Abstract: ACTIVATION of the N-methyl-d-aspartate (NMDA) receptor is important for certain forms of activity-dependent synaptic plasticity, such as long-term potentiation (reviewed in ref. 1), and the patterning of connections during development of the visual system (reviewed in refs 2, 3). Several subunits of the NMDA receptor have been cloned: these are NMDAR1 (NR1), and NMDAR2A, 2B, 2C and 2D (NR2A-D)4–8. Based on heterologous co-expression studies, it is inferred that NR1 encodes an essential subunit of NMDA receptors and that functional diversity of NMDA receptors in vivo is effected by differential incorporation of subunits NR2A–NR2D5–8. Little is known, however, about the actual subunit composition or heterogeneity of NMDA receptors in the brain. By co-immunoprecipitation with subunit-specific antibodies, we present here direct evidence that NMDA receptors exist in rat neocortex as heteromeric complexes of considerable heterogeneity, some containing both NR2A and NR2B subunits. A progressive alteration in subunit composition seen postnatally could contribute to NMDA-receptor variation and changing synaptic plasticity during cortical development.

Abstract: Circadian rhythms of mammals are timed by an endogenous clock with a period of about 24 hours located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Light synchronizes this clock to the external environment by daily adjustments in the phase of the circadian oscillation. The mechanism has been thought to involve the release of excitatory amino acids from retinal afferents to the SCN. Brief treatment of rat SCN in vitro with glutamate (Glu), N-methyl-D-aspartate (NMDA), or nitric oxide (NO) generators produced lightlike phase shifts of circadian rhythms. The SCN exhibited calcium-dependent nitric oxide synthase (NOS) activity. Antagonists of NMDA or NOS pathways blocked Glu effects in vitro, and intracerebroventricular injection of a NOS inhibitor in vivo blocked the light-induced resetting of behavioral rhythms. Together, these data indicate that Glu release, NMDA receptor activation, NOS stimulation, and NO production link light activation of the retina to cellular changes within the SCN mediating the phase resetting of the biological clock.

Abstract: Glutamate is the principal excitatory neurotransmitter in the brain and, as such, it inevitably plays a role in the initiation and spread of seizure activity. It also plays a critical role in epileptogenesis. The process of "kindling" limbic seizures in rodents by repeated electrical stimulation is dependent on activation of N-methyl-D-aspartate (NMDA) receptors. The function of these receptors is enhanced in the hippocampus of kindled rats and in the cerebral cortex of patients with focal epilepsy. Microdialysis studies show an increase in the extracellular concentration of glutamate and aspartate before or during seizure onset, suggesting that either enhanced amino acid release or impaired uptake contributes to seizure initiation. Glutamate antagonists selective for NMDA or non-NMDA receptors are potent anticonvulsants when given systemically in a wide variety of animal models of epilepsy. They are of limited efficacy against kindled seizures in rats and (on the basis of preliminary evidence) in patients with drug-refractory complex partial seizures. Cognitive side effects appear to be a significant problem with competitive, as well as noncompetitive, NMDA antagonists. Glutamate receptor antagonists provide significant protection against brain damage following global or focal cerebral ischemia or acute traumatic injury in rodent models. Anticonvulsant compounds of the lamotrigine type, which act on sodium channels and reduce ischemia-induced glutamate release, are cerebroprotective in rodent ischemia models and are free from the cognitive side effects of NMDA-receptor antagonists.

Abstract: The relationship between four pharmacologically distinct NMDA receptor subtypes, identified in radioligand binding studies, and the recently identified NMDA receptor subunits (NR1a-g, NR2A-D) has not been determined. In this report, we demonstrate that the anatomical distribution of the four NMDA receptor subtypes strikingly parallels the distribution of mRNA encoding NR2A-D subunits. The distribution of NR2A mRNA was very similar to that of “antagonist-preferring” NMDA receptors [defined by high-affinity 3H-2-carboxypiperazine-4-yl-propyl- 1-phosphonic (3H-CPP) binding sites; correlation coefficient = 0.88]. Agonist-preferring NMDA receptors localized to brain regions expressing both NR2B mRNA and NR1- mRNA (NR1 splice variant lacking insert 1). NR2C mRNA was largely restricted to the cerebellar granule cell layer, a region that displays a unique pharmacological profile. NR2D mRNA localized exclusively to those diencephalic nuclei that have a fourth, distinct pharmacological profile (typified by the midline thalamic nuclei). The pharmacology of native NMDA receptors was compared to that of heteromeric NMDA receptors expressed in Xenopus oocytes (NR1/NR2A, NR1/NR2B, NR1/NR2C). The oocyte-expressed NR1/NR2A receptor displayed a higher affinity for antagonists and a slightly lower affinity for agonists than the NR1/NR2B receptor. These patterns are analogous to those found for radioligand binding to native receptors in the lateral thalamus and medial striatum, respectively. NMDA receptors in the lateral thalamus (with a high density of NR2A subunit mRNA) displayed higher affinity for antagonists and a lower affinity for agonists than did NMDA receptors of the medial striatum (a region rich in NR2B mRNA). Relative to the NR1/NR2A and NR1/NR2B receptors, oocyte-expressed NR1/NR2C receptors had a lower affinity specifically for both D-3-(2- carboxypiperazin-4-yl)-1-propenyl-1-phosphonic acid (D-CPPene) and homoquinolinate (HQ). This pattern was identical to that observed for cerebellar (NR2C-containing) versus forebrain (NR2A- and NR2B- containing) NMDA receptors. Taken together, the data in this report suggest that the four previously identified native NMDA receptor subtypes differ in their NR2 composition. Furthermore, the NR2 subunits significantly contribute to the anatomical and pharmacological diversity of NMDA receptor subtypes.

Abstract: The regional, cellular, and subcellular distributions of N-methyl-D-aspartate (NMDA) receptor subunit 1, NMDAR-1, were investigated in monkey hippocampus by using a monoclonal antibody directed against a fusion protein corresponding to aa 660-811 of NMDAR-1. The data indicate that many neurons in each subfield of the hippocampus contain NMDAR-1 protein, although the intensity and distribution of immunoreactivity varied across regions, strata, and cellular compartments. In stratum lucidum of CA3, mossy fiber axons were immunoreactive for NMDAR-1, which may correspond to previously hypothesized presynaptic receptors. NMDAR-1-labeled postsynaptic profiles were present in stratum radiatum of CA3 but were largely absent from stratum lucidum. Such intraneuronal segregation of glutamate receptor subunits or classes may be spatially correlated with afferent systems that exhibit laminar segregation and terminate in different portions of the postsynaptic dendritic tree. For example, in CA3 pyramidal cells, NMDA receptors are postsynaptic in distal apical dendrites (stratum radiatum) where NMDA-dependent long-term potentiation in rats is mediated by associational/commissural afferents, and are absent from proximal apical dendrites (stratum lucidum), where NMDA-independent long-term potentiation is mediated by the mossy fiber input.

Abstract: Adenosine is present in the mammalian brain in large amounts and has potent effects on neuronal activity, but its role in neural signaling is poorly understood. The glutamate receptor agonist N-methyl-D-aspartate (NMDA) caused a presynaptic depression of excitatory synaptic transmission in the CA1 region of guinea pig hippocampal slices. This depression was blocked by an adenosine A1 receptor antagonist, which suggests that activation of the NMDA subtype of glutamate receptor raises the concentration of extracellular adenosine, which acts on presynaptic inhibitory A1 receptors. Strong tetanic stimulation caused a heterosynaptic inhibition that was blocked by both NMDA and A1 receptor antagonists. Enkephalin, which selectively inhibits interneurons, antagonized the heterosynaptic inhibition. These findings suggest that synaptically released glutamate activates NMDA receptors, which in turn releases adenosine, at least in part from interneurons, that acts at a distance to inhibit presynaptically the release of glutamate from excitatory synapses. Thus, interneurons may mediate a widespread purinergic presynaptic inhibition.

Abstract: The neurotoxic effects of methamphetamine (METH) on striatal dopaminergic neurons have been hypothesized to be mediated by excess dopamine (DA) release. In addition, N-methyl-D-aspartate (NMDA) receptor antagonists block METH-induced DA depletions. This suggests that glutamate also mediates the toxic effects of METH. The purpose of this study is to demonstrate that DA and glutamate efflux contribute to METH-induced neurotoxicity. In vivo microdialysis in rats was used to measure extracellular concentrations of striatal DA and glutamate following 3 injections of METH (10 mg/kg, i.p.), each injection given 2 hours apart. One week following the dialysis experiment, rats were sacrificed and the ventral lateral striata were assayed for DA content. Glutamate concentrations in the dialysate increased by over 4-fold after the third METH injection. In these same animals, striatal DA tissue content was significantly depleted. In separate groups of rats, pretreatment with haloperidol (2 mg/kg at the first METH injection) significantly increased METH-induced DA efflux. The haloperidol pretreatment attenuated the extracellular increase in glutamate produced by METH and blocked subsequent neurotoxicity to DA neurons. In contrast, pretreatment with the DA uptake blocker, GBR-12909 (10 mg/kg, 30 min before each METH injection) significantly attenuated the increased DA release produced by METH but did not change glutamate efflux. However, pretreatment with GBR-12909 did protect against the tissue content depletion of DA in the striatum. Based on these findings, it appears that increased DA and glutamate release in the striatum are important and possibly interact in the development of METH-induced neurotoxicity.

Abstract: Once daily s.c. administration of 5 mg/kg morphine, a mu-opioid agonist, or U50488H (U50), a kappa 1-opioid agonist, for 5 days in male CD-1 mice results in a 2-3-fold shift to the right of the respective analgesic (tail flick) dose-response curves, indicating the development of tolerance. Concurrent s.c. administration of the competitive NMDA receptor antagonist, LY274614 (LY), at 24 mg/kg/24 h infusion (osmotic pump) or 6 mg/kg i.p. once daily attenuates the development of morphine tolerance, when the response to saline plus morphine is compared on day 5 with LY plus morphine. Using this paradigm, once daily administration of either the non-competitive NMDA antagonist, MK-801, at 0.3 mg/kg i.p. or the nitric oxide synthase inhibitor, NG-nitro-L-arginine (NorArg), at 1 mg/kg i.p. twice daily attenuated the development of morphine tolerance. None of these drugs modify the tail-flick response or alter the ED50 for morphine. In contrast, co-administration of LY, MK-801 or NorArg, as above, failed to attenuate the development of tolerance to U50 or to the kappa 3-opioid agonist, naloxone benzoylhydrazone (NalBzoH). These results suggest that mu-opioid tolerance but not kappa 1- or kappa 3-opioid tolerance involves the mediation of NMDA receptors and the nitric oxide system.

Abstract: Chronic (14 day) but not acute (1 day) treatment of mice with clinically active antidepressants produces a significant (approximately 1.8-4.3 fold) reduction in the potency of glycine to inhibit [3H]-5,7-dichlorkynurenic acid (5,7-DCKA) binding to strychnine-insensitive glycine receptors in neocortical membranes. Moreover, these effects were not observed following chronic treatment with a variety of nonantidepressant drugs such as D-deprenyl, chlorpromazine, salbutamol, scopolamine and chlordiazepoxide. The time course and dose-response relationships for this effect were examined after treatment with two representative antidepressant drugs (imipramine and citalopram) and electriconvulsive shock (ECS). Increases in the IC50 of glycine to inhibit [3H]-5,7-DCKA binding were observed after treatment for 7 days with ECS, 10 days with citalopram and 14 days with imipramine, respectively, and were no longer apparent by the 10th day after cessation of treatment. These findings indicate that the antidepressant-induced reduction in the IC50 of glycine to inhibit [3H]-5,7-DCKA binding is: 1) a slowly developing, adaptive phenomenon; 2) remarkably persistent after cessation of treatment; and 3) a significantly better predictor of antidepressant activity (22 of 23 drugs) than either beta adrenoceptor down-regulation (15 of 23 drugs) or efficacy in the forced swim test (13 of 23 drugs) [P < .01 vs. each measure, Fisher's Exact Test]. The ability of antidepressants drawn from every principal therapeutic class to effect adaptive changes in the N-methyl-D-aspartate receptor complex is consistent with the hypothesis that this ligand-gated ion channel serves as a final common pathway of antidepressant action and indicates that glutamatergic pathways may be involved in the pathophysiology of depression.

Abstract: Glutamate is the predominant excitatory neurotransmitter in the mammalian CNS. The neurotransmitter pool of glutamate is stored in synaptic vesicles and, upon depolarization, is released into the synaptic cleft in a Ca(2+)-dependent fashion. Glutamate is cleared from the synaptic cleft by high-affinity, Na(+)-dependent uptake carriers located in both neurons and glia. Glutamate acts on several distinct families of receptors, each of which has multiple subtypes with distinct pharmacologic and physiologic properties. Under some conditions, glutamate and related compounds act as excitotoxins and might participate in the events leading to neuronal damage and death in a variety of acute and chronic neurologic disorders. The potential for glutamate to become an excitotoxin is highly dependent upon neuronal metabolic status. A great deal of interest in developing selective, well-tolerated glutamate receptor antagonists for the treatment of a variety of neurologic disorders exists.

Abstract: NMDA receptor activation can alter synaptic strength, cause cell death, and may modulate the release of glutamate and other neurotransmitters. Using a specific and selective antiserum directed against the R1 subunit of the NMDA receptor, we examined (1) whether NMDA receptors in the adult rat visual cortex are exclusively postsynaptic or also presynaptic and (2) whether NMDA-R1 subunits are incorporated into the plasma membrane prior to, contemporaneously, or following the formation of synapses during postnatal development. By light microscopy, NMDA-R1 immunoreactivity in the adult visual cortex is easily detectable within perikarya and proximal dendrites in laminae 2–6. Many of them have the morphological features of pyramidal neurons. In addition, fine punctate labeling is evident throughout the neuropil. Electron microscopy reveals these puncta to reside at postsynaptic densities of axospinous junctions and at fine astrocytic processes and axon terminals. In the deeper laminae, the majority of labeled profiles are astrocytic. Visual cortices of animals in their first postnatal week show concentrated immunoreactivity in a few nonpyramidal neurons within laminae that have just differentiated from the cortical plate. Electron microscopy reveals diffuse labeling along the plasma membrane of dendritic shafts lacking morphologically identifiable synaptic junctions or appositions to axons. Immunoreactivity is detectable in dendritic processes by postnatal day (PND) 2, in axonal processes by PND 4, and in astrocytic profiles by PND 14. Immunoreactivity also is detectable along the postsynaptic membrane of presumably transient axosomatic junctions. At all ages, the prevalence of NMDA-R1-immunoreactive profiles is lamina 1 > 4/5 > 6/6B. These results provide the cellular basis for NMDA receptors' participation in (1) postsynaptic membrane excitability, (2) regulation of transmitter release, (3) and, in the deeper laminae, astrocyte responses. During development, NMDA-R1 subunits are associated with the plasma membrane prior to axons' arrival while clustering of receptors to junctions may be promoted by axonal contact. Finally, spatial segregation of axonal growth cones may be mediated by NMDA-R1 subunits on these axonal processes.

Abstract: Cytokines may play a pathogenetic role in the brain. Using human fetal brain cell cultures, we investigated whether cytokines released during inflammation modulate neuronal injury. Exposure of human fetal neuronal cells to the excitatory amino acid neurotransmitter, glutamate, for 6 days resulted in a dose-dependent cell loss. Tumor necrosis factor (TNF)-a potentiated glutamate neurotoxicity. This TNFα-potentiated glutamate neurotoxicity was blocked by the glutamate receptor antagonists, 2-APV and MK-801, suggesting that the potentiating effect of TNFα is predominantly mediated by a glutamate receptor mechanism. Exposure of neuronal cultures to TNFα for 5 days resulted in a 27% decrease in astrocyte glutamine synthetase and in a 50% inhibition of 3H-glutamate uptake, suggesting that the effect of TNFα indirectly involves glutamate metabolism. These findings suggest that under pathologic conditions, TNFα may impair embryonic development of the brain by exacerbating excitotoxicity.

Abstract: The endogenous polyamine spermine has multiple effects on the N-methyl-D-aspartate (NMDA) receptor. These include an increase in the magnitude of NMDA-induced whole-cell currents that is seen in the presence of saturating concentrations of glycine ("glycine-independent" stimulation), an increase in the affinity of the receptor for glycine ("glycine-dependent" stimulation), and voltage-dependent inhibition. Although many of the properties of native NMDA receptors are seen with homomeric NR1 receptors expressed in Xenopus oocytes, we have found that the effects of spermine are differentially regulated by NR2 subunits in heteromeric NR1/NR2 receptors. Glycine-independent stimulation by spermine occurred at homomeric NR1A receptors, which lack the amino-terminal insert in NR1, and at heteromeric NR1A/NR2B receptors but not at heteromeric NR1A/NR2A or NR1A/NR2C receptors. Glycine-independent stimulation was not seen at homomeric NR1B receptors, which include the amino-terminal insert in NR1, or at heteromeric receptors containing NR1B. Thus, glycine-independent stimulation by polyamines requires the presence of an NR1 variant, such as NR1A, that lacks the amino-terminal insert, but the manifestation of the stimulatory effect is controlled by the type of NR2 subunit present in a heteromeric complex. Glycine-dependent stimulation was seen at NR1A/NR2A and NR1A/NR2B receptors and may therefore involve a second polyamine binding site distinct from that which produces glycine-independent stimulation. The voltage-dependent inhibitory effect of spermine, which is more pronounced at hyperpolarized membrane potentials, occurred with similar magnitudes at NR1A/NR2A and NR1A/NR2B receptors but was absent at NR1A/NR2C receptors. Thus, NR2 subunits control both the stimulatory and inhibitory effects of spermine at NMDA receptors. Stimulation but not inhibition by spermine was seen at NR1A/NR2B receptors in the presence of extracellular Mg2+. Stimulation, seen in the presence of physiological concentrations of Ca2+ and Mg2+, may be the predominant effect of polyamines at NMDA receptors in the intact nervous system.

Abstract: Phosphorylation of glutamate receptors is probably an important mechanism for modulating excitatory transmission. However, there is little direct evidence to indicate which protein phosphatases can dephosphorylate glutamate or other ligand-gated channels, although it is known that protein phosphatases 1 and 2A play a major part in modulating voltage and second-messenger-gated channels. Here we report that in cultured hippocampal neurons, the N-methyl-D-aspartate (NMDA) receptor can be regulated by endogenous and exogenous serine/threonine protein phosphatases. Phosphatase inhibitors enhanced NMDA currents recorded using the perforated patch technique or in cell-attached patches, whereas protein phosphatases 1 or 2A decreased the open probability of these channels in inside-out patches.

Abstract: Since ischemic damage in the brain is linked to glutamate excitotoxicity, the effects of an acute exposure to glutamate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) or N-methyl-D aspartate (NMDA) on the radial dendrites were compared with those occurring after a severe cochlear ischemia. Glutamate and AMPA, but not NMDA, produced a drastic swelling restricted to the radial dendrites below the inner hair cells (IHCs). At a concentration of 20 microM AMPA, a full electrophysiological recovery could be observed in some cochleas after washing the drug out. A prior perfusion of 6-7-dinitroquinoxaline-2,3-dione (DNQX, 50 microM) prevented the 25 microM AMPA-induced dendritic swelling. No protective effect of D-2-amino-5-phosphonopentanoate (D-AP5) could be observed. In the same way, ischemia (5-40 minutes) resulted in a clear swelling of the radial dendrites. While D-AP5 had no protective effects, 50 microM DNQX protected most of the radial dendrites from the ischemia-induced swelling, excepting those contacting the modiolar side of the IHCs. Finally, 50 microM DNQX + 50 microM D-AP5 resulted in a nearly complete protection of all the radial dendrites. Altogether, these results suggest that the acute swelling of radial dendrites primarily occurs via AMPA/kainate receptors. However, in radial dendrites contacting the inner hair cells on their modiolar side, NMDA receptors may be also involved.

Abstract: The nucleus accumbens (NA), a ventral extension of the striatum, plays a role in several complex behaviour patterns and also is a major site of action of drugs of abuse such as cocaine. Intrinsic NA cells are predominantly quiescent and their activity depends on excitatory input from cortical and subcortical limbic afferents. Here we examine the mechanisms of synaptic plasticity at the synapse between prelimbic cortical afferents and cells in the core region of the NA. Manipulations that induce a Ca(2+)-dependent long-term potentiation (LTP) of non-NMDA (N-methyl-D-aspartate)-receptor-mediated responses also produce a simultaneous long-term depression (LTD) of NMDA-receptor-mediated responses. These results indicate that in a single cell the same change in postsynaptic Ca2+ concentration can have opposite effects on non-NMDA- and NMDA-receptor-mediated synaptic responses. This may be particularly important in the NA, where NMDA receptors are critical for mediating the behavioural actions of drugs of abuse.

Abstract: Phosphorylation of glutamate receptors (GluRs) is emerging as an important regulatory mechanism. In this study 32P labeling of non-NMDA GluRs was investigated in cultured hippocampal neurons stimulated 2–15 min with agonists that selectively stimulate either Ca2+/calmodulin- dependent protein kinase II (CaM-kinase II), Ca2+/phospholipid- dependent protein kinase C (PKC), or cAMP-dependent protein kinase A (PKA). Treatment of hippocampal neurons with glutamate/glycine (Glu/Gly), ionomycin, or 12-O-tetradecanoylphorbol 13-acetate (TPA) increased 32P labeling of immunoprecipitated alpha-amino-3-hydroxy-5- methyl-4-isoxazoleproprionate (AMPA)-type GluRs by 145%, 180%, and 227%, respectively, of control values. This increased phosphorylation of GluRs was predominantly 32P-Ser with little 32P-Thr and no detectable 32P-Tyr. Glu/Gly and ionomycin, but not TPA, also increased 32P labeling of CaM-kinase II by 175% and 195%, respectively, of control values. Of these three agonists, only TPA stimulated phosphorylation of MARCKS (225% of control), a specific substrate of PKC. Forskolin treatment gave a three- to fourfold increase in the active catalytic subunit of PKA but did not result in the 32P labeling of AMPA-type GluRs, CaM-kinase II, or MARCKS. Phosphorylation of GluRs in response to Glu/Gly was blocked by a specific NMDA receptor/ion channel antagonist (DL-2-amino-5-phosphonovaleric acid) or by a cell- permeable inhibitor of CaM-kinase II (1-[N,O-bis(1,5- isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4- phenylpiperazine, KN-62). These results are consistent with the hypothesis that Ca2+ influx through the NMDA-type ion channel can activate CaM-kinase II, which in turn can phosphorylate and regulate AMPA-type GluR ion channels (McGlade-McCulloh et al., 1993).(ABSTRACT TRUNCATED AT 250 WORDS)