Effects of NMDA-R1 antisense oligodeoxynucleotide administration: behavioral and radioligand binding studies

The effects of an antisense phosphodiester oligodeoxynucleotide (ODN) directed to the NR1 subunit of the NMDA receptor mRNA and of its corresponding sense ODN were investigated in mice. Treatment with the antisense ODN significantly increased the time mice spent in the open arms of an elevated maze while the total number of arm entries was unaltered. Furthermore, seizure latencies after the administration of an ED100 dose of NMDA (150 mg/kg) were significantly higher in antisense treated animals compared to vehicle controls. At the same time, treatment with NR1 antisense ODN significantly reduced the Bmax of [3H]CGS-19755 binding (2101 fmol/mg protein) compared to both vehicle (2787 fmol/mg protein) and sense (2832 +/- 39 fmol/mg protein) controls without any significant change in KD (33 nM). A corresponding reduction of [3H]CGP-39653 binding was also observed after treatment with NR1 antisense compared to both sense and vehicle controls. In contrast, neither antisense nor sense ODNs altered the proportion of high affinity glycine sites or the potency of glycine at either high or low affinity glycine binding sites to inhibit [3H]CGP-39653 binding. These results show that in vivo treatment with NR1 antisense ODNs to the NMDA receptor complex reduces antagonist binding at NMDA receptors and has pharmacological effects similar to those observed with some NMDA receptor antagonists. These results also suggest that treatment with antisense ODNs may provide another means to investigate allosteric modulation of receptor subtypes in vivo.     

Liposomal amikacin: improved treatment of Mycobacterium avium complex infection in the beige mouse model

1. Binding of D,L-(E)-2-amino-4-[3H]-propyl-5-phosphono-3-pentenoic acid ([3H]-CGP 39653), a high affinity, selective antagonist at the glutamate site of the N-methyl-D-aspartate (NMDA) receptor, was investigated in rat brain by means of receptor binding and quantitative autoradiography techniques. 2. [3H]-CGP 39653 interacted with striatal and cerebellar membranes in a saturable manner and to a single binding site, with KD values of 15.5 nM and 10.0 nM and receptor binding densities (Bmax values) of 3.1 and 0.5 pmol mg-1 protein, respectively. These KD values were not significantly different from that previously reported in the cerebral cortex (10.7 nM). 3. Displacement analyses of [3H]-CGP 39653 in striatum and cerebellum, performed with L-glutamic acid, 3-((+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) and glycine showed a pharmacological profile similar to that reported in the cerebral cortex. L-Glutamic acid and CPP produced complete displacement of specific binding with Ki values not significantly different from the cerebral cortex. Glycine inhibited [3H]CGP 39653 binding with shallow, biphasic curves, characterized by a high and a low affinity component. Furthermore, glycine discriminated between these regions (P < 0.005, one-way ANOVA), since the apparent Ki of the high affinity component of the glycine inhibition curve (KiH) was significantly lower (Fisher's protected LSD) in the striatum than the cortex (33 nM and 104 nM, respectively). 4. Regional binding of [3H]-CGP 39653 to horizontal sections of rat brain revealed a heterogeneous distribution of binding sites, similar to that reported for other radiolabelled antagonists at the NMDA site (D-2-[3H]-amino-5-phosphonopentanoic acid ([3H]-D-AP5) and [3H]-CPP). High values of binding were detected in the hippocampal formation, cerebral cortex and thalamus, with low levels in striatum and cerebellum. 5. [3H]-CGP 39653 binding was inhibited by increasing concentrations of L-glutamic acid, CPP and glycine. L-Glutamic acid and CPP completely displaced specific binding in all regions tested, with similar IC50 values throughout. Similarly, glycine was able to inhibit the binding in all areas considered: 10 microM and 1 mM glycine reduced the binding to 80% and 65% of control (average between areas) respectively. The percentage of specific [3H]-CGP 39653 binding inhibited by 1 mM glycine varied among regions (P < 0.05, two-ways ANOVA). Multiple comparison, performed by Fisher's protected LSD method, showed that the inhibition was lower in striatum (72% of control), with respect to cortex (66% of control) and hippocampal formation (58% of control). 6. The inhibitory action of 10 microM glycine was reversed by 100 microM 7-chloro-kynurenic acid (7-CKA), a competitive antagonist of the glycine site of the NMDA receptor channel complex, in all areas tested. Moreover, reversal by 7-CKA was not the same in all regions (P < 0.05, two-ways ANOVA). In fact, in the presence of 10 microM glycine and 100 microM 7-KCA, specific [3H]-CGP 39653 binding in the striatum was 131% of control, which was significantly greater (Fisher's protected LSD) than binding in the hippocampus and the thalamus (104% and 112% of control, respectively). 7. These results demonstrate that [3H]-CGP 39653 binding can be inhibited by glycine in rat brain regions containing NMDA receptors; moreover, they suggest the existence of regionally distinct NMDA receptor subtypes with a different allosteric mechanism of [3H]-CGP 39653 binding modulation through the associated glycine site.     

Developmental changes in NMDA receptor glycine affinity and ifenprodil sensitivity reveal three distinct populations of NMDA receptors in individual rat cortical neurons

Previous work with recombinant receptors has shown that the identity of the NMDA NR2 subunit influences receptor affinity for both glutamate and glycine. We have investigated the developmental change in NMDA receptor affinity for both glutamate and glycine in acutely dissociated parietal cortex neurons of the rat, together with the expression during ontogeny of NR2A and NR2B mRNA and protein. Whereas there is little change in NMDA receptor glutamate affinity with age, a population of NMDA receptors emerges in 14- and 28-d-old animals with a markedly reduced affinity for glycine (mKD = approximately 800 nM) and a reduced sensitivity to the NR2B subunit-selective NMDA antagonist ifenprodil. These changes are paralleled by a developmental increase in the expression of NR2A. Thus, in mature animals a population of NMDA receptors appears with a lower affinity for glycine that might not be saturated under normal physiological conditions. Ifenprodil (10 microM) inhibits virtually all of the NMDA receptor-evoked current in very young neurons that contain a single population of receptors exhibiting a high affinity for glycine (mKD = approximately 20 nM). In older neurons, which contain NMDA receptors with both high and low affinities for glycine, ifenprodil (10 microM) inhibits both the high-affinity population and a significant proportion of the low-affinity component, thus revealing three pharmacologically distinct populations of NMDA receptors in single neurons. Moreover, these observations suggest that ifenprodil might bind with high affinity to NMDA receptors containing both NR2A and NR2B subunits as well as those containing only NR2B.     

Adrenergic blockade in diabetic and uninephrectomized rats: effects on renal size and on renal and urinary contents of epidermal growth factor

The glutamatergic transmission system plays a key role in afferent and efferent pathways involved in micturition. By in situ hybridization combined with retrograde Fast Blue labeling, expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor (GluR-A to -D) and N-methyl-D-aspartate (NMDA) receptor (NR1 and NR2A-D) subunit mRNAs were examined in visceromotor and somatomotor neurons of the rat lumbosacral spinal cord. Parasympathetic preganglionic neurons (PGNs) in the intermediolateral nucleus highly expressed GluR-A and GluR-B subunit mRNAs, with very low levels for GluR-C and GluR-D subunits. As for the NMDA receptor, PGNs were associated with abundant signals for NR1 subunit mRNA, but without any NR2 subunit mRNAs. On the other hand, somatomotor neurons in the ventral horn (dorsolateral nucleus) express all four AMPA receptor subunit mRNAs, showing relatively abundant expressions of GluR-C and GluR-D subunit mRNA compared with PGNs. In addition to high levels of NR1 subunit mRNA, dorsolateral nucleus neurons moderately expressed NR2A and NR2B subunit mRNAs. These results suggest that molecular organization of both AMPA and NMDA receptor channels are distinct between PGNs and dorsolateral nucleus neurons. Considering that native NMDA receptors are heteromeric channels composed of NR1 and NR2 subunits, it seems likely that dorsolateral nucleus neurons, not PGNs, are provided with functional NMDA receptors, which could induce activity-dependent changes in synaptic transmission in the efferent pathway for the lower urinary tract.     

Expression of mRNAs encoding subunits of the N-methyl-D-aspartate receptor in cultured cortical neurons

The expression of mRNAs encoding subunits of the N-methyl-D-aspartate (NMDA) receptor was examined in cortical neurons maintained in primary culture. Cultures were prepared from embryonic day 17 rat neocortex. At this developmental age, levels of NR1, NR2A, NR2B, and NR2C mRNA were low or undetectable. Expression of NR1 mRNA increased progressively between days 1 and 21 in vitro. The amount of NR2A mRNA did not change between days 1 and 7 but increased between days 7 and 21. In contrast, levels of NR2B mRNA increased between days 1 and 7, with little further change after day 7. The level of NR2B mRNA was approximately 4-fold higher than that of NR2A mRNA in 21-day cultures. Using ligand binding assays, the proportion of NMDA receptors having a low affinity for ifenprodil was also found to increase over time in culture. The increase in the expression of receptors having a low affinity for ifenprodil and the increase in NR1 and NR2A mRNAs were reduced or prevented by maintaining cells in medium with a low concentration of serum. The results are consistent with the hypothesis that inclusion of the NR2A subunit in native NMDA receptors is responsible for their low affinity for ifenprodil. Splice variants of NR1 lacking the 5' (amino-terminal) insert were found to be the predominant forms of NR1 in cultured neurons. Variants containing the 5' insert represented only a small (< or = 5%) fraction of total NR1 mRNA, and their proportion was not altered as a function of time in culture. Time-dependent changes in the properties of NMDA receptors and in the expression of subunit mRNA occurring in cultured neurons are similar to changes observed in developing rat brain. Thus, the developmental sequence of NMDA receptor expression that occurs in vivo is partially retained in neurons maintained in vitro.     

NMDA-NR1 receptor subunit mRNA expression in rat brain after 6-OH-dopamine induced lesions: a non-isotopic in situ hybridization study

Antisense digoxigenin-labeled deoxyoligonucleotides probes and non-isotopic in situ hybridization (HIS) techniques have been used to explore the NMDA-NR1 receptor subunit mRNA distribution in different brain areas of rats which had their dopaminergic nigrostriatal pathway previously lesioned with intracerebral administration of 6-OH-dopamine (6-OH-DA). Intense and significant hybridization signals for NR1 mRNA were found in dentate gyrus and regions CA1-CA2-CA3 of the hippocampus, in layers II-III and V-VI of the cerebral cortex, and in the cerebellum of sham-treated rats. Basal ganglia structures such as the striatum exhibited few NR1 mRNA hybridization signals as compared to the hippocampus and cerebral cortex. In contrast, both zona compacta and reticulata of substantia nigra (SN) showed a reduced number of cells with nevertheless intense NR1 mRNA HIS signals. The NR1 mRNA distribution in the brain was affected in a brain regional selective manner by 6-OH-DA induced lesions of DA neuronal systems. A striking increase in NR1 mRNA HIS signals was observed in both striata after unilateral lesioning with 6-OH-DA. Instead, in SN compacta but not in reticulata, a moderate but significant bilateral reduction of NR1 mRNA was observed after unilateral 6-OH-DA injection. No significant changes in NR1 mRNA were detected in cerebral cortex and other brain regions after 6-OH-DA treatment. These studies, and others reported in the literature, support the view that extensive lesions of nigrostriatal DA-containing neurons in the brain may trigger compensatory or adaptative responses in basal ganglia structures such as striatum and substantia nigra which involve glutamateric neurons and the genic expression of NMDA receptors.     

Dilated fallopian tubes: MR imaging characteristics

A complex of four proteins isolated from neuronal membranes has ligand binding sites for N-methyl-d-aspartate (NMDA) receptor agonists and antagonists and forms NMDA-activated ion channels upon reconstitution into lipid membranes. In this study, the cDNA of a subunit of this complex containing binding sites for the competitive antagonists of NMDA receptors was cloned. The cDNA clone coded for a protein of 719 amino acids (78.9 kDa). The expressed protein had binding activity for the agonists l-[3H]glutamate and [3H]glycine, the antagonist (+/-)-[3H]-(E)-2-amino-4-propyl-5-phosphonopentanoic acid ([3H]CGP 39653), but not the ion channel inhibitors. The cloned cDNA had no homology to other cloned cDNAs. Northern blot analyses indicated high expression of an 3.8 kb poly(A+) RNA in brain, but not in other tissues. These findings indicate that proteins that have recognition sites for NMDA receptor activators and inhibitors and that differ from the well-characterized NMDA receptor proteins NR1-3 are expressed in mammalian brain.     

Immunoblot analyses on the differential distribution of NR2A and NR2B subunits in the adult rat brain

Quantitative immunoblot analyses were carried out to study the distribution of N-methyl-D-aspartate (NMDA) receptor subunit 2A and 2B (NR2A and NR2B, respectively) at the protein level in the adult rat brain. Highest levels of NR2A were detected in cerebral cortex and hippocampus, followed at more or less similar levels (about 36-72% of cerebral cortex) by striatum, thalamus, olfactory bulb, superior and inferior colliculi, and cerebellum. The lowest levels were detected in midbrain and lower brain stem (30-31% of cerebral cortex). The NR2B was more dramatic in differential distribution than the NR2A. Highest levels of NR2B were found in telencephalic (olfactory bulb, cerebral cortex, hippocampus, and striatum) and thalamic regions, and expression in superior and inferior colliculi, midbrain, lower brain stem, and cerebellum were significantly lower (4-25% of cerebral cortex). Interestingly, NR2B proteins were barely detectable in the cerebellum. When the postsynaptic density (PSD) fractions were compared, the amount of NR2B in the cerebellar PSD fraction was only 1.8% of that present in the cerebral PSD fraction where the subunit is highly enriched. Immunoblot analyses with a phosphotyrosine-specific antibody showed that the molecular sizes of major phosphotyrosine-containing proteins in forebrain and hindbrain are 180 and 45 kDa, respectively. The regional distribution of the 180 kDa major phosphotyrosine protein was very similar to that of NR2B, and the protein could be immunoprecipitated by NR2B antibody. Our data shows that NR2A and NR2B subunits are differentially distributed in the brain in an overlapping manner, and that the major phosphotyrosine-containing protein of 180 kDa in forebrain is the NR2B.     

High level expression of the NMDAR1 glutamate receptor subunit in electroporated COS cells

The rat N-methyl-D-aspartate (NMDA) glutamate receptor subunit NR1-1a was transiently expressed in COS cells using the technique of electroporation, which was fivefold more efficient than the calcium phosphate precipitation method of transfection. The glycine site antagonist 5,7-[3H]dichlorokynurenic acid labeled a single high-affinity site (KD = 29.6 +/- 6 nM; Bmax = 19.4 +/- 1.6 pmol/mg of protein) in membranes derived from COS cells electroporated with NR1-1a. In contrast to previous reports using transiently transfected human embryonic kidney 293 cells, binding of the noncompetitive antagonist (+)-5-[3H]methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5, 10-imine ([3H]MK-801) was not detected in NR1-1a-transfected COS cells. Although immunofluorescent labeling of electroporated COS cells demonstrated that the NR1-1a protein appears to be associated with the cell membrane, neither NMDA nor glutamate effected an increase in intracellular calcium concentration in fura-2-loaded cells, suggesting that homomeric NR1-1a receptors do not act as functional ligand-gated ion channels. Therefore, COS cells appear to differ from Xenopus oocytes with respect to the transient expression of functional homomeric NR1 receptors. Although expression of NR1-1a is sufficient to reconstitute a glycine binding site with wild-type affinity for antagonists in COS cells, recombinant homomeric NR1-1a receptors do not display properties that are characteristic of native NMDA receptors, such as permeability to Ca2+ and channel occupancy by MK-801, when expressed in this mammalian cell line.     

Inhibition of ornithine decarboxylase by ifenprodil

The present study investigated the changes in NMDA receptor subunit proteins in diazepam-withdrawn rat cerebral cortex, using Western blotting analysis. The protein levels of the NR1 and NR2B, but not NR2A, subunits were significantly increased in diazepam-withdrawn rats compared to those in control rats. Therefore, an increase in the NR1 and NR2B subunit proteins may be responsible for both the previously observed upregulation of [3H]dizocilpine binding in the cerebral cortex and the appearance of diazepam withdrawal signs.     

Chronic administration of a glycine partial agonist alters the expression of N-methyl-D-aspartate receptor subunit mRNAs

Both acute and chronic treatments with the glycine partial agonist 1-aminocyclopropanecarboxylic acid (ACPC) are neuroprotective in animal models of focal, global and spinal ischemia. After a chronic regimen of ACPC, brain and plasma levels were undetectable at the time of ischemic insult, which suggests that the neuroprotective effects of acute and chronic ACPC are mediated by different mechanisms. To investigate the possibility that chronic administration of ACPC alters N-methyl-D-aspartate (NMDA) receptor composition, the levels of mRNAs encoding zeta and epsilon subunits were quantified by in situ hybridization histochemistry with 35S-labeled riboprobes. Chronic ACPC administered to mice (200 mg/kg for 14 days) increased the level of epsilon-1 mRNA in the hippocampus (particularly CA1 and CA2 regions) and cerebral cortex (frontal, parietal and occipital regions), without altering levels in cerebellum. In contrast, this regimen decreased epsilon-3 subunit mRNA levels in the hippocampus (especially CA1 and dentate gyrus) and frontal and occipital cortices. Decreases in epsilon-2 subunit mRNA levels in cerebral cortex (especially frontal and parietal cortices) were also observed without accompanying alterations in the cerebellum, hippocampus or dentate gyrus. The levels of zeta subunit mRNA (determined with a probe that detects all splice variants) were not altered in any brain areas examined. Based on studies in recombinant receptors, these region-specific changes in mRNAs produced by a chronic regimen of ACPC could result in NMDA receptors with reduced affinities for glycine and glutamate. It is hypothesized that such alterations in NMDA receptor subunit composition may explain the neuroprotective effects produced by chronic ACPC.     

Functional change of NMDA receptors related to enhancement of susceptibility to neurotoxicity in the developing pontine nucleus

The developing neurons have been reported to be extremely susceptible to toxicity of NMDA during a restricted developmental period. Pontosubicular neuronal necrosis is a typical type of perinatal human brain lesion and often coexists with other forms of cerebral hypoxic and ischemic injuries. To determine whether functional changes of NMDA receptors related to the susceptibility to NMDA toxicity are involved in developing neurons in the pontine nucleus, we have examined the lesion produced by in vivo direct injection of NMDA into the pontine nucleus of rats at postnatal days 1-30, recorded NMDA-induced whole-cell currents from neurons in the pontine nucleus in the developing rat brainstem slices, and performed in situ hybridization for NMDA receptor subunit mRNAs in the pontine nucleus. The susceptibility to NMDA neurotoxicity peaked near postnatal day 15, and the NMDA-induced currents showed prominent reduction of the voltage-dependent block by Mg2+ near postnatal day 15. The pontine nucleus near postnatal day 15 showed distinct expression of the NMDA receptor subunit NR2C mRNA. These results suggest that the susceptibility to NMDA neurotoxicity that is enhanced in the rat pontine nucleus near postnatal day 15 is mediated by the NMDA receptor channels that are relatively insensitive to Mg2+ and that the reduction in the sensitivity of NMDA receptors to Mg2+ correlates with the expression of the NR2C. We present the possibility that functional changes in the NMDA receptor channels play a crucial role in the occurrence of developmentally specific neuronal injury.     

The Role of NMDA Receptor Binding Sites in Ethanol Place Conditioning.

Little is known about the specific role of glutamate, in particular its actions at N-methyl-D-aspartate (NMDA) receptors, in ethanol reward. Pretreatment with channel blockers MK-801 and ketamine, NMDA NR2B receptor subunit antagonists ifenprodil and CP-101,606, and the glycineB partial agonist (+)-HA-966 did not alter acquisition of ethanol-induced conditioned place preference (CPP) in mice. However, pretreatment with the competitive antagonist CGP-37849 attenuated acquisition of ethanol-induced CPP. Follow-up experiments indicated that CGP-37849 also blocked acquisition of ethanol-induced and lithium chloride-induced conditioned place aversion but did not produce rewarding or aversive effects on its own. These results suggest that the NMDA receptor glutamate binding site is important for ethanol place conditioning. Moreover, these results suggest CGP-37849 modulates ethanol place conditioning by impairing the ability to learn these tasks. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Pharmacological properties of acquired excitotoxicity in Chinese hamster ovary cells transfected with N-methyl-D-aspartate receptor subunits

The cytotoxicity induced by the transient expression of functional N-methyl-D-aspartate (NMDA) receptors has been examined with the use of a luciferase reporter assay in Chinese hamster ovary cells. Various NMDA receptor antagonists, in a dose-dependent manner, prevented a loss of luciferase activity 24 to 48 hr post-transfection of either the NR1/NR2A or NR1/ NR2B subunit receptor configurations, likely correlating to the time required to express functionally these receptors. Both glutamate and NMDA were potently cytotoxic to transfected cells previously protected by antagonists. The novel ifenprodil analog (1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol (CP101,606-27) protected cells expressing NR1/NR2B, but not those cells expressing either NR1/NR2A or, putatively, NR1/NR2A/NR2B. Decreased cytotoxicity was observed when a mutated NR1 subunit (N616R) with reduced Ca++ permeability was used in coexpression studies with NR2A or NR2B. In contrast to our results with NR1/NR2A or NR1/NR2B, cells expressing NR1/NR2C did not perish. Our studies suggest that expression of functional NMDA receptors in non-neuronal cells leads to a form of excitotoxicity similar to that observed in mammalian neurons in vitro.     

Identification of amino acid residues of the NR2A subunit that control glutamate potency in recombinant NR1/NR2A NMDA receptors

The NMDA type of ligand-gated glutamate receptor requires the presence of both glutamate and glycine for gating. These receptors are hetero-oligomers of NR1 and NR2 subunits. Previously it was thought that the binding sites for glycine and glutamate were formed by residues on the NR1 subunit. Indeed, it has been shown that the effects of glycine are controlled by residues on the NR1 subunit, and a "Venus flytrap" model for the glycine binding site has been suggested by analogy with bacterial periplasmic amino acid binding proteins. By analysis of 10 mutant NMDA receptors, we now show that residues on the NR2A subunit control glutamate potency in recombinant NR1/NR2A receptors, without affecting glycine potency. Furthermore, we provide evidence that, at least for some mutated residues, the reduced potency of glutamate cannot be explained by alteration of gating but has to be caused primarily by impairing the binding of the agonist to the resting state of the receptor. One NR2A mutant, NR2A(T671A), had an EC50 for glutamate 1000-fold greater than wild type and a 255-fold reduced affinity for APV, yet it had single-channel openings very similar to those of wild type. Therefore we propose that the glutamate binding site is located on NR2 subunits and (taking our data together with previous work) is not on the NR1 subunit. Our data further imply that each NMDA receptor subunit possesses a binding site for an agonist (glutamate or glycine).     

An acidic amino acid in the N-methyl-D-aspartate receptor that is important for spermine stimulation

The polyamine spermine has multiple effects on N-methyl-D-aspartate (NMDA) receptors, including "glycine-independent" stimulation, which is seen in the presence of saturating concentrations of glycine; "glycine-dependent" stimulation, which is due to an increase in the affinity of the receptor for glycine; and voltage-dependent block. These effects may involve three separate polyamine binding sites on the receptor. To identify amino acid residues that are important for spermine binding, we used site-directed mutagenesis to alter amino acids in and around a region of the NR1 subunit of the NMDA receptor that shows homology with PotD, a polyamine binding protein from Escherichia coli. Mutated subunits, expressed in heteromeric and homomeric NMDA receptors, were studied by voltage-clamp recording in Xenopus oocytes. Mutation of two acidic residues (E339-E342) to neutral amino acids reduced or abolished glycine-independent stimulation by spermine without affecting glycine-dependent stimulation or voltage-dependent block by spermine. Mutation of these residues also had modest effects on sensitivity to protons and to ifenprodil but did not alter sensitivity to glutamate and glycine or to voltage-dependent block by Mg2+. Residue E342 in NR1 appears to be critical for glycine-independent spermine stimulation. Mutations at equivalent positions in NR2A(E352Q) or NR2B(E353Q) had no effect on sensitivity to spermine, pH, or ifenprodil. Residue E342 in NR1 may form part of a discrete spermine binding site on the NMDA receptor or be involved in the mechanism of modulation by polyamines. This residue may also be involved in modulation by protons and ifenprodil.     

Functional expression of a recombinant unitary glutamate receptor from Xenopus, which contains N-methyl-D-aspartate (NMDA) and non-NMDA receptor subunits

A cDNA encoding a 100-kDa subunit (XenNR1) of the N-methyl-D-aspartate (NMDA) glutamate receptor type has been cloned from Xenopus central nervous system. When XenNR1 is coexpressed in a mammalian cell line with a recently cloned 51-kDa non-NMDA receptor subunit (XenU1), also from Xenopus, it forms a functional unitary receptor exhibiting the pharmacological properties characteristic of both NMDA and non-NMDA receptors. Firstly, XenU1 can replace NR2 subunits, in complementing XenNR1 to introduce the ligand binding properties of a complete NMDA receptor. Second, responses to both NMDA and non-NMDA receptor agonists and antagonists were obtained in patch-clamp recordings from the cotransfected cells, but no significant responses were recorded when the cells were singly transfected. Third, from solubilized cell membranes from the cotransfected cells, an antibody to the NR1 subunit coprecipitated the binding sites of the non-NMDA receptor subunit. The unitary glutamate receptor has a unique set of properties that denote intersubunit interaction, including a glycine requirement for the responses to non-NMDA as well as to NMDA receptor agonists and voltage-dependent block by Mg2+ of the non-NMDA agonist responses.     

Adaptation of cortical NMDA receptors by chronic treatment with specific serotonin reuptake inhibitors

Glycine displaces [3H]CGP-39653 ([3H]D,L-(E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid) binding to the glutamate recognition site with both high and low affinity. We reported previously that chronic treatment with antidepressants reduced the proportion of high to low affinity sites, or, even eliminated the high affinity sites in case of citalopram. Here, we compared the effects of citalopram with another serotonin specific reuptake inhibitor, fluoxetine on this measure. Chronic administration of citalopram or fluoxetine eliminated high affinity glycine-displaceable [3H]CGP-39653 binding to the mouse cortex in 78 and 56% of animals, respectively, indicating that selective serotonin reuptake inhibitors produce qualitatively similar adaptive changes at NMDA receptors, that differ from other antidepressants in this neurochemical measure.     

Attenuation of focal ischemic brain injury in mice deficient in the epsilon1 (NR2A) subunit of NMDA receptor

The role of glutamate neurotoxicity in cerebral ischemia has long been advocated but still remains controversial, because various glutamate receptor (GluR) antagonists showed inconsistent protective efficacy in brain ischemia models. To address this central issue of ischemic brain damage more directly, we used mutant mice deficient in the GluRepsilon1 (NR2A) subunit of NMDA receptor with or without additional heterozygous mutation in the GluRepsilon2 (NR2B) subunit. Those mutant mice, as well as their littermates, were subjected to focal cerebral ischemia by introducing a 6-0 nylon suture from left common carotid artery. Brain injury volumes after 2 hr of suture insertion, as evaluated by 2,3,5-triphenyltetrazolium chloride staining at 24 hr after ischemia, revealed significantly smaller injury size in GluRepsilon1 subunit knock-out mice compared with their wild-type littermates. The reduction in injury volume was not attributable to differences in body temperature or in blood flow during ischemia. Additional heterozygous GluRepsilon2 subunit disruption did not result in further reduction in injury volume. These data directly demonstrate relevance of NMDA receptor-mediated tissue injury after brain ischemia and provide evidence that GluRepsilon1 subunit is involved in these injurious mechanisms.