised that rapamycin may extend the antidepressant effects of ketamine via an mTORC1-dependent anti-inflammatory mechanism, protecting newly made synapses from inflammatory processes that cause synaptic elimination and undermine the antidepressant effects of ketamine, or by enhancing autophagy (a crucial mTORC1 regulated process involved in normal cellular plasticity, which involves degrading and recycling toxic or dysfunctional cellular components). Alternatively, it is possible to speculate rapamycin may have mTORC1-independent effects that contribute to the antidepressant effects of ketamine. For example, rapamycin may attenuate an unacknowledged homeostatic mechanism that normally contributes to relapse. A final speculative consideration is whether rapamycin may promote longer-lasting antidepressant effects of (R)-ketamine via increased ERK signalling as low concentrations of rapamycin have been shown to increase Akt and ERK activation in vitro through an mTORC1-dependent mechanism. Although interesting, these preliminary findings should be interpreted with caution and replication in future studies is needed, before back translation to animal studies, alongside work to determine any differential effects of rapamycin on each of ketamine’s enantiomers.

24Activation of mTORC1 signalling has been linked to phosphorylation (deactivation) of glycogen synthase kinase-3 (GSK-3) and inhibition of GSK-3 has been shown to be necessary for the rapid antidepressant-like effects of (R,S)-ketamine in mice. Furthermore, administration of (R,S)-ketamine in combination with lithium, a non-selective GSK-3 inhibitor, resulted in rapid activation of the mTORC1 signalling pathway, increased inhibitory phosphorylation of GSK-3, increased synaptic spine density and potentiated antidepressant-like responses in rodents. Ketamine-induced inhibition of GSK-3 has also been linked to AMPA receptor upregulation and stabilisation at the cell surface. In a preclinical study it was demonstrated that (R,S)-ketamine-induced inhibition of GSK-3 resulted in reduced phosphorylation of post-synaptic density-95 (which regulates AMPA receptor trafficking), diminishing the internalisation of AMPA GluA1 subunits. This could ultimately allow for augmented signalling through AMPA receptors following ketamine treatment.

25In clinical work, testing the GSK-3 inhibition hypothesis, lithium continuation therapy showed no benefit over placebo at 2 weeks following the cessation of four (R,S)-ketamine infusions in individuals with TRD. In patients with treatment-resistant bipolar depression, maintained on either therapeutic-dose lithium or valproate before receiving (R,S)-ketamine versus placebo, a significant improvement in depressive symptoms was seen in both mood stabiliser groups, and although ketamine’s antidepressant effect size relative to placebo was larger for lithium (d=2.27) than valproate (d=0.79), there was no significant difference observed between these two agents. Furthermore, neither serum lithium nor valproate levels correlated with ketamine’s antidepressant efficacy.

26Although some evidence highlights GSK-3 as an important regulatory target for ketamine’s antidepressant effects, clinical studies have not yet confirmed preclinical findings. Further evaluation of the role of GSK-3 in the antidepressant effects of ketamine’s individual enantiomers and metabolites is still required.

27An increase in intracellular cyclic adenosine monophosphate (cAMP) that acts to upregulate neurotrophic factors and increase synaptogenesis has been associated with conventional antidepressant action. Gαs is a subunit of the G protein GS that stimulates the generation of cAMP by activating adenylyl cyclase. Localisation of Gαs within lipid raft microdomains in the plasma membrane acts to regulate cellular signalling, and indeed production of cAMP is diminished when Gαs is localised to lipid raft microdomains. A number of preclinical studies have demonstrated increases in cAMP through translocation of Gαs from lipid raft domains into non-raft regions, augmenting interaction between Gαs and adenylyl cyclase, following administration of various classes of antidepressants.

28have since reported that (R,S)-ketamine administration to C6 glioma cells led to immediate translocation of Gαs from lipid raft domains to non-raft domains and an increase in cAMP, followed by an increase in BDNF expression after 24 hours. The (R,S)-ketamine induced increase in cAMP was found to persist after knocking out the NMDA receptor indicating an NMDA receptor-independent mechanism. Further, administration of the ketamine metabolite (2R,6R)-HNK also resulted in redistribution of Gαs from lipid rafts and an increase cAMP production. These findings suggest the translocation of Gαs from lipid rafts is a reliable hallmark of antidepressant action; however, further research is needed to examine to what degree this mechanism contributes to the antidepressant effect of the individual enantiomers of ketamine.

29Several studies suggest that 5-hydroxytryptamine (5-HT) signalling plays a role in the antidepressant effects of ketamine. Preclinical work has demonstrated that the antidepressant-like action of (R,S)-ketamine is blocked by pre-treatment with a 5-HT-depleting agent. (R,S)-ketamine has been found to inhibit serotonin transporter (SERT) function in vitro and a positron emission tomography (PET) study in conscious monkeys further reported that subanaesthetic (R,S)-ketamine selectively enhanced serotonergic transmission by inhibition of SERT activity. Alongside SERT inhibition, increased mPFC 5-HT release via AMPA receptor stimulation in the dorsal raphe nucleus may be involved in the antidepressant effects of (R,S)-ketamine. Moreover, it has been demonstrated that an mPFC infusion of a 5-HT1a receptor antagonist blocks the antidepressant-like effects of (R,S)-ketamine in mice and attenuates ketamine-induced increases in phosphorylation of Akt. (R,S)-ketamine antidepressant effects were mimicked by intra-mPFC, but not systemic, administration of a 5-HT1a receptor agonist and both the antidepressant effects of ketamine and the 5-HT1a receptor agonist were blocked by the mTORC1 inhibitor rapamycin. Finally, in a recent study, infusion of a selective 5-HT1A receptor agonist into the mPFC produced ketamine-like rapid synaptic and antidepressant-like behavioural responses in a rodent model that were blocked by co-infusion of an AMPA receptor antagonist. Taken together, it appears another route via which (R,S)-ketamine may cause its antidepressant effects is through 5-HT1A receptor activation in the mPFC, by AMPA receptor-dependent 5-HT release, with downstream convergence on signalling mechanisms. These include the Akt/mTORC1 pathway but may also include ERK signalling, which is also activated by direct 5HT1A receptor stimulation.

30Considering the individual enantiomers, an in vivo microdialysis study has shown that both (R)- and (S)-ketamine acutely increase 5-HT release in the prefrontal cortex, with (R)-ketamine causing a greater increase than (S)-ketamine. Although the (S)-ketamine-induced 5-HT release was attenuated by an AMPA receptor antagonist, (R)-ketamine-induced 5-HT release was not affected by AMPA receptor blockade. Although preclinical work has demonstrated that 5-HT depletion abolishes the antidepressant-like actions of (S)-ketamine in a genetic model of depression, other work has shown that 5-HT depletion does not alter the antidepressant effects of (R)-ketamine in a chronic social defeat stress model. This suggests that 5-HT may not play as major a role in antidepressant effects of (R)-ketamine. The reason for these differences is not entirely clear and further work is needed to explore the role of 5-HT in the effects of ketamine and its enantiomers.

31The dopamine system has also been implicated in depression and the antidepressant effects of ketamine; however, the mechanism underlying the action of ketamine or its enantiomers on this system has not been fully established. Acute subanaesthetic (R,S)-ketamine administration is associated with significantly increased dopamine levels in the cortex, striatum and nucleus accumbens in rodents and there is also in vivo PET imaging evidence that (R,S)-ketamine and (S)-ketamine administration leads to increased striatal dopamine release in humans as indexed by D2/D3 receptor tracer binding. A further PET study found that IV (S)-ketamine administration, but not (R)-ketamine, led to a significant reduction of binding availability of dopamine D2/D3 receptor in the monkey striatum and suggests that unlike (R)-ketamine, (S)-ketamine can cause dopamine release in the striatum that may contribute to the psychotomimetic/dissociative side effects in humans. Other groups found that (R,S)-ketamine-induced reductions of D2/D3 binding in humans only occurred in combination with amphetamine, suggesting ketamine may enhance the sensitivity of the dopamine system but not lead to direct dopamine release.

32In a study examining the effects of (R,S)-ketamine and metabolites on evoked striatal dopamine release and dopamine receptors, (R,S)-ketamine did not alter the magnitude or kinetics of electrical stimulation-evoked dopamine release in the nucleus accumbens of anesthetised mice and neither ketamine’s enantiomers nor its metabolites had affinity for dopamine receptors or the dopamine transporter. This suggests the side effects and antidepressant actions of ketamine (or its metabolites) may not be associated with direct effects on mesolimbic dopaminergic neurotransmission. An alternative hypothesis is that ketamine produces indirect effects through NMDA receptor antagonism on GABAergic interneurons, resulting in disinhibition of glutamatergic projections on to dopamine neurons in the midbrain, an increase in glutamate release, subsequent activation of dopaminergic neurons and increased dopamine levels in targets such as the striatum and cortex.

33Additional research findings indicate that dopamine D1 receptor activity in the mPFC are necessary for the rapid antidepressant actions of (R,S)-ketamine using optogenetic stimulation in a mouse model. Another potential convergence from a signal transduction perspective may involve NMDA and D1 receptor-dependent induction of mTORC/ERK and inactivation of eEF2 kinase resulting in increased protein synthesis. Further work found that pre-treatment with a dopamine D1 receptor antagonist did not block the antidepressant effects of (R)-ketamine in a chronic social defeat stress model. Furthermore, although (S)-ketamine has been shown to cause a robust increase in dopamine release compared with (R)-ketamine, the antidepressant-like effects were more potent and longer acting following (R)-ketamine administration in a mouse model. Taken together, these findings suggest that activation of the dopamine system may be required for the antidepressant actions of (S)- but not (R)-ketamine.

34Increasing lines of preclinical and clinical evidence highlight a major role for the lateral habenula, an anti-reward centre, in the pathophysiology of depression. It is suggested that abnormal increases in neuronal activity in this region signal downregulation of brainstem dopaminergic and serotonergic firing, resulting in depressive symptomatology including anhedonia, helplessness and excessive focus on negative experiences.

35Recent work from found that blockade of NMDA receptor-dependent bursting activity in the lateral habenula mediated the antidepressant actions of (R,S)-ketamine in rodent models of depression. It was demonstrated that lateral habenula bursting required both NMDA receptors and low-voltage-sensitive T-type calcium channels. Furthermore, administration of T-type calcium channel inhibitors (ethosuximide and mibefradil) caused rapid antidepressant-like effects in both the forced swim test and sucrose preference test. In contrast, preclinical work utilising a chronic social defeat stress model failed to demonstrate antidepressant effects of ethosuximide whereas (R)-ketamine showed rapid and long-lasting antidepressant actions in this model. Furthermore, a recent double-blind RCT in medication-free patients with depression found no significant reductions in depression and anxiety scores following treatment with ethosuximide, suggesting T-type calcium channel inhibitors are unlikely to exert ketamine-like robust antidepressant actions.

36It should be noted that inhibition of lateral habenula bursting as a mechanism of antidepressant action has only been assessed acutely at 1 hour post (R,S)-ketamine infusion. Whether this mechanism is active later during (R,S)-ketamine’s antidepressant effects (>24 hours) or indeed if there are differential effects of (S)- and (R)-ketamine on lateral habenula bursting remains unknown.

37Ketamine interacts with mu, kappa and, to a lesser extent, delta-opioid receptors (Ki = 42.1, 28.1, and 272 mΜ, respectively). The affinity of (S)-ketamine for the mu and kappa opioid receptors is two to fourfold that of (R)-ketamine. Recent work demonstrated that pre-treatment with naltrexone, an opioid receptor antagonist, significantly blocked the antidepressant and anti-suicidal effects of (R,S)-ketamine in TRD, suggesting that opioid system activation was necessary for the rapid-acting antidepressant and antisuicidal effects of (R,S)-ketamine. There are a number of important limitations to these studies including the small sample size (only 12 participants completing both naltrexone and placebo pre-treatment conditions and only seven of the 12 meeting response criteria during the ketamine plus placebo condition), lack of a placebo control arm for the (R,S)-ketamine infusion (i.e., naltrexone + IV saline and placebo + IV saline) and finally that participants may have experienced a noxious, nocebo type of response to the naltrexone + ketamine treatment, which influenced subsequent depression ratings. Other work has demonstrated that naltrexone pre-treatment did not affect the antidepressant effects of (R,S)-ketamine in depressed individuals with alcohol use disorder and an earlier study in healthy individuals found that the behavioural effects of an antidepressant dose of ketamine were potentiated by pre-treatment with naltrexone. Finally, in patients with TRD, concurrent use of buprenorphine and methadone (high affinity mu opioid receptor agonists) was not associated with reductions in antidepressant efficacy of (R,S)-ketamine.

38In rodent models of depression (chronic social defeat stress and inflammation induced), it was shown that naltrexone pre-treatment did not block the antidepressant effects of (R,S)-ketamine. However, in a subsequent preclinical study, it was shown that opioid antagonists abolish the ability of (R,S)-ketamine to reduce depression-like behavioural and lateral habenula cellular hyperactivity. The authors suggested the opioid system is ‘necessary but not sufficient’ for the antidepressant actions of (R,S)-ketamine in rodents as activation by morphine, a mu-opioid agonist, at a dose high enough to induce a hedonic response, did not mimic the rapid antidepressant-like effects of ketamine or reduce lateral habenula neuronal activity. The authors argued that in their studies of lateral habenula cellular activity, (R,S)-ketamine did not appear to act as a mu-opioid agonist but that some mu-opioid receptor activity was necessary for NMDA receptor antagonism. In brain regions, including the habenula, NMDA receptors and opioid receptors display colocalisation and NMDA receptor activation can be modulated by actions of opioid receptors. Taken together, this suggests a potential interaction that may be explained by direct ‘crosstalk’ between the glutamatergic and the opioid receptor systems, or by convergence at downstream signalling pathways.

39There are several potential convergences between opioid signalling and other mechanisms implicated in the antidepressant action of ketamine. For example, administration of endogenous opioids has been shown to upregulate BDNF expression in the frontal cortex, hippocampus and amygdala. Moreover, these effects were reversed by naltrexone administration. Acute mu-opioid receptor activation has been shown to result in rapid activation of ERK signalling and acute treatment with ketamine enhances the levels of opioid-induced ERK phosphorylation in cells that endogenously express mu-opioid receptors. Further, administration of an opioid receptor antagonist, naloxone, has been shown to inhibit mu-opioid-induced ERK activation in a dose-dependent manner in C6 glioma cell lines. Finally, there are a number of functional interactions between opioid receptors and monoaminergic systems relevant to mood control. Specifically, activation of mu-opioid receptors expressed in the dorsal raphe nucleus and ventral tegmental area, via GABAergic interneurons, disinhibit 5-HT and dopamine neurons with projections including the prefrontal cortex and nucleus accumbens (Figure 4).

40The role of the opioid system in the antidepressant effect of ketamine remains controversial and a topic of debate. Further work with rigorous trial design and parallel mechanistic studies are required to understand the function of the ketamine-opioid receptor interaction and subsequent signalling cascades in the antidepressant effect of each of ketamine and its individual enantiomers.

41The discovery of the rapid antidepressant effects of (R,S)-ketamine, including in treatment-resistant patients, has appropriately been hailed ‘the most important discovery in half a century’ in depression research. Through the drug development and clinical trials process, the (S)-ketamine nasal spray, SpravatoTM, has been approved in both the United States and Europe, although some concerns remain regarding efficacy and side effects. The first pilot study of (R)-ketamine in TRD has demonstrated encouraging results and, considering preclinical findings, it appears (R)-ketamine may have a more favourable safety profile than (S)-ketamine. Accumulating preclinical evidence also suggests (R)-ketamine to have more potent and longer-lasting antidepressant effects than both (R,S)-ketamine and (S)-ketamine. As studies of (R)-ketamine progress through Phase I and Phase II, results from direct comparison studies of the safety and efficacy of (R)-ketamine and (S)-ketamine in TRD will be crucial. Other key outstanding questions are outlined in Figure 5.

42Although NMDA receptor inhibition and subsequent AMPA receptor activation have a role in the antidepressant effects of ketamine, further mechanistic work is building a more nuanced understanding of the distinct molecular and cellular mechanisms of ketamine, its enantiomers and metabolites, including BDNF-TrkB, mTORC1 and ERK signalling. Although there may be a role for monoaminergic and opioid receptor systems in the antidepressant effects or detrimental side effects of ketamine, further work examining the effects of each of the component enantiomers on these systems is required. All the while, new pieces of the ketamine puzzle are being discovered and other potential future directions of enquiry include examining the role of the transforming growth factor β1 system and the brain-gut-microbiome axis in the antidepressant effects of ketamine and its enantiomers.

43As we further our understanding of the similarities and differences in the signalling pathways associated with (S)-ketamine, (R)-ketamine and their metabolites, we should bear in mind potential complementary or synergistic antidepressant effects that might arise via distinct mechanisms. A deeper understanding of the precise molecular and cellular mechanisms underlying the antidepressant effects and negative side effects of (R,S)-ketamine, (S)-ketamine and (R)-ketamine will be invaluable as we seek to develop future rapid-acting antidepressants with favourable safety profiles, alongside treatment strategies to maintain adequate response.