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1.  Dynamic Loss of Surface-Expressed AMPA Receptors in Mouse Cortical and Striatal Neurons During Anesthesia 
Journal of neuroscience research  2011;90(1):315-323.
Ionotropic glutamate receptors, especially the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subtype, undergo dynamic trafficking between the surface membrane and intracellular organelles. This trafficking activity determines the efficacy and strength of excitatory synapses and is subject to modulation by changing synaptic inputs. Given the possibility that glutamate receptors in the central nervous system might be a sensitive target of anesthetic agents, this study investigated the possible impact of anesthesia on trafficking and subcellular expression of AMPA receptors in adult mouse brain neurons in vivo. We found that anesthesia induced by a systemic injection of pentobarbital did not alter total protein levels of three AMPA receptor subunits (GluR1–3) in cortical neurons. However, an anesthetic dose of pentobarbital reduced GluR1 and GluR3 proteins in the surface pool and elevated these proteins in the intracellular pool of cortical neurons. The similar redistribution of GluR1/3 was observed in mouse striatal neurons. Pentobarbital did not significantly alter GluR2 expression in the two pools. Chloral hydrate at an anesthetic dose also reduced surface GluR1/3 expression and increased intracellular levels of these proteins. The effect of pentobarbital on subcellular distribution of AMPA receptors was reversible. Altered subcellular distribution of GluR1/3 returned to normal levels after the anesthesia subsided. These data indicate that anesthesia induced by pentobarbital and chloral hydrate can alter AMPA receptor trafficking in both cortical and striatal neurons. This alteration is characterized by the concurrent loss and addition of GluR1/3 subunits in the respective surface and intracellular pools.
PMCID: PMC3218204  PMID: 21932367
pentobarbital; chloral hydrate; glutamate; GluR1; GluR3; trafficking
2.  Natural Reward Experience Alters AMPA and NMDA Receptor Distribution and Function in the Nucleus Accumbens 
PLoS ONE  2012;7(4):e34700.
Natural reward and drugs of abuse converge upon the mesolimbic system which mediates motivation and reward behaviors. Drugs induce neural adaptations in this system, including transcriptional, morphological, and synaptic changes, which contribute to the development and expression of drug-related memories and addiction. Previously, it has been reported that sexual experience in male rats, a natural reward behavior, induces similar neuroplasticity in the mesolimbic system and affects natural reward and drug-related behavior. The current study determined whether sexual experience causes long-lasting changes in mating, or ionotropic glutamate receptor trafficking or function in the nucleus accumbens (NAc), following 3 different reward abstinence periods: 1 day, 1 week, or 1 month after final mating session. Male Sprague Dawley rats mated during 5 consecutive days (sexual experience) or remained sexually naïve to serve as controls. Sexually experienced males displayed facilitation of initiation and performance of mating at each time point. Next, intracellular and membrane surface expression of N-methyl-D-aspartate (NMDA: NR1 subunit) and α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA: GluA1, GluA2 subunits) receptors in the NAc was determined using a bis(sulfosuccinimidyl)suberate (BS3) protein cross-linking assay followed by Western Blot analysis. NR1 expression was increased at 1 day abstinence both at surface and intracellular, but decreased at surface at 1 week of abstinence. GluA2 was increased intracellularly at 1 week and increased at the surface after 1 month of abstinence. Finally, whole-cell patch clamp electrophysiological recordings determined reduced AMPA/NMDA ratio of synaptic currents in NAc shell neurons following stimulation of cortical afferents in sexually experienced males after all reward abstinence periods. Together, these data show that sexual experience causes long-term alterations in glutamate receptor expression and function in the NAc. Although not identical, this sex experience-induced neuroplasticity has similarities to that caused by psychostimulants, suggesting common mechanisms for reinforcement of natural and drug reward.
PMCID: PMC3329487  PMID: 22529926
3.  Changes in surface expression of N-methyl-D-aspartate receptors in the striatum in a rat model of Parkinson’s disease 
N-methyl-D-aspartate (NMDA) receptors play a central role in glutamatergic synaptic transmission in the mammalian brain and are linked to the pathophysiology and symptomatology of Parkinson’s disease (PD). However, changes in NMDA receptor expression in distinct subcellular compartments in PD have not been elucidated. In this study, we investigated changes in subcellular expression of NMDA receptors in striatal neurons in a rodent PD model.
Intracranial injection of the neurotoxin 6-hydroxydopamine (6-OHDA) was selectively lesioned into the nigrostriatal dopaminergic pathway in adult Sprague Dawley rats, which is a common rat model of PD. A surface receptor crosslinking assay was conducted to examine the response of individual NMDA receptor subunits to dopamine depletion in isolated and confined surface and intracellular compartments of striatal neurons.
In PD rats where 6-OHDA was selectively lesioned, surface expression of NMDA receptor GluN1 subunits as detected by surface protein crosslinking assays was increased in the striatum. In contrast, intracellular levels of GluN1 were decreased in the lesioned region. The NMDA receptor GluN2B subunit was elevated in its abundance in the surface pool of the lesioned striatum, while intracellular GluN2B levels were not altered. GluN2A subunits in both surface and intracellular fractions remained stable. In addition, total cellular levels of striatal GluN1 and GluN2A were not changed in lesioned tissue, while total GluN2B proteins showed an increase.
These results demonstrate the differential sensitivity of principal NMDA receptor subunits to dopamine depletion. GluN1 and GluN2B expression in the distinct surface compartment underwent upregulation in striatal neurons after selective lesions of the dopaminergic pathway by 6-OHDA.
PMCID: PMC3900317  PMID: 24465126
glutamate; excitatory amino acid; NMDA; GluN; dopamine; 6-hydroxydopamine; caudate putamen; nucleus accumbens
4.  Caldendrin–Jacob: A Protein Liaison That Couples NMDA Receptor Signalling to the Nucleus 
PLoS Biology  2008;6(2):e34.
NMDA (N-methyl-D-aspartate) receptors and calcium can exert multiple and very divergent effects within neuronal cells, thereby impacting opposing occurrences such as synaptic plasticity and neuronal degeneration. The neuronal Ca2+ sensor Caldendrin is a postsynaptic density component with high similarity to calmodulin. Jacob, a recently identified Caldendrin binding partner, is a novel protein abundantly expressed in limbic brain and cerebral cortex. Strictly depending upon activation of NMDA-type glutamate receptors, Jacob is recruited to neuronal nuclei, resulting in a rapid stripping of synaptic contacts and in a drastically altered morphology of the dendritic tree. Jacob's nuclear trafficking from distal dendrites crucially requires the classical Importin pathway. Caldendrin binds to Jacob's nuclear localization signal in a Ca2+-dependent manner, thereby controlling Jacob's extranuclear localization by competing with the binding of Importin-α to Jacob's nuclear localization signal. This competition requires sustained synapto-dendritic Ca2+ levels, which presumably cannot be achieved by activation of extrasynaptic NMDA receptors, but are confined to Ca2+ microdomains such as postsynaptic spines. Extrasynaptic NMDA receptors, as opposed to their synaptic counterparts, trigger the cAMP response element-binding protein (CREB) shut-off pathway, and cell death. We found that nuclear knockdown of Jacob prevents CREB shut-off after extrasynaptic NMDA receptor activation, whereas its nuclear overexpression induces CREB shut-off without NMDA receptor stimulation. Importantly, nuclear knockdown of Jacob attenuates NMDA-induced loss of synaptic contacts, and neuronal degeneration. This defines a novel mechanism of synapse-to-nucleus communication via a synaptic Ca2+-sensor protein, which links the activity of NMDA receptors to nuclear signalling events involved in modelling synapto-dendritic input and NMDA receptor–induced cellular degeneration.
Author Summary
Long-lasting changes in communication between nerve cells require the regulation of gene expression. The influx of calcium ions into the cell, particularly through membrane protein called NMDA receptors, plays a crucial role in this process by determining the type of gene expression induced. NMDA receptors can exert multiple and very divergent effects within neuronal cells by impacting opposing phenomena such as synaptic plasticity and neuronal degeneration. We identified a protein termed Jacob that appears to play a pivotal role in such processes by entering the nucleus in response to NMDA receptor activation and controlling gene expression that governs cell survival and the stability of synaptic cell contacts. Removal of Jacob from the nucleus protects neurons from NMDA receptor–induced cell death and increases phosphorylation of the transcription factor CREB, whereas the opposite occurs after targeting Jacob exclusively to the nucleus. The work defines a novel pathway of synapse-to-nucleus communication involved in modelling synapto-dendritic input and NMDA receptor–induced cellular degeneration.
A new signaling mechanism from NMDA receptors to the nucleus plays an important role in the phosphorylation of the transcription factor CREB and neuronal cell survival.
PMCID: PMC2253627  PMID: 18303947
5.  GluN2B-containing NMDA receptors regulate depression-like behavior and are critical for the rapid antidepressant actions of ketamine 
eLife  null;3:e03581.
A single, low dose of the NMDA receptor antagonist ketamine produces rapid antidepressant actions in treatment-resistant depressed patients. Understanding the cellular mechanisms underlying this will lead to new therapies for treating major depression. NMDARs are heteromultimeric complexes formed through association of two GluN1 and two GluN2 subunits. We show that in vivo deletion of GluN2B, only from principal cortical neurons, mimics and occludes ketamine's actions on depression-like behavior and excitatory synaptic transmission. Furthermore, ketamine-induced increases in mTOR activation and synaptic protein synthesis were mimicked and occluded in 2BΔCtx mice. We show here that cortical GluN2B-containing NMDARs are uniquely activated by ambient glutamate to regulate levels of excitatory synaptic transmission. Together these data predict a novel cellular mechanism that explains ketamine's rapid antidepressant actions. In this model, basal glutamatergic neurotransmission sensed by cortical GluN2B-containing NMDARs regulates excitatory synaptic strength in PFC determining basal levels of depression-like behavior.
eLife digest
Depression is the leading cause of disability worldwide, with hundreds of millions of people living with the condition. The ‘gold standard’ for depression treatment involves a combination of psychotherapy and medication. Unfortunately, current antidepressant medications do not help everyone, waiting lists for psychotherapy are often long, and both normally take a number of weeks of regular treatment before they begin to have an effect. As patients are often at a high risk of suicide, it is crucial that treatments that act more quickly, and that are safe and effective, are developed.
One substance that may fulfill these requirements is a drug called ketamine. Studies have shown that depression symptoms can be reduced within hours by a single low dose of ketamine, and this effect on mood can last for more than a week. However, progress has been hindered by a lack of knowledge about what ketamine actually does inside the brain.
Neurons communicate with one another by releasing chemicals known as neurotransmitters, which transfer information by binding to receptor proteins on the surface of other neurons. Drugs such as ketamine also bind to these receptors. Ketamine works by blocking a specific receptor called the n-methyl d-aspartate (NMDA) receptor, but how this produces antidepressant effects is not fully understood.
The NMDA receptor is actually formed from a combination of individual protein subunits, including one called GluN2B. Now Miller, Yang et al. have created mice that lack receptors containing these GluN2B subunits in neurons in their neocortex, including the prefrontal cortex, a brain region involved in complex mental processes such as decision-making. This allowed Miller, Yang et al. to discover that when the neurotransmitter glutamate binds to GluN2B-containing NMDA receptors, it limits the production of certain proteins that make it easier for signals to be transmitted between neurons. Suppressing the synthesis of these proteins too much may cause depressive effects by reducing communication between the neurons in the prefrontal cortex.
Both mice lacking GluN2B-containing receptors in their cortical neurons and normal mice treated with ketamine showed a reduced amount of depressive-like behavior. This evidence supports Miller, Yang et al.'s theory that by blocking these NMDA receptors, ketamine restricts their activation. This restores normal levels of protein synthesis, improves communication between neurons in the cortex, and reduces depression.
Understanding how ketamine works to alleviate depression is an important step towards developing it into a safe and effective treatment. Further research is also required to determine the conditions that cause overactivation of the GluN2B-containing NMDA receptors.
PMCID: PMC4270067  PMID: 25340958
depression; cortex; synapse; ketamine; electrophysiology; protein synthesis; mouse; rat
6.  Increased Response to Glutamate in Small Diameter Dorsal Root Ganglion Neurons after Sciatic Nerve Injury 
PLoS ONE  2014;9(4):e95491.
Glutamate in the peripheral nervous system is involved in neuropathic pain, yet we know little how nerve injury alters responses to this neurotransmitter in primary sensory neurons. We recorded neuronal responses from the ex-vivo preparations of the dorsal root ganglia (DRG) one week following a chronic constriction injury (CCI) of the sciatic nerve in adult rats. We found that small diameter DRG neurons (<30 µm) exhibited increased excitability that was associated with decreased membrane threshold and rheobase, whereas responses in large diameter neurons (>30 µm) were unaffected. Puff application of either glutamate, or the selective ionotropic glutamate receptor agonists alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainic acid (KA), or the group I metabotropic receptor (mGluR) agonist (S)-3,5-dihydroxyphenylglycine (DHPG), induced larger inward currents in CCI DRGs compared to those from uninjured rats. N-methyl-D-aspartate (NMDA)-induced currents were unchanged. In addition to larger inward currents following CCI, a greater number of neurons responded to glutamate, AMPA, NMDA, and DHPG, but not to KA. Western blot analysis of the DRGs revealed that CCI resulted in a 35% increase in GluA1 and a 60% decrease in GluA2, the AMPA receptor subunits, compared to uninjured controls. mGluR1 receptor expression increased by 60% in the membrane fraction, whereas mGluR5 receptor subunit expression remained unchanged after CCI. These results show that following nerve injury, small diameter DRG neurons, many of which are nociceptive, have increased excitability and an increased response to glutamate that is associated with changes in receptor expression at the neuronal membrane. Our findings provide further evidence that glutamatergic transmission in the periphery plays a role in nociception.
PMCID: PMC3991716  PMID: 24748330
7.  LRP1 is critical for the surface distribution and internalization of the NR2B NMDA receptor subtype 
The N-methyl-D-aspartate receptors are key mediators of excitatory transmission and are implicated in many forms of synaptic plasticity. These receptors are heterotetrameres consisting of two obligatory NR1 and two regulatory subunits, usually NR2A or NR2B. The NR2B subunits are abundant in the early postnatal brain, while the NR2A/NR2B ratio increases during early postnatal development. This shift is driven by NMDA receptor activity. A functional interplay of the Low Density Lipoprotein Receptor Related Protein 1 (LRP1) NMDA receptor has already been reported. Such abilities as interaction of LRP1 with NMDA receptor subunits or its important role in tPa-mediated NMDA receptor signaling were already demonstrated. Moreover, mice harboring a conditional neuronal knock-out mutation of the entire Lrp1 gene display NMDA-associated behavioral changes. However, the exact role of LRP1 on NMDA receptor function remains still elusive.
To provide a mechanistic explanation for such effects we investigated whether an inactivating knock-in mutation into the NPxY2 motif of LRP1 might influence the cell surface expression of LRP1 and NMDA receptors in primary cortical neurons. Here we demonstrate that a knock-in into the NPxY2 motif of LRP1 results in an increased surface expression of LRP1 and NR2B NMDA receptor subunit due to reduced endocytosis rates of LRP1 and the NR2B subunit in primary neurons derived from LRP1ΔNPxY2 animals. Furthermore, we demonstrate an altered phosphorylation pattern of S1480 and Y1472 in the NR2B subunit at the surface of LRP1ΔNPxY2 neurons, while the respective kinases Fyn and casein kinase II are not differently regulated compared with wild type controls. Performing co-immunoprecipitation experiments we demonstrate that binding of LRP1 to NR2B might be linked by PSD95, is phosphorylation dependent and this regulation mechanism is impaired in LRP1ΔNPxY2 neurons. Finally, we demonstrate hyperactivity and changes in spatial and reversal learning in LRP1ΔNPxY2 mice, confirming the mechanistic interaction in a physiological readout.
In summary, our data demonstrate that LRP1 plays a critical role in the regulation of NR2B expression at the cell surface and may provide a mechanistic explanation for the behavioral abnormalities detected in neuronal LRP1 knock-out animals reported earlier.
PMCID: PMC3722104  PMID: 23866919
LRP1; NPxY2 motif; NMDA receptor; NR1; NR2B receptor subunit; PSD95; Cell surface expression
8.  Downregulation of Early Ionotrophic Glutamate Receptor Subunit Developmental Expression as a Mechanism for Observed Plasticity Deficits Following Gestational Exposure to Benzo(a)pyrene 
Neurotoxicology  2007;28(5):965-978.
The focus of this study was to characterize the impact of gestational exposure to benzo(a)pyrene, [B(a)P] on modulation of glutamate receptor subunit expression that is critical for the maintenance of synaptic plasticity mechanisms during hippocampal or cortical development in offspring. Previous studies have demonstrated that hippocampal and/or cortical synaptic plasticity (as measured by long-term potentiation and S1-cortex spontaneous/evoked neuronal activity) and learning behavior (as measured by fixed-ratio performance operant testing) is significantly impaired in polycyclic aromatic or halogenated aromatic hydrocarbon-exposed offspring as compared to controls. These previous studies have also revealed that brain to body weight ratios are greater in exposed offspring relative to controls indicative of intrauterine growth retardation which has been shown to manifest as low birth weight in offspring. Recent epidemiological studies have identified an effect of prenatal exposure to airborne polycyclic aromatic hydrocarbons on neurodevelopment in the first 3 Years of life among inner-city children (Perera et al., 2006). The present study utilizes a well-characterized animal model to test the hypothesis that gestational exposure to B(a)P causes dysregulation of developmental ionotropic glutamate receptor subunit expression, namely the N-methyl-D-aspartate receptor (NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptor (AMPAR) both critical to the expression of synaptic plasticity mechanisms. To mechanistically ascertain the basis of B(a)P-induced plasticity perturbations, timed pregnant Long-Evans rats were exposed in an oral subacute exposure regimen to 0, 25 and 150µg/kg BW B(a)P on gestation days 14–17. The first sub-hypothesis tested whether gestational exposure to B(a)P would result in significant disposition in offspring. The second sub-hypothesis tested whether gestational exposure to B(a)P would result in downregulation of early developmental expression of NMDA and AMPA receptor subunits in the hippocampus of offspring as well as in primary neuronal cultures. The results of these studies revealed significant: 1) disposition to the hippocampus and cortex, 2) down-regulation of developmental glutamate receptor mRNA and protein subunit expression and 3) voltage-dependent decreases in the amplitude of inward currents at negative potentials in B(a)P-treated cortical neuronal membranes.
These results suggest that plasticity and behavioral deficits produced as a result of gestational B(a)P exposure are at least, in part, a result of down-regulation of early developmental glutamate receptor subunit expression and function at a time when excitatory synapses are being formed for the first time in the developing central nervous system. The results also predict that in B(a)P-exposed offspring with reduced early glutamate receptor subunit expression, a parallel deficit in behaviors that depend on normal hippocampal or cortical functioning will be observed and that these deficits will be present throughout life.
PMCID: PMC2276633  PMID: 17606297
9.  Ethanol Inhibition of Recombinant NMDA Receptors Is Not Altered by Co-Expression of CaMKII-α or CaMKII-β 
Alcohol (Fayetteville, N.Y.)  2008;42(5):425-432.
Previous studies have shown that the N-methyl-D-aspartate (NMDA) receptor is an important target for the actions of ethanol in the brain. NMDA receptors are glutamate-activated ion channels that are highly expressed in neurons. They are activated during periods of significant glutamatergic synaptic activity and are an important source of the signaling molecule calcium in the post-synaptic spine. Alterations in the function of NMDA receptors by drugs or disease are associated with deficits in motor, sensory and cognitive processes of the brain. Acutely, ethanol inhibits ion flow through NMDA receptors while sustained exposure to ethanol can induce compensatory changes in the density and localization of the receptor. Defining factors that govern the acute ethanol sensitivity of NMDA receptors is an important step in how an individual responds to ethanol. In the present study, we investigated the effect of calcium-calmodulin dependent protein kinase II (CaMKII) on the ethanol sensitivity of recombinant NMDA receptors. CaMKII is a major constituent of the post-synaptic density and is critically involved in various forms of learning and memory. NMDA receptor subunits were transiently expressed in human embryonic kidney 293 cells (HEK 293) along with CaMKII-α or CaMKII-β tagged with the green fluorescent protein (GFP). Whole cell currents were elicited by brief exposures to glutamate and were measured using patchclamp electrophysiology. Neither CaMKII-α or CaMKII-β had any significant effect on the ethanol inhibition of NR1/2A or NR1/2B receptors. Ethanol inhibition was also unaltered by deletion of CaMKII binding domains in NR1 or NR2 subunits or by phospho-site mutants that mimic or occlude CaMKII phosphorylation. Chronic treatment of cortical neurons with ethanol had no significant effect on the expression of CaMKII-α or CaMKII-β. The results of this study suggest that CaMKII is not involved in regulating the acute ethanol sensitivity of NMDA receptors.
PMCID: PMC2629600  PMID: 18562151
electrophysiology; alcohol; ion channel; kinase; phosphorylation
10.  Diabetes changes expression of genes related to glutamate neurotransmission and transport in the Long-Evans rat retina 
Molecular Vision  2013;19:1538-1553.
This study investigated changes in the transcript levels of genes related to glutamate neurotransmission and transport as diabetes progresses in the Long-Evans rat retina. Transcript levels of vascular endothelial growth factor (VEGF), erythropoietin, and insulin-like growth factor binding protein 3 (IGFBP3) were also measured due to their protective effects on the retinal vasculature and neurons.
Diabetes was induced in Long-Evans rats with a single intraperitoneal (IP) injection of streptozotocin (STZ; 65 mg/kg) in sodium citrate buffer. Rats with blood glucose >300 mg/dl were deemed diabetic. Age-matched controls received a single IP injection of sodium citrate buffer only. The retinas were dissected at 4 and 12 weeks after induction of diabetes, and mRNA and protein were extracted from the left and right retinas of each rat, respectively. Gene expression was analyzed using quantitative real-time reverse-transcription PCR. Enzyme-linked immunosorbent assay was used to quantify the concentration of VEGF protein in each retina. Statistical significance was determined using 2×2 analysis of variance followed by post-hoc analysis using Fisher’s protected least squares difference.
Transcript levels of two ionotropic glutamate receptor subunits and one glutamate transporter increased after 4 weeks of diabetes. In contrast, 12 weeks of diabetes decreased the transcript levels of several genes, including two glutamate transporters, four out of five N-methyl-D-aspartate (NMDA) receptor subunits, and all five kainate receptor subunits. Diabetes had a greater effect on gene expression of NMDA and kainate receptor subunits than on the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunits, for which only GRIA4 significantly decreased after 12 weeks. VEGF protein levels were significantly increased in 4-week diabetic rats compared to age-matched control rats whereas the increase was not significant after 12 weeks. Transcript levels of VEGF and VEGF receptors were unchanged with diabetes. Erythropoietin and IGFBP3 mRNA levels significantly increased at both time points, and IGFBP2 mRNA levels increased after 12 weeks.
Diabetes caused significant changes in the transcriptional expression of genes related to ionotropic glutamate neurotransmission, especially after 12 weeks. Most genes with decreased transcript levels after 12 weeks were expressed by retinal ganglion cells, which include glutamate transporters and ionotropic glutamate receptors. Two genes expressed by retinal ganglion cells but unrelated to glutamate neurotransmission, γ-synuclein (SNCG) and adenosine A1 receptor (ADORA1), also had decreased mRNA expression after 12 weeks. These findings may indicate ganglion cells were lost as diabetes progressed in the retina. Decreased expression of the glutamate transporter SLC1A3 would lead to decreased removal of glutamate from the extracellular space, suggesting that diabetes impairs this function of Müller cells. These findings suggest that ganglion cells were lost due to glutamate excitotoxicity. The changes at 12 weeks occurred without significant changes in retinal VEGF protein or mRNA, although higher VEGF protein levels at 4 weeks may be an early protective response. Increased transcript levels of erythropoietin and IGFBP3 may also be a protective response.
PMCID: PMC3716414  PMID: 23878504
11.  Hippocampal N-Methyl-D-Aspartate Receptor Subunit Expression Profiles in a Mouse Model of Prenatal Alcohol Exposure 
Although several reports have been published showing prenatal ethanol exposure is associated with alterations in N-methyl-D-aspartate (NMDA) receptor subunit levels and, in a few cases, subcellular distribution, results of these studies are conflicting.
We used semi-quantitative immunoblotting techniques to analyze NMDA receptor NR1, NR2A, and NR2B subunit levels in the adult mouse hippocampal formation isolated from offspring of dams who consumed moderate amounts of ethanol throughout pregnancy. We employed subcellular fractionation and immunoprecipitation techniques to isolate synaptosomal membrane- and postsynaptic density protein-95 (PSD-95)-associated pools of receptor subunits.
We found that, compared to control animals, fetal alcohol-exposed (FAE) adult mice had: (i) increased synaptosomal membrane NR1 levels with no change in association of this subunit with PSD-95 and no difference in total NR1 expression in tissue homogenates; (ii) decreased NR2A subunit levels in hippocampal homogenates, but no alterations in synaptosomal membrane NR2A levels and no change in NR2A-PSD-95 association; and (iii) no change in tissue homogenate or synaptosomal membrane NR2B levels but a reduction in PSD-95-associated NR2B subunits. No alterations were found in mRNA levels of NMDA receptor subunits suggesting that prenatal alcohol-associated differences in subunit protein levels are the result of differences in post-transcriptional regulation of subunit localization.
Our results demonstrate that prenatal alcohol exposure induces selective changes in NMDA receptor subunit levels in specific subcellular locations in the adult mouse hippocampal formation. Of particular interest is the finding of decreased PSD-95-associated NR2B levels, suggesting that synaptic NR2B-containing NMDA receptor concentrations are reduced in FAE animals. This result is consistent with various biochemical, physiological, and behavioral findings that have been linked with prenatal alcohol exposure.
PMCID: PMC3600588  PMID: 19951292
NMDA Receptor; Prenatal Alcohol; Hippocampus; NR2A; NR2B
12.  CXCL12 inhibits expression of the NMDA receptor's NR2B subunit through a histone deacetylase-dependent pathway contributing to neuronal survival 
Cell Death & Disease  2010;1(4):e33-.
Homeostatic chemokines, such as CXCL12, can affect neuronal activity by the regulation of inhibitory and excitatory neurotransmission, but the mechanisms involved are still undefined. Our previous studies have shown that CXCL12 protects cortical neurons from excitotoxicity by promoting the function of the gene-repressor protein Rb, which is involved in the recruitment of chromatin modifiers (such as histone deacetylases (HDACs)) to gene promoters. In neurons, Rb controls activity-dependent genes essential to neuronal plasticity and survival, such as the N-methyl--aspartic acid (NMDA) receptor's subunit NR2B, the expression of which in the tetrameric ion channel largely affects calcium signaling by glutamate. In this study, we report that CXCL12 differentially modulates intracellular responses after stimulation of synaptic and extrasynaptic NMDA receptors, by a specific regulation of the NR2B gene that involves HDACs. Our results show that CXCL12 selectively inhibits NR2B expression in vitro and in vivo altering NMDA-induced calcium responses associated with neuronal death, while promoting prosurvival pathways that depend on stimulation of synaptic receptors. Along with previous studies, these findings underline the role of CXCL12/CXCR4 in the regulation of crucial components of glutamatergic transmission. These novel effects of CXCL12 may be involved in the physiological function of the chemokine in both developing and mature brains.
PMCID: PMC3032300  PMID: 21364640
chemokine; neuron; CXCR4; cell death; calcium
13.  Ethanol self-administration modulation of NMDA receptor subunit and related synaptic protein mRNA expression in prefrontal cortical fields 
Brain Research  2010;1318:144-154.
Functional impairment of the orbital and medial prefrontal cortex underlies deficits in executive control that characterize addictive disorders, including alcohol addiction. Previous studies indicate that alcohol alters glutamate neurotransmission and one substrate of these effects may be through the reconfiguration of the subunits constituting ionotropic glutamate receptor (iGluR) complexes. Glutamatergic transmission is integral to cortico-cortical and cortico-subcortical communication, and alcohol-induced changes in the abundance of the receptor subunits and/or their splice variants may result in critical functional impairments of prefrontal cortex in the alcohol-addicted state.
Methods and results
The effects of chronic ethanol self-administration on glutamate receptor ionotropic NMDA (GRIN), as well as GRIN1 splice variant mRNA expression was studied in the orbitofrontal cortex (OFC; Area 13), dorsolateral prefrontal cortex (DLPFC; Area 46) and anterior cingulate cortex (ACC; Area 24) of male cynomolgus monkeys. Chronic ethanol self-administration resulted in significant changes in the expression of NMDA subunit mRNA expression in the DLPFC and OFC, but not the ACC. In DLPFC, the overall expression of NMDA subunits was significantly decreased in ethanol treated monkeys. Slight but significant changes were observed for synaptic associated protein 102 kD (SAP102) and neuronal nitric oxide synthase (nNOS) mRNAs. In OFC, the NMDAR1 variant GRIN1-1 was reduced while GRIN1-2 was increased. Furthermore, no significant changes in GFAP protein levels were observed in either the DLPFC or OFC.
Results from these studies provide the first demonstration of post-transcriptional regulation of iGluR subunits in the primate brain following long-term ethanol self-administration. Furthermore, changes in these transcripts do not appear to reflect changes in glial activation or loss. Further studies examining the expression and cellular localization of subunit proteins and receptor pharmacology would shed more light on the findings reported here.
PMCID: PMC3272763  PMID: 20043891
Ethanol; Glutamate; messenger RNA; Prefrontal Cortex; qPCR; Primate
14.  Diabetes changes the levels of ionotropic glutamate receptors in the rat retina 
Molecular Vision  2009;15:1620-1630.
Diabetic retinopathy (DR) is a leading cause of vision loss and blindness among adults between the age 20 to 74. Changes in ionotropic glutamate receptor subunit composition can affect retinal glutamatergic neurotransmission and, therefore, contribute to visual impairment. The purpose of this study was to investigate whether diabetes leads to changes in ionotropic glutamate receptor subunit expression at the protein and mRNA level in the rat retina.
Changes in the expression of ionotropic glutamate receptor subunits were investigated at the mRNA and protein levels in retinas of streptozotocin (STZ)-induced diabetic and age-matched control rats. Animals were euthanized one, four and 12 weeks after the onset of diabetes. Retinal protein extracts were prepared, and the receptor subunit levels were assessed by western blotting. Transcript levels were assessed by real-time quantitative PCR.
Transcript levels of most ionotropic glutamate receptor subunits were not significantly changed in the retinas of diabetic rats, as compared to age-matched controls but protein levels of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA), kainate, and N-methyl-D-aspartic acid receptors (NMDA) receptors were found to be altered.
The results provide evidence that diabetes affects the retinal content of ionotropic glutamate receptor subunits at the protein level. The possible implications of these changes on retinal physiology and visual impairment in DR are discussed.
PMCID: PMC2728563  PMID: 19693289
15.  Acute 5-HT7 receptor activation increases NMDA-evoked currents and differentially alters NMDA receptor subunit phosphorylation and trafficking in hippocampal neurons 
Molecular Brain  2013;6:24.
N-methyl-D-aspartate (NMDA) receptors are regulated by several G protein-coupled receptors (GPCRs) as well as receptor tyrosine kinases. Serotonin (5-HT) type 7 receptors are expressed throughout the brain including the thalamus and hippocampus. Long-term (2–24 h) activation of 5-HT7 receptors promotes the expression of neuroprotective growth factor receptors, including the platelet-derived growth factor (PDGF) β receptors which can protect neurons against NMDA-induced neurotoxicity.
In contrast to long-term activation of 5-HT7 receptors, acute (5 min) treatment of isolated hippocampal neurons with the 5-HT7 receptor agonist 5-carboxamidotryptamine (5-CT) enhances NMDA-evoked peak currents and this increase in peak currents is blocked by the 5-HT7 receptor antagonist, SB 269970. In hippocampal slices, acute 5-HT7 receptor activation increases NR1 NMDA receptor subunit phosphorylation and differentially alters the phosphorylation state of the NR2B and NR2A subunits. NMDA receptor subunit cell surface expression is also differentially altered by 5-HT7 receptor agonists: NR2B cell surface expression is decreased whereas NR1 and NR2A surface expression are not significantly altered.
In contrast to the negative regulatory effects of long-term activation of 5-HT7 receptors on NMDA receptor signaling, acute activation of 5-HT7 receptors promotes NMDA receptor activity. These findings highlight the potential for temporally differential regulation of NMDA receptors by the 5-HT7 receptor.
PMCID: PMC3661375  PMID: 23672716
5-HT7; NMDA; Hippocampus; Isolated neurons; Phosphorylation; Trafficking
16.  Direct pharmacological monitoring of the developmental switch in NMDA receptor subunit composition using TCN 213, a GluN2A-selective, glycine-dependent antagonist 
British Journal of Pharmacology  2012;166(3):924-937.
Developmental switches in NMDA receptor subunit expression have been inferred from studies of GluN2 expression levels, changes in kinetics of glutamatergic synaptic currents and sensitivity of NMDA receptor-mediated currents to selective GluN2B antagonists. Here we use TCN 213, a novel GluN2A-selective antagonist to identify the presence of this subunit in functional NMDA receptors in developing cortical neurones.
Two-electrode voltage-clamp (TEVC) recordings were made from Xenopus laevis oocytes to determine the pharmacological activity of TCN 213 at recombinant NMDA receptors. TCN 213 antagonism was studied in cultures of primary cortical neurones, assessing the NMDA receptor dependency of NMDA-induced excitotoxicity and monitoring developmental switches in NMDA receptor subunit composition.
TCN 213 antagonism of GluN1/GluN2A NMDA receptors was dependent on glycine but independent of glutamate concentrations in external recording solutions. Antagonism by TCN 213 was surmountable and gave a Schild plot with unity slope. TCN 213 block of GluN1/GluN2B NMDA receptor-mediated currents was negligible. In cortical neurones, at a early developmental stage predominantly expressing GluN2B-containing NMDA receptors, TCN 213 failed to antagonize NMDA receptor-mediated currents or to prevent GluN2B-dependent, NMDA-induced excitoxicity. In older cultures (DIV 14) or in neurones transfected with GluN2A subunits, TCN 213 antagonized NMDA-evoked currents. Block by TCN 213 of NMDA currents inversely correlated with block by ifenprodil, a selective GluN2B antagonist.
TCN 213 selectively blocked GluN1/GluN2A over GluN1/GluN2B NMDA receptors allowing direct dissection of functional NMDA receptors and pharmacological profiling of developmental changes in native NMDA receptor subunit composition.
PMCID: PMC3417419  PMID: 22022974
NMDA; glutamate; glycine; antagonism; oocyte; two-electrode voltage clamp; electrophysiology; neurotoxicity; development
17.  Direct pharmacological monitoring of the developmental switch in NMDA receptor subunit composition using TCN 213, a GluN2A-selective, glycine-dependent antagonist 
British Journal of Pharmacology  2012;166(3):924-937.
Developmental switches in NMDA receptor subunit expression have been inferred from studies of GluN2 expression levels, changes in kinetics of glutamatergic synaptic currents and sensitivity of NMDA receptor-mediated currents to selective GluN2B antagonists. Here we use TCN 213, a novel GluN2A-selective antagonist to identify the presence of this subunit in functional NMDA receptors in developing cortical neurones.
Two-electrode voltage-clamp (TEVC) recordings were made from Xenopus laevis oocytes to determine the pharmacological activity of TCN 213 at recombinant NMDA receptors. TCN 213 antagonism was studied in cultures of primary cortical neurones, assessing the NMDA receptor dependency of NMDA-induced excitotoxicity and monitoring developmental switches in NMDA receptor subunit composition.
TCN 213 antagonism of GluN1/GluN2A NMDA receptors was dependent on glycine but independent of glutamate concentrations in external recording solutions. Antagonism by TCN 213 was surmountable and gave a Schild plot with unity slope. TCN 213 block of GluN1/GluN2B NMDA receptor-mediated currents was negligible. In cortical neurones, at a early developmental stage predominantly expressing GluN2B-containing NMDA receptors, TCN 213 failed to antagonize NMDA receptor-mediated currents or to prevent GluN2B-dependent, NMDA-induced excitoxicity. In older cultures (DIV 14) or in neurones transfected with GluN2A subunits, TCN 213 antagonized NMDA-evoked currents. Block by TCN 213 of NMDA currents inversely correlated with block by ifenprodil, a selective GluN2B antagonist.
TCN 213 selectively blocked GluN1/GluN2A over GluN1/GluN2B NMDA receptors allowing direct dissection of functional NMDA receptors and pharmacological profiling of developmental changes in native NMDA receptor subunit composition.
PMCID: PMC3417419  PMID: 22022974
NMDA; glutamate; glycine; antagonism; oocyte; two-electrode voltage clamp; electrophysiology; neurotoxicity; development
18.  The Effects of NR2 Subunit-Dependent NMDA Receptor Kinetics on Synaptic Transmission and CaMKII Activation 
PLoS Computational Biology  2008;4(10):e1000208.
N-Methyl-d-aspartic acid (NMDA) receptors are widely expressed in the brain and are critical for many forms of synaptic plasticity. Subtypes of the NMDA receptor NR2 subunit are differentially expressed during development; in the forebrain, the NR2B receptor is dominant early in development, and later both NR2A and NR2B are expressed. In heterologous expression systems, NR2A-containing receptors open more reliably and show much faster opening and closing kinetics than do NR2B-containing receptors. However, conflicting data, showing similar open probabilities, exist for receptors expressed in neurons. Similarly, studies of synaptic plasticity have produced divergent results, with some showing that only NR2A-containing receptors can drive long-term potentiation and others showing that either subtype is capable of driving potentiation. In order to address these conflicting results as well as open questions about the number and location of functional receptors in the synapse, we constructed a Monte Carlo model of glutamate release, diffusion, and binding to NMDA receptors and of receptor opening and closing as well as a model of the activation of calcium-calmodulin kinase II, an enzyme critical for induction of synaptic plasticity, by NMDA receptor-mediated calcium influx. Our results suggest that the conflicting data concerning receptor open probabilities can be resolved, with NR2A- and NR2B-containing receptors having very different opening probabilities. They also support the conclusion that receptors containing either subtype can drive long-term potentiation. We also are able to estimate the number of functional receptors at a synapse from experimental data. Finally, in our models, the opening of NR2B-containing receptors is highly dependent on the location of the receptor relative to the site of glutamate release whereas the opening of NR2A-containing receptors is not. These results help to clarify the previous findings and suggest future experiments to address open questions concerning NMDA receptor function.
Author Summary
Information processing in the brain is carried out by networks of neurons connected by synapses. Synapses can change strength, allowing these networks to adapt and learn, in a process known as synaptic plasticity. At a synapse, an electrical signal in one neuron is converted into a chemical signal, carried by a neurotransmitter, which is in turn converted into electrical and chemical signals in another neuron by specialized proteins called receptors. One such protein, the N-methyl-d-aspartic acid (NMDA) receptor, is particularly important for plasticity, due to its ability to detect the voltage of the cell receiving the neurotransmitter signal and to the fact that it allows calcium, an important signaling molecule, to enter the cell. Here we use computational modeling to investigate the role of one part of the NMDA receptor: the NR2 subunit. The subunit has various forms, and which of these forms are present in the NMDA receptor can strongly affect the kinetics and other properties of the receptor. We show that, along with changing the kinetics of the receptor, changing the NR2 subunit affects the reliability of the receptor, its ability to respond to large stimuli, and its spatial response properties. These results have implications for synaptic transmission and plasticity.
PMCID: PMC2563690  PMID: 18974824
19.  Coactivation of NMDA receptors by glutamate and -serine induces dilation of isolated middle cerebral arteries 
N-methyl--aspartate (NMDA) receptors are glutamate-gated cation channels that mediate excitatory neurotransmission in the central nervous system. In addition to glutamate, NMDA receptors are also activated by coagonist binding of the gliotransmitter, -serine. Neuronal NMDA receptors mediate activity-dependent blood flow regulation in the brain. Our objective was to determine whether NMDA receptors expressed by brain endothelial cells can induce vasodilation of isolated brain arteries. Adult mouse middle cerebral arteries (MCAs) were isolated, pressurized, and preconstricted with norepinephrine. N-methyl--aspartate receptor agonists, glutamate and NMDA, significantly dilated MCAs in a concentration-dependent manner in the presence of -serine but not alone. Dilation was significantly inhibited by NMDA receptor antagonists, -2-amino-5-phosphonopentanoate and 5,7-dichlorokynurenic acid, indicating a response dependent on NMDA receptor glutamate and -serine binding sites, respectively. Vasodilation was inhibited by denuding the endothelium and by selective inhibition or genetic knockout of endothelial nitric oxide synthase (eNOS). We also found evidence for expression of the pan-NMDA receptor subunit, NR1, in mouse primary brain endothelial cells, and for the NMDA receptor subunit NR2C in cortical arteries in situ. Overall, we conclude that NMDA receptor coactivation by glutamate and -serine increases lumen diameter in pressurized MCA in an endothelial and eNOS-dependent mechanism.
PMCID: PMC3293118  PMID: 22068228
-serine; eNOS; glutamate; middle cerebral artery; NMDA receptor; NR2C
20.  Activation of group I mGlu receptors contributes to facilitation of NMDA receptor membrane current in spinal dorsal horn neurons after hind paw inflammation in rats 
European journal of pharmacology  2011;670(2-3):509-518.
The interaction between the group I metabotropic glutamate (mGlu) receptors and N-methyl-D-aspartate (NMDA) receptors plays a critical role in spinal hyperexcitability and hyperalgesia. The cellular mechanisms underlying this interaction remain unknown. Utilizing an ex vivo spinal slice preparation from young adult rats, we investigated the group I mGlu receptor modulation of NMDA receptor-mediated current in superficial dorsal horn neurons by patch clamp recording after complete Freund’s adjuvant (CFA)-induced hind paw inflammation. We show that NMDA receptor-mediated dorsal root stimulation-evoked EPSC (eEPSC) and NMDA-induced current was enhanced in the inflamed rats, compared to naïve rats and this effect was attenuated by AIDA (1 mM), a group I mGlu receptor antagonist. There were also increases in the frequency and amplitude of miniature excitatory postsynaptic currents in the presence of tetrodotoxin, suggesting enhanced presynaptic glutamate release probability and postsynaptic membrane responsiveness in inflamed rats. DHPG (10 µM), a selective group I mGlu receptor agonist, further facilitated NMDA receptor-mediated eEPSC and NMDA-induced current in inflamed rats. The DHPG-produced facilitation of NMDA-induced current was blocked by intracellular dialysis of GDP-beta-S (1 mM), a G protein antagonist, and BAPTA (15 mM), an intracellular calcium chelating agent; and by pretreatment with U73,122 (10 µM), a PLC inhibitor, or 2-APB (100 µM), an IP3-receptor antagonist. These findings support the hypothesis that signal transduction coupling between group I mGlu receptors and NMDA receptors underlies the activation of NMDA receptors in spinal hyperexcitability and hyperalgesia.
PMCID: PMC3220411  PMID: 21951968
N-methyl-D-aspartate receptors; Metabotropic glutamate receptors; Spinal cord slice; Electrophysiology; Pain; Hyperexcitability
21.  Oligodendrocyte Precursor Cells Modulate the Neuronal Network by Activity-Dependent Ectodomain Cleavage of Glial NG2 
PLoS Biology  2014;12(11):e1001993.
This study shows that the activity of neurons can trigger shedding of a protein, NG2, from the surface of oligodendrocyte precursor cells; this protein in turn modulates synaptic transmission, revealing a two-way conversation between neurons and glia.
The role of glia in modulating neuronal network activity is an important question. Oligodendrocyte precursor cells (OPC) characteristically express the transmembrane proteoglycan nerve-glia antigen 2 (NG2) and are unique glial cells receiving synaptic input from neurons. The development of NG2+ OPC into myelinating oligodendrocytes has been well studied, yet the retention of a large population of synapse-bearing OPC in the adult brain poses the question as to additional functional roles of OPC in the neuronal network. Here we report that activity-dependent processing of NG2 by OPC-expressed secretases functionally regulates the neuronal network. NG2 cleavage by the α-secretase ADAM10 yields an ectodomain present in the extracellular matrix and a C-terminal fragment that is subsequently further processed by the γ-secretase to release an intracellular domain. ADAM10-dependent NG2 ectodomain cleavage and release (shedding) in acute brain slices or isolated OPC is increased by distinct activity-increasing stimuli. Lack of NG2 expression in OPC (NG2-knockout mice), or pharmacological inhibition of NG2 ectodomain shedding in wild-type OPC, results in a striking reduction of N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) in pyramidal neurons of the somatosensory cortex and alterations in the subunit composition of their α-amino-3-hydroxy-5-methyl-4-isoxazolepr opionicacid (AMPA) receptors. In NG2-knockout mice these neurons exhibit diminished AMPA and NMDA receptor-dependent current amplitudes; strikingly AMPA receptor currents can be rescued by application of conserved LNS protein domains of the NG2 ectodomain. Furthermore, NG2-knockout mice exhibit altered behavior in tests measuring sensorimotor function. These results demonstrate for the first time a bidirectional cross-talk between OPC and the surrounding neuronal network and demonstrate a novel physiological role for OPC in regulating information processing at neuronal synapses.
Author Summary
Although glial cells substantially outnumber neurons in the mammalian brain, much remains to be discovered regarding their functions. Among glial cells, oligodendrocyte precursors differentiate into oligodendrocytes, whose function is to enwrap nerves with myelin to ensure proper impulse conduction. However, oligodendrocyte precursors also comprise a stable population in all major regions of the adult brain, making up around 5% of the total number of neurons and glia. Synapses are classically formed between neurons. Nonetheless, oligodendrocyte precursors are unique among glial cells in that they receive direct synaptic input from different types of neurons; whether OPC also send signals to neurons is still unknown. Here we show a bidirectional communication between neurons and oligodendrocyte precursors: neuronal activity regulates the cleavage of a glial membrane protein and the release of an extracellular domain that in turn modulates synaptic transmission between neurons. Our data thus show that a particular subtype of glial cells, oligodendrocyte precursors, functionally integrate into the neuronal network and we link this bidirectional signaling to mouse behavior and disease.
PMCID: PMC4227637  PMID: 25387269
22.  Activation of NMDA receptors leads to phosphorylation of TRPV1 S800 by protein kinase C and A-Kinase anchoring protein 150 in rat trigeminal ganglia 
A-Kinase anchoring protein 150 (AKAP150) is required for the phosphorylation of transient receptor potential cation channel subfamily V member 1 (TRPV1) by PKA or PKC in sensory neurons and, hence, affects TRPV1-dependent hyperalgesia under pathological conditions. Recently, we showed that the activation of N-methyl-d-aspartate (NMDA) receptors sensitizes TRPV1 by enhancing serine phosphorylation through PKC in trigeminal nociceptors. In this study, we extended this observation by investigating whether AKAP150 mediates NMDA-induced phosphorylation of TRPV1 via PKC in native sensory neurons in the rat. By adopting a phospho-specific antibody combined with a surface biotinylation assay, we first assessed NMDA-induced changes in the phosphorylation level of serine 800 residues (S800) in TRPV1 delimited to cell surface membrane in cultured trigeminal ganglia (TG). The biotinylation assay yielded that the application of NMDA significantly increased the phosphorylation of S800 (p-S800) of TRPV1 at time points correlating with the development of NMDA-induced mechanical hyperalgesia [10]. We then obtained a siRNA sequence against AKAP150 that dose-dependently down-regulated the AKAP150 protein. Pretreatment of TG culture with the siRNA, but not mismatch sequences, prevented the NMDA-induced phosphorylation of serine residues of total TRPV1 as well as S800 of membrane bound TRPV1. We confirmed that AKAP150 coimmunoprecipitated with TRPV1 and demonstrated that it also co-immunoprecipitated with NMDA receptor subunits (NR1 and NR2B) in TG. These data offer novel information that the activation of NMDA-induced TRPV1 sensitization involves p-S800 of TRPV1 in cell surface membrane in native sensory neurons and that AKAP150 is required for NMDA-and PKC-mediated phosphorylation of TRPV1 S800. Therefore, we propose that the NMDA receptor, AKAP150, and TRPV1 forms a signaling complex that underlies the sensitization of trigeminal nociceptors by modulating phosphorylation of specific TRPV1 residues.
PMCID: PMC3408820  PMID: 22789851
Nociceptor; Sensitization; Peripheral; Rat
23.  Activation of mGluR5 Attenuates NMDA-Induced Neurotoxicity through Disruption of the NMDAR-PSD-95 Complex and Preservation of Mitochondrial Function in Differentiated PC12 Cells 
Glutamate-mediated toxicity is implicated in various neuropathologic conditions, and activation of ionotropic and metabotropic glutamate receptors is considered to be the most important mechanism. It has been reported that pharmacological saturation of metabotropic glutamate receptors (mGluRs) can facilitate N-methyl-d-aspartate receptor (NMDAR) related signaling cascades, but the mechanism leading to mGluR-NMDAR interactions in excitotoxic neuronal injury has remained unidentified. In the present study, we investigated the role of mGluR5 in the regulation of N-methyl-d-aspartate (NMDA)-induced excitotoxicity in differentiated PC12 cells. We found that activation of mGluR5 with the specific agonist R,S-2-chloro-5-hydroxyphenylglycine (CHPG) increased cell viability and inhibited lactate dehydrogenase (LDH) release in a dose-dependent manner. CHPG also inhibited an increase in the Bax/Bcl-2 ratio, attenuated cleavage of caspase-9 and caspase-3, and reduced apoptotic cell death after NMDA treatment. The NMDA-induced mitochondrial dysfunction, as indicated by mitochondrial reactive oxygen species (ROS) generation, collapse of mitochondrial membrane potential (MMP), and cytochrome c release, was also partly prevented by CHPG treatment. Furthermore, CHPG blocked the NMDA-induced interaction of NMDAR with postsynaptic density protein-95 (PSD-95), but had no effects on intracellular calcium concentrations. All these results indicated that activation of mGluR5 protects differentiated PC12 cells from NMDA-induced neuronal excitotoxicity by disrupting NMDAR-PSD-95 interaction, which might be an ideal target for investigating therapeutic strategies in various neurological diseases where excitotoxicity may contribute to their pathology.
PMCID: PMC4100187  PMID: 24941251
glutamate; mGlu receptor; NMDAR (N-methyl-d-aspartate receptor) receptor; post-synaptic density protein 95; mitochondrial dysfunction
24.  Expression of mRNA for glutamate receptor subunits distinguishes the major classes of retinal neurons, but is less specific for individual cell types 
Molecular Vision  2007;13:933-948.
To investigate the expression of ionotropic glutamate receptor subunits by retinal neurons, to assess the extent to which different functional types of retinal neurons are characterized by the expression of the receptor subtypes.
Rod photoreceptor cells and bipolar cells were identified in retina dissociates. Amacrine cells were identified in dissociates from transgenic mice or by staining with an antibody against the extracellular carbohydrate epitope CD15. Ganglion cells were identified by retrograde axonal transport of FITC-dextran or by green fluorescent protein (GFP) fluorescence in a transgenic strain. We examined the receptors simultaneously using non-quantitative single-cell reverse transcriptase polymerase chain reaction for GluR1-R4 (α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors), GluR5-R7, and KA1 and 2 (kainate receptors), δ1 and δ2 subunits, and the N-methyl-D-aspartate (NMDA) receptor subunits NR1, 2a-d, and 3a.
The expression of glutamate receptors on bipolar cells and rod photoreceptors was limited: Neither expressed functional NMDA receptors, and rods were also negative for AMPA receptors. The sample of ganglion cells included examples of many ganglion cell types; these were distinguished morphologically using quantitative parameters defined in a previous cluster analysis. All types of ionotropic glutamate receptors were found to be expressed on ganglion cells. The iGluR subunits GluR4, KA2, δ1, and NR1 were expressed on almost all ganglion cells examined.
Despite the heterogeneity of ganglion cell types, differences among them in this PCR-based method were minor. Thus, retinal interneurons are characterized by expression of distinctive glutamate receptor types, but functional differences among ganglion cells seem to be reflected instead in the amounts as well as spatial distributions of a widely expressed group of receptors.
PMCID: PMC2774459  PMID: 17653033
25.  Depolarization and CaM Kinase IV Modulate NMDA Receptor Splicing through Two Essential RNA Elements 
PLoS Biology  2007;5(2):e40.
Alternative splicing controls the activity of many proteins important for neuronal excitation, but the signal-transduction pathways that affect spliced isoform expression are not well understood. One particularly interesting system of alternative splicing is exon 21 (E21) of the NMDA receptor 1 (NMDAR1 E21), which controls the trafficking of NMDA receptors to the plasma membrane and is repressed by Ca++/calmodulin-dependent protein kinase (CaMK) IV signaling. Here, we characterize the splicing of NMDAR1 E21. We find that E21 splicing is reversibly repressed by neuronal depolarization, and we identify two RNA elements within the exon that function together to mediate the inducible repression. One of these exonic elements is similar to an intronic CaMK IV–responsive RNA element (CaRRE) originally identified in the 3′ splice site of the BK channel STREX exon, but not previously observed within an exon. The other element is a new RNA motif. Introduction of either of these two motifs, called CaRRE type 1 and CaRRE type 2, into a heterologous constitutive exon can confer CaMK IV–dependent repression on the new exon. Thus, either exonic CaRRE can be sufficient for CaMK IV–induced repression. Single nucleotide scanning mutagenesis defined consensus sequences for these two CaRRE motifs. A genome-wide motif search and subsequent RT-PCR validation identified a group of depolarization-regulated alternative exons carrying CaRRE consensus sequences. Many of these exons are likely to alter neuronal function. Thus, these two RNA elements define a group of co-regulated splicing events that respond to a common stimulus in neurons to alter their activity.
Alternative splicing of NMDA receptor 1 exon 21 is reversibly repressed by depolarization in a CaMK IV-dependent manner in neurons. This suggests splicing is finely tuned by dynamic activity inputs.
Author Summary
Multiple mechanisms direct changes in neuronal activity in response to external stimuli, ranging from short-acting modifications of membrane proteins to longer-acting changes in gene expression. A frequently regulated step in gene expression is the pre-mRNA splicing reaction in which the inclusion of exons (protein-coding sequences) or the position of splice sites produces alternatively spliced mRNA isoforms encoding functionally different proteins. Here, we study splicing of the NMDA receptor, which responds to the neurotransmitter glutamate to modify neuronal activity. We show that the splicing of an important exon (E21) in the NMDA receptor subunit NR1 mRNA is repressed by cell depolarization and activation of the intracellular signaling molecule, CaMK IV. We find that this splicing repression is mediated by two regulatory sequences within the exon itself. One sequence is similar to a previously described regulatory element that had not been known to function in an exon. The other is a new element. The characterization of these elements as a family of degenerate sequences allowed the identification of a group of exons sharing responsiveness to cell depolarization and CamK IV. These results define a new set of gene expression changes that may occur in modulating neuronal activity.
PMCID: PMC1790950  PMID: 17298178

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