NMDA Subtype Of Glutamate Receptor Research Articles

CNS4 causes subtype-specific changes in agonist efficacy and reversal potential of permeant cations in NMDA receptors.

The NMDA subtype of glutamate receptor serves as an attractive drug target for the treatment of disorders evolving from hyper- or hypoglutamatergic conditions. Compounds that optimize the function of NMDA receptors are of great clinical significance. Here, we present the pharmacological characterization of a biased allosteric modulator, CNS4. Results indicate that CNS4 sensitizes ambient levels of agonists and reduces higher-concentration glycine & glutamate efficacy in 1/2AB receptors, but minimally alters these parameters in diheteromeric 1/2A or 1/2B receptors. Glycine efficacy is increased in both 1/2C and 1/2D, while glutamate efficacy is decreased in 1/2C and unaltered in 1/2D. CNS4 does not affect the activity of competitive antagonist binding at glycine (DCKA) and glutamate (DL-AP5) sites; however, it decreases memantine potency in 1/2A receptors but not in 1/2D receptors. Current-voltage (I-V) relationship studies indicate that CNS4 potentiates 1/2A inward currents, a phenomenon that was reversed in the absence of permeable Na+ ions. In 1/2D receptors, CNS4 blocks inward currents based on extracellular Ca2+ concentration. Further, CNS4 positively modulates glutamate potency on E781A_1/2A mutant receptors, indicating its role at the distal end of the 1/2A agonist binding domain interface. Together, these findings reveal that CNS4 sensitizes ambient agonists and allosterically modulates agonist efficacy by altering Na+ permeability based on the GluN2 subunit composition. Overall, the pharmacology of CNS4 aligns with the need for drug candidates to treat hypoglutamatergic neuropsychiatric conditions such as loss function GRIN disorders and anti-NMDA receptor encephalitis.

Upregulation of Connexins in the Rat Hippocampal and Cortical Neurons Following Blockade of NMDA Receptors During Postnatal Development.

Interneural gap junctional coupling represents neural development that decreases during the postnatal period. The decrease of gap junction function coincides with the main period of chemical synapse creation and increment of synaptic activity during postnatal weeks 1 to 3. Here, we have assessed the role of chemical synapses on connexin (Cx) expression in neurons and glial cells of hippocampal and cortical neurons. We characterized the impact of NMDA receptors blockade on the expression of Cx36 and Cx43 proteins by western blot analysis in postnatal day (PND)14 and PND28. MK801 was injected subcutaneously from the first day of birth until 14 or 28 days, depending on the experimental groups. Saline was injected in the same volumes in the control group. Early postnatal blockade of the NMDA subtype of glutamate receptors by the non-competitive antagonist dizocilpine maleate (MK801) arrested the developmental reduction in gap junctions during the initial postnatal weeks. Expression of Cx43 declined in PND28 compared to PND14 in visual cortex (VC) neurons. Also, we found that the expression of Cx36 and Cx43 augmented in the rats' VC in PND28 following the blockade of NMDA receptors. Expression of Cx36 declined in PND28 compared to PND14 in hippocampal neurons. Also, we found that the expression of Cx36 augmented in the rats' hippocampal neurons in PND14 and PND28 following a blockade of NMDA receptors. These results suggest that the postnatal enhancement in glutamatergic synaptic activity is associated with the loss of gap junctional connections and downregulation of Cx36 and Cx43 between developing neurons and glial cells.

Improved NMDA Receptor Activation by the Secreted Amyloid-Protein Precursor-α in Healthy Aging: A Role for D-Serine?

Impaired activation of the N-methyl-D-aspartate subtype of glutamate receptors (NMDAR) by D-serine is linked to cognitive aging. Whether this deregulation may be used to initiate pharmacological strategies has yet to be considered. To this end, we performed electrophysiological extracellular recordings at CA3/CA1 synapses in hippocampal slices from young and aged mice. We show that 0.1 nM of the soluble N-terminal recombinant fragment of the secreted amyloid-protein precursor-α (sAPPα) added in the bath significantly increased NMDAR activation in aged but not adult mice without impacting basal synaptic transmission. In addition, sAPPα rescued the age-related deficit of theta-burst-induced long-term potentiation. Significant NMDAR improvement occurred in adult mice when sAPPα was raised to 1 nM, and this effect was drastically reduced in transgenic mice deprived of D-serine through genetic deletion of the synthesizing enzyme serine racemase. Altogether, these results emphasize the interest to consider sAPPα treatment targeting D-serine-dependent NMDAR deregulation to alleviate cognitive aging.

Effects of high glutamate concentrations on mitochondria of human neuroblastoma SH-SY5Y cells

The excess amount of glutamate in neurons is associated with the excitotoxicity and neurodegenerative diseases. Glutamate induces neurotoxicity primarily by immense influx of Ca2+arising from overstimulation of the NMDA subtype of glutamate receptors. The neuronal death induced by the overstimulation of glutamate receptors depends critically on a sustained increase in mitochondrial Ca2+influx and impairment in mitochondrial functions. The mitochondrial impairment is an important contributor to the glutamate-induced neuronal toxicity and thus provides an important target for the intervention. The present study investigates the effects of high glutamate concentrations on mitochondrial functions. Here, we have shown that the higher concentration of glutamate treatment caused a significant elevation in the N-methyl-D-aspartate (NMDA) receptors expression and elevated the intra-mitochondrial calcium accumulation in SHSY5Y neuronal cells. As a result of an accumulation of intra-mitochondrial calcium, there is a concentration-dependent elevation in ROS in the mitochondria. Tyrosine nitration of several mitochondrial proteins was increased while the mitochondrial membrane potential was dissipated. Furthermore, glutamate treatments also resulted in mitochondrial membrane permeability transition. These findings suggest that treatment of high glutamate concentration causes impairment of mitochondrial functions by an increase in intra-mitochondrial calcium, ROS production, dissipation of mitochondrial membrane potential and mitochondrial permeability transition pore opening in human neuroblastoma SHSY5Y cells.

Tyrosine 121 moves revealing a ligandable pocket that couples catalysis to ATP-binding in serine racemase

Human serine racemase (hSR) catalyses racemisation of L-serine to D-serine, the latter of which is a co-agonist of the NMDA subtype of glutamate receptors that are important in synaptic plasticity, learning and memory. In a ‘closed’ hSR structure containing the allosteric activator ATP, the inhibitor malonate is enclosed between the large and small domains while ATP is distal to the active site, residing at the dimer interface with the Tyr121 hydroxyl group contacting the α-phosphate of ATP. In contrast, in ‘open’ hSR structures, Tyr121 sits in the core of the small domain with its hydroxyl contacting the key catalytic residue Ser84. The ability to regulate SR activity by flipping Tyr121 from the core of the small domain to the dimer interface appears to have evolved in animals with a CNS. Multiple X-ray crystallographic enzyme-fragment structures show Tyr121 flipped out of its pocket in the core of the small domain. Data suggest that this ligandable pocket could be targeted by molecules that inhibit enzyme activity.

LY235959 attenuates development phase of Methamphetamine-Induced behavioral sensitization through the PP2A/B - AKT cascade in the dorsal striatum of C57/BL6 mice

Drug addiction can be described as a chronic and relapsing brain disease. Behavioral sensitization is common animal model in the study of addiction and N-Methyl-D-aspartate subtype of glutamate receptor (NMDAR) is believed play key role in this process. LY235959 is a competitive NMDAR antagonist, however, its effect on methamphetamine (METH)-induced behavioral sensitization is not been reported yet. In this study, we choose three doses (0.33 mg/kg, 1.0 mg/kg, and 3.0 mg/kg) of LY235959 to investigate its effect on locomotor activity, METH-induced behavioral sensitization and different phases of it in C57/BL6 mice. We also used western blotting to examine the PP2A/B – AKT cascade which had been proved involved in METH-induced behavioral sensitization in the dorsal striatum (DS). The results showed that only 0.33 mg/kg LY235959 increased locomotor activity dramatically, however, 1.0 mg/kg and 3.0 mg/kg of LY235959 could attenuate METH-induced behavioral sensitization markedly. We also found that LY235959 only disrupted the development phase of METH-induced behavioral sensitization and the following western blotting results further indicated that PP2A/B – AKT cascade might involve in this process. Taken together, these results indicated that LY235959 attenuates development phase of METH-induced behavioral sensitization through the PP2A/B - AKT cascade in the DS.

Extrasynaptic NMDA Receptors Bidirectionally Modulate Intrinsic Excitability of Inhibitory Neurons.

The NMDA subtype glutamate receptors (NMDARs) play important roles in both physiological and pathologic processes in the brain. Compared with their critical roles in synaptic modifications and excitotoxicity in excitatory neurons, much less is understood about the functional contributions of NMDARs to the inhibitory GABAergic neurons. By using selective NMDAR inhibitors and potentiators, we here show that NMDARs bidirectionally modulate the intrinsic excitability (defined as spontaneous/evoked spiking activity and EPSP-spike coupling) in inhibitory GABAergic neurons in adult male and female mice. This modulation depends on GluN2C/2D- but not GluN2A/2B-containing NMDARs. We further show that NMDAR modulator EU1794-4 mostly enhances extrasynaptic NMDAR activity, and by using it we demonstrate a significant contribution of extrasynaptic NMDARs to the modulation of intrinsic excitability in inhibitory neurons. Together, this bidirectional modulation of intrinsic excitability reveals a previously less appreciated importance of NMDARs in the second-to-second functioning of inhibitory GABAergic neurons.SIGNIFICANCE STATEMENT NMDA subtype of glutamate receptors (NMDARs) have important roles in brain functions, including both physiological and pathologic ones. The role of NMDARs in inhibitory neurons has been less elucidated compared with that in excitatory neurons. Our results demonstrate the importance of GluN2C/GluN2D-containing but not GluN2A/GluN2B-containing extrasynaptic NMDARs in modulating the intrinsic excitability of inhibitory neurons. These results further suggest distinct contributions of subsynaptic locations and subunit compositions of NMDARs to their functions in excitatory and inhibitory neurons. The above findings have implications for better understanding of brain diseases, such as schizophrenia.

Regulation of neuronal excitability by reactive oxygen species and calcium signaling: Insights into brain aging.

:Altered cognition and inefficient learning and memory are hallmarks of brain aging resulting from many small changes in the structure and function of neurons. One such change is a decrease in excitatory synaptic transmission mediated by glutamate and its binding to the AMPA and NMDA subtypes of glutamate receptors. Why there is decreased glutamatergic transmission in aging is not well understood. Interestingly, in aged excitatory neurons, abnormal calcium homeostasis and energy production are reliably observed. These processes have also been shown to modulate the transport and delivery of glutamate receptors to synapses. Most of these channels are translated in the cell body and must be transported to synapses by molecular motors and then transferred to the synaptic surface for proper function. Despite there being little to no research on how aging impacts these transport processes, a detailed understanding of the mechanisms regulating long-distance and local transport of these channels is coming together. Here, we review recent research on how synaptic content, specifically of glutamate receptors and voltage-gated calcium channels, is normally regulated by calcium and energy production. In addition, we discuss how that regulation may change in the aged nervous system. These advances begin to detail a mechanistic explanation in which an interplay between calcium signaling and metabolism are impacted by and, in-turn, regulate the strength of excitatory synapses.

Leptin modulates pancreatic β-cell membrane potential through Src kinase–mediated phosphorylation of NMDA receptors

The adipocyte-derived hormone leptin increases trafficking of KATP and Kv2.1 channels to the pancreatic β-cell surface, resulting in membrane hyperpolarization and suppression of insulin secretion. We have previously shown that this effect of leptin is mediated by the NMDA subtype of glutamate receptors (NMDARs). It does so by potentiating NMDAR activity, thus enhancing Ca2+ influx and the ensuing downstream signaling events that drive channel trafficking to the cell surface. However, the molecular mechanism by which leptin potentiates NMDARs in β-cells remains unknown. Here, we report that leptin augments NMDAR function via Src kinase-mediated phosphorylation of the GluN2A subunit. Leptin-induced membrane hyperpolarization diminished upon pharmacological inhibition of GluN2A but not GluN2B, indicating involvement of GluN2A-containing NMDARs. GluN2A harbors tyrosine residues that, when phosphorylated by Src family kinases, potentiate NMDAR activity. We found that leptin increases phosphorylation of Tyr-418 in Src, an indicator of kinase activation. Pharmacological inhibition of Src or overexpression of a kinase-dead Src mutant prevented the effect of leptin, whereas a Src kinase activator peptide mimicked it. Using mutant GluN2A overexpression, we show that Tyr-1292 and Tyr-1387 but not Tyr-1325 are responsible for the effect of leptin. Importantly, β-cells from db/db mice, a type 2 diabetes mouse model lacking functional leptin receptors, or from obese diabetic human donors failed to respond to leptin but hyperpolarized in response to NMDA. Our study reveals a signaling pathway wherein leptin modulates NMDARs via Src to regulate β-cell excitability and suggests NMDARs as a potential target to overcome leptin resistance.

Electroacupuncture Pretreatment Elicits Tolerance to Cerebral Ischemia/Reperfusion through Inhibition of the GluN2B/m-Calpain/p38 MAPK Proapoptotic Pathway.

Background As one of the first steps in the pathology of cerebral ischemia, glutamate-induced excitotoxicity progresses too fast to be the target of postischemic intervention. However, ischemic preconditioning including electroacupuncture (EA) might elicit cerebral ischemic tolerance through ameliorating excitotoxicity. Objective To investigate whether EA pretreatment based on TCM theory could elicit cerebral tolerance against ischemia/reperfusion (I/R) injury, and explore its potential excitotoxicity inhibition mechanism from regulating proapoptotic pathway of the NMDA subtype of glutamate receptor (GluN2B). Methods The experimental procedure included 5 consecutive days of pretreatment stage and the subsequent modeling stage for one day. All rats were evenly randomized into three groups: sham MCAO/R, MCAO/R, and EA+MCAO/R. During pretreatment procedure, only rats in the EA+MCAO/R group received EA intervention on GV20, SP6, and PC6 once a day for 5 days. Model preparation for MCAO/R or sham MCAO/R started 2 hours after the last pretreatment. 24 hours after model preparation, the Garcia neurobehavioral scoring criteria was used for the evaluation of neurological deficits, TTC for the measurement of infarct volume, TUNEL staining for determination of neural cell apoptosis at hippocampal CA1 area, and WB and double immunofluorescence staining for expression and the cellular localization of GluN2B and m-calpain and p38 MAPK. Results This EA pretreatment regime could improve neurofunction, decrease cerebral infarction volume, and reduce neuronal apoptosis 24 hours after cerebral I/R injury. And EA pretreatment might inhibit the excessive activation of GluN2B receptor, the GluN2B downstream proapoptotic mediator m-calpain, and the phosphorylation of its transcription factor p38 MAPK in the hippocampal neurons after cerebral I/R injury. Conclusion The EA regime might induce tolerance against I/R injury partially through the regulation of the proapoptotic GluN2B/m-calpain/p38 MAPK pathway of glutamate.

Glutamate receptors from diverse animal species exhibit unexpected structural and functional diversity.

The identification of AMPA, kainate and NMDA glutamate receptor subtypes by Watkins and colleagues underlies much of our understanding of excitatory synaptic transmission in the central nervous system of animals. Ongoing large scale genome sequencing projects in species for which physiological analysis of receptor function is challenging are resulting in identification of numerous eukaryotic glutamate receptor ion channels in the animal kingdom of life. On the basis of sequence similarity, these are frequently classified into the three vertebrate subtypes, initially identified using subtype selective ligands. Recent work reveals unexpected ligand binding profiles for these newly identified glutamate receptors, for example, kainate receptors on which NMDA acts as a competitive antagonist, and high affinity homomeric glycine activated glutamate receptors. Structural studies reveal that only subtle changes in the ligand binding domain, often identified only in retrospect, underlie different patterns of ligand binding, and that the biology of glutamate receptors is more complex than first anticipated.

SNAP25/syntaxin4/VAMP2/Munc18-1 Complexes in Spinal Dorsal Horn Contributed to Inflammatory Pain

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) have been implicated in the trafficking of postsynaptic glutamate receptors, including N-methyl-d-aspartate (NMDA)-subtype glutamate receptors (NMDARs) that are critical for nociceptive plasticity and behavioral sensitization. However, the components of SNAREs complex involved in spinal nociceptive processing remain largely unknown. Here we found that SNAP25, syntaxin4, VAMP2 and Munc18-1 were localized at postsynaptic sites and formed the complex in the superficial lamina of spinal cord dorsal horn of rats. The complex formation between these SNAREs components were accelerated after intraplantar injection of complete Freund’s adjuvant (CFA), pharmacological removal of GABAergic inhibition or activation of NMDAR in intact rats. The increased SNAP25/syntaxin4/VAMP2/Munc18-1 interaction facilitated the surface delivery and synaptic accumulation of NMDAR during inflammatory pain. Disruption of the molecular interaction between SNAP25 with its SNARE partners by using a blocking peptide derived from the C-terminus of SNAP25 effectively repressed the surface and synaptic accumulation of GluN2B-containing NMDARs in CFA-injected rats. This peptide also alleviated inflammatory mechanical allodynia and thermal hypersensitivity. These data suggested that SNAREs complex assembly in spinal cord dorsal horn was involved in the inflammatory pain hypersensitivity through promoting NMDAR synaptic trafficking.

Development of Depotentiation in Adult-Born Dentate Granule Cells.

Activity-dependent synaptic plasticity, i.e., long-term potentiation (LTP), long-term depression (LTD) and LTP reversal, is generally thought to make up the cellular mechanism underlying learning and memory in the mature brain, in which N-methyl-D-aspartate subtype of glutamate (NMDA) receptors and neurogenesis play important roles. LTP reversal may be the mechanism of forgetting and may mediate many psychiatric disorders, such as schizophrenia, but the specific mechanisms underlying these disorders remain unclear. In addition, LTP reversal during the development of adult-born dentate granule cells (DGCs) remains unknown. We found that the expression of the NMDA receptor subunits NR2A and NR2B displayed dynamic changes during the development of postnatal individuals and the maturation of adult-born neurons and was coupled with the change in LTP reversal. The susceptibility of LTP reversal progressively increases with the rise in the expression of NR2A during the development of postnatal individual and adult-born neurons. In addition, NMDA receptor subunits NR2A, but not NR2B, mediated LTP reversal in the DGCs of the mouse hippocampus.

Asc-1 transporter activation: an alternative to rescue age-related alterations in functional plasticity at rat hippocampal CA3/CA1 synapses.

Activation of the N-methyl-D-aspartate subtype of glutamate receptor (NMDA-R) represents a key functional process for memory formation. A decreased synthesis of the NMDA-R co-agonist d-serine was recently proposed to contribute to alterations of hippocampus-dependent memory mechanisms with ageing. Nevertheless, other pathways could also be involved and thus considered to be targets of interest to prevent cognitive ageing. Herein, we demonstrate that the Asc-1 subtype of neutral amino acid (nAA) transporters that regulates d-serine and glycine release from neurons could be viewed as one of these targets. At CA3/CA1 hippocampal synapses, Asc-1 activation did not modify basal glutamate neurotransmission either in adult or aged rats. In contrast, Asc-1 activation significantly increased NMDA-R-dependent long-term potentiation (LTP) in both groups of animals and fully rescued the age-related LTP deficits. This rescue in aged animals was observed only when Asc-1 activation was selectively managed by d-Isoleucine (d-Ile), but not when less specifically driven by a mixture of nAA. Similarly, while any activation of Asc-1 improved the isolated NMDA-R-induced synaptic potentials in adult rats, only d-Ile was efficient in aged animals. Taken together, these results strengthen the interest in specifically targeting Asc-1 transporters to better cure age-associated memory decline. OPEN PRACTICES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Reactive Sulfur Species Emerge as Gliotransmitters to Support Memory via Sulfuration-Dependent Gating of NR2A-Containing N-Methyl-d-Aspartate Subtype Glutamate Receptor Function.

Astrocytes have been revealed as a controller of synaptic plasticity and memory via releasing gliotransmitters. Our recent findings showed that reactive sulfur species (RSS), including hydrogen sulfide (H2S) and polysulfide (H2Sn), regulated the availability of d-serine, which is a well-known gliotransmitter that is involved in synaptic plasticity. An interesting question is whether RSS, which are small molecules, can function as direct gliotransmitters to integrate astrocyte-neuron interactions throughout the memory process. We found that hippocampal RSS level increased significantly in response to learning. We further demonstrated that the activity-triggered RSS signal controlled memory formation by using pharmacological and genetic approaches. The RSS-supporting memory was primarily conferred by enzymes that were mainly located in astrocytes, including cystathionine β-synthase (CBS) and mercaptopyruvate sulfurtransferase (3-MST), and the memory-promoting effects were mostly dependent on sulfration of the NR2A subunit of N-methyl-d-aspartate subtype glutamate receptors (NMDARs). Further, RSS were demonstrated to buffer the strong inhibitory effect of synaptically released zinc on NR2A-containing NMDARs. Innovation and Conclusion: These results suggest that glial-derived RSS signals can serve as direct gliotransmitters that regulate memory formation through the redox modulation of postsynaptic receptors; this conclusion will enrich the gliotransmission hypothesis.

Corrigendum: Chronic MK-801 Application in Adolescence and Early Adulthood: A Spatial Working Memory Deficit in Adult Long-Evans Rats But No Changes in the Hippocampal NMDA Receptor Subunits

[This corrects the article on p. 42 in vol. 9, PMID: 29487522.].

Inhibition of Acid Sensing Ion Channel 3 Aggravates Seizures by Regulating NMDAR Function.

The existing data about whether acid sensing ion channels (ASICs) are proconvulsant or anticonvulsant are controversial. Particularly, acid sensing ion channel 3 (ASIC3) is the most sensitive to extracellular pH and has the characteristic ability to generate a biphasic current, but few studies have focused on the role of ASIC3 in seizure. Here we found ASIC3 expression was increased in the hippocampus of pilocarpine induced seizure rats, as well as in hippocampal neuronal cultures undergoing epileptiform discharge elicited by Mg2+-free media. Furthermore, ASIC3 blockade by the selective inhibitor APETx2 shortened seizure onset latency and increased seizure severity compared with the control in the pilocarpine induced seizure model. Incubation with APETx2 enhanced the excitability of primary cultured hippocampal neurons in Mg2+-free media. Notably, the aggravated seizure was associated with upregulation of the N-methyl-D-aspartate subtype of glutamate receptors (NMDARs), increased NMDAR mediated excitatory neurotransmission and subsequent activation of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) and cAMP-response element binding protein (CREB) signaling pathway. Moreover, co-immunoprecipitation confirmed the interaction between ASIC3 and NMDAR subunits, and NMDARs blockade prevented the aggravated seizure caused by ASIC3 inhibition. Taken together, our findings suggest that ASIC3 inhibition aggravates seizure and potentiates seizure induced hyperexcitability at least partly by the NMDAR/CaMKII/CREB signaling pathway, which implies that ASIC3 agonists may be a promising approach for seizure treatment.

Lipid Raft Dynamics in the Adolescent Brain under the Influence of Chronic Ethanol and Caffeine

Simultaneous consumption of alcohol with stimulants such as caffeine and amphetamine has become increasingly prevalent, particularly among adolescents already prone to binge drinking. Initial work in this lab has modeled prolonged alcohol‐stimulant co‐use in behavioral studies of adolescent Long‐Evans rats. This work suggested that stimulants mask or block symptoms associated with alcohol withdrawal. Thus, stimulants may lessen the awareness of growing dependence on alcohol when consumed regularly with alcohol. The goal of the present study was to determine whether lipid raft dynamics play a role in the cellular mechanisms by which caffeine and ethanol interact in this model. Lipid rafts were isolated as detergent resistant membrane fragments from forebrain regions of adolescent rats fed liquid diets with alcohol alone or in combination with caffeine. Pair‐fed rats with no additions to the diet or with caffeine alone were used as controls. All procedures involving rats were reviewed and approved by the Institutional Animal Care and Use Committee for compliance with regulations and best practices. Western blotting was used to analyze fractions for the raft marker flotilin and the NMDA‐subtype of glutamate receptor. Two fractions were found to contain flotilin with a relatively small percentage (<20%) of the marker found in the less buoyant of the two fractions. However, there was a highly significant 2–3 fold increase in this less buoyant fraction from membranes derived from the ethanol‐fed rats. This increase did not occur when rats were consuming caffeine along with alcohol under conditions that result in decreased alcohol withdrawal severity. Looking at NMDA receptor expression, we first confirmed that there was an upregulation of NMDA receptors during chronic alcohol consumption. NMDA receptors were expressed in both raft fractions in all treatment groups. However, paralleling the increase in flotilin, NMDA receptor expression was significantly higher in the less buoyant fraction following chronic ethanol consumption. This elevated expression of NMDA receptor was not observed when the rats consumed caffeine and ethanol. Neither flotilin nor NMDA receptor expression differed by treatment group in the more prominent higher buoyancy raft fraction. Location of NMDA receptors in what appears to be an “alcohol responsive” raft fraction may help account for its upregulation during chronic alcohol consumption. Moreover, the shift does not occur with caffeine “co‐use” suggesting lipid raft dynamics may be a site associated with the ability of caffeine to antagonize adaptive responses of the brain to chronic ethanol. Given that the appearance of withdrawal symptoms can be a cue to emerging dependency, understanding the ability of stimulants to mask or block these symptoms is crucial to both education and treatment strategies related to adolescent alcohol abuse.Support or Funding InformationSupported by a Grant‐in‐Aid from Monmouth University and by the Department of Biology.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chronic MK-801 Application in Adolescence and Early Adulthood: A Spatial Working Memory Deficit in Adult Long-Evans Rats But No Changes in the Hippocampal NMDA Receptor Subunits

The role of NMDA receptors in learning, memory and hippocampal function has long been recognized. Post-mortem studies have indicated that the expression or subunit composition of the NMDA glutamate receptor subtype might be related to the impaired cognitive functions found in schizophrenia patients. NMDA receptor antagonists have been used to develop animal models of this disorder. There is accumulating evidence showing that not only the acute but also the chronic application of NMDA receptor antagonists may induce schizophrenia-like alterations in behavior and brain functions. However, limited evidence is available regarding the consequences of NMDA receptor blockage during periods of adolescence and early adulthood. This study tested the hypothesis that a 2-week treatment of male Long-Evans and Wistar rats with dizocilpine (MK-801; 0.5 mg/kg daily) starting at postnatal days (PD) 30 and 60 would cause a long-term cognitive deficit and changes in the levels of NMDA receptor subunits. The working memory version of the Morris water maze (MWM) and active place avoidance with reversal on a rotating arena (Carousel) requiring cognitive coordination and flexibility probed cognitive functions and an elevated-plus maze (EPM) was used to measure anxiety-like behavior. The western blot method was used to determine changes in NMDA receptor subunit levels in the hippocampus. Our results showed no significant changes in behaviors in Wistar rats. Slightly elevated anxiety-like behavior was observed in the EPM in Long-Evans rats with the onset of treatment on PD 30. Furthermore, Long-Evans rats treated from PD 60 displayed impaired working memory in the MWM. There were; however, no significant changes in the levels of NMDA receptor subunits because of MK-801 administration. These findings suggest that a 2-week treatment starting on PD 60 in Long-Evans rats leads to long-term changes in working memory, but this deficit is not paralleled by changes in NMDA receptor subunits. These results support the face validity, but not construct validity of this model. We suggest that chronic treatment of adolescent and adult rats does not constitute a plausible animal model of schizophrenia.

Human d-amino acid oxidase: The inactive G183R variant

In the brain, the enzyme d-amino acid oxidase (DAAO) catalyzes the oxidative deamination of d-serine, a main positive modulator of the N-methyl-d-aspartate subtype of glutamate receptors (NMDAR). Dysregulation in d-serine signaling is implicated in the NMDAR dysfunctions observed in various brain diseases, such as amyotrophic lateral sclerosis, Alzheimer's disease, schizophrenia. A strain of ddY mice lacking DAAO activity due to the G181R substitution (DAAOG181R mice) and exhibiting increased d-serine concentration as compared to wild-type mice shows altered pain response, improved adaptative learning and cognitive functions, and larger hippocampal long-term potentiation. In past years, this mice line has been used to shed light on physiological and pathological brain functions related to NMDAR. Here, we decided to introduce the corresponding substitution in human DAAO (hDAAO). The recombinant G183R hDAAO is produced as an inactive apoprotein: the substitution alters the protein conformation that negatively affects the ability to bind the flavin cofactor in the orientation required for hydride-transfer during catalysis. At the cellular level, the overexpressed G183R hDAAO is not fully targeted to peroxisomes, forms protein aggregates showing a strong colocalization with ubiquitin, and significantly (7-fold) increases both the d-serine cellular concentration and the D/(D+L)-serine ratio. Taken together, our investigation warrants caution in using DAAOG181R mice: the abolition of enzymatic activity is coupled to DAAO aggregation, a central process in different pathological conditions. The effect due to G181R substitution in DAAO could be misleading: the effects due to impairment of d-serine degradation overlap with those related to aggregates accumulation.