NMDA Receptor Agonist Research Articles

Effect of NMDA receptor agonist and antagonist on spermatogonial stem cells proliferation in 2- and 3- dimensional culture systems.

The main purpose of this study was to investigate the effect of D-serine (DS) and Dizocilpine (MK-801) on the proliferation of spermatogonial stem cells (SSCs) in two-dimensional (2D) and three-dimensional (3D) culture systems. The SSCs of male NMRI mice were isolated by enzymatic digestion and cultured for two weeks. Then, the identity of SSCs was validated by anti-Plzf and anti-GFR-α1 antibodies via immunocytochemistry (ICC). The proliferation capacity of SSCs was evaluated by their culture on a layer of the decellularized testicular matrix (DTM) prepared from mouse testis, as well as two-dimensional (2D) with different mediums. After two weeks of the initiation of proliferation culture on 3D and 2D medium, the pre-meiotic at the mRNA and protein levels were evaluated via qRT-PCR and flow cytometry methods, respectively. The results showed that the proliferation rate of SSCs in 3D culture with 50mM glutamic acid and 20mM D-serine was significantly different from other groups after 14days treatment. mRNA expression levels of promyelocytic leukemia zinc finger (Plzf) in 3D cultures supplemented by 20mM D-serine and 50mM glutamic acid were considerably higher than the 3D control group (p < 0.001). The flow cytometry analysis revealed that the amount of Plzf in the 2D-culture groups of SSCs with 20mM MK-801 was considerably lower compared to the 2D-culture control group (p < 0.001). This study indicated that decellularized testicular matrix supplemented with D-serine and glutamic acid could be considered a promising vehicle to support cells and provide an appropriate niche for the proliferation of SSCs.

Astrocytic N-Methyl-D-Aspartate Receptors Protect the Hippocampal Neurons Against Amyloid-β142-Induced Synaptotoxicity by Regulating Nerve Growth Factor.

Soluble oligomeric amyloid-β (Aβ)-induced synaptic dysfunction is an early event in Alzheimer's disease (AD) pathogenesis. Mounting evidence has suggested N-methyl-D-aspartate receptors (NMDARs) play an important role in Aβ-induced synaptotoxicity. Originally NMDARs were believed to be expressed exclusively in neurons; however, recent two decades studies have demonstrated functional NMDARs present on astrocytes. Neuronal NMDARs are modulators of neurodegeneration, while our previous initial study found that astrocytic NMDARs mediated synaptoprotection and identified nerve growth factor (NGF) secreted by astrocytes, as a likely mediator, but how astrocytic NMDARs protect neurons against Aβ-induced synaptotoxicity through regulating NGF remains unclear. To achieve further insight into the mechanism of astrocytic NMDARs oppose Aβ-induced synaptotoxicity through regulating NGF. With the primary hippocampal neuronal and astrocytic co-cultures, astrocytes were pretreated with agonist or antagonist of NMDARs before Aβ142 oligomers application to neuron-astrocyte co-cultures. Western blot, RT-PCR, etc., were used for the related proteins evaluation. Activation of astrocytic NMDARs can significantly mitigate Aβ142-induced loss of PSD-95 and synaptophysin through increasing NGF release. Blockade of astrocytic NMDARs inhibited Aβ-induced compensatory protective NGF increase in protein and mRNA levels through modulating NF-κB of astrocytes. Astrocytic NMDARs activation can enhance Aβ-induced Furin increase, and blockade of astrocytic NMDARs inhibited Aβ-induced immunofluorescent intensity elevation of vesicle trafficking protein VAMP3 and NGF double-staining. Astrocytic NMDARs oppose Aβ-induced synaptotoxicity through modulating the synthesis, maturation, and secretion of NGF in astrocytes. This new information may contribute to the quest for specific targeted strategy of intervention to delay the onset of AD.

Serotonin 1A receptor agonist modulation of motor deficits and cortical oscillations by NMDA receptor interaction in parkinsonian rats

Although serotonin 1A (5-HT1A) receptor agonists are widely used as the additive compound to reduce l-dopa-induced dyskinesia in Parkinson's disease (PD), few studies focused on the effect and mechanism of 5-HT1A receptor agonist on the motor symptoms of PD. Unilateral 6-hydroxydopamine (6-OHDA)-lesioned rats were used and implantation of electrodes was performed in the motor cortex of these rats. So the effect of 5-HT1A receptor agonist 8-OH-DPAT on motor behaviors and oscillatory activities were evaluated. In addition, 8-OH-DPAT combined with D2 receptor antagonist raclopride, NMDA receptor antagonist MK-801, or its agonist d-cycloserine (DCS) were co-administrated. 8-OH-DPAT administration significantly improved spontaneous locomotor activity and asymmetric forepaw function in 6-OHDA-lesioned rats. Meanwhile, 8-OH-DPAT identified selective modulation of the abnormal high beta oscillations (25–40 Hz) in the motor cortex of 6-OHDA-lesioned rats, without inducing pathological finely tuned gamma around 80 Hz. Different from 8-OH-DPAT, l-dopa treatment produced a prolonged improvement on motor performances and differential regulation of high beta and gamma oscillations. However, dopamine D2 receptor antagonist had no influence on the 8-OH-DPAT-mediated-motor behaviors and beta oscillations in 6-OHDA-lesioned rats. In contrast, subthreshold NMDA receptor antagonist MK-801 obviously elevated the 8-OH-DPAT-mediated-motor behaviors, while NMDA receptor agonist DCS partially impaired the 8-OH-DPAT-mediated symptoms in 6-OHDA-lesioned rats. This study suggests that 5-HT1A receptor agonist 8-OH-DPAT improves motor activity and modulates the oscillations in the motor cortex of parkinsonian rats. Different from l-dopa, 8-OH-DPAT administration ameliorates motor symptoms of PD through glutamatergic rather than the dopaminergic pathway.

Amyloid-β disrupts unitary calcium entry through endothelial NMDA receptors in mouse cerebral arteries

Transient increases in intracellular Ca2+ activate endothelium-dependent vasodilatory pathways. This process is impaired in cerebral amyloid angiopathy, where amyloid-β(1-40) accumulates around blood vessels. In neurons, amyloid-β impairs the Ca2+-permeable N-methyl-D-aspartate receptor (NMDAR), a mediator of endothelium-dependent dilation in arteries. We hypothesized that amyloid-β(1-40) reduces NMDAR-elicited Ca2+ signals in mouse cerebral artery endothelial cells, blunting dilation. Cerebral arteries isolated from 4-5 months-old, male and female cdh5:Gcamp8 mice were used for imaging of unitary Ca2+ influx through NMDAR (NMDAR sparklets) and intracellular Ca2+ transients. The NMDAR agonist NMDA (10 µmol/L) increased frequency of NMDAR sparklets and intracellular Ca2+ transients in endothelial cells; these effects were prevented by NMDAR antagonists D-AP5 and MK-801. Next, we tested if amyloid-β(1-40) impairs NMDAR-elicited Ca2+ transients. Cerebral arteries incubated with amyloid-β(1-40) (5 µmol/L) exhibited reduced NMDAR sparklets and intracellular Ca2+ transients. Lastly, we observed that NMDA-induced dilation of pial arteries is reduced by acute intraluminal amyloid-β(1-40), as well as in a mouse model of Alzheimer’s disease, the 5x-FAD, linked to downregulation of Grin1 mRNA compared to wild-type littermates. These data suggest that endothelial NMDAR mediate dilation via Ca2+-dependent pathways, a process disrupted by amyloid-β(1-40) and impaired in 5x-FAD mice.

Effect 1 of NMDA Receptor Agonist and Antagonist on Nicotine withdrawal induced Hyperexcitability in Mice

Kynurenic acid is a recognized broad-spectrum antagonist of excitatory amino acid receptors with a particularly high affinity for the glycine co-agonist site of the N-methyl-D-aspartate (NMDA) receptor complex. D- Cycloserine is a NMDA receptor partial agonist which facilitate in an initiation of nicotine withdrawal symptoms and dependence. Thus, the influence of kynurenic acid treatment on the development and expression of nicotine dependence was tested by using the nicotine withdrawal-induced hyperexcitability paradigm. Mice were provided with a nutritionally balanced control liquid diet as the sole nutrient source on day 0; from day 1–4 (nicotine 25µg), from day 5–7 (nicotine, 50ug) and from day 8–10 (nicotine, 100ug) was incorporated into the liquid diet. On day 11, the nicotine liquid diet was replaced with nutritionally balanced control liquid diet, and nicotine withdrawal-induced hyperexcitability signs were recorded. The results revealed that acute administration of kunurenic acid (50 and 100mg/kg, i.p.) dose-dependently attenuated nicotine withdrawal-induced hyperexcitability signs, and these results were comparable to D- Cycloserine (50 and 100mg/kg, i.p.) Further, chronic administration of kunurenic acid (50 and 100mg/kg, i.p.) to the nicotine diet fed mice markedly attenuated the nicotine withdrawal-induced hyperexcitability signs. In conclusion, the results and evidence suggest that kinurenic acid exhibited an inhibitory influence against nicotine withdrawal-induced hyperexcitability signs, which could be mediated through its neuromodulatory action.

THC-induced behavioral stereotypy in zebrafish as a model of psychosis-like behavior

High doses of the Cannabis constituent Δ9-tetrahydrocannabinol (THC) increase the risk of psychosis in humans. Highly accessible animal models are needed to address underlying mechanisms. Using zebrafish with a conserved endocannabinoid system, this study investigates the acute effects of THC on adult zebrafish behavior and the mechanisms involved. A concentration-dependent THC-induced behavioral stereotypy akin to THC’s effect in rats and the psychotropics phencyclidine and ketamine in zebrafish was established. Distinctive circular swimming during THC-exposure was measured using a novel analytical method that we developed, which detected an elevated Repetition Index (RI) compared to vehicle controls. This was reduced upon co-administration of N-methyl-D-aspartate (NMDA) receptor agonist NMDA, suggesting that THC exerts its effects via biochemical or neurobiological mechanisms associated with NMDA receptor antagonism. Co-treatment of γ‐aminobutyric acid receptor antagonist pentylenetetrazol also showed signs of reducing the RI. Since THC-induced repetitive behavior remained in co-administrations with cannabinoid receptor 1 inverse agonist AM251, the phenotype may be cannabinoid receptor 1-independent. Conversely, the inverse cannabinoid receptor 2 agonist AM630 significantly reduced THC-induced behavioral stereotypy, indicating cannabinoid receptor 2 as a possible mediator. A significant reduction of the THC-RI was also observed by the antipsychotic sulpiride. Together, these findings highlight this model’s potential for elucidating the mechanistic relationship between Cannabis and psychosis.

NMDAR in bladder smooth muscle is not a pharmacotherapy target for overactive bladder in mice.

Overactive bladder (OAB) is a common condition that affects a significant patient population. The N-methyl-D-aspartate receptor (NMDAR) has a role in developing bladder overactivity, pharmacological inhibition of which inhibits bladder overactivity. The common pathogenesis of OAB involves bladder smooth muscle (BSM) overactivity. In this study, a smooth muscle–specific NMDAR knockout (SMNRKO) mouse model was generated. The bladders from SMNRKO mice displayed normal size and weight with an intact bladder wall and well-arranged BSM bundles. Besides, SMNRKO mice had normal voiding patterns and urodynamics and BSM contractility, indicating that NMDAR in BSM was not essential for normal physiological bladder morphology and function. Unexpectedly, cyclophosphamide (CYP)-treated SMNRKO and wild-type (WT) mice had similar pathological changes in the bladder. Furthermore, SMNRKO mice displayed similar altered voiding patterns and urodynamic abnormalities and impaired BSM contractility compared with WT mice after CYP treatment. MK801 partially reversed the pathological bladder morphology and improved bladder dysfunction induced by CYP, but did not cause apparent differences between WT mice and SMNRKO mice, suggesting that NMDAR in BSM was not involved in pathological bladder morphology and function. Moreover, the direct instillation of NMDAR agonists or antagonists into the CYP-induced OAB did not affect bladder urodynamic function, indicating that NMDAR in BSM was not the pharmacotherapy target of MK801 for CYP-induced cystitis. The findings indicated that NMDAR in BSM was not essential for normal physiological or pathological bladder morphology and function, and MK801 improving pathological bladder function was not mediated by an action on NMDAR in BSM.

GluN2D NMDA Receptors Gate Fear Extinction Learning and Interneuron Plasticity.

The cerebellum is critically involved in the formation of associative fear memory and in subsequent extinction learning. Fear conditioning is associated with a long-term potentiation at both excitatory and inhibitory synapses onto Purkinje cells. We therefore tested whether fear conditioning unmasks novel forms of synaptic plasticity, which enable subsequent extinction learning to reset cerebellar circuitry. We found that fear learning enhanced GABA release from molecular layer interneurons and this was reversed after fear extinction learning. Importantly an extinction-like stimulation of parallel fibers after fear learning is sufficient to induce a lasting decrease in inhibitory transmission (I-LTDstim) in the cerebellar cortex, a form of plasticity that is absent in naïve animals. While NMDA (N-methyl-D-aspartate) receptors are required for the formation and extinction of associative memory, the role of GluN2D, one of the four major NMDA receptor subunits, in learning and memory has not been determined. We found that fear conditioning elevates spontaneous GABA release in GluN2D KO as shown in WT mice. Deletion of GluN2D, however, abolished the I-LTDstim induced by parallel fiber stimulation after learning. At the behavioral level, genetic deletion of GluN2D subunits did not affect associative learning and memory retention, but impaired subsequent fear extinction learning. D-cycloserine, a partial NMDA receptor (NMDAR) agonist, failed to rescue extinction learning in mutant mice. Our results identify GluN2D as a critical NMDAR subunit for extinction learning and reveal a form of GluN2D-dependent metaplasticity that is associated with extinction in the cerebellum.

Protective effects of calcium ions via L-type calcium channels and NMDA receptors on prostaglandin E2-induced apoptosis in rat cortical cells.

Calcium ions mediate a variety of physiological responses of developing neurons including survival. The purpose of this study was to examine the effect of calcium influx through L-type calcium channels (LTCCs) or NMDA receptors on prostaglandin E2 (PGE2)-induced apoptosis in rat cortical cells. Cultures of rat cortical cells were prepared from an embryonic day 18 rat neocortex. After culturing for 2 or 8days in vitro (DIV), the cells were subjected to PGE2 treatment for 48h. FPL64176, an LTCC agonist, protected the cells at 2 and 8 DIV from PGE2-induced apoptosis. On the other hand, N-methyl-D-aspartate (NMDA), an agonist of NMDA receptor, protected the cells from PGE2-induced apoptosis only at 8 DIV. FPL64176 increased the calcium levels at 2 and 8 DIV, whereas NMDA increased the calcium levels only at 8 DIV. The protective effects of the LTCC agonist and NMDA on PGE2-induced apoptosis were blocked following treatment of the cells with protein kinase C inhibitors. Our results suggest that LTCCs and NMDA receptors modulate the cell death of developing cortical neurons possibly through a protein kinase C pathway.

Effects of D-Cycloserine on the Primary Motor Cortex Excitability in Healthy Human Subjects: A Paired-Pulse-Tms Protocol

Paired-pulse transcranial magnetic stimulation (ppTMS) can probe the human primary motor cortex excitability. Short interstimulus intervals (ISI<5ms) between a subthreshold and a suprathreshold pulse cause short-term intracortical inhibition (SICI), whereas longer ISI causes intracortical facilitation (ICF). NMDA-receptor antagonists decrease ICF, a proxy for glutamatergic transmission, and increase ICI and cortical silent period (CSP), for GABAergic transmission. The effect of NMDA-receptor agonists (e.g.

Synergistic enhancing-memory effect of D-serine and RU360, a mitochondrial calcium uniporter blocker in rat model of Alzheimer's disease

BackgroundAlthough Amyloid beta (Aβ) and N - methyl d- aspartate receptors (NMDARs are involved in Ca2+ neurotoxicity, the function of mitochondrial calcium uniporter in cognition deficit remain uncertain. Here, we examined the effect of mitochondrial calcium uniporter (MCU) blocker, together with NMDA receptor agonist d-cycloserine (DCS) on memory impairment in a rat model of AD. MethodsForty adult male Wistar rats underwent stereotaxic cannulation for inducing AD by intracerebroventricular (ICV) injection of Aβ1–42 (5 μg /8 μl/rat). Then animals were divided into 5 groups of: Saline + Saline, Aβ + Saline, Aβ + RU360, Aβ + DCS, Aβ + RU360 + DCS. Two weeks after the treatments, Morris Water Maze (MWM) and step through passive avoidance learning (SPL) were undertaken for evaluating of spatial and associative memories, respectively. Hippocampal level of cyclic-AMP response element binding protein (CREB) and brain-derived neurotrophic factor (BDNF) were measured by western blot and ELISA. ResultsCo - administration of RU360 and DCS significantly improved both acquisition and retrieval of spatial memory as evident by decreased escape latency and increased time spent in the target quadrant (TTS) in MWM, together with increase in step-through latency, but reduced time spent in the dark compartment in SPL. Furthermore, there was a significant rise in the hippocampal level of CREB and BDNF in comparison with Aβ + Saline. ConclusionThe present study supports the idea that co- administration of RU360 and DCS ameliorate memory impairment induced by Aβ 1–42 probably via CREB / BDNF signaling.

The Role of D-Amino Acids in Alzheimer's Disease.

Alzheimer's disease (AD), the main cause of dementia worldwide, is characterized by a complex and multifactorial etiology. In large part, excitatory neurotransmission in the central nervous system is mediated by glutamate and its receptors are involved in synaptic plasticity. The N-methyl-D-aspartate (NMDA) receptors, which require the agonist glutamate and a coagonist such as glycine or the D-enantiomer of serine for activation, play a main role here. A second D-amino acid, D-aspartate, acts as agonist of NMDA receptors. D-amino acids, present in low amounts in nature and long considered to be of bacterial origin, have distinctive functions in mammals. In recent years, alterations in physiological levels of various D-amino acids have been linked to various pathological states, ranging from chronic kidney disease to neurological disorders. Actually, the level of NMDA receptor signaling must be balanced to promote neuronal survival and prevent neurodegeneration: this signaling in AD is affected mainly by glutamate availability and modulation of the receptor's functions. Here, we report the experimental findings linking D-serine and D-aspartate, through NMDA receptor modulation, to AD and cognitive functions. Interestingly, AD progression has been also associated with the enzymes related to D-amino acid metabolism as well as with glucose and serine metabolism. Furthermore, the D-serine and D-/total serine ratio in serum have been recently proposed as biomarkers of AD progression. A greater understanding of the role of D-amino acids in excitotoxicity related to the pathogenesis of AD will facilitate novel therapeutic treatments to cure the disease and improve life expectancy.

Role of D-serine Binding to the NMDA Receptor Ligand-Binding Domain

The N-methyl-D-aspartate receptor (NMDAR) is a ligand-gated ion channel that promotes synaptic plasticity critical for learning and memory. NMDAR activation is initiated by binding of two different neurotransmitter agonists to the ligand-binding domains (LBDs). Specifically, the GluN2 subunit binds glutamate, while the GluN1 subunit binds either glycine or D-serine. While the mechanisms of glycine and glutamate binding to NMDAR LBDs have already been determined, little is known about the mechanisms by which D-serine can bind to and activate the receptor. To probe binding mechanisms, we performed multi-microsecond molecular dynamics simulations of the GluN1/GluN2A LBD dimer in the presence of freely diffusing D-serine and glutamate and observed multiple binding and unbinding events. While D-serine is primarily a GluN1 agonist, we surprisingly observed that D-serine binds to both GluN1 and GluN2A NMDAR subunits. Computing interaction frequencies between LBD residues and D-serine revealed residues important for agonist binding. However, we observed that binding pathways are made up of sequences of interactions that guide D-serine into the binding site. To identify properties of these pathways, we developed a framework for analyzing binding pathways by geometric and spatial similarity, which allowed us to cluster them according to the region of the LBD with which D-serine interacts. This allowed us to identify groups of residues that work together to contribute to particular binding pathways. We determined that D-serine and glutamate bind by similar pathways and found that D-serine is able to stabilize the active LBD conformation. Lastly, we determined that N-linked glycans at physiologically relevant glycosylation sites generally do not interfere with agonist-binding pathways to modulate LBD activity. These insights highlight the versatility of D-serine as an NMDAR agonist and its unique role in receptor activation.

Facilitation of GluN2C-containing NMDA receptors in the external globus pallidus increases firing of fast spiking neurons and improves motor function in a hemiparkinsonian mouse model

Globus pallidus externa (GPe) is a nucleus in the basal ganglia circuitry involved in the control of movement. Recent studies have demonstrated a critical role of GPe cell types in Parkinsonism. Specifically increasing the function of parvalbumin (PV) neurons in the GPe has been found to facilitate motor function in a mouse model of Parkinson's disease (PD). The knowledge of contribution of NMDA receptors to GPe function is limited. Here, we demonstrate that fast spiking neurons in the GPe express NMDA receptor currents sensitive to GluN2C/GluN2D-selective inhibitors and glycine site agonist with higher efficacy at GluN2C-containing receptors. Furthermore, using a novel reporter model, we demonstrate the expression of GluN2C subunits in PV neurons in the GPe which project to subthalamic nuclei. GluN2D subunit was also found to localize to PV neurons in GPe. Ablation of GluN2C subunit does not affect spontaneous firing of fast spiking neurons. In contrast, facilitating the function of GluN2C-containing receptors using glycine-site NMDA receptor agonists, D-cycloserine (DCS) or AICP, increased the spontaneous firing frequency of PV neurons in a GluN2C-dependent manner. Finally, we demonstrate that local infusion of DCS or AICP into the GPe improved motor function in a mouse model of PD. Together, these results demonstrate that GluN2C-containing receptors and potentially GluN2D-containing receptors in the GPe may serve as a therapeutic target for alleviating motor dysfunction in PD and related disorders.

Melatonin attenuated the behavioral despair induced by acute neurogenic stress through blockade of N-methyl D-aspartate receptors in mice

It has been well documented that administration of melatonin could reveal antidepressant-like effect in rodents. However, the protective effect of melatonin on stress-induced depression/anxiety and its underlying mechanism is yet to be understood. In this regard, in the current study, acute foot-shock stress (FSS) was used to evaluate the antidepressant-like effect of melatonin on neurogenic stress-induced depression in mice. Behavioral evaluation was done by using the forced swimming test (FST) and Open-field test (OFT). Melatonin, MK-801, and ketamine (NMDA receptor antagonists), and NMDA (NMDA receptor agonist) were used to elucidate any association between melatonin and NMDA pathway in behavioral despair induced by acute-FSS. Applying acute-FSS to mice significantly induced depressant-like behavior in FST without any significant impact on locomotor activity in the OFT. We observed that melatonin (dose-dependently) significantly improved the depressant-like effect of FSS, but it did not impact the locomotion in animals. Acute injection of MK-801 at sub-effective doses (0.01 mg/kg) or ketamine (0.1 mg/kg) potentiated the antidepressant-like effect of a sub-effective dose of melatonin. However, the sub-effective dose of NMDA (30 mg/kg) abolished the protective effect of melatonin on the behavioral profile of stressed animals. Our results could reflect the antidepressant-like effect of melatonin on neurogenic stress-induced depressive behaviors in mice. Also, our results showed that NMDA receptors could be involved in the antidepressant-like effect of melatonin.

Real-time fMRI neurofeedback amygdala training may influence kynurenine pathway metabolism in major depressive disorder

Real-time fMRI neurofeedback (rtfMRI-nf) left amygdala (LA) training is a promising intervention for major depressive disorder (MDD). We have previously proposed that rtfMRI-nf LA training may reverse depression-associated regional impairments in neuroplasticity and restore information flow within emotion-regulating neural circuits. Inflammatory cytokines as well as the neuroactive metabolites of an immunoregulatory pathway, i.e. the kynurenine pathway (KP), have previously been implicated in neuroplasticity. Therefore, in this proof-of-principle study, we investigated the association between rtfMRI-nf LA training and circulating inflammatory mediators and KP metabolites. Based on our previous work, the primary variable of interest was the ratio of the NMDA-receptor antagonist, kynurenic acid to the NMDA receptor agonist, quinolinic acid (KynA/QA), a putative neuroprotective index. We tested two main hypotheses. i. Whether rtfMRI-nf acutely modulates KynA/QA, and ii. whether baseline KynA/QA predicts response to rtfMRI-nf.Twenty-nine unmedicated participants who met DSM-5 criteria for MDD based on the Mini-International Neuropsychiatric Interview and had current depressive symptoms (Montgomery-Åsberg Depression Rating Scale (MADRS) score > 6) completed two rtfMRI-nf sessions to upregulate LA activity (Visit1 and 2), as well as a follow-up (Visit3) without rtfMRI-nf. All visits occurred at two-week intervals. At all three visits, the MADRS was administered to participants and serum samples for the quantification of inflammatory cytokines and KP metabolites were obtained. First, the longitudinal changes in the MADRS score and immune markers were tested by linear mixed effect model analysis. Further, utilizing a linear regression model, we investigated the relationship between rtfMRI-nf performance and immune markers. After two sessions of rtfMRI-nf, MADRS scores were significantly reduced (t[58] = −4.07, p = 0.009, d = 0.56). Thirteen participants showed a ≥ 25% reduction in the MADRS score (the partial responder group). There was a significant effect of visit (F[2,58] = 3.17, p = 0.05) for the neuroprotective index, KynA to 3-hydroxykynurenine (3-HK), that was driven by a significant increase in KynA/3-HK between Visit1 and Visit3 (t[58] = 2.50, p = 0.03, d = 0.38). A higher baseline level of KynA/QA (β = 5.23, p = 0.06; rho = 0.49, p = 0.02) was associated with greater ability to upregulate the LA. Finally, for exploratory purposes correlation analyses were performed between the partial responder and the non-responder groups as well as in the whole sample including all KP metabolites and cytokines. In the partial responder group, greater ability to upregulate the LA was correlated with an increase in KynA/QA after rtfMRI-nf (rho = 0.75, p = 0.03). The results are consistent with the possibility that rtfMRI-nf decreases metabolism down the so-called neurotoxic branch of the KP. Nevertheless, non-specific effects cannot be ruled out due to the lack of a sham control. Future, controlled studies are needed to determine whether the increase in KynA/3HK and KynA/QA is specific to rtfMRI-nf or whether it is a non-specific correlate of the resolution of depressive symptoms. Similarly, replication studies are needed to determine whether KynA/QA has clinical utility as a treatment response biomarker.

Stereoselective synthesis of novel 2'-(S)-CCG-IV analogues as potent NMDA receptor agonists.

We developed a versatile stereoselective route for the synthesis of new 2'-(S)-CCG-IV analogues. The route allows for late stage diversification and thereby provides access to a great variety of conformationally restricted cyclopropyl glutamate analogues. A selection of the 2'-(S)-CCG-IV analogues were evaluated using two-electrode voltage-clamp electrophysiology at recombinant GluN1/GluN2A-D receptors, demonstrating that agonists can be developed with GluN2 subunit-dependent potency and agonist efficacy. We also describe a crystal structure of the GluN2A agonist binding domain in complex with 2'-butyl-(S)-CCG-IV that determines the position of 2'-substituents in (S)-CCG-IV agonists in the glutamate binding site and provides further insight to the structural determinants of their agonist efficacy. The stereoselective synthesis described here enables versatile and straight-forward modifications to diverse analogues of interest for the development of potent subtype-specific NMDA receptor agonists and other applications.

A randomized cross-over trial to define neurophysiological correlates of AV-101 N-methyl-d-aspartate receptor blockade in healthy veterans

The kynurenine pathway (KP) is a strategic metabolic system that combines regulation of neuronal excitability via glutamate receptor function and neuroinflammation via other KP metabolites. This pathway has great promise in treatment of depression and suicidality. The KP modulator AV-101 (4-chlorokynurenine, 4-Cl-KYN), an oral prodrug of 7-chlorokynurenic acid (7-Cl-KYNA), an N-methyl-d-aspartate receptor (NMDAR) glycine site antagonist, and of 4-chloro-3-hydroxyanthranilic acid (4-Cl-3-HAA), a suppressor of NMDAR agonist quinolinic acid (QUIN), is a promising potential antidepressant that targets glutamate functioning via the KP. However, a recent placebo-controlled clinical trial of AV-101 in depression found negative results. This raises the question of whether AV-101 can penetrate the brain and engage the NMDAR and KP effectively. To address this problem, ten healthy US military veterans (mean age = 32.6 years ± 6.11; 1 female) completed a phase-1 randomized, double-blind, placebo-controlled, crossover study to examine dose-related effects of AV-101 (720 and 1440 mg) on NMDAR engagement measured by γ-frequency band auditory steady-state response (40 Hz ASSR) and resting EEG. Linear mixed models revealed that 1440 mg AV-101, but not 720 mg, increased 40 Hz ASSR and 40 Hz ASSR γ-inter-trial phase coherence relative to placebo. AV-101 also increased 4-Cl-KYN, 7-Cl-KYNA, 4-Cl-3-HAA, 3-HAA, and KYNA in a dose-dependent manner, without affecting KYN and QUIN. AV-101 was safe and well tolerated. These results corroborate brain target engagement of 1440 mg AV-101 in humans, consistent with blockade of interneuronal NMDAR blockade. Future studies should test higher doses of AV-101 in depression. Suicidal behavior, which has been associated with high QUIN and low KYNA, is also a potential target for AV-101.

Description of amyloid beta influence on synaptic plasticity and tau hyperphosphorylation by mechanistic quantitative systems pharmacology model

AbstractBackgroundLong‐term potentiation (LTP) is a dependent increase in synaptic plasticity that may be an appropriate experimental and theoretical model for learning and memory. In animal models of Alzheimer’s disease (AD), amyloid beta (Aβ) impacts the LTP decline and tau hyperphosphorylation. Increase of the intracellular calcium level, mediated by NMDA and nACh receptors, is responsible for appearance of LTP, which depends on increased in AMPA phosphorylation. Quantitative systems pharmacology (QSP) model of synaptic plasticity was developed to investigate influence of Aβ on impairment of LTP and tau hyperphosphorylation.MethodThe model describes a calcium influx in postsynapse through acetylcholine and glutamate receptors, activation of efflux from intracellular stores, activation of kinase/phosphatase cascade, tau phosphorylation and level of phosphorylation of AMPA receptors, which is associated with LTP. AD pathology is modeled by Aβ impact on receptors (inhibits nACh) and glial glutamate uptake. Model is developed step by step from simpler variants by consecutive addition of new mechanistic details, using in vitro data. In vitro data from the literature on synaptic plasticity in presence of different effectors, are used for verification.ResultThe model qualitatively describes impairment of LTP and tau hyperphosphorylation by Aβ influence and effect of receptor blockers, kinase and phosphatase inhibitors. It demonstrates that: 1) cholinergic and glutamate stimulation leads to LTP, depending on the timing of cholinergic input relative to glutamate input; 2) impaired LTP may recover due to extrasynaptic NMDA receptor blockers (e.g. memantine) or agonists of nACh receptors (e.g. SSR180711); 3) inhibitors of calcineurin decreased tau phosphorylation and recover LTPConclusionThe QSP model describes an influence Aβ on different postsynaptic mechanisms (receptors activation and kinase/phosphatase cascade, phosphorylation and dephosphorylation of AMPA receptors), which seems to underlie LTP and tau hyperphosphorylation.

Neuron-derived factors negatively modulate ryanodine receptor-mediated calcium release in cultured mouse astrocytes

Changes in intracellular Ca2+ concentration ([Ca2+]i) produced by ryanodine receptor (RyR) agonist, caffeine (caf), and ionotropic agonists: N-methyl-d-aspartate (NMDA) receptor (NMDAR) agonist, NMDA and P2X7 receptor (P2X7R) agonist, 3′-O-(4-benzoyl)benzoyl adenosine 5′-triphosphate (BzATP) were measured in cultured mouse cortical astrocytes loaded with the fluorescent calcium indicator Fluo3-AM in a confocal laser scanning microscope. In mouse astrocytes cultured in standard medium (SM), treatment with caf increased [Ca2+]i, with a peak response occurring about 10 min after stimulus application. Peak responses to NMDA or BzATP were observed about <1 min and 4.5 min post stimulus, respectively. Co-treatment with NMDA or BzATP did not alter the peak response to caf in astrocytes cultured in SM, the absence of the effects being most likely due to asynchrony between the response to caf, NMDA and BzATP. Incubation of astrocytes with neuron-condition medium (NCM) for 24 h totally abolished the caf-evoked [Ca2+]i increase. In NCM-treated astrocytes, peak of [Ca2+]i rise evoked by NMDA was delayed to about 3.5 min, and that induced by BzATP occurred about three minutes earlier than in SM. The results show that neurons secrete factors that negatively modulate RyR-mediated Ca2+-induced Ca2+ release (CICR) in astrocytes and alter the time course of Ca2+ responses to ionotropic stimuli.