Rapid anti-depressant-like effects of ketamine and other candidates: Molecular and cellular mechanisms.

Abstract Major depressive disorder takes at least 3 weeks for clinical anti‐depressants, such as serotonin selective reuptake inhibitors, to take effect, and only one‐third of patients remit. Ketamine, a kind of anaesthetic, can alleviate symptoms of major depressive disorder patients in a short time and is reported to be effective to treatment‐resistant depression patients. The rapid and strong anti‐depressant‐like effects of ketamine cause wide concern. In addition to ketamine, caloric restriction and sleep deprivation also elicit similar rapid anti‐depressant‐like effects. However, mechanisms about the rapid anti‐depressant‐like effects remain unclear. Elucidating the mechanisms of rapid anti‐depressant effects is the key to finding new therapeutic targets and developing therapeutic patterns. Therefore, in this review we summarize potential molecular and cellular mechanisms of rapid anti‐depressant‐like effects based on the pre‐clinical and clinical evidence, trying to provide new insight into future therapy.


| INTRODUC TI ON
Major depressive disorder (MDD) is a mental disorder associated with mood disorders, characterized by depressed mood, decreased interest, cognitive impairment and even suicidal ideation. It is the main cause of global disability, 1 and almost 20% of people will suffer one episode of depression at some point in their lifetime. 2 Treatments of depression mainly include cognitive behavioural therapy and drug intervention. The pathogenesis of depression is associated with disorder of monoamine neurotransmitter levels. Based on the pathogenesis, drug treatments include selective serotonin reuptake inhibitors (SSRIs), serotonin and norepinephrine reuptake inhibitors (SNRIs), tricyclic anti-depressants (TCAs) and monoamine oxidase inhibitors (MAOIs). Though traditional medications may alleviate depressive symptoms in some degree, they work slowly. It takes weeks to months for patients to benefit from drug treatment when up to 30% of those patients still do not relieve symptoms and even develop resistance after receiving medication. 3 Unlike traditional anti-depressants, ketamine could reduce suicidal ideation and improve mood in a short period of time 4 (Tables 1 and 2). Ketamine is a commonly used anaesthetic and analgesic drug. Clinical study showed that intravenous injection of 0.5 mg/kg of ketamine for 40 minutes could induce a strong and rapid anti-depressant-like response in patients with depression, 5 even in those who failed to treatment with traditional drugs. This effect could last 1-2 weeks. 6,7 (R,S)-ketamine is a racemic mixture comprising equal parts of (R)-ketamine (arketamine) and (S)-ketamine (esketamine). Esketamine has five times greater affinity for N-methyl-d-aspartate receptor (NMDAR) than arketamine. 8 9 The response arose at 28 days 10 and appeared to persist for more than 2 months. 11 However, the clinical application of esketamine still needs to be concerned. On the one hand, the efficacy of esketamine is controversial. It was found that in the phase 3 clinical trials, the grouping criteria were not strict. About 22% of the patients only resisted to one class of drugs, which meant that they were not strictly defined TRD. Patients participated in the randomized withdrawal trial were those who had been previously randomly assigned to esketamine and achieved stable remission, leading to a statistically higher response to the drug.
In addition, in the sole positive phase 3 trial, the mean decrease on the Montgomery-Åsberg Depression Rating Scale (MADRS) was 20.8 for esketamine vs 16.8 for placebo. Besides, the result of meta-analysis showed that the standardized mean difference (SMD) of esketamine was similar to the olanzapine-fluoxetine combination, and less than the SMD of aripiprazole and quetiapine. These suggest that esketamine shows no significant advantage over placebo or other drugs approved by FDA. Moreover, one of the trials involved older patients and showed non-significant results, indicating that the efficacy of esketamine in this demographic remained unclear. Finally, the rapid onset of response was not demonstrated formally. About 8%-10% of patients who took esketamine achieved a rapid clinical response, compared with 5% of placebo. On the other hand, the results of the study 3003 were not consistent with the FDA requirement for substantial evidence of effectiveness. One site in Poland drives the overall study result due to a 100% of placebo arm relapses in this study.
Removement of the outlier site changed the results from significant to non-significant. 12 So far, the use of esketamine has been limited to certified medical offices or clinics in America. Another isomer (R)-ketamine is also a potential anti-depressant which is undergoing clinical trials. 13 It is worth noting that (R)-ketamine has greater potency and longer-lasting anti-depressant effects than (S)-ketamine in rodents. [14][15][16] In fMRI test, it was shown that (R,S)ketamine and (S)-ketamine significantly activated the cortex, nucleus accumbens and striatum of conscious rats, so as the NMDAR antagonist MK-801. On the contrary, (R)-ketamine produced negative response. 17 Similar pattern could be observed in clinical test. 18 These indicate that NMDAR may not be the primary target of (R)-ketamine. 19 (S)-ketamine and (R)-ketamine are also agonists of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor (AMPAR) and both activated brain-derived neurotrophic factor (BDNF)-tropomyosin receptor kinase B (TrkB) pathway. It is worth noting that their mechanisms may be different. Study showed that (S)-ketamine activated BDNF-TrkB pathway through mTOR signalling pathway while (R)-ketamine activated MEK-ERK pathway, mediating the activation of BDNF-TrkB pathway. 20 In another study, it was shown that (R)-ketamine could activate BDNF-TrkB pathway and reverse the decrease in dendritic spine density, inducing synaptogenesis in the pre-frontal cortex (PFC), CA3 and dentate gyrus (DG) of the hippocampus and eliciting sustained anti-depressant effects in depressed rodents. 15 Nevertheless, neither isomer attenuated the reduced BDNF in the PFC of susceptible chronic social defeat stress (CSDS) mice after 30 minutes, indicating that neither isomer improved the level of BDNF or induced synaptogenesis. 20 Whether the long-lasting anti-depressant effects of (R)-ketamine is related to MERK-ERK signalling is unknown. Besides, detrimental side effects of (R)-ketamine are fewer than (R,S)-ketamine and (S)-ketamine. 15,21 It was observed that (S)-ketamine caused a reduction in parvalbumin (PV)-positive cells in the medial pre-frontal cortex (mPFC) and DG, while (R)ketamine did not. PV-positive cell is related to schizophrenia, and this may be the reason why (S)-ketamine produces psychotomimetic side effects. 15 In addition, side effects of (S)-ketamine are associated with mechanistic target of rapamycin (mTOR). The activation of mTOR signalling after drug abuse contributes to drug-related behaviours such as excessive drug intake. 22 (S)-ketamine activates mTOR signalling in the brain regions, and this may lead to drug abuse. Moreover, a study using positron emission tomography showed that in the conscious monkey, (S)-ketamine but not (R)-ketamine could reduce dopamine D2/3 receptor binding in striatum. 23 It is possible that (S)-ketamine-induced dopamine release relates to acute psychotomimetic side effects in humans.

| NEUR AL CIRCU IT
Depression is associated with multiple brain regions including prefrontal cortex (PFC), hippocampus (HP) and amygdala. 30 These regions do not play a separate role in the onset of depression but are connected by nerve fibres, forming different neural circuits. The structure and function of these circuits are abnormal under a condition of depression. [31][32][33] Restoring normal connections of neural pathways may be an effective and fast way to alleviate depression symptoms. Ketamine is a non-specific NMDAR antagonist. It can change the local activities of relevant brain regions and reshape the brain circuit in a short time (Figure 1).

| Neural circuits associated with prefrontal cortex
Pre-frontal cortex is related to cognitive function and emotional regulation. 34 Reduced activity of PFC has been observed both in depressed patients and in rodent models of depression. Dysfunction of the pre-frontal-hippocampal (PFC-HP) circuit is associated with major depression. It was demonstrated that in rat brain, functional connectivity within the PFC-HP system is increased by acute ketamine stimulation in a dose-and exposure-dependent manner. 35 In the same way, the activation of ventral hippocampus (vHipp)-mPFC pathway was proved to be necessary in anti-depressant responses of ketamine. 36 Abnormal functional connection within dorsal PFC and anterior cingulate gyrus (ACC) is highly correlated with depression. 37 Ketamine has a positive effect on this connection. Study showed that functional connection between the right PFC and subgenual cingulate was increased in depressed patients 1 day after a single infusion of ketamine. 38 Besides, the functional connection between the PFC and the amygdala also relates to depressive behaviour. It was reported that ketamine strengthens amygdala inputs to basal dendrites of layer V cells in mPFC and reversed depression-like behaviours. 39 Optogenetic experiment showed that light-activated mPFC-basolateral amygdala (BLA) projection produced rapid anti-depressant-like effects. Light stimulation to D1 dopamine receptor (Drd1) neurons in the brain region of mPFC increased the neuronal activity in the BLA area exclusively, indicating that the Drd1 neurons mediated BLA area to participate in the rapid anti-depressant-like effects. 40 However, whether ketamine stimulates mPFC and amygdala in the same time has not been proved.

TA B L E 2 Summary of the rapid anti-depressant-like effects of ketamine in animal
In addition, the PFC-dorsal raphe nucleus (DRN) circuit has been confirmed to be implicated in depression. [41][42][43] The mPFC is one of the various areas projecting densely to the DRN, 44 which has abundant 5-HT cell bodies located in. Activation of 5-HT neurons can improve depression-like behaviours in elevated plus maze and forced swim test (FST). 45 Combining whole-cell recordings with optogenetic approaches, it was found that the mPFC axon monosynapse was con-

| Neural circuits associated with ventral tegmental area
Anhedonia, which is related to structure and function abnormalities of the reward circuit, is a core clinical feature of award-control disorder and also a core symptom of depression. The ventral tegmental area (VTA) is a heterogeneous brain region, mainly composed of dopaminergic (DAergic) neurons (60%-65%). 49   More than that, the internal environment of the human body is more complicated than that of animal. Even one pathway is affected by ketamine, other alternatives can be activated instead. Studies on other T-VSCCs blockers and the possible targets need to be done.

| Neural circuits associated with amygdala
The amygdala is involved in coordinating the function of cortical networks when evaluating the biological significance of affective stimuli. Liu et al 39 discovered that ketamine activated amygdala and increased the amygdala output to the PFC through the anterior marginal area in the chronic unpredictable stress (CUS) model of rats. By using fMRI and resting-state fMRI (rsfMRI), it was found that in healthy subjects without any mental, neurological or medical illness, ketamine reduced neural reactivity in the bilateral amygdalohippocampal complex during emotional stimulation, which was different from amygdala-PFC circuit. 60 It is hypothesized that the amygdala and its interaction with the pre-genual anterior cingulate cortex (pgACC) could predict the response of patients to ketamine. Clinical studies have demonstrated that MDD patients were either in working memory task mode or stimulated by rapidly presenting fearful faces, and the pgACC was highly activated but could be inactivated by ketamine within 4 hours. Pre

| SYNAP TIC PL A S TICIT Y
Another crucial mechanism of ketamine rapid anti-depressant-like effects is synaptic plasticity. Synapse is the basic structure of in- and clinical tests. 73,74 However, a meta-analysis showed that nonketamine NMDAR antagonists were superior to placebo only on days 5-8, while ketamine reduced depression in 40 minutes. 75 Not all non-ketamine NMDAR antagonists elicit robust anti-depressant effects such as ketamine, suggesting that NMDAR may not be the key role in the anti-depressant mechanisms of ketamine. Inhibition of NMDAR causes changes in its downstream molecules and signalling pathways, and these changes can be seen in depression-related brain regions. 76 But now, there are more and more reports of rapid anti-depressants that are less related to NMDAR. Maybe we should stop focusing on NMDAR only and begin to pay more attention to other potential targets. Other mechanisms of anti-depressant effects of ketamine will be discussed below.

| BDNF in synaptic plasticity
Brain-derived neurotrophic factor is a vital protein in the process of synaptic transmission. It regulates neural plasticity, synaptic production, neurogenesis and cell survival. BDNF is necessary for the formation and maintenance of activity-dependent synaptic connections. It has been found that the expression of BDNF in the pre-frontal cortex and hippocampus was downregulated in animal depression models, so as the level of BDNF in depressed patients. 90,91 Evidence showed that ketamine administration increases BDNF levels and improves depressive-like behaviours. [92][93][94] More importantly, BDNF is indispensable in anti-depressant effects. In the BDNF Met gene knock-in mice, especially Met/Met mice, synaptogenesis was significantly weakened, 95 consisted of depressed patients. 96 Clinical study showed that either 0.5 or 0.2 mg/kg of ketamine injection could reduce suicidal ideation of patients who had the Val allelic genes.
However, patients with genotype Met/Met only responded at a dose of 0.5 mg/kg ketamine. 96 Sufficient BDNF content regulates synaptic plasticity and participates in reversing depression. [97][98][99] Except for ketamine, acute caloric restriction (CR) is also able to elevate BDNF level. CR refers to a 30%-40% reduction in calorie intake while retaining protein, vitamins, minerals and water intake to maintain proper nutrition. Some mental illnesses, such as the typical major depression and anorexia nervosa, are characterized by reduced calorie intake. Previous studies showed that long-term strict energy limitation (5 weeks, 50% intake of the control group) may cause brain 5-HT system dysfunction, leading to the development of depression and anxiety. 100 Otherwise, strict energy limitation might lead to malnutrition 101  could partially reverse the anti-depressant effects of imipramine and 9-hour CR. 28 We also found that DOI could suppress the increase in BDNF level and 5-HT 2A R antagonist ketanserin inhibited the effects of DOI on BDNF. 102 There is a possibility that acute fasting may exert anti-depressant effects by blocking 5-HT 2A R.
Evidence shows that the activation of 5-HTergic system leads to an activation of glutamatergic system. Activated by 5-HT receptors, glutamate pyramidal cells in mPFC release BDNF rapidly and activate BDNF signalling pathway, resulting in synaptogenesis accompanied by rapid anti-depressant effects. [103][104][105] These studies suggest that monoamine manner (5-HT) and non-monoamine manner (BDNF) are not separated in anti-depressant effects. This suggests us that combining monoamine with non-monoamine may be a new strategy for treating MDD. Some studies showed that CR regulated the release of orexin [106][107][108][109] and ghrelin, [110][111][112][113][114][115][116][117] producing some anti-depressant effects. But this evidence on synaptic plasticity is weak, and we mention here only for reference ( Figure 3).
Additionally, scopolamine has similar pharmacological mechanisms to ketamine for its anti-depressant effects. Scopolamine activates AMPARs, promotes BDNF release rapidly and stimulates BDNF-mTOR signalling pathway. 118 The difference is that scopolamine acts on cholinergic system. Scopolamine inhibits GABAergic neuron function by combining with M1-AChR on GABA interneurons in mPFC. 119

| Neuroglia in synaptic plasticity
Ketamine also affects glial cells in the central nervous system to regulate synaptic plasticity. Glial cells are mainly divided into three categories: astroglia, microglia and oligodendroglia. Among them, the former two are associated with depression. Astroglia is the most abundant glial cell. Its main functions are to regulate regional blood flow and energy metabolism, immune defence and amino acid neurotransmitter clearance. It is also associated with the stabilization and dissection of synaptic connections 120 and participates in antidepressant effects. 121 Pre-treated with ketamine 1 day after, immobility time in FST was significantly reduced. The volume of CA1 stratum radiatum and molecular layer of the dentate gyrus in the hippocampus and the volume of astrocytes of rats increased significantly, so as the number and length. 122 Ketamine modified the morphology of astrocytes and astrocytes, regulating the synaptic microenvironment, neurogenesis and angiogenesis. 123 Microglia is a kind of immunocompetent cell. Excessive microglial activation would cause inflammatory process, leading to astrocyte glutamatergic dysfunction and activation of microglial function in turn. 124 Evidence showed that ketamine inactivates microglial due to inhibition of ERK1/2 phosphorylation. 125 Besides, ketamine regulated STAT3 and the type I interferon pathway in microglia through eEF2, increasing the BDNF expression and promoting the synthesis of PSD95 and synapsin I (SYN1). 126 Additionally, microglial cells induce immune dysfunction by producing quinolinic acid (QUIN). QUIN is an endogenous modulator with agonistic properties on NMDA. It was observed that in acutely depressed patients, QUIN increased in subregions of the anterior cingulate gyrus. 127 Increase in QUIN comes along with decrease in kynurenic acid (KYNA), a NMDA receptor antagonist synthesized by astrocytes. 128 Ketamine could modulate the microglial reactivity and decrease QUIN production. It was reported that KYNA-to-QUIN ratio was a predictor of ketamine response in treatment-resistant depressed patients, while the reduction in QUIN after treated by ketamine was a predictor to the reduction in MADRS score. 129 Ketamine regulates functions of astrocytes and microcytes to maintain synaptic complement.

| Neuroinflammation in synaptic plasticity
Depression is considered to be relevant with the activation of chronic, low-grade inflammatory responses and cell-mediated immunity. 130 137 Changes in peripheral IL-6 and gut microbiota may be vital for the pathogenesis of depression. It was found that baseline serum levels of IL-6 were both higher in ketamine responder and non-responder groups than control group. More than that, serum level of IL-6 is significantly higher in the responder group than non-responder group. 138 Another clinical study also demonstrated that higher baseline interleukin-6 (IL-6) in serum predicted better response to ketamine. 139 Serum IL-6 may be a predictive biomarker for the anti-depressant effects of ketamine in TRD patients.
Therapeutic SD is a direct and rapid treatment for MDD, reducing the depressive symptoms of 50%-60% of MDD patients significantly within a few hours, 140 consistent with animal experiment. 141 It was reported that SD produced rapid anti-depressant effects by activating adenosine A1R in astrocytes and could be mim- and activity of AMPAR were elevated. 145 The molecular change was consistent with those caused by ketamine and also SD. In animal studies, AMPAR level in the cerebral cortex and hippocampus was about 40% higher after arousal than after sleep. The change in AMPAR phosphorylation and other enzymes important for plasticity was consistent with synaptic strengthening during wakefulness and contraction during sleep. 146 These evidence indicates that synaptic homeostasis is regulated by wakefulness and sleep.
Synaptic homeostasis refers to the ability of neurons to regulate their own excitability and synaptic strength, connected closely with synaptic plasticity. The core of the synaptic homeostasis hypothesis is that the number and intensity of cortical synapses vary widely throughout the sleep-wake cycle. It is believed that wakefulness leads to a net increase in synaptic strength of the cortical circuits, while a basic function of sleep is to reduce the proportion of cortical synapses. 147 Given to that, circadian rhythms also regulate synaptic plasticity. Circadian rhythms are reset by the transcription of clock genes, including the cycle genes PER1, PER2 and PER3. After 2-hour SD treatment on mice, the expression levels of PER1 and PER2 significantly increased. 148 Similarly, ketamine regulated circadian rhythms by affecting clock genes accompanied by a rapid anti-depressant effect. In animal experiment, it was seen that clock genes including PER2, neuronal PAS domain protein 4 and D-Box binding protein, were downregulated in mice treated with ketamine and SD. 149 Reviewing data from human, animal and neuronal cell, both low-dose SD and ketamine could regulate circadian rhythms. 150 It is hypothesized that A1R ameliorates the depression-like behaviours through regulating cycle genes and then affecting synaptic homeostasis. 151 However, we still lack evidence for that so far (Figure 4).

| CON CLUS ION
In this review, we summarized the mechanisms of rapid anti-depres-

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

AUTH O R CO NTR I B UTI O N S
FP wrote the first draft. JF, TG and QL participated in the discussion of the manuscript. BL provided critical revisions. All authors approved the final version of the manuscript for submission.