Abstract: OBJECTIVE: Research has implicated dysfunction of glutamatergic neurotransmission in the pathophysiology of schizophrenia. This review evaluates evidence from preclinical and clinical studies that brain glutamatergic neurotransmission is altered in schizophrenia, may affect symptom expression, and is modulated by antipsychotic drugs. METHOD: A comprehensive review of scientific articles published over the last decade that address the role of glutamate in the pathophysiology of schizophrenia was carried out. RESULTS: Glutamatergic neurons are the major excitatory pathways linking the cortex, limbic system, and thalamus, regions that have been implicated in schizophrenia. Postmortem studies have revealed alterations in pre- and postsynaptic markers for glutamatergic neurons in several brain regions in schizophrenia. The N-methyl-d-aspartic acid (NMDA) subtype of glutamate receptor may be particularly important as blockade of this receptor by the dissociative anesthetics reproduces in normal subjects the sym...

Abstract: The tryptophan metabolite kynurenic acid (KYNA) has long been recognized as an NMDA receptor antagonist. Here, interactions between KYNA and the nicotinic system in the brain were investigated using the patch-clamp technique and HPLC. In the electrophysiological studies, agonists were delivered via a U-shaped tube, and KYNA was applied in admixture with agonists and via the background perfusion. Exposure (≥4 min) of cultured hippocampal neurons to KYNA (≥100 nm) inhibited activation of somatodendritic α7 nAChRs; the IC50 for KYNA was ∼7 μm. The inhibition of α7 nAChRs was noncompetitive with respect to the agonist and voltage independent. The slow onset of this effect could not be accounted for by an intracellular action because KYNA (1 mm) in the pipette solution had no effect on α7 nAChR activity. KYNA also blocked the activity of preterminal/presynaptic α7 nAChRs in hippocampal neurons in cultures and in slices. NMDA receptors were less sensitive than α7 nAChRs to KYNA. The IC50 values for KYNA-induced blockade of NMDA receptors in the absence and presence of glycine (10 μm) were ∼15 and 235 μm, respectively. Prolonged (3 d) exposure of cultured hippocampal neurons to KYNA increased their nicotinic sensitivity, apparently by enhancing α4β2 nAChR expression. Furthermore, as determined by HPLC with fluorescence detection, repeated systemic treatment of rats with nicotine caused a transient reduction followed by an increase in brain KYNA levels. These results demonstrate that nAChRs are targets for KYNA and suggest a functionally significant cross talk between the nicotinic cholinergic system and the kynurenine pathway in the brain.

Abstract: Tissue-plasminogen activator (t-PA) is now available for the treatment of thrombo-embolic stroke but adverse effects have been reported in some patients, particularly hemorrhaging. In contrast, the results of animal studies have indicated that t-PA could increase neuronal damage after focal cerebral ischemia. Here we report for the first time that t-PA potentiates signaling mediated by glutamatergic receptors by modifying the properties of the N-methyl-D-aspartate (NMDA) receptor. When depolarized, cortical neurons release bio-active t-PA that interacts with and cleaves the NR1 subunit of the NMDA receptor. Moreover, the treatment with recombinant t-PA leads to a 37% increase in NMDA-stimulated fura-2 fluorescence, which may reflect an increased NMDA-receptor function. These results were confirmed in vivo by the intrastriatal injection of recombinant-PA, which potentiated the excitotoxic lesions induced by NMDA. These data provide insight into the regulation of NMDA-receptor-mediated signaling and could initiate therapeutic strategies to improve the efficacy of t-PA treatment in man.

Abstract: Calcium- and calmodulin-dependent protein kinase II (CaMKII) and glutamate receptors are integrally involved in forms of synaptic plasticity that may underlie learning and memory. In the simplest model for long-term potentiation, CaMKII is activated by Ca2+ influx through NMDA (N-methyl-D-aspartate) receptors and then potentiates synaptic efficacy by inducing synaptic insertion and increased single-channel conductance of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors. Here we show that regulated CaMKII interaction with two sites on the NMDA receptor subunit NR2B provides a mechanism for the glutamate-induced translocation of the kinase to the synapse in hippocampal neurons. This interaction can lead to additional forms of potentiation by: facilitated CaMKII response to synaptic Ca2+; suppression of inhibitory autophosphorylation of CaMKII; and, most notably, direct generation of sustained Ca2+/calmodulin (CaM)-independent (autonomous) kinase activity by a mechanism that is independent of the phosphorylation state. Furthermore, the interaction leads to trapping of CaM that may reduce down-regulation of NMDA receptor activity. CaMKII-NR2B interaction may be prototypical for direct activation of a kinase by its targeting protein.

Abstract: A compelling model of experience-dependent plasticity is the long-lasting sensitization to the locomotor stimulatory effects of drugs of abuse. Adaptations in the nucleus accumbens (NAc), a component of the mesolimbic dopamine system, are thought to contribute to this behavioral change. Here we examine excitatory synaptic transmission in NAc slices prepared from animals displaying sensitization 10-14 days after repeated in vivo cocaine exposure. The ratio of AMPA (alpha-amino-3-hydroxy-5-methyl-4- isoxazole propionic acid) receptor- to NMDA (N-methyl-d-aspartate) receptor-mediated excitatory postsynaptic currents (EPSCs) was decreased at synapses made by prefrontal cortical afferents onto medium spiny neurons in the shell of the NAc. The amplitude of miniature EPSCs at these synapses also was decreased, as was the magnitude of long-term depression. These data suggest that chronic in vivo administration of cocaine elicits a long-lasting depression of excitatory synaptic transmission in the NAc, a change that may contribute to behavioral sensitization and addiction.

Abstract: Analysis of excitatory synaptic transmission in the rat hypothalamic supraoptic nucleus revealed that glutamate clearance and, as a consequence, glutamate concentration and diffusion in the extracellular space, is associated with the degree of astrocytic coverage of its neurons. Reduction in glutamate clearance, whether induced pharmacologically or associated with a relative decrease of glial coverage in the vicinity of synapses, affected transmitter release through modulation of presynaptic metabotropic glutamate receptors. Astrocytic wrapping of neurons, therefore, contributes to the regulation of synaptic efficacy in the central nervous system.

Abstract: Glutamate neurotoxicity has been implicated in stroke, head trauma, multiple sclerosis and neurodegenerative diseases. Although recent data show that cultured glioma cells secrete glutamate, the growth potential of brain tumors has not yet been linked to an excitotoxic mechanism. Using bioluminescence detection of glutamate release from freshly prepared brain slices, we show that implanted glioma cells continue to secrete glutamate. Moreover, gliomas with high glutamate release have a distinct growth advantage in host brain that is not present in vitro. Treatment with the NMDA receptor antagonists MK801 or memantine slowed the growth of glutamate-secreting tumors in situ, suggesting that activation of NMDA receptors facilitates tumor expansion. These findings support a new approach for therapy of brain tumors, based upon antagonizing glutamate secretion or its target receptors.

Abstract: Although synaptic AMPA receptors have been shown to rapidly internalize, synaptic NMDA receptors are reported to be static. It is not certain whether NMDA receptor stability at synaptic sites is an inherent property of the receptor, or is due to stabilization by scaffolding proteins. In this study, we demonstrate that NMDA receptors are internalized in both heterologous cells and neurons, and we define an internalization motif, YEKL, on the distal C-terminus of NR2B. In addition, we show that the synaptic protein PSD-95 inhibits NR2B-mediated internalization, and that deletion of the PDZ-binding domain of NR2B increases internalization in neurons. This suggests an involvement for PSD-95 in NMDA receptor regulation and an explanation for NMDA receptor stability at synaptic sites.

Abstract: Excitotoxicity is one of the most extensively studied processes of neuronal cell death, and plays an important role in many central nervous system (CNS) diseases, including CNS ischemia, trauma, and neurodegenerative disorders. First described by Olney, excitotoxicity was later characterized as an excessive synaptic release of glutamate, which in turn activates postsynaptic glutamate receptors. While almost every glutamate receptor subtype has been implicated in mediating excitotoxic cell death, it is generally accepted that the N-methyl-D-aspartate (NMDA) subtypes play a major role, mainly owing to their high calcium (Ca2+) permeability. However, other glutamate receptor subtypes such as 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) propionate (AMPA) or kainate receptors have also been attributed a critical role in mediating excitotoxic neuronal cell death. Although the molecular basis of glutamate toxicity is uncertain, there is general agreement that it is in large part Ca2+-dependent. The present review is aimed at summarizing the molecular mechanisms of NMDA receptor and AMPA/kainate receptor-mediated excitotoxic neuronal cell death.

Abstract: NMDA receptors (NMDARs) are highly calcium-permeable and are negatively regulated by intracellular calcium during prolonged exposure to agonist. We have investigated whether calcium-mediated feedback occurs during transient exposure to glutamate during single synaptic events. Examination of miniature EPSCs (mEPSCs) indicated that the decay kinetics of the NMDAR component was markedly slowed by the intracellular perfusion of exogenous calcium buffers (BAPTA or Fluo-3). In contrast, the AMPA receptor component of the miniature EPSC was unaffected. Slow on-rate calcium buffers, such as EGTA, did not alter kinetics of the NMDAR component of the mEPSC. Addition of exogenous fast calcium buffers did not slow the decay kinetics of glutamate-evoked currents mediated by NR1/NR2A heteromers expressed in HEK 293 cells, suggesting that the effect we observed in neurons may be specific to processes associated with synaptically activated receptors. Trial-to-trial amplitude variability of miniature calcium transients mediated by NMDARs increased with the injection of exogenous calcium buffers, suggesting that the amplitude of synaptic calcium transients are maintained at a rather constant level by a calcium-mediated feedback mechanism.

Abstract: The activation of group I metabotropic glutamate receptors (mGluRs) produces a variety of actions that lead to alterations in excitability and synaptic transmission in the CA1 region of the hippocampus. The group I mGluRs, mGluR1 and mGluR5, are activated selectively by (S)-3,5-dihydroxyphenylglycine (DHPG). To identify which of these mGluR subtypes are responsible for the various actions of DHPG in area CA1, we took advantage of two novel subtype-selective antagonists. (S)-(+)-alpha-amino-a-methylbenzeneacetic acid (LY367385) is a potent competitive antagonist that is selective for mGluR1, whereas 2-methyl-6-(phenylethynyl)-pyridine (MPEP) is a potent noncompetitive antagonist that is selective for mGluR5. The use of these compounds in experiments with whole-cell patch-clamp recording and Ca(2+)-imaging techniques revealed that each group I mGluR subtype plays distinct roles in regulating the function of CA1 pyramidal neurons. The block of mGluR1 by LY367385 suppressed the DHPG-induced increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) and the direct depolarization of CA1 hippocampal neurons. In addition, the increase in the frequency of spontaneous IPSCs (sIPSCs) caused by the DHPG-induced depolarization of inhibitory interneurons also was blocked by LY367385, as was the DHPG-induced inhibition of transmission at the Schaffer collateral-->CA1 synapse. On the other hand, the block of mGluR5 by MPEP antagonized the DHPG-induced suppression of the Ca(2+)-activated potassium current (I(AHP)) and potentiation of the NMDA receptor. Finally, antagonism of the DHPG-induced suppression of evoked IPSCs required the blockade of both mGluR1 and mGluR5. These data suggest that mGluR1 and mGluR5 play distinct roles in the regulation of the excitability of hippocampal CA1 pyramidal neurons.

Abstract: Dopamine acts mainly through the D1/D5 receptor in the prefrontal cortex (PFC) to modulate neural activity and behaviors associated with working memory. To understand the mechanism of this effect, we examined the modulation of excitatory synaptic inputs onto layer V PFC pyramidal neurons by D1/D5 receptor stimulation. D1/D5 agonists increased the size of N-methyl-d-aspartate (NMDA) component of excitatory postsynaptic currents (EPSCs) through a postsynaptic mechanism. In contrast, D1/D5 agonists caused a slight reduction in the size of the non-NMDA component of EPSCs through a small decrease in release probability. With 20 Hz synaptic trains, we found that the D1/D5 agonists increased depolarization of summating the NMDA component of excitatory postsynaptic potential (EPSP). By increasing the NMDA component of EPSCs, yet slightly reducing release, D1/D5 receptor activation selectively enhanced sustained synaptic inputs and equalized the sizes of EPSPs in a 20-Hz train.

Abstract: Recent work has shown substantial alterations in NMDA receptor subunit expression, assembly, and phosphorylation in the dopamine-depleted striatum of a rodent 6-hydroxydopamine model of Parkinson's disease. These modifications are hypothesized to result from the trafficking of NMDA receptors between subcellular compartments. Here we show that in rat striatal tissues the NR2A and NR2B subunits in the synaptosomal membrane, and not those in the light membrane and synaptic vesicle-enriched compartments, are tyrosine phosphorylated. The dopamine D1 receptor agonist SKF-82958 produces (1) an increase in NR1, NR2A, and NR2B proteins in the synaptosomal membrane fraction; (2) a decrease in NR1, NR2A, and NR2B proteins in the light membrane and synaptic vesicle-enriched fractions; and (3) an increase in the tyrosine phosphorylation of NR2A and NR2B in the synaptosomal membrane compartment. The protein phosphatase inhibitor pervanadate reproduces the alterations in subcellular distribution and phosphorylation, whereas the effects of the dopamine D1 receptor agonist are blocked by genistein, a protein tyrosine kinase inhibitor. Dopamine D1 receptor agonist treatment does not change the subcellular distribution of the AMPA receptor subunits GluR1 or GluR2/3 in the striatum and has no effect on cortical or cerebellar NMDA receptor subunits. These data reveal a rapid dopamine D1 receptor- and tyrosine kinase-dependent trafficking of striatal NMDA receptors between intracellular and postsynaptic sites. The subcellular trafficking of striatal NMDA receptors may play a significant role both in the pathogenesis of Parkinson's disease and in the development of adverse effects of chronic dopaminergic therapy in parkinsonian patients.

Abstract: Glutamate is the primary excitatory amino acid neurotransmitter in the human brain. It is important in synaptic plasticity, learning, and development. Its activity at the synaptic cleft is carefully balanced by receptor inactivation and glutamate reuptake. When this balance is upset, excess glutamate can itself become neurotoxic.

Abstract: N-methyl-D-aspartate (NMDA) receptors contribute to many brain functions. We studied the effect of forebrain-targeted overexpression of the NMDA receptor subunit NR2B on the response of mice to tissue injury and inflammation. Transgenic mice exhibited prominent NR2B expression and enhanced NMDA receptor-mediated synaptic responses in two pain-related forebrain areas, the anterior cingulate cortex and insular cortex, but not in the spinal cord. Although transgenic and wild type mice were indistinguishable in tests of acute pain, transgenic mice exhibited enhanced responsiveness to peripheral injection of two inflammatory stimuli, formalin and complete Freund's adjuvant. Genetic modification of forebrain NMDA receptors can therefore influence pain perception, which suggests that forebrain-selective NMDA receptor antagonists, including NR2B-selective agents, may be useful analgesics for persistent pain.

Abstract: The interactions between N-methyl-D-aspartate (NMDA) and D(1) dopamine receptors in the rat prefrontal cortex were examined using whole-cell recordings from pyramidal neurons. The effects of NMDA, the D(1) agonist SKF38393, or both compounds combined were tested on measures of cell excitability. Both NMDA (10-100 microM) and SKF38393 (5-10 microM) independently increased the number of spikes and decreased the latency of the first spike evoked by intracellular depolarizing current pulses. Combining low doses of NMDA (5 microM) and SKF38393 (2 microM) resulted in a marked increase of cell excitability. This synergism was blocked by SCH23390, protein kinase A (PKA) inhibitors, and the Ca(2+) chelator BAPTA, and reduced by nifedipine. These results indicate the presence of a dopamine- glutamate interaction in the prefrontal cortex at the postsynaptic level, by which D(1) dopamine receptors may maintain NMDA- mediated responses in prefrontal cortical pyramidal neurons through both a PKA-dependent pathway and Ca(2+)-dependent mechanisms.

Abstract: Huntington disease (HD) is an autosomal dominant disorder in which degeneration of medium-sized spiny striatal neurons occurs. The HD gene and the protein it encodes, huntingtin, have been identified but their functions remain unknown. Transgenic mouse models for HD have been developed and we examined responses of medium-sized striatal neurons recorded in vitro to application of N-methyl-D-aspartate (NMDA) in two of these. The first model (R6/2) expresses exon 1 of the human HD gene with approximately 150 CAG repeats. In the R6/2 an enhancement of currents induced by selective activation of NMDA receptors as well as an enhancement of intracellular Ca(2+) flux occurred in both presymptomatic and symptomatic mice. These alterations appeared specific for the NMDA receptor because alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor-mediated currents were reduced in symptomatic R6/2s. In R6/2 animals there were parallel increases in NMDA-R1 and decreases in NMDA-R2A/B subunit proteins as established by immunohistochemistry. The second model (YAC72) contains human genomic DNA spanning the full-length gene and all its regulatory elements with 72 CAG repeats. The phenotypical expression of the disorder develops more gradually than in the R6/2. In YAC72 mice we found similar but less marked increases in responses of medium-sized striatal neurons to NMDA. These findings indicate that alterations in NMDA receptor function may predispose striatal neurons to excitotoxic damage, leading to subsequent neuronal degeneration and underscore the functional importance of NMDA receptors in HD.

Abstract: Members of the N-methyl-d-aspartate (NMDA) class of glutamate receptors (NMDARs) are critical for development, synaptic transmission, learning and memory; they are targets of pathological disorders in the central nervous system. NMDARs are phosphorylated by both serine/threonine and tyrosine kinases. Here, we demonstrate that cyclin dependent kinase-5 (Cdk5) associates with and phosphorylates NR2A subunits at Ser-1232 in vitro and in intact cells. Moreover, we show that roscovitine, a selective Cdk5 inhibitor, blocks both long-term potentiation induction and NMDA-evoked currents in rat CA1 hippocampal neurons. These results suggest that Cdk5 plays a key role in synaptic transmission and plasticity through its up-regulation of NMDARs.

Abstract: Although accumulating evidence indicates that cAMP response element-binding protein (CREB) phosphorylation mediates not only synaptic plasticity but also survival of certain neurons, it remains uncertain whether CREB phosphorylation induced after metabolic insult leads to CRE-mediated gene transcription and is involved in cell survival or not. In the present study, we clarified that (1) CREB phosphorylation and ischemic tolerance induced after preconditioning ischemia in the hippocampal neurons was abolished by MK801 administration in gerbil global ischemia model, (2) CREB phosphorylation induced after exposure to glutamate in cultured neurons was inhibited by removal of extracellular calcium, by MK801 and by an inhibitor of calcium–calmodulin-dependent protein kinase (CaMK) II and IV, (3) inhibitor of CaMK II–IV or CRE-decoy oligonucleotide suppressed upregulation of BCL-2 expression and accelerated neuronal damage after exposure to glutamate, and (4) CREB phosphorylation induced in the hippocampal neurons after ischemia and in cultured neurons after exposure to glutamate was followed by CRE-mediated gene transcription in transgenic mice with a CRE–LacZ reporter. Our results suggest that CREB phosphorylation in neurons after ischemia and exposure to glutamate is induced by NMDA receptor-gated calcium influx and subsequent activation of CaMK II–IV and that CREB phosphorylation after metabolic stress might show a neuroprotective response through CRE-mediated gene induction.

Abstract: Glutamate released at synapses in the CA1 region of the hippocampus escapes the synaptic cleft and activates extrasynaptic targets; it also may “spill over” into neighboring synapses and activate receptors there Glutamate transporters in glial membranes restrict extrasynaptic diffusion, but it is unclear whether neuronal glutamate transporters also limit transmitter diffusion and receptor activation by spillover I examined the effects of a low-affinity competitive NMDA receptor antagonist on EPSCs in acute hippocampal slices to distinguish receptors activated within active synapses from those activated by spillover Glutamate spillover is observed between Schaffer collateral fiber synapses onto CA1 pyramidal cells only when transporters in the postsynaptic neuron are inhibited Because glutamate transporters operate most effectively at negative membrane potentials, these results suggest that activation of NMDA receptors by spillover may depend on postsynaptic activity

Abstract: Toxic effects of HIV-1 proteins contribute to altered function and decreased survival of select populations of neurons in HIV-1-infected brain. One such HIV-1 protein, Tat, can activate calcium release from IP3-sensitive intracellular pools, induce calcium influx in neural cells, and, as a result, can increase neuronal cell death. Here, we provide evidence that Tat potentiates excitatory amino acid (glutamate and NMDA) triggered calcium flux, as well as glutamate- and staurosporine-mediated neurotoxicity. Calcium flux in cultured rat hippocampal neurons triggered by the transient application of glutamate or NMDA was facilitated by pre-exposure to Tat. Facilitation of glutamate-triggered calcium flux by Tat was prevented by inhibitors of ADP-ribosylation of Gi/Go proteins (pertussis toxin), protein kinase C (H7 and bisindolymide), and IP3-mediated calcium release (xestospongin C), but was not prevented by an activator of Gs (cholera toxin) or an inhibitor of protein kinase A (H89). Facilitation of NMDA-triggered calcium flux by Tat was reversed by inhibitors of tyrosine kinase (genestein and herbimycin A) and by an inhibitor of NMDA receptor function (zinc). Tat increased 32P incorporation into NMDA receptor subunits NR2A and NR2B and this effect was blocked by genestein. Subtoxic concentrations of Tat combined with subtoxic concentrations of glutamate or staurosporine increased neuronal cell death significantly. Together, these findings suggest that NMDA receptors play an important role in Tat neurotoxicity and the mechanisms identified may provide additional therapeutic targets for the treatment of HIV-1 associated dementia.