Investigating the shared genetic basis and causal relationships between mucosa-associated lymphoid tissue inflammation and psychiatric disorders.

Background: Chronic and acute inflammation of the mucosa-associated lymphoid tissue have been positively linked to the development of psychiatric disorders in observational studies. However, it remains unclear whether this association is causal. In the present study, we investigated this association, using as proxies genetically predicted tonsillectomy, appendectomy and appendicitis on psychiatric disorders including major depressive disorder (MDD), schizophrenia (SCZ), bipolar depression (BD) and anxiety (ANX) via a two-sample Mendelian randomization (MR) analysis. Methods: Genetic association summary statistics for tonsillectomy, appendectomy and appendicitis were sourced from FinnGen Consortium, comprising data from 342,000 participants. Genetic correlations between all exposures and outcome were calculated with Linkage Disequilibrium Score (LDSC) Regression analysis. MR estimates were then calculated to assess their impact on the risk of developing psychiatric disorders. Sensitivity analysis was employed to test for any directional pleiotropy. Results: Our results suggest that there is no direct causal association between tonsillectomy, appendectomy or appendicitis with a heightened risk for development of psychiatric disorders. The robustness of the results of the main MR analysis was further confirmed with additional sensitivity analyses. However, a moderate inverse genetic correlation was observed between tonsillectomy and MDD traits (rg=-0.39, p-value (P)=7.5x10-5). Conclusion: Our findings provide, for the first time, evidence that there is no causal association between tonsillectomy or appendectomy on subsequent vulnerability of developing psychiatric disorders. Future studies using larger sample size GWAS should focus on unraveling the confounding factors and mediators to investigate this relationship further.

Transcriptomic analysis reveals sex-specific patterns in the hippocampus in Alzheimer's disease.

Background: The hippocampus, vital for memory and learning, is among the first brain regions affected in Alzheimer's Disease (AD) and exhibits adult neurogenesis. Women face twice the risk of developing AD compare to men, making it crucial to understand sex differences in hippocampal function for comprehending AD susceptibility. Methods: We conducted a comprehensive analysis of bulk mRNA postmortem samples from the whole hippocampus (GSE48350, GSE5281) and its CA1 and CA3 subfields (GSE29378). Our aim was to perform a comparative molecular signatures analysis, investigating sex-specific differences and similarities in the hippocampus and its subfields in AD. This involved comparing the gene expression profiles among: (a) male controls (M-controls) vs. female controls (F-controls), (b) females with AD (F-AD) vs. F-controls, (c) males with AD (M-AD) vs. M-controls, and (d) M-AD vs. F-AD. Furthermore, we identified AD susceptibility genes interacting with key targets of menopause hormone replacement drugs, specifically the ESR1 and ESR2 genes, along with GPER1. Results: The hippocampal analysis revealed contrasting patterns between M-AD vs. M-controls and F-AD vs. F-controls, as well as M-controls vs. F-controls. Notably, BACE1, a key enzyme linked to amyloid-beta production in AD pathology, was found to be upregulated in M-controls compared to F-controls in both CA1 and CA3 hippocampal subfields. In M-AD vs. M-controls, the GABAergic synapse was downregulated, and the Estrogen signaling pathway was upregulated in both subfields, unlike in F-AD vs. F-controls. Analysis of the whole hippocampus also revealed upregulation of the GABAergic synapse in F-AD vs. F-controls. While direct comparison of M-AD vs. F-AD, revealed a small upregulation of the ESR1 gene in the CA1 subfield of males. Conversely, F-AD vs. F-controls exhibited downregulation of the Dopaminergic synapse in both subfields, while the Calcium signaling pathway showed mixed regulation, being upregulated in CA1 but downregulated in CA3, unlike in M-AD vs. M-controls. The upregulated Estrogen signaling pathway in M-AD, suggests a compensatory response to neurodegenerative specifically in males with AD. Our results also identified potential susceptibility genes interacting with ESR1 and ESR2, including MAPK1, IGF1, AKT1, TP53 and CD44. Conclusion: These findings underscore the importance of sex-specific disease mechanisms in AD pathogenesis. Region-specific analysis offers a more detailed examination of localized changes in the hippocampus, enabling to capture sex-specific molecular patterns in AD susceptibility and progression.

The impact of estradiol on serotonin, glutamate, and dopamine systems.

Estradiol, the most potent and prevalent member of the estrogen class of steroid hormones and is expressed in both sexes. Functioning as a neuroactive steroid, it plays a crucial role in modulating neurotransmitter systems affecting neuronal circuits and brain functions including learning and memory, reward and sexual behaviors. These neurotransmitter systems encompass the serotonergic, dopaminergic, and glutamatergic signaling pathways. Consequently, this review examines the pivotal role of estradiol and its receptors in the regulation of these neurotransmitter systems in the brain. Through a comprehensive analysis of current literature, we investigate the multifaceted effects of estradiol on key neurotransmitter signaling systems, namely serotonin, dopamine, and glutamate. Findings from rodent models illuminate the impact of hormone manipulations, such as gonadectomy, on the regulation of neuronal brain circuits, providing valuable insights into the connection between hormonal fluctuations and neurotransmitter regulation. Estradiol exerts its effects by binding to three estrogen receptors: estrogen receptor alpha (ERα), estrogen receptor beta (ERß), and G protein-coupled receptor (GPER). Thus, this review explores the promising outcomes observed with estradiol and estrogen receptor agonists administration in both gonadectomized and/or genetically knockout rodents, suggesting potential therapeutic avenues. Despite limited human studies on this topic, the findings underscore the significance of translational research in bridging the gap between preclinical findings and clinical applications. This approach offers valuable insights into the complex relationship between estradiol and neurotransmitter systems. The integration of evidence from neurotransmitter systems and receptor-specific effects not only enhances our understanding of the neurobiological basis of physiological brain functioning but also provides a comprehensive framework for the understanding of possible pathophysiological mechanisms resulting to disease states. By unraveling the complexities of estradiol's impact on neurotransmitter regulation, this review contributes to advancing the field and lays the groundwork for future research aimed at refining understanding of the relationship between estradiol and neuronal circuits as well as their involvement in brain disorders.

Exploring the neurofunctional impairments and cognitive biases concerning food and body related stimuli in anorexia nervosa: An integrated EEG and eye-tracking study protocol.

BACKGROUND: Patients with Anorexia Nervosa (AN) exhibit significant cognitive and neural disturbances compared to healthy individuals when processing food and body-related stimuli. These disturbances not only contribute to the manifestation and chronification of their pathological eating behaviour but also underscore the complex interplay of cognitive, emotional, and neurobiological factors in AN. However, the precise underlying cognitive and neural mechanisms of these disturbances remain a compelling area of investigation. METHODS: This study presents a protocol developed for conducting a cross-sectional quasi-experimental study using a mixed model ANOVA approach with a crossover design. Our participants will consist of 20 patients with an active diagnosis of AN, 20 Overweight/obese individuals, and 20 Healthy Controls (HCs) with a normal BMI. An integrated eye-tracking and EEG methodology will be used in conjunction, with the primary aim of assessing participants' cognitive and neural processing towards high and low-calorie food stimuli. On an exploratory level, by utilizing the same methods, the present study will also investigate AN patients' responses towards high weight, normal weight, low weight, and self-body pictures, as well as towards images from the International Affective Picture System (IAPS) characterized by elevated valence and arousal levels. Additionally, behavioural methods such as yes or no questions, and self-reported questionnaires will be administered. The EEG and eye-tracking data will be analysed at early (50-300 ms) and late (350-500 ms) time intervals. DISCUSSION: The investigation of the underlying cognitive and neural processes employed by patients with AN during the processing of food and body-related stimuli can help us develop a better understanding of the cognitive and neural mechanisms that contribute to the manifestation and maintenance of the disorder and assist in the development of more effective screening methods. ETHICAL APPROVAL AND CONSENT TO PARTICIPATE: Ethical approval for the study has been obtained by the Cyprus National Bioethics Committee on 27.04.2023 (ΕΕΒΚ/ΕΠ/2023/19), and by the University of Cyprus (20.02.2023). Written informed consent will be obtained from all participants.

Unraveling the transcriptomic signatures of Parkinson's disease and major depression using single-cell and bulk data.

Background: Motor symptoms are well-characterized in Parkinson's disease (PD). However, non-motor symptoms, such as depression, are commonly observed and can appear up to 10 years before motor features, resulting in one-third of individuals being misdiagnosed with a neuropsychiatric disorder. Thus, identifying diagnostic biomarkers is crucial for accurate PD diagnosis during its prodromal or early stages. Methods: We employed an integrative approach, combining single nucleus RNA and bulk mRNA transcriptomics to perform comparative molecular signatures analysis between PD and major depressive disorder (MDD). We examined 39,834 nuclei from PD (GSE202210) and 32,707 nuclei from MDD (GSE144136) in the dorsolateral prefrontal cortex (dlPFC) of Brodmann area 9. Additionally, we analyzed bulk mRNA peripheral blood samples from PD compared to controls (GSE49126, GSE72267), as well as MDD compared to controls (GSE39653). Results: Our findings show a higher proportion of astrocytes, and oligodendrocyte cells in the dlPFC of individuals with PD vs. MDD. The excitatory to inhibitory neurons (E/I) ratio analysis indicates that MDD has a ratio close to normal 80/20, while PD has a ratio of 62/38, indicating increased inhibition in the dlPFC. Microglia displayed the most pronounced differences in gene expression profiles between the two conditions. In PD, microglia display a pro-inflammatory phenotype, while in MDD, they regulate synaptic transmission through oligodendrocyte-microglia crosstalk. Analysis of bulk mRNA blood samples revealed that the COL5A, MID1, ZNF148, and CD22 genes were highly expressed in PD, whereas the DENR and RNU1G2 genes were highly expressed in MDD. CD22 is involved in B-cell activation and the negative regulation of B-cell receptor signaling. Additionally, CD86, which provides co-stimulatory signals for T-cell activation and survival, was found to be a commonly differentially expressed gene in both conditions. Pathway analysis revealed several immune-related pathways common in both conditions, including the complement and coagulation cascade, and B-cell receptor signaling. Discussion: This study demonstrates that bulk peripheral immune cells play a role in both conditions, but neuroinflammation in the dlPFC specifically manifests in PD as evidenced by the analysis of single nucleus dlPFC datasets. Integrating these two omics levels offers a better understanding of the shared and distinct molecular pathophysiology of PD and MDD in both the periphery and the brain. These findings could lead to potential diagnostic biomarkers, improving accuracy and guiding pharmacological treatments.

Role of group II metabotropic glutamate receptors in ketamine's antidepressant actions.

Major Depressive Disorder (MDD) is a serious neuropsychiatric disorder afflicting around 16-17 % of the global population and is accompanied by recurrent episodes of low mood, hopelessness and suicidal thoughts. Current pharmacological interventions take several weeks to even months for an improvement in depressive symptoms to emerge, with a significant percentage of individuals not responding to these medications at all, thus highlighting the need for rapid and effective next-generation treatments for MDD. Pre-clinical studies in animals have demonstrated that antagonists of the metabotropic glutamate receptor subtype 2/3 (mGlu2/3 receptor) exert rapid antidepressant-like effects, comparable to the actions of ketamine. Therefore, it is possible that mGlu2 or mGlu3 receptors to have a regulatory role on the unique antidepressant properties of ketamine, or that convergent intracellular mechanisms exist between mGlu2/3 receptor signaling and ketamine's effects. Here, we provide a comprehensive and critical evaluation of the literature on these convergent processes underlying the antidepressant action of mGlu2/3 receptor inhibitors and ketamine. Importantly, combining sub-threshold doses of mGlu2/3 receptor inhibitors with sub-antidepressant ketamine doses induce synergistic antidepressant-relevant behavioral effects. We review the evidence supporting these combinatorial effects since sub-effective dosages of mGlu2/3 receptor antagonists and ketamine could reduce the risk for the emergence of significant adverse events compared with taking normal dosages. Overall, deconvolution of ketamine's pharmacological targets will give critical insights to influence the development of next-generation antidepressant treatments with rapid actions.

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.

Ketamine preservative benzethonium chloride potentiates hippocampal synaptic transmission and binds neurotransmitter receptors and transporters.

Benzethonium chloride (BZT) is an excipient used in numerous products including (R,S)-ketamine (ketamine) drug formulations for human and veterinary use. Emerging evidence indicates BZT is pharmacologically active. BZT may therefore contribute to some of the clinical or preclinical effects observed with ketamine. In the present study, we evaluated: (i) the affinity of BZT for neurotransmitter receptors and transporters, (ii) the effects of BZT on hippocampal synaptic transmission in vitro, and (iii) plasma and brain concentrations of BZT following its intraperitoneal administration to male CD1 mice. Radioligand binding assays determined the affinity of BZT for neurotransmitter targets. Effects of BZT on field excitatory postsynaptic potentials (fEPSPs) were established via electrophysiological recordings from slices collected from male C57BL/6J mice. The binding assays revealed that BZT binds to numerous receptors (e.g., σ2 Ki = 7 nM) and transporters (e.g., dopamine transporter Ki = 545 nM). Bath application of BZT potentiated hippocampal fEPSPs in mouse hippocampal slices with an EC50 of 2.03 nM. Following intraperitoneal administration, BZT was detected in the plasma, but not in the brain of mice. These data highlight that studies measuring peripheral endpoints or directly exposing systems, in vitro, intracerebroventricularly, or intracortically, to BZT-containing formulations should account for the direct effects of BZT. Our findings also suggest that earlier data attributing pharmacological effects to ketamine may be confounded by BZT and that additional investigation into the functional impact of BZT is warranted. This article is part of the Special Issue on 'Ketamine and its Metabolites'.

Experimenters' sex modulates mouse behaviors and neural responses to ketamine via corticotropin releasing factor.

We show that the sex of human experimenters affects mouse behaviors and responses following administration of the rapid-acting antidepressant ketamine and its bioactive metabolite (2R,6R)-hydroxynorketamine. Mice showed aversion to the scent of male experimenters, preference for the scent of female experimenters and increased stress susceptibility when handled by male experimenters. This human-male-scent-induced aversion and stress susceptibility was mediated by the activation of corticotropin-releasing factor (CRF) neurons in the entorhinal cortex that project to hippocampal area CA1. Exposure to the scent of male experimenters before ketamine administration activated CA1-projecting entorhinal cortex CRF neurons, and activation of this CRF pathway modulated in vivo and in vitro antidepressant-like effects of ketamine. A better understanding of the specific and quantitative contributions of the sex of human experimenters to study outcomes in rodents may improve replicability between studies and, as we have shown, reveal biological and pharmacological mechanisms.

Target deconvolution studies of (2R,6R)-hydroxynorketamine: an elusive search.

The off-label use of racemic ketamine and the FDA approval of (S)-ketamine are promising developments for the treatment of depression. Nevertheless, racemic ketamine and (S)-ketamine are controlled substances with known abuse potential and their use is associated with undesirable side effects. For these reasons, research efforts have focused on identifying alternatives. One candidate is (2R,6R)-hydroxynorketamine ((2R,6R)-HNK), a ketamine metabolite that in preclinical models lacks the dissociative and abuse properties of ketamine while retaining its antidepressant-like behavioral efficacy. (2R,6R)-HNK's mechanism of action however is unclear. The main goals of this study were to perform an in-depth pharmacological characterization of (2R,6R)-HNK at known ketamine targets, to use target deconvolution approaches to discover novel proteins that bind to (2R,6R)-HNK, and to characterize the biodistribution and behavioral effects of (2R,6R)-HNK across several procedures related to substance use disorder liability. We found that unlike (S)- or (R)-ketamine, (2R,6R)-HNK did not directly bind to any known or proposed ketamine targets. Extensive screening and target deconvolution experiments at thousands of human proteins did not identify any other direct (2R,6R)-HNK-protein interactions. Biodistribution studies using radiolabeled (2R,6R)-HNK revealed non-selective brain regional enrichment, and no specific binding in any organ other than the liver. (2R,6R)-HNK was inactive in conditioned place preference, open-field locomotor activity, and intravenous self-administration procedures. Despite these negative findings, (2R,6R)-HNK produced a reduction in immobility time in the forced swim test and a small but significant increase in metabolic activity across a network of brain regions, and this metabolic signature differed from the brain metabolic profile induced by ketamine enantiomers. In sum, our results indicate that (2R,6R)-HNK does not share pharmacological or behavioral profile similarities with ketamine or its enantiomers. However, it could still be possible that both ketamine and (2R,6R)-HNK exert antidepressant-like efficacy through a common and previously unidentified mechanism. Given its pharmacological profile, we predict that (2R,6R)-HNK will exhibit a favorable safety profile in clinical trials, and we must wait for clinical studies to determine its antidepressant efficacy.

Comparative metabolomic analysis in plasma and cerebrospinal fluid of humans and in plasma and brain of mice following antidepressant-dose ketamine administration.

Subanesthetic-dose racemic (R,S)-ketamine (ketamine) produces rapid, robust, and sustained antidepressant effects in major depressive disorder (MDD) and bipolar disorder (BD) and has also been shown to effectively treat neuropathic pain, complex regional pain syndrome, and post-traumatic stress disorder (PTSD). However, to date, its mechanism of action remains unclear. Preclinical studies found that (2 R,6 R;2 S,6 S)-hydroxynorketamine (HNK), a major circulating metabolite of ketamine, elicits antidepressant effects similar to those of ketamine. To help determine how (2 R,6 R)-HNK contributes to ketamine's mechanism of action, an exploratory, targeted, metabolomic analysis was carried out on plasma and CSF of nine healthy volunteers receiving a 40-minute ketamine infusion (0.5 mg/kg). A parallel targeted metabolomic analysis in plasma, hippocampus, and hypothalamus was carried out in mice receiving either 10 mg/kg of ketamine, 10 mg/kg of (2 R,6 R)-HNK, or saline. Ketamine and (2 R,6 R)-HNK both affected multiple pathways associated with inflammatory conditions. In addition, several changes were unique to either the healthy human volunteers and/or the mouse arm of the study, indicating that different pathways may be differentially involved in ketamine's effects in mice and humans. Mechanisms of action found to consistently underlie the effects of ketamine and/or (2 R,6 R)-HNK across both the human metabolome in plasma and CSF and the mouse arm of the study included LAT1, IDO1, NAD+, the nitric oxide (NO) signaling pathway, and sphingolipid rheostat.

Hydroxynorketamine Pharmacokinetics and Antidepressant Behavioral Effects of (2,6)- and (5R)-Methyl-(2R,6R)-hydroxynorketamines.

(R,S)-Ketamine is rapidly metabolized to form a range of metabolites in vivo, including 12 unique hydroxynorketamines (HNKs) that are distinguished by a cyclohexyl ring hydroxylation at the 4, 5, or 6 position. While both (2R,6R)- and (2S,6S)-HNK readily penetrate the brain and exert rapid antidepressant-like actions in preclinical tests following peripheral administration, the pharmacokinetic profiles and pharmacodynamic actions of 10 other HNKs have not been examined. We assessed the pharmacokinetic profiles of all 12 HNKs in the plasma and brains of male and female mice and compared the relative potencies of four (2,6)-HNKs to induce antidepressant-relevant behavioral effects in the forced swim test in male mice. While all HNKs were readily brain-penetrable following intraperitoneal injection, there were robust differences in peak plasma and brain concentrations and exposures. Forced swim test immobility rank order of potency, from most to least potent, was (2R,6S)-, (2S,6R)-, (2R,6R)-, and (2S,6S)-HNK. We hypothesized that distinct structure-activity relationships and the resulting potency of each metabolite are linked to unique substitution patterns and resultant conformation of the six-membered cyclohexanone ring system. To explore this, we synthesized (5R)-methyl-(2R,6R)-HNK, which incorporates a methyl substitution on the cyclohexanone ring. (5R)-Methyl-(2R,6R)-HNK exhibited similar antidepressant-like potency to (2R,6S)-HNK. These results suggest that conformation of the cyclohexanone ring system in the (2,6)-HNKs is an important factor underlying potency and that additional engineering of this structural feature may improve the development of a new generation of HNKs. Such HNKs may represent novel drug candidates for the treatment of depression.

Negative Allosteric Modulation of Gamma-Aminobutyric Acid A Receptors at α5 Subunit-Containing Benzodiazepine Sites Reverses Stress-Induced Anhedonia and Weakened Synaptic Function in Mice.

BACKGROUND: Abnormal reward processing, typically anhedonia, is a hallmark of human depression and is accompanied by altered functional connectivity in reward circuits. Negative allosteric modulators of GABAA (gamma-aminobutyric acid A) receptors (GABA-NAMs) have rapid antidepressant-like properties in rodents and exert few adverse effects, but molecular targets underlying their behavioral and synaptic effects remain undetermined. We hypothesized that GABA-NAMs act at the benzodiazepine site of GABAA receptors containing α5 subunits to increase gamma oscillatory activity, strengthen synapses in reward circuits, and reverse anhedonia. METHODS: Anhedonia was induced by chronic stress in male mice and assayed by preferences for sucrose and female urine (n = 5-7 mice/group). Hippocampal slices were then prepared for electrophysiological recording (n = 1-6 slices/mouse, 4-6 mice/group). Electroencephalography power was quantified in response to GABA-NAM and ketamine administration (n = 7-9 mice/group). RESULTS: Chronic stress reduced sucrose and female urine preferences and hippocampal temporoammonic-CA1 synaptic strength. A peripheral injection of the GABA-NAM MRK-016 restored hedonic behavior and AMPA-to-NMDA ratios in wild-type mice. These actions were prevented by pretreatment with the benzodiazepine site antagonist flumazenil. MRK-016 administration increased gamma power over the prefrontal cortex in wild-type mice but not α5 knockout mice, whereas ketamine promoted gamma power in both genotypes. Hedonic behavior and AMPA-to-NMDA ratios were only restored by MRK-016 in stressed wild-type mice but not α5 knockout mice. CONCLUSIONS: α5-Selective GABA-NAMs exert rapid anti-anhedonic actions and restore the strength of synapses in reward regions by acting at the benzodiazepine site of α5-containing GABAA receptors. These results encourage human studies using GABA-NAMs to treat depression by providing readily translatable measures of target engagement.

Sex-dependent metabolism of ketamine and (2R,6R)-hydroxynorketamine in mice and humans.

BACKGROUND: Ketamine is rapidly metabolized to norketamine and hydroxynorketamine (HNK) metabolites. In female mice, when compared to males, higher levels of (2R,6R;2S,6S)-HNK have been observed following ketamine treatment, and higher levels of (2R,6R)-HNK following the direct administration of (2R,6R)-HNK. AIM: The objective of this study was to evaluate the impact of sex in humans and mice, and gonadal hormones in mice on the metabolism of ketamine to form norketamine and HNKs and in the metabolism/elimination of (2R,6R)-HNK. METHODS: In CD-1 mice, we utilized gonadectomy to evaluate the role of circulating gonadal hormones in mediating sex-dependent differences in ketamine and (2R,6R)-HNK metabolism. In humans (34 with treatment-resistant depression and 23 healthy controls) receiving an antidepressant dose of ketamine (0.5 mg/kg i.v. infusion over 40 min), we evaluated plasma levels of ketamine, norketamine, and HNKs. RESULTS: In humans, plasma levels of ketamine and norketamine were higher in males than females, while (2R,6R;2S,6S)-HNK levels were not different. Following ketamine administration to mice (10 mg/kg i.p.), Cmax and total plasma concentrations of ketamine and norketamine were higher, and those of (2R,6R;2S,6S)-HNK were lower, in intact males compared to females. Direct (2R,6R)-HNK administration (10 mg/kg i.p.) resulted in higher levels of (2R,6R)-HNK in female mice. Ovariectomy did not alter ketamine metabolism in female mice, whereas orchidectomy recapitulated female pharmacokinetic differences in male mice, which was reversed with testosterone replacement. CONCLUSION: Sex is an important biological variable that influences the metabolism of ketamine and the HNKs, which may contribute to sex differences in therapeutic antidepressant efficacy or side effects.

Post-weaning A1/A2 ß-casein milk intake modulates depressive-like behavior, brain µ-opioid receptors, and the metabolome of rats.

The postnatal period is critical for brain and behavioral development and is sensitive to environmental stimuli, such as nutrition. Prevention of weaning from maternal milk was previously shown to cause depressive-like behavior in rats. Additionally, loss of dietary casein was found to act as a developmental trigger for a population of brain opioid receptors. Here, we explore the effect of exposure to milk containing A1 and A2 ß-casein beyond weaning. A1 but not A2 ß-casein milk significantly increased stress-induced immobility in rats, concomitant with an increased abundance of Clostridium histolyticum bacterial group in the caecum and colon of A1 ß-casein fed animals, brain region-specific alterations of µ-opioid and oxytocin receptors, and modifications in urinary biochemical profiles. Moreover, urinary gut microbial metabolites strongly correlated with altered brain metabolites. These findings suggest that consumption of milk containing A1 ß-casein beyond weaning age may affect mood via a possible gut-brain axis mechanism.

A comparison of the pharmacokinetics and NMDAR antagonism-associated neurotoxicity of ketamine, (2R,6R)-hydroxynorketamine and MK-801.

With the increasing use of ketamine as an off-label treatment for depression and the recent FDA approval of (S)-ketamine for treatment-resistant depression, there is an increased need to understand the long-term safety profile of chronic ketamine administration. Of particular concern is the neurotoxicity previously observed in rat models following acute exposure to high doses of ketamine, broadly referred to as 'Olney's lesions'. This type of toxicity presents as abnormal neuronal cellular vacuolization, followed by neuronal death and has been associated with ketamine's inhibition of the N-methyl-d-aspartate receptor (NMDAR). In this study, a pharmacological and neuropathological analysis of ketamine, the potent NMDAR antagonist MK-801, and the ketamine metabolite (2R,6R)-hydroxynorketamine [(2R,6R)-HNK)] in rats is described following both single dose and repeat dose drug exposures. Ketamine dosing was studied up to 20 mg/kg intravenously for the single-dose neuropathology study and up to 60 mg/kg intraperitoneally for the multiple-dose neuropathology study. MK-801 dosing was studied up to 0.8 mg/kg subcutaneously for both the single and multiple-dose neuropathology studies, while (2R,6R)-HNK dosing was studied up to 160 mg/kg intravenously in both studies. These studies confirm dose-dependent induction of 'Olney's lesions' following both single dose and repeat dosing of MK-801. Ketamine exposure, while showing common behavioral effects, did not induce wide-spread Olney's lesions. Treatment with (2R,6R)-HNK did not produce behavioral effects, toxicity or any evidence of Olney's lesion formation. Based on these results, future NMDAR-antagonist neurotoxicity studies should strongly consider taking pharmacokinetics more thoroughly into account.

Hydroxynorketamines: Pharmacology and Potential Therapeutic Applications.

Hydroxynorketamines (HNKs) are formed in vivo after (R,S)-ketamine (ketamine) administration. The 12 HNK stereoisomers are distinguished by the position of cyclohexyl ring hydroxylation (at the 4, 5, or 6 position) and their unique stereochemistry at two stereocenters. Although HNKs were initially classified as inactive metabolites because of their lack of anesthetic effects, more recent studies have begun to reveal their biologic activities. In particular, (2R,6R)- and (2S 6)-HNK exert antidepressant-relevant behavioral and physiologic effects in preclinical models, which led to a rapid increase in studies seeking to clarify the mechanisms by which HNKs exert their pharmacological effects. To date, the majority of HNK research has focused on the actions of (2R,6R)-HNK because of its robust behavioral actions in tests of antidepressant effectiveness and its limited adverse effects. This review describes HNK pharmacokinetics and pharmacodynamics, as well as the putative cellular, molecular, and synaptic mechanisms thought to underlie their behavioral effects, both following their metabolism from ketamine and after direct administration in preclinical studies. Converging preclinical evidence indicates that HNKs modulate glutamatergic neurotransmission and downstream signaling pathways in several brain regions, including the hippocampus and prefrontal cortex. Effects on other neurotransmitter systems, as well as possible effects on neurotrophic and inflammatory processes, and energy metabolism, are also discussed. Additionally, the behavioral effects of HNKs and possible therapeutic applications are described, including the treatment of unipolar and bipolar depression, post-traumatic stress disorder, chronic pain, neuroinflammation, and other anti-inflammatory and analgesic uses. SIGNIFICANCE STATEMENT: Preclinical studies indicate that hydroxynorketamines (HNKs) exert antidepressant-relevant behavioral actions and may also have analgesic, anti-inflammatory, and other physiological effects that are relevant for the treatment of a variety of human diseases. This review details the pharmacokinetics and pharmacodynamics of the HNKs, as well as their behavioral actions, putative mechanisms of action, and potential therapeutic applications.

Ketamine metabolite (2R,6R)-hydroxynorketamine reverses behavioral despair produced by adolescent trauma.

Early life trauma dramatically increases the risk of developing major depressive disorder (MDD), and is associated with a markedly decreased adult treatment response to antidepressants. Novel treatment approaches are required to treat childhood trauma-associated MDD. Recent studies suggest that the (R,S)-ketamine (ketamine) metabolite, (2R,6R)-hydroxynorketamine (HNK), exerts fast- and long-lasting antidepressant-like effects without ketamine's NMDAR-inhibition-associated adverse side-effect profile. We investigated the therapeutic potential of (2R,6R)-HNK against behavioral despair produced by a novel live-predator stress exposure during adolescence. Male and female C57BL/6J mice were exposed to a live snake or control conditions at post-natal (PND) days 31, 45 and 61. In order to assess the enduring consequences of trauma-exposure, at a minimum of 14 days following the last exposure, mice received inescapable shocks followed by a session with available escape options twenty-four hours later. Mice that manifested enduring escape deficits (helplessness) were treated with vehicle or (2R,6R)-HNK (20 mg/kg, i.p.), 24 h prior to retesting for reversal of escape deficits. We found that a significantly greater number of mice developed the helpless phenotype when they were exposed to the live predator and that the helpless phenotype was reversed in mice treated with (2R,6R)-HNK. There were no sex differences in the response to predator-stress exposure or (2R,6R)-HNK treatment. The live-predator model developed in this study provides an opportunity to further refine our understanding of the neurobiological substrates impacted by adolescent trauma and improve treatment strategies. The demonstrated efficacy of (2R,6R)-HNK in this model suggests a novel therapeutic intervention for a treatment-resistant population.

A Randomized Trial of the N-Methyl-d-Aspartate Receptor Glycine Site Antagonist Prodrug 4-Chlorokynurenine in Treatment-Resistant Depression.

BACKGROUND: Ketamine has rapid-acting antidepressant effects but is associated with psychotomimetic and other adverse effects. A 7-chlorokynurenic acid is a potent and specific glycine site N-methyl-d-aspartate receptor antagonist but crosses the blood-brain barrier inefficiently. Its prodrug, L-4-chlorokynurenine (4-Cl-KYN), exerts acute and sustained antidepressant-like effects in rodents and has no reported psychotomimetic effects in either rodents or healthy volunteers. This study examined whether 4-Cl-KYN has rapid antidepressant effects in individuals with treatment-resistant depression. METHODS: After a 2-week drug-free period, 19 participants with treatment-resistant depression were randomized to receive daily oral doses of 4-Cl-KYN monotherapy (1080 mg/d for 7 days, then 1440 mg/d for 7 days) or placebo for 14 days in a randomized, placebo-controlled, double-blind, crossover manner. The primary outcome measure was the Hamilton Depression Rating Scale score, assessed at several time points over a 2-week period; secondary outcome measures included additional rating scale scores. Pharmacokinetic measures of 7-chlorokynurenic acid and 4-Cl-KYN and pharmacodynamic assessments were obtained longitudinally and included 1H-magnetic resonance spectroscopy brain glutamate levels, resting-state functional magnetic resonance imaging, and plasma and cerebrospinal fluid measures of kynurenine metabolites and neurotrophic factors. RESULTS: Linear mixed models detected no treatment effects, as assessed by primary and secondary outcome measures. No difference was observed for any of the peripheral or central biological indices or for adverse effects at any time between groups. A 4-Cl-KYN was safe and well-tolerated, with generally minimal associated adverse events. CONCLUSIONS: In this small crossover trial, 4-Cl-KYN monotherapy exerted no antidepressant effects at the doses and treatment duration studied.ClinicalTrials.gov identifier: NCT02484456.

Psychological stress enhances tumor growth and diminishes radiation response in preclinical model of lung cancer.

BACKGROUND AND PURPOSE: Patients with life-threatening illnesses, such as cancer, experience emotional distress. This study was to investigate the molecular and cellular mechanisms of relevant psychological stressor on tumor growth and therapeutic resistance. MATERIALS AND METHODS: Stress was induced in C57BL/6J mice bearing LLC lung tumors by exposure to a conspecific mice receiving inescapable foot shocks. Mice were irradiated at 7 Gy for 3 consecutive days. Behaviors were monitored by open field test (OFT), elevated plus maze (EPM), sucrose preference test (SPT), and learned helplessness (LH) test. Protein expression in tissues and cultured cells were measured by Western blot. RESULTS: This study in animals showed that observing a conspecific mouse receiving foot shocks induced depression like behaviors with increased plasma corticosterone and adrenaline levels which increased tumor growth and radioresistance. Stress increased Wnt1, Drosha, and vimentin expression and decreased E-cadherin expression in tumor tissues. The combination of stress and irradiation enhanced radioresistance along with the increase in vimentin expression. The in vitro study showed that a ß2-adrenergic receptor (ß2-AR) agonist blocked irradiation-induced cell apoptosis and decreased cell viability, while silencing ß2-AR expression reduced the protective effects of ß2-AR agonist. ß2-AR agonist obviously increased Wnt1 and Drosha expression in LLC-1 cells. CONCLUSION: Psychological stress increased tumor growth and enhanced radioresistance associated with the activation of epithelial-mesenchymal transition by stress hormone-stimulated adrenergic receptors.