Abstract: The N-methyl-d-aspartate receptor (NMDAR) antagonist ketamine has attracted enormous interest in mental health research owing to its rapid antidepressant actions, but its mechanism of action has remained elusive. Here we show that blockade of NMDAR-dependent bursting activity in the 'anti-reward center', the lateral habenula (LHb), mediates the rapid antidepressant actions of ketamine in rat and mouse models of depression. LHb neurons show a significant increase in burst activity and theta-band synchronization in depressive-like animals, which is reversed by ketamine. Burst-evoking photostimulation of LHb drives behavioural despair and anhedonia. Pharmacology and modelling experiments reveal that LHb bursting requires both NMDARs and low-voltage-sensitive T-type calcium channels (T-VSCCs). Furthermore, local blockade of NMDAR or T-VSCCs in the LHb is sufficient to induce rapid antidepressant effects. Our results suggest a simple model whereby ketamine quickly elevates mood by blocking NMDAR-dependent bursting activity of LHb neurons to disinhibit downstream monoaminergic reward centres, and provide a framework for developing new rapid-acting antidepressants.

Abstract: NMDA-type glutamate receptors are ligand-gated ion channels that mediate a Ca2+-permeable component of excitatory neurotransmission in the central nervous system (CNS). They are expressed throughout the CNS and play key physiological roles in synaptic function, such as synaptic plasticity, learning, and memory. NMDA receptors are also implicated in the pathophysiology of several CNS disorders and more recently have been identified as a locus for disease-associated genomic variation. NMDA receptors exist as a diverse array of subtypes formed by variation in assembly of seven subunits (GluN1, GluN2A-D, and GluN3A-B) into tetrameric receptor complexes. These NMDA receptor subtypes show unique structural features that account for their distinct functional and pharmacological properties allowing precise tuning of their physiological roles. Here, we review the relationship between NMDA receptor structure and function with an emphasis on emerging atomic resolution structures, which begin to explain unique features of this receptor.

Abstract: The NMDA (N-methyl-d-aspartate) receptor transduces the binding of glutamate and glycine, coupling it to the opening of a calcium-permeable ion channel 1 . Owing to the lack of high-resolution structural studies of the NMDA receptor, the mechanism by which ion-channel blockers occlude ion permeation is not well understood. Here we show that removal of the amino-terminal domains from the GluN1–GluN2B NMDA receptor yields a functional receptor and crystals with good diffraction properties, allowing us to map the binding site of the NMDA receptor blocker, MK-801. This crystal structure, together with long-timescale molecular dynamics simulations, shows how MK-801 and memantine (a drug approved for the treatment of Alzheimer’s disease) bind within the vestibule of the ion channel, promote closure of the ion channel gate and lodge between the M3-helix-bundle crossing and the M2-pore loops, physically blocking ion permeation.

Abstract: Ketamine and its enantiomer S-ketamine (esketamine) are promising candidates to produce a rapid-onset antidepressant effect in treatment-resistant depression. Ketamine causes continued blockade of the glutamate N-methyl-d-aspartate (NMDA) receptor, though this might not primarily mediate the antidepressant effect. Alternative hypotheses include selectivity for the NMDA receptor subtype containing the NMDA receptor subunit 2B (NR2B), inhibition of the phosphorylation of the eukaryotic elongation factor 2 (eEF2) kinase, increased expression of brain-derived neurotrophic factor (BDNF) and tropomyosin receptor kinase B (TrKB), and activation of the mammalian target of rapamycin (mTOR) signaling pathway, alongside other independent actions attributed to the ketamine metabolism to R-hydroxynorketamine (R-HNK). The enantiomer S-ketamine (esketamine) displays approximately fourfold greater affinity for the glutamate NMDA receptor in vitro than R-ketamine. Proof-of-concept single-dose and repeat-dose studies with intravenous ketamine show a significant antidepressant and probably antisuicidal effect in the short term, with response rates over 60% as early as 4.5 h after a single dose, with a sustained effect after 24 h, and over 40% after 7 days. This response can be further sustained over several weeks with repeated doses (two to three doses per week). Tolerability seems acceptable in the short term, with transient elevation of blood pressure and mild and transient dissociative and psychotomimetic effects. Intranasal esketamine has shown a comparable antidepressant effect, which has resulted in the US FDA granting the drug a “breakthrough therapy” designation, and theoretically it may offer an improved tolerability profile. However, major concerns remain regarding an effective protocol to maintain the clinical antidepressant effect of ketamine seen with acute administration and the safety of ketamine and esketamine in the long term, specifically related to potential neurocognitive and urologic toxicity, together with the potential induction of substance use disorders. Ketamine and esketamine are not currently approved treatments for depression, but the clinical use of ketamine is increasing in a variety of practice settings internationally.

Abstract: Therapeutic medications for the treatment of depression have serious limitations, particularly delayed onset and low rates of efficacy. However, the discovery that a single subanesthetic dose of ketamine, a glutamate NMDA receptor channel blocker, can produce a rapid (within hours) antidepressant response that is sustained (about 1 week), even in patients considered treatment-resistant, has invigorated the field. In addition to these remarkable actions, ketamine has proven effective for the treatment of suicidal ideation. Efforts are under way to develop ketamine-like drugs with fewer side effects as well as agents that act at other sites within the glutamate neurotransmitter system. This includes ketamine metabolites and stereoisomers, drugs that act as NMDA allosteric modulators or that block mGluR2/3 autoreceptors. In addition, targets that enhance glutamate neurotransmission or synaptic function (or both), which are essential for the rapid and sustained antidepressant actions of ketamine in rodent models, are being investigated; examples are the muscarinic cholinergic antagonist scopolamine and activators of mechanistic target of rapamycin complex 1 (mTORC1) signaling, which is required for the actions of ketamine. The discovery of ketamine and its unique mechanisms heralds a new era with tremendous promise for the development of novel, rapid, and efficacious antidepressant medications.

Abstract: Traditional pharmacological treatments for depression have a delayed therapeutic onset, ranging from several weeks to months, and there is a high percentage of individuals who never respond to treatment. In contrast, ketamine produces rapid-onset antidepressant, anti-suicidal, and anti-anhedonic actions following a single administration to patients with depression. Proposed mechanisms of the antidepressant action of ketamine include N-methyl-d-aspartate receptor (NMDAR) modulation, gamma aminobutyric acid (GABA)-ergic interneuron disinhibition, and direct actions of its hydroxynorketamine (HNK) metabolites. Downstream actions include activation of the mechanistic target of rapamycin (mTOR), deactivation of glycogen synthase kinase-3 and eukaryotic elongation factor 2 (eEF2), enhanced brain-derived neurotrophic factor (BDNF) signaling, and activation of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors (AMPARs). These putative mechanisms of ketamine action are not mutually exclusive and may complement each other to induce potentiation of excitatory synapses in affective-regulating brain circuits, which results in amelioration of depression symptoms. We review these proposed mechanisms of ketamine action in the context of how such mechanisms are informing the development of novel putative rapid-acting antidepressant drugs. Such drugs that have undergone pre-clinical, and in some cases clinical, testing include the muscarinic acetylcholine receptor antagonist scopolamine, GluN2B-NMDAR antagonists (i.e., CP-101,606, MK-0657), (2R,6R)-HNK, NMDAR glycine site modulators (i.e., 4-chlorokynurenine, pro-drug of the glycineB NMDAR antagonist 7-chlorokynurenic acid), NMDAR agonists [i.e., GLYX-13 (rapastinel)], metabotropic glutamate receptor 2/3 (mGluR2/3) antagonists, GABAA receptor modulators, and drugs acting on various serotonin receptor subtypes. These ongoing studies suggest that the future acute treatment of depression will typically occur within hours, rather than months, of treatment initiation.

Abstract: CaMKII is one of the most studied synaptic proteins, but many critical issues regarding its role in synaptic function remain unresolved. Using a CRISPR-based system to delete CaMKII and replace it with mutated forms in single neurons, we have rigorously addressed its various synaptic roles. In brief, basal AMPAR and NMDAR synaptic transmission both require CaMKIIα, but not CaMKIIβ, indicating that, even in the adult, synaptic transmission is determined by the ongoing action of CaMKIIα. While AMPAR transmission requires kinase activity, NMDAR transmission does not, implying a scaffolding role for the CaMKII protein instead. LTP is abolished in the absence of CaMKIIα and/or CaMKIIβ and with an autophosphorylation impaired CaMKIIα (T286A). With the exception of NMDAR synaptic currents, all aspects of CaMKIIα signaling examined require binding to the NMDAR, emphasizing the essential role of this receptor as a master synaptic signaling hub.

Abstract: Cognitive impairment occurs in 40-90% of patients with systemic lupus erythematosus (SLE), which is characterized by autoantibodies to nuclear antigens, especially DNA We discovered that a subset of anti-DNA antibodies, termed DNRAbs, cross reacts with the N-methyl-d-aspartate receptor (NMDAR) and enhances NMDAR signaling In patients, DNRAb presence associates with spatial memory impairment In a mouse model, DNRAb-mediated brain pathology proceeds through an acute phase of excitotoxic neuron loss, followed by persistent alteration in neuronal integrity and spatial memory impairment The latter pathology becomes evident only after DNRAbs are no longer detectable in the brain Here we investigate the mechanism of long-term neuronal dysfunction mediated by transient exposure to antibody We show that activated microglia and C1q are critical mediators of neuronal damage We further show that centrally acting inhibitors of angiotensin-converting enzyme (ACE) can prevent microglial activation and preserve neuronal function and cognitive performance Thus, ACE inhibition represents a strong candidate for clinical trials aimed at mitigating cognitive dysfunction

Abstract: Long-term potentiation (LTP) and depression (LTD) at glutamatergic synapses are intensively investigated processes for understanding the synaptic basis for learning and memory, but the underlying molecular mechanisms remain poorly understood. We have made three mouse lines where the C-terminal domains (CTDs) of endogenous AMPA receptors (AMPARs), the principal mediators of fast excitatory synaptic transmission, are specifically exchanged. These mice display profound deficits in synaptic plasticity without any effects on basal synaptic transmission. Our study reveals that the CTDs of GluA1 and GluA2, the key subunits of AMPARs, are necessary and sufficient to drive NMDA receptor–dependent LTP and LTD, respectively. In addition, these domains exert differential effects on spatial and contextual learning and memory. These results establish dominant roles of AMPARs in governing bidirectional synaptic and behavioral plasticity in the CNS.

Abstract: Glutamate is the most abundant neurotransmitter of the central nervous system, as the majority of neurons use glutamate as neurotransmitter. It is also well known that this neurotransmitter is not restricted to synaptic clefts, but found in the extrasynaptic regions as ambient glutamate. Extrasynaptic glutamate originates from spillover of synaptic release, as well as from astrocytes and microglia. Its concentration is magnitudes lower than in the synaptic cleft, but receptors responding to it have higher affinity for it. Extrasynaptic glutamate receptors can be found in neuronal somatodendritic location, on astroglia, oligodendrocytes or microglia. Activation of them leads to changes of neuronal excitability with different amplitude and kinetics. Extrasynaptic glutamate is taken up by neurons and astrocytes mostly via EAAT transporters, and astrocytes, in turn metabolize it to glutamine. Extrasynaptic glutamate is involved in several physiological phenomena of the central nervous system. It regulates neuronal excitability and synaptic strength by involving astroglia; contributing to learning and memory formation, neurosecretory and neuromodulatory mechanisms, as well as sleep homeostasis.The extrasynaptic glutamatergic system is affected in several brain pathologies related to excitotoxicity, neurodegeneration or neuroinflammation. Being present in dementias, neurodegenerative and neuropsychiatric diseases or tumor invasion in a seemingly uniform way, the system possibly provides a common component of their pathogenesis. Although parts of the system are extensively discussed by several recent reviews, in this review I attempt to summarize physiological actions of the extrasynaptic glutamate on neuronal excitability and provide a brief insight to its pathology for basic understanding of the topic.

Abstract: Low doses of ketamine trigger rapid and lasting antidepressant effects after one injection in treatment-resistant patients with major depressive disorder. Modulation of AMPA receptors (AMPARs) in the hippocampus and prefrontal cortex is suggested to mediate the antidepressant action of ketamine and of one of its metabolites (2R,6R)-hydroxynorketamine ((2R,6R)-HNK). We have examined whether ketamine and (2R,6R)-HNK affect glutamatergic transmission and plasticity in the mesolimbic system, brain regions known to have key roles in reward-motivated behaviors, mood and hedonic drive. We found that one day after the injection of a low dose of ketamine, long-term potentiation (LTP) in the nucleus accumbens (NAc) was impaired. Loss of LTP was maintained for 7 days and was not associated with an altered basal synaptic transmission mediated by AMPARs and N-methyl-D-aspartate receptors (NMDARs). Inhibition of mammalian target of rapamycin signaling with rapamycin did not prevent the ketamine-induced loss of LTP but inhibited LTP in saline-treated mice. However, ketamine blunted the increase in the phosphorylation of the GluA1 subunit of AMPARs at a calcium/calmodulin-dependent protein kinase II/protein kinase C site induced by an LTP induction protocol. Moreover, ketamine caused a persistent increased phosphorylation of GluA1 at a protein kinase A site. (2R,6R)-HNK also impaired LTP in the NAc. In dopaminergic neurons of the ventral tegmental area from ketamine- or (2R,6R)-HNK-treated mice, AMPAR-mediated responses were depressed, while those mediated by NMDARs were unaltered, which resulted in a reduced AMPA/NMDA ratio, a measure of long-term synaptic depression. These results demonstrate that a single injection of ketamine or (2R,6R)-HNK induces enduring alterations in the function of AMPARs and synaptic plasticity in brain regions involved in reward-related behaviors.

Abstract: NMDA ionotropic glutamate receptors (NMDARs) are crucial in activity-dependent synaptic changes and in learning and memory. NMDARs are composed of two GluN1 essential subunits and two regulatory subunits which define their pharmacological and physiological profile. In CNS structures involved in cognitive functions as the hippocampus and prefrontal cortex, GluN2A and GluN2B are major regulatory subunits; their expression is dynamic and tightly regulated, but little is known about specific changes after plasticity induction or memory acquisition. Data strongly suggest that following appropriate stimulation, there is a rapid increase in surface GluN2A-NMDAR at the postsynapses, attributed to lateral receptor mobilization from adjacent locations. Whenever synaptic plasticity is induced or memory is consolidated, more GluN2A-NMDARs are assembled likely using GluN2A from a local translation and GluN1 from local ER. Later on, NMDARs are mobilized from other pools, and there are de novo syntheses at the neuron soma. Changes in GluN1 or NMDAR levels induced by synaptic plasticity and by spatial memory formation seem to occur in different waves of NMDAR transport/expression/degradation, with a net increase at the postsynaptic side and a rise in expression at both the spine and neuronal soma. This review aims to put together that information and the proposed hypotheses.

Abstract: Men are generally superior to women in remembering spatial relationships, whereas the reverse holds for semantic information, but the neurobiological bases for these differences are not understood. Here we describe striking sexual dimorphism in synaptic mechanisms of memory encoding in hippocampal field CA1, a region critical for spatial learning. Studies of acute hippocampal slices from adult rats and mice show that for excitatory Schaffer-commissural projections, the memory-related long-term potentiation (LTP) effect depends upon endogenous estrogen and membrane estrogen receptor α (ERα) in females but not in males; there was no evident involvement of nuclear ERα in females, or of ERβ or GPER1 (G-protein-coupled estrogen receptor 1) in either sex. Quantitative immunofluorescence showed that stimulation-induced activation of two LTP-related kinases (Src, ERK1/2), and of postsynaptic TrkB, required ERα in females only, and that postsynaptic ERα levels are higher in females than in males. Several downstream signaling events involved in LTP were comparable between the sexes. In contrast to endogenous estrogen effects, infused estradiol facilitated LTP and synaptic signaling in females via both ERα and ERβ. The estrogen dependence of LTP in females was associated with a higher threshold for both inducing potentiation and acquiring spatial information. These results indicate that the observed sexual dimorphism in hippocampal LTP reflects differences in synaptic kinase activation, including both a weaker association with NMDA receptors and a greater ERα-mediated kinase activation in response to locally produced estrogen in females. We propose that male/female differences in mechanisms and threshold for field CA1 LTP contribute to differences in encoding specific types of memories.SIGNIFICANCE STATEMENT There is good evidence for male/female differences in memory-related cognitive function, but the neurobiological basis for this sexual dimorphism is not understood. Here we describe sex differences in synaptic function in a brain area that is critical for learning spatial cues. Our results show that female rodents have higher synaptic levels of estrogen receptor α (ERα) and, in contrast to males, require membrane ERα for the activation of signaling kinases that support long-term potentiation (LTP), a form of synaptic plasticity thought to underlie learning. The additional requirement of estrogen signaling in females resulted in a higher threshold for both LTP and hippocampal field CA1-dependent spatial learning. These results describe a synaptic basis for sexual dimorphism in encoding spatial information.

Abstract: d-serine is a physiologic coagonist of NMDA receptors, but little is known about the regulation of its synthesis and synaptic turnover The amino acid exchangers ASCT1 (Slc1a4) and ASCT2 (Slc1a5) are candidates for regulating d-serine levels Using ASCT1 and ASCT2 KO mice, we report that ASCT1, rather than ASCT2, is a physiologic regulator of d-serine metabolism ASCT1 is a major d-serine uptake system in astrocytes and can also export l-serine via heteroexchange, supplying neurons with the substrate for d-serine synthesis ASCT1-KO mice display lower levels of brain d-serine along with higher levels of l-alanine, l-threonine, and glycine Deletion of ASCT1 was associated with neurodevelopmental alterations including lower hippocampal and striatal volumes and changes in the expression of neurodevelopmental-relevant genes Furthermore, ASCT1-KO mice exhibited deficits in motor function, spatial learning, and affective behavior, along with changes in the relative contributions of d-serine vs glycine in mediating NMDA receptor activity In vivo microdialysis demonstrated lower levels of extracellular d-serine in ASCT1-KO mice, confirming altered d-serine metabolism These alterations are reminiscent of some of the neurodevelopmental phenotypes exhibited by patients with ASCT1 mutations ASCT1-KO mice provide a useful model for potential therapeutic interventions aimed at correcting the metabolic impairments in patients with ASCT1 mutations

Abstract: Cannabidiol (CBD), the major non-psychotomimetic compound present in the Cannabis sativa plant, exhibits therapeutic potential for various human diseases, including chronic neurodegenerative diseases, such as Alzheimer’s and Parkinson’s, ischemic stroke, epilepsy and other convulsive syndromes, neuropsychiatric disorders, neuropathic allodynia and certain types of cancer. CBD does not bind directly to endocannabinoid receptors 1 and 2, and despite research efforts, its specific targets remain to be fully identified. Notably, sigma 1 receptor (σ1R) antagonists inhibit glutamate N-methyl-D-aspartate acid receptor (NMDAR) activity and display positive effects on most of the aforesaid diseases. Thus, we investigated the effects of CBD on three animal models in which NMDAR overactivity plays a critical role: opioid analgesia attenuation, NMDA-induced convulsive syndrome and ischemic stroke. In an in vitro assay, CBD disrupted the regulatory association of σ1R with the NR1 subunit of NMDAR, an effect shared by σ1R antagonists, such as BD1063 and progesterone, and prevented by σ1R agonists, such as 4-IBP, PPCC and PRE084. The in vivo administration of CBD or BD1063 enhanced morphine-evoked supraspinal antinociception, alleviated NMDA-induced convulsive syndrome, and reduced the infarct size caused by permanent unilateral middle cerebral artery occlusion. These positive effects of CBD were reduced by the σ1R agonists PRE084 and PPCC, and absent in σ1R−/− mice. Thus, CBD displays antagonist-like activity toward σ1R to reduce the negative effects of NMDAR overactivity in the abovementioned experimental situations.

Abstract: Long-term potentiation (LTP) at hippocampal CA1 synapses is classically triggered by the synaptic activation of NMDA receptors (NMDARs). More recently, it has been shown that calcium-permeable (CP) AMPA receptors (AMPARs) can also trigger synaptic plasticity at these synapses. Here, we review this literature with a focus on recent evidence that CP-AMPARs are critical for the induction of the protein kinase A (PKA)- and protein synthesis-dependent component of LTP.

Abstract: The protein kinase C (PKC) enzymes have long been established as critical for synaptic plasticity. However, it is unknown whether Ca2+-dependent PKC isozymes are activated in dendritic spines during plasticity and, if so, how this synaptic activity is encoded by PKC. Here, using newly developed, isozyme-specific sensors, we demonstrate that classical isozymes are activated to varying degrees and with distinct kinetics. PKCα is activated robustly and rapidly in stimulated spines and is the only isozyme required for structural plasticity. This specificity depends on a PDZ-binding motif present only in PKCα. The activation of PKCα during plasticity requires both NMDA receptor Ca2+ flux and autocrine brain-derived neurotrophic factor (BDNF)-TrkB signaling, two pathways that differ vastly in their spatiotemporal scales of signaling. Our results suggest that, by integrating these signals, PKCα combines a measure of recent, nearby synaptic plasticity with local synaptic input, enabling complex cellular computations such as heterosynaptic facilitation of plasticity necessary for efficient hippocampus-dependent learning.

Abstract: Several preclinical studies have reported the rapid antidepressant effects of N-methyl-D-aspartate receptor (NMDAR) antagonists, although the underlying mechanisms are still unclear. Death-associated protein kinase 1 (DAPK1) couples GluN2B subunits at extrasynaptic sites to regulate NMDAR channel conductance. In the present study, we found that chronic unpredictable stress (CUS) induced extracellular glutamate accumulation, accompanied by an increase in the DAPK1-NMDAR interaction, the high expression of DAPK1 and phosphorylated GluN2B at Ser1303, a decrease in phosphorylated DAPK1 at Ser308 and synaptic protein deficits in the rat medial prefrontal cortex (mPFC). CUS also enhanced GluN2B-mediated NMDA currents and extrasynaptic responses that were induced by bursts of high-frequency stimulation, which may be associated with the loss of astrocytes and low expression of glutamate transporter-1 (GLT-1). The blockade of GLT-1 in the mPFC was sufficient to induce depressive-like behavior and cause similar molecular changes. Selective GluN2B antagonist, DAPK1 knockdown by adeno-associated virus-mediated short-hairpin RNA or a pharmacological inhibitor, and the uncoupling of DAPK1 from the NMDAR GluN2B subunit produced rapid antidepressant-like effects and reversed CUS-induced alterations in the mPFC. The inhibition of DAPK1 and its interaction with GluN2B subunit in the mPFC also rescued CUS-induced depressive-like behavior 7 days after treatment. A selective GluN2B antagonist did not have rewarding effects in the conditioned place preference paradigm. Altogether, our findings suggest that the DAPK1 interaction with the NMDAR GluN2B subunit acts as a critical component in the pathophysiology of depression and is a potential target for new antidepressant treatments.

Abstract: Background: Excessive proliferation and apoptosis resistance in pulmonary vascular cells underlie vascular remodeling in pulmonary arterial hypertension (PAH). Specific treatments for PAH exist, mostly targeting endothelial dysfunction, but high pulmonary arterial pressure still causes heart failure and death. Pulmonary vascular remodeling may be driven by metabolic reprogramming of vascular cells to increase glutaminolysis and glutamate production. The N -methyl-d-aspartate receptor (NMDAR), a major neuronal glutamate receptor, is also expressed on vascular cells, but its role in PAH is unknown. Methods: We assessed the status of the glutamate-NMDAR axis in the pulmonary arteries of patients with PAH and controls through mass spectrometry imaging, Western blotting, and immunohistochemistry. We measured the glutamate release from cultured pulmonary vascular cells using enzymatic assays and analyzed NMDAR regulation/phosphorylation through Western blot experiments. The effect of NMDAR blockade on human pulmonary arterial smooth muscle cell proliferation was determined using a BrdU incorporation assay. We assessed the role of NMDARs in vascular remodeling associated to pulmonary hypertension, in both smooth muscle-specific NMDAR knockout mice exposed to chronic hypoxia and the monocrotaline rat model of pulmonary hypertension using NMDAR blockers. Results: We report glutamate accumulation, upregulation of the NMDAR, and NMDAR engagement reflected by increases in GluN1-subunit phosphorylation in the pulmonary arteries of human patients with PAH. K v channel inhibition and type A-selective endothelin receptor activation amplified calcium-dependent glutamate release from human pulmonary arterial smooth muscle cell, and type A-selective endothelin receptor and platelet-derived growth factor receptor activation led to NMDAR engagement, highlighting crosstalk between the glutamate-NMDAR axis and major PAH-associated pathways. The platelet-derived growth factor-BB-induced proliferation of human pulmonary arterial smooth muscle cells involved NMDAR activation and phosphorylated GluN1 subunit localization to cell-cell contacts, consistent with glutamatergic communication between proliferating human pulmonary arterial smooth muscle cells via NMDARs. Smooth-muscle NMDAR deficiency in mice attenuated the vascular remodeling triggered by chronic hypoxia, highlighting the role of vascular NMDARs in pulmonary hypertension. Pharmacological NMDAR blockade in the monocrotaline rat model of pulmonary hypertension had beneficial effects on cardiac and vascular remodeling, decreasing endothelial dysfunction, cell proliferation, and apoptosis resistance while disrupting the glutamate-NMDAR pathway in pulmonary arteries. Conclusions: These results reveal a dysregulation of the glutamate-NMDAR axis in the pulmonary arteries of patients with PAH and identify vascular NMDARs as targets for antiremodeling treatments in PAH.

Abstract: Lactate, a product of aerobic glycolysis in astrocytes, is required for memory formation and consolidation, and has recently emerged as a signaling molecule for neurons and various cell types in peripheral tissues. In particular lactate stimulates mRNA expression of a few plasticity-related genes. Here, we describe a RNA-seq study that unravels genome-wide transcriptomic responses to this energy metabolite in cortical neurons. Our results show that mRNA expression of 20 immediate-early genes involved in the MAPK signaling pathway and in synaptic plasticity were increased by more than twofold following 1 h of lactate stimulation. This effect was dependent on NMDA receptor (NMDAR) activity since it was prevented by pre-treatment with MK-801. Comparison with published datasets showed that a significant proportion of genes modulated by lactate were similarly regulated by a stimulation protocol activating specifically synaptic NMDARs known to result in upregulation of pro-survival and downregulation of pro-death genes. Remarkably, transcriptional responses to lactate were reproduced by NADH (for 74 of the 113 genes, FDR < 0.05), suggesting a redox-dependent mechanism of action. Longer-term gene expression changes observed after 6 h of lactate treatment affected genes involved in regulating neuronal excitability and genes coding for proteins localized at synapses. Gene set enrichment analyses performed with ranked lists of expressed genes revealed effects on molecular functions involved in epigenetic modulation, and on processes relevant to sleep physiology and behavioral phenotypes such as anxiety and hyperactivity. Overall, these results strengthen the notion that lactate effectively regulates activity-dependent and synaptic genes, and highlight new signaling effects of lactate in plasticity and neuroprotection.

Abstract: GluN3A and GluN3B are glycine-binding subunits belonging to the NMDA receptor (NMDAR) family that can assemble with the GluN1 subunit to form unconventional receptors activated by glycine alone. Functional characterization of GluN1/GluN3 NMDARs has been difficult. Here, we uncover two modalities that have transformative properties on GluN1/GluN3A receptors. First, we identify a compound, CGP-78608, which greatly enhances GluN1/GluN3A responses, converting small and rapidly desensitizing currents into large and stable responses. Second, we show that an endogenous GluN3A disulfide bond endows GluN1/GluN3A receptors with distinct redox modulation, profoundly affecting agonist sensitivity and gating kinetics. Under reducing conditions, ambient glycine is sufficient to generate tonic receptor activation. Finally, using CGP-78608 on P8-P12 mouse hippocampal slices, we demonstrate that excitatory glycine GluN1/GluN3A NMDARs are functionally expressed in native neurons, at least in the juvenile brain. Our work opens new perspectives on the exploration of excitatory glycine receptors in brain function and development.

Abstract: Chronic infection with the neurotropic parasite Toxoplasma gondii has been implicated in the risk for several neuropsychiatric disorders. The mechanisms, by which the parasite may alter neural function and behavior of the host, are not yet understood completely. Here, a novel proteomic approach using mass spectrometry was employed to investigate the alterations in synaptic protein composition in a murine model of chronic toxoplasmosis. In a candidate-based strategy, immunoblot analysis and immunohistochemistry were applied to investigate the expression levels of key synaptic proteins in glutamatergic signaling. A comparison of the synaptosomal protein composition revealed distinct changes upon infection, with multiple proteins such as EAAT2, Shank3, AMPA receptor, and NMDA receptor subunits being downregulated, whereas inflammation-related proteins showed an upregulation. Treatment with the antiparasitic agent sulfadiazine strongly reduced tachyzoite levels and diminished neuroinflammatory mediators. However, in both conditions, a significant number of latent cysts persisted in the brain. Conversely, infection-related alterations of key synaptic protein levels could be partly reversed by the treatment. These results provide evidence for profound changes especially in synaptic protein composition in T. gondii-infected mice with a downregulation of pivotal components of glutamatergic neurotransmission. Our results suggest that the detected synaptic alterations are a consequence of the distinct neuroinflammatory milieu caused by the neurotropic parasite.

Abstract: Despite the characteristic etiologies and phenotypes, different brain disorders rely on common pathogenic events. Glutamate-induced neurotoxicity is a pathogenic event shared by different brain disorders. Another event occurring in different brain pathological conditions is the increase of the extracellular ATP levels, which is now recognized as a danger and harmful signal in the brain, as heralded by the ability of P2 receptors (P2Rs) to affect a wide range of brain disorders. Yet, how ATP and P2R contribute to neurodegeneration remains poorly defined. For that purpose, we now examined the contribution of extracellular ATP and P2Rs to glutamate-induced neurodegeneration. We found both in vitro and in vivo that ATP/ADP through the activation of P2Y1R contributes to glutamate-induced neuronal death in the rat hippocampus. We found in cultured rat hippocampal neurons that the exposure to glutamate (100 µM) for 30 min triggers a sustained increase of extracellular ATP levels, which contributes to NMDA receptor (NMDAR)-mediated hippocampal neuronal death through the activation of P2Y1R. We also determined that P2Y1R is involved in excitotoxicity in vivo as the blockade of P2Y1R significantly attenuated rat hippocampal neuronal death upon the systemic administration of kainic acid or upon the intrahippocampal injection of quinolinic acid. This contribution of P2Y1R fades with increasing intensity of excitotoxic conditions, which indicates that P2Y1R is not contributing directly to neurodegeneration, rather behaving as a catalyst decreasing the threshold from which glutamate becomes neurotoxic. Moreover, we unraveled that such excitotoxicity process began with an early synaptotoxicity that was also prevented/attenuated by the antagonism of P2Y1R, both in vitro and in vivo. This should rely on the observed glutamate-induced calpain-mediated axonal cytoskeleton damage, most likely favored by a P2Y1R-driven increase of NMDAR-mediated Ca2+ entry selectively in axons. This may constitute a degenerative mechanism shared by different brain diseases, particularly relevant at initial pathogenic stages.

Abstract: Depression is a common mental disorder that is associated with memory dysfunction. Ketamine has recently been demonstrated to be a rapid antidepressant. The mechanisms underlying how depression induces memory dysfunction and how ketamine relieves depressive symptoms remain poorly understood. This work compared three groups of male C57BL/6J mice: mice exposed to chronic social defeat stress (CSDS) to induce a depression-like phenotype, depression-like mice treated with ketamine, and control mice that were not exposed to CSDS or treated with ketamine. Spatial working memory and long term memory were assessed by spontaneous alternation Y-maze and fear conditioning tests, respectively. We used western blot to analyze the density of N-methyl-D-aspartate receptor (NMDAR) subunits in the hippocampus. We recorded long term potentiation (LTP) and NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) in hippocampal slices. We observed that compared with control mice, depression-like mice had significant reductions in spatial working memory and contextual fear memory. The level of NR2B, LTP and NMDA receptor-mediated EPSCs of depression-like mice were decreased. Ketamine treatment attenuated the memory impairment, and increased the density of NR2B and the amplitude of LTP and NMDA receptor-mediated EPSCs in the hippocampus of depression-like mice. In conclusion, depression-like mice have deficits in working memory and contextual fear memory. The decrease of NR2B, LTP induction and NMDA receptor-mediated EPSCs in the hippocampus may be involved in this process. Ketamine can improve expression of NR2B, LTP induction and NMDA receptor-mediated EPSCs in the hippocampus of depression-like mice, which might be part of the reason why ketamine can alleviate the memory dysfunction induced by depression.