Differential regulation of STP, LTP and LTD by structurally diverse NMDA receptor subunit-specific positive allosteric modulators.

Different types of memory are thought to rely on different types of synaptic plasticity, many of which depend on the activation of the N-Methyl-D Aspartate (NMDA) subtype of glutamate receptors. Accordingly, there is considerable interest in the possibility of using positive allosteric modulators (PAMs) of NMDA receptors (NMDARs) as cognitive enhancers. Here we firstly review the evidence that NMDA receptor-dependent forms of synaptic plasticity: short-term potentiation (STP), long-term potentiation (LTP) and long-term depression (LTD) can be pharmacologically differentiated by using NMDAR ligands. These observations suggest that PAMs of NMDAR function, depending on their subtype selectivity, might differentially regulate STP, LTP and LTD. To test this hypothesis, we secondly performed experiments in rodent hippocampal slices with UBP714 (a GluN2A/2B preferring PAM), CIQ (a GluN2C/D selective PAM) and UBP709 (a pan-PAM that potentiates all GluN2 subunits). We report here, for the first time, that: (i) UBP714 potentiates sub-maximal LTP and reduces LTD; (ii) CIQ potentiates STP without affecting LTP; (iii) UBP709 enhances LTD and decreases LTP. We conclude that PAMs can differentially regulate distinct forms of NMDAR-dependent synaptic plasticity due to their subtype selectivity.

Functional heterogeneity of NMDA receptors in rat substantia nigra pars compacta and reticulata neurones.

The nigra substantia nigra pars compacta (SNc) and substantia pars reticulata (SNr) form two major basal ganglia components with different functional roles. SNc dopaminergic (DA) neurones are vulnerable to cell death in Parkinson's disease, and NMDA receptor activation is a potential contributing mechanism. We have investigated the sensitivity of whole-cell and synaptic NMDA responses to intracellular ATP and GTP application in the SNc and SNr from rats on postnatal day (P) 7 and P28. Both NMDA current density (pA/pF) and desensitization to prolonged or repeated NMDA application were greater in the SNr than in the SNc. When ATP levels were not supplemented, responses to prolonged NMDA administration desensitized in P7 SNc DA neurones but not at P28. At P28, SNr neurones desensitized more than SNc neurones, with or without added ATP. Responses to brief NMDA applications and synaptic NMDA currents were not sensitive to inclusion of ATP in the pipette solution. To investigate these differences between the SNc and SNr, NR2 subunit-selective antagonists were tested. NMDA currents were inhibited by ifenprodil (10 microM) and UBP141 (4 microM), but not by Zn(2+) (100 nm), in both the SNr and SNc, suggesting that SNc and SNr neurones express similar receptor subunits; NR2B and NR2D, but not NR2A. The different NMDA response properties in the SNc and SNr may be caused by differences in receptor modulation and/or trafficking. The vulnerability of SNc DA neurones to cell death is not correlated with NMDA current density or receptor subtypes, but could in part be related to inadequate NMDA receptor desensitization.

NR2B- and NR2D-containing synaptic NMDA receptors in developing rat substantia nigra pars compacta dopaminergic neurones.

NMDA receptors are present at glutamatergic synapses throughout the brain, and are important for the development and plasticity of neural circuits. Their subunit composition is developmentally regulated. We have investigated the developmental profile of functional synaptic NMDA receptor subunits in dopaminergic neurones of the substantia nigra pars compacta (SNc). In SNc dopaminergic neurones from rats aged postnatal day (P)7, ifenprodil inhibited NMDA-EPSCs with an estimated IC(50) of 0.36 microm and a maximum inhibition of 73.5 +/- 2.7% (10 microm), consistent with a substantial population of NR1/NR2B-containing diheteromeric receptors. UBP141, a novel NR2D-preferring antagonist, inhibited NMDA-EPSCs with an estimated IC(50) of 6.2 microm. During postnatal development, the maximum inhibitory effect of 10 microm ifenprodil significantly decreased. However, NMDA-EPSCs were not inhibited by Zn(2+) (200 nM) or potentiated by the Zn(2+) chelator TPEN (1 microm), and the effect of UBP141 did not increase during development, indicating that NR2B subunits are not replaced with diheteromeric NR2A or NR2D subunits. The time course of the decay of NMDA-EPSCs was not significantly changed in ifenprodil at any age tested. Together, these data suggest that diheteromeric NR1/NR2A or NR1/NR2D receptors do not account for the ifenprodil-resistant component of the NMDA-EPSC. We propose that NR1/NR2B/NR2D triheteromers form a significant fraction of synaptic NMDA receptors during postnatal development. This is the first report of data suggesting NR2D-containing triheteromeric NMDA receptors at a brain synapse.

Distinct NMDA receptor subpopulations contribute to long-term potentiation and long-term depression induction.

Long-term potentiation (LTP) and long-term depression (LTD) are persistent modifications of synaptic strength that have been implicated in learning, memory, and neuronal development. Despite their opposing effects, both forms of plasticity can be triggered by the activation of NMDA receptors. One mechanism proposed for this bidirectional response is that the specific patterns of afferent stimulation producing LTP and LTD activate to different degrees a uniform receptor population. A second possibility is that these patterns activate separate receptor subpopulations composed of different NMDA receptor (NR) subunits. To test this hypothesis we examined the inhibition of LTP and LTD by a series of competitive NMDA receptor antagonists that varied in their affinities for NR2A/B and NR2C/D subunits. The potency for the inhibition of LTP compared with inhibition of LTD varied widely among the agents. Antagonists with higher affinity for NR2A/B subunits relative to NRC/D subunits showed more potent inhibition of LTP than of LTD. D-3-(2-carboxypiperazine-4-yl)-1-propenyl-1-phosphonic acid, which binds to NR2A/B with very high affinity relative to NR2C/D, showed an approximately 1000-fold higher potency for LTP than for LTD. These results show that distinct subpopulations of NMDA receptors characterized by different NR2 subunits contribute to the induction mechanisms of potentiation and depression.

Interaction of the N-methyl-D-aspartic acid receptor NR2D subunit with the c-Abl tyrosine kinase.

The COOH-terminal domain of the NR2D subunit of the NMDA receptor contains proline-rich regions that show striking homology to sequences known to bind to Src homology 3 (SH3) domains. To determine whether the proline-rich region of the NR2D subunit interacts with specific SH3 domains, in vitro SH3 domain binding assays were performed. A proline-rich fragment of the NR2D subunit (2D(866-1064)) bound to the Abl SH3 domain but not to the SH3 domains from Src, Fyn, Grb2, GAP, or phospholipase C-gamma (PLCgamma). Co-immunoprecipitation of NR2D with Abl suggests stable association of NR2D and Abl in transfected cells. The SH3 domain plays an important role in the negative regulation of Abl kinase activity. To determine whether the interaction of NR2D with the Abl SH3 domain alters Abl kinase activity, Abl was expressed alone or with NR2D in 293T cells. Autophosphorylation of Abl was readily observed when Abl was expressed alone. However, co-expression of Abl with 2D(866-1064) or full-length NR2D inhibited autophosphorylation. 2D(866-1064) did not inhibit DeltaSH3 Abl, indicating a requirement for the Abl SH3 domain in the inhibitory effect. Similarly, 2D(866-1064) did not inhibit the catalytic activity of Abl-PP, which contains two point mutations in the SH2-kinase linker domain that release the negative kinase regulation by the SH3 domain. In contrast, the full-length NR2D subunit partially inhibited the autokinase activity of both DeltaSH3 Abl and Abl-PP, suggesting that NR2D and Abl may interact at multiple sites. Taken together, the data in this report provide the first evidence for a novel inhibitory interaction between the NR2D subunit of the NMDA receptor and the Abl tyrosine kinase.

Native N-methyl-D-aspartate receptors containing NR2A and NR2B subunits have pharmacologically distinct competitive antagonist binding sites.

The pharmacological properties of native N-methyl-D-aspartate (NMDA) receptors were determined in rat brain sections with quantitative autoradiography of [(3)H](E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid (CGP39653) binding. With five competitive antagonists as displacers, two subpopulations of binding sites were observed in the horizontal plane of section examined. These two populations corresponded anatomically to NR2A and NR2B subunits. Quantitative analysis of NR2A-like and NR2B-like binding sites was enabled by examining the cerebellar granule cell layer, which expresses NR2A and NR2C subunits, and the medial striatum, which predominately expresses NR2B subunits. The antagonists (R)-(E)-4-(3-phosphonoprop-2-enyl)piperazine-2-carboxylic acid and (R)-2-amino-5-phosphonopentanoate (D-AP5) displayed similar affinities at cerebellar NMDA receptors and medial striatal NMDA receptors. In contrast, the NMDA receptor antagonists (+/-)-6-(1H-Tetrazol-5-ylmethyl)decahydroisoquinoline- 3-carboxylic acid, (S)-alpha-amino-5-(phosphonomethyl)[1,1'-biphenyl]-3-propanoic acid, and (+/-)-cis-4-(4-phenylbenzoyl) piperazine-2,3-dicarboxylic acid displayed varied, higher affinities at medial striatal NMDA receptors than at cerebellar NMDA receptors. For the five antagonists, there was a strong correlation (r = 0.9) between the cerebellar K(i)/medial striatum K(i) ratio and the NR2A K(i)/NR2B K(i) ratio for recombinant receptors. Thus, [(3)H]CGP39653 labels two pharmacologically distinct populations of NMDA receptors that have pharmacological and anatomical properties consistent with NR2A and NR2B subunits. Because native NR2A- and NR2B-containing receptors are pharmacologically distinct, it should be possible to develop NR2A- and NR2B-selective glutamate site antagonists.

Insulin causes a transient tyrosine phosphorylation of NR2A and NR2B NMDA receptor subunits in rat hippocampus.

NMDA receptors play a critical role in various aspects of CNS function. Hence, it is important to identify mechanisms that regulate NMDA receptor activity. We have shown previously that insulin rapidly potentiates NMDA receptor activity in both native and recombinant expression systems. Here we report that insulin causes a transient phosphorylation of NR2A and NR2B NMDA receptor subunits on tyrosine residues. Rat hippocampal slices were exposed to 1 microM insulin for 20 and 60 min and then solubilized. NR2A and NR2B subunits were immunoprecipitated and probed for tyrosine phosphorylation. Insulin incubation of hippocampal slices for 20 min elicited an increase in tyrosine phosphorylation to 176 +/- 16% (NR2A) and 203 +/- 15% (NR2B) of control levels. In contrast, 60 min of insulin incubation did not alter NR2 tyrosine phosphorylation levels (NR2A: 85 +/- 13% of control; NR2B: 93 +/- 10% of control). Although the consequence of insulin-stimulated tyrosine phosphorylation is unknown, it is possible that this site(s) is responsible for insulin potentiation of NMDA receptor activity. This possibility is consistent with our earlier finding that insulin potentiates hippocampal NMDA receptor activity after 20 min, but not after 60 min, of insulin exposure.

ERK2 activation by homocysteine in vascular smooth muscle cells.

Homocysteine at abnormally high levels is an independent risk factor for atherosclerosis and may be a key factor in atherogenesis. Since homocysteine (Hcys) has been shown to promote cell proliferation and induction of the gene transcription factor c-fos in vascular smooth muscle cells (VSMCs), effects which can be mediated by MAP kinase, we hypothesized that homocysteine activates a MAP kinase-dependent signal transduction pathway. In this study, we find that homocysteine transiently activates MAP kinase (ERK2 isoform) in cultured VSMCs from chick embryos. Homocysteine activation of ERK2 is dose-dependent with an EC50 of approximately 500 nM and blocked by the MAP/Erk kinase (MEK) inhibitor PD98059. VSMC embryonic lineage is another determinant of homocysteine sensitivity. These findings demonstrate that homocysteine activates the MAP kinase signal transduction pathway and thus support the hypothesis that homocysteine may promote atherosclerosis by stimulation of growth promoting signal transduction pathways.

Evidence for a causative role of N-methyl-D-aspartate receptors in an in vitro model of alcohol withdrawal hyperexcitability.

Synaptic mechanisms underlying hyperexcitability due to withdrawal from chronic ethanol exposure were investigated in a hippocampal explant model system using electrophysiological techniques. Whole-cell voltage clamp recordings from CA1 pyramidal cells demonstrated that acute ethanol exposure inhibited N-methyl-D-aspartate receptor (NMDAR)-mediated excitatory postsynaptic currents by over 40%. Chronic ethanol exposure for 6 to 11 days at 35 or 75 mM induced no differences from control explants in the fast component of the population synaptic response (non-NMDAR-mediated). Prolonged field potential recordings (to 10 hr) were used to monitor the withdrawal process in vitro. Ethanol-exposed explants from both 35 and 75 mM groups displayed an increase (60% and 89%, respectively) in the NMDAR-mediated component of synaptic transmission on withdrawal from chronic exposure. Prolonged tonic-clonic electrographic seizure activity was consistently observed after ethanol withdrawal only after the increase in NMDAR function. This hyperexcitability was inhibited by the NMDAR antagonist D-2-amino-5-phosphonovaleric acid and returned once the NMDAR component was reestablished after antagonist washout. In situ hybridization studies suggest that expression of NR2B subunit mRNA may be enhanced in explants after chronic ethanol exposure. No lasting differences were observed in the NMDAR component after acute in vitro ethanol exposure and withdrawal. These data suggest that the occurance of ethanol withdrawal hyperexcitability in this system may be directly dependent on alterations in NMDAR function after chronic exposure. Since this region and others that contain ethanol sensitive NMDARs may serve as epileptic foci, long term alterations in NMDAR function may be expected to generate paroxysmal depolarizing shifts underlying ictal events after withdrawal from ethanol exposure.

[3H]homoquinolinate binds to a subpopulation of NMDA receptors and to a novel binding site.

NMDA receptors mediate several important functions in the CNS; however, little is known about the pharmacology, biochemistry, and function of distinct NMDA receptor subtypes in brain tissue. To facilitate the study of native NMDA receptor subpopulations, we have determined the radioligand binding properties of [3H]homoquinolinate, a potential subtype-selective ligand. Using quantitative receptor autoradiography, NMDA-specific [3H]homoquinolinate binding selectively labeled brain regions expressing NR2B mRNA (layers I-III of cerebral cortex, striatum, hippocampus, and septum). NMDA-specific [3H]homoquinolinate binding was low in brain regions that express NR2C and NR2D mRNA (cerebellar granular cell layer, NR2C; glomerular layer of olfactory bulb, NR2C/NR2D; and midline thalamic nuclei, NR2D). In forebrain, the pattern of NMDA-specific [3H]homoquinolinate binding paralleled NR2B and not NR2A distribution. In addition to NMDA-displaceable binding, there was a subpopulation of [3H]homoquinolinate binding sites in the forebrain, cerebellum, and choroid plexus that was not displaced by NMDA or L-glutamate. In contrast, we found that the derivative of homoquinolinate, 2-carboxy-3-carboxymethylquinoline, markedly inhibited the NMDA-insensitive binding of [3H]homoquinolinate without inhibiting the NMDA-sensitive population. [3H]Homoquinolinate may be useful for selectively characterizing NR2B-containing NMDA receptors in a preparation containing multiple receptor subtypes and for characterizing a novel binding site of unknown function.

Survival and functional demonstration of interregional pathways in fore/midbrain slice explant cultures.

An important general question in neurobiology concerns the development and expression of the rich context of neuronal phenotypes, especially in relation to the diverse patterns of connectivity. Organotypic cultures of brain slices may offer distinct advantages for such studies if such a preparation survives, maintains a wide diversity of neuronal phenotypes and displays appropriate synaptic connections between regions. To address these requirements, we utilized long-term organotypic cultures of intact horizontal slices of rat forebrain and midbrain and assessed a variety of markers of phenotype in combination with functional tests of connectivity. This explant preparation displayed a distinct viability requirement such that the greatest explant survival was seen in slices taken from pups of less than postnatal day 7 and was independent of N-methyl-D-aspartate channel blockade. The anatomical features of the major brain regions (e.g., neocortex, striatum, septum, hippocampus, diencephalon and midbrain) were observed in their normal boundaries. The presence of cholinergic and catecholaminergic neurons was demonstrated with acetylcholinesterase histochemistry and tyrosine hydroxylase immunohistochemistry. Labelled neurons displayed multiple, regionally-appropriate cytoarchitectures and, in some cases, could be seen to project to brain regions in a manner quite similar to that seen in vivo. Finally, the direct demonstration of spontaneous and evoked interregional excitatory synaptic transmission was made using whole-cell patch-clamp recordings from striatal neurons which revealed an intact glutamate-using corticostriatal pathway. This simple explant preparation appears to contain a rich diversity of neuronal types and synaptic organization. Therefore, this preparation appears to have several distinct advantages for basic neurobiologic research since it combines long-term culture viability and many features of mature brain including complex interregional neuronal systems.

Organotypic brain slice cultures for functional analysis of alcohol-related disorders: novel versus conventional preparations.

Assessment of long-term alterations in neural function and phenotype has usually involved culture techniques that utilize dissociated preparations. Recently, we have approached such topics in alcohol research by using brain slice cultures, also known as explant or organotypic preparations. In this symposium presentation, two preparations will be discussed, and examples of the particular advantages of these preparations will be presented in relation to alcohol research. First, we use the hippocampal explant preparation for assessment of long-term alterations in N-methyl-D-aspartate receptor (NMDAR) function due to chronic ethanol exposure and subsequent withdrawal. This preparation displays many synaptic, structural, and enzymatic phenotypes indicative of normal neural preparations. Patch clamp recordings reveal NMDAR-mediated excitatory postsynaptic current (EPSC) elicited upon stimulation of Schaffer collateral fibers and recorded from CA1 pyramidal cells. Long-term ethanol exposure followed by subsequent withdrawal resulted in a specific enhancement of NMDAR-mediated synaptic responses which preceded the expression of epileptiform events that occurred after prolonged withdrawal periods. Second, we describe a novel explant preparation, derived from horizontal slices of the entire forebrain and midbrain of the rat. These long-term explants displayed multiple normal phenotypes including Nissl, AChE, TH, and GFAP staining. Electrophysiologically, these explants displayed a functional corticostriatal pathway recorded with field and patch clamp techniques and elicited by synaptic stimulation. Taken together, these explant preparations display utility for long-term study of ethanol effects on neural systems, especially relating to withdrawal hyperexcitability as well as systems involved in drug-seeking behavior.

Dextromethorphan and other N-methyl-D-aspartate receptor antagonists are teratogenic in the avian embryo model.

N-Methyl-D-aspartate (NMDA) receptors are a calcium-conducting class of excitatory amino acid receptors that are involved in neuronal development and migration. Certain well known teratogens (e.g. homocysteine, ethanol, and chloroform) that induce congenital neural tube and neural crest defects also have the capacity to act as NMDA receptor antagonists. We hypothesized that teratogenicity was a general property of NMDA receptor antagonists, and that high affinity NMDA receptor antagonists would induce neural tube and neural crest defects. Chicken embryos were given 5, 50, or 500 nmol/d of selected NMDA receptor antagonists for 3 consecutive days during the process of neural tube closure, beginning 4 h after the beginning of incubation. Selected NMDA receptor antagonists represented three classes of antagonists: ion channel blockers, glycine site antagonists, and glutamate site agonists and antagonists. All classes of NMDA receptor antagonists induced embryonic death and congenital defects of the neural crest and neural tube; however, the channel blockers were the most potent teratogens. Dextromethorphan at 500 nmol/embryo/d killed more than half the embryos and induced congenital defects in about one-eighth of the survivors; dextromethorphan was also highly lethal at 50 nmol/embryo/d. Glutamate site NMDA receptor agonists (NMDA and homoquinolinic acid) displayed weak toxicity relative to their known NMDA receptor potency. Taken together, these data indicate that NMDA receptor antagonists, particularly channel blockers, are potent teratogens in the chicken embryo model. Because dextromethorphan is a widely used nonprescription antitussive, its strong teratogeneticity using this model is particularly noteworthy.

Methylation of the NMDA receptor agonist L-trans-2,3-pyrrolidine-dicarboxylate: enhanced excitotoxic potency and selectivity.

This study investigated the excitotoxic properties of a novel series of NMDA analogues in which a methyl group was introduced to the 5-position of the pyrrolidine ring of L-trans-2,3-PDC, a previously identified NMDA receptor agonist. While all of these compounds induced NMDA-receptor-mediated injury, methylation increased in vivo excitotoxic potency 1000-fold. Injections (1 mu 1) in rat dorsal hippocampus of cis- and trans-5-methyl-L-trans-2,3-PDC (0.1 nmol) induced 50-70% neuronal damage to areas CA1 and CA4, comparable to that induced by 100 nmol of L-trans-2,3-PDC. Further, cis- and trans-methylated analogues induced distinct patterns of hippocampal pathology consistent with differential excitotoxic vulnerability of neurons expressing NMDA receptors. Neuronal damage produced by the 5-methyl-L-trans-2,3-PDCs could be blocked by coadministration of MK-801 (3 mg/kg ip), but not NBQX (25 nmol). Biochemical and physiological assays confirmed the action of the analogues as NMDA agonists, but did not provide an explanation for differences in excitotoxic potency between the methylated and nonmethylated 2,3-PDCs. or example, the activity of the compounds as inhibitors of 3H-glutamate binding (IC50 values: 0.4, 1.4, and 1.2 microM for cis-5-methyl-,trans-5-methyl-, and L-trans-2,3-PDC, respectively), agonists at NR1A/NR2B receptors (EC50 values: 5, 49, and 16 microM for cis-5-methyl-,trans-5-methyl-, and L-trans-2,3-PDC, respectively), and in vitro excitotoxins in cortical cultures varied only two- to fivefold as a consequence of methylation. Potential roles of NMDA receptor subtypes and transport in these effects are discussed. As potent and selective NMDA excitotoxins, cis- and trans-5-methyl-L-trans-2,3-PDC will be of value studying excitotoxic mechanisms, MDA-receptor-mediated pathology, and NMDA receptor heterogeneity.

NR1 and NR2 subunit contributions to N-methyl-D-aspartate receptor channel blocker pharmacology.

The potencies of various N-methyl-D-aspartate(NMDA) receptor channel blockers were determined at recombinant NMDA receptors containing differing combinations of NR1 and NR2 subunits expressed in Xenopus laevis oocytes. When the NR1 subunit was varied (NR1e/NR2A or NR1b/NR2A), none of the 9 channel blockers tested displayed a statistically different affinity. In contrast, altering NR2 composition changed the affinities of several channel blockers. Three of 10 compounds displayed significantly higher affinities for NR1b/NR2C receptors than NR1b/NR2A receptors, and three of five compounds had higher affinity at NR1b/NR2C than NR1b/NR2B receptors. Both MK-801 and N-[1-(2-thienyl)cyclohyxyl]piperidine displayed identical affinities at all receptor subunit combinations tested. However, these two compounds displayed significantly slower rates of blockade and unblockade at NR1b/NR2C than at NR1b/NR2A receptors, perhaps reflecting the shorter mean open times of NR1/NR2C receptors. NR1b/NR2B and NR1b/NR2A were distinguished by one of five compounds tested. Taken together, these results indicate that NR2 subunits impart differing pharmacological profiles to NMDA receptors; thus, it may be possible to develop NMDA receptor channel blocker antagonists of greater subtype selectivity.

Pharmacological heterogeneity of NMDA receptors: characterization of NR1a/NR2D heteromers expressed in Xenopus oocytes.

The pharmacology of recombinant NR1a/NR2D NMDA receptors expressed in Xenopus oocytes was examined and compared to the pharmacology of NR1a/NR2A, NR1a/NR2B and NR1a/NR2C heteromers. The NR1/NR2D heteromer showed a pharmacological profile distinct from each of the other NR1/NR2 heteromers. This unique pharmacological profile was characterized by a relatively lower affinity for the agonist homoquinolinate and the antagonists 2-amino-5-phosphonopentanoate (D-AP5) and (R,E)-4-(3-phosphonoprop-2-enyl)piperazine-2-carboxylic acid (D-CPPene) but not for the antagonists (+/-)-4-(4-phenylbenzoyl) piperazine-2,3-dicarboxylic acid (PBPD) and alpha-amino-5-(phosphonomethyl)[1,1'-biphenyl]-3-propanoic acid (EAB515). NR2D-containing receptors displayed a pharmacological profile most similar to that observed for receptors containing the genetically related NR2C subunit. These findings parallel observations obtained for native NMDA receptors in the medial thalamus (presumed to contain NR2D subunits) and forebrain (presumed to contain NR2A and NR2B subunits). Thus, only compounds that discriminate between either NR2A- or NR2B-containing heteromers and NR2D-containing heteromers also discriminate between forebrain and medial thalamic NMDA receptors. While the pharmacology of the NR1a/NR2D receptor shows many parallels to the medial thalamic NMDA receptor, some differences were observed. Certain compounds which discriminate between medial thalamic and cerebellar (presumed to contain NR2C subunits) receptors (e.g., homoquinolinate, D-CPPene) did not show a similar selectivity for NR1a/NR2D receptors relative to NR1/NR2C receptors. Co-expression of NR1a, NR2B and NR2D subunits in Xenopus oocytes resulted in the formation of heteromeric complexes with unique pharmacological properties, suggesting the co-existence of these two distinct NR2 subunits in the same receptor complex.

Glycine modulates ethanol inhibition of heteromeric N-methyl-D-aspartate receptors expressed in Xenopus oocytes.

Ethanol inhibits N-methyl-D-aspartate (NMDA) receptor-mediated responses at pharmacologically relevant concentrations, suggesting that inhibition of NMDA receptors may underlie some of the actions of ethanol in the central nervous system. We examined the ability of glycine to modulate ethanol inhibition of four recombinant heteromeric NMDA receptors (NR1a/NR2A through NR2D) expressed in Xenopus oocytes. Ethanol dose-response analysis revealed enhanced inhibitory efficacy of ethanol in the presence of subsaturating glycine concentrations at the NR1/NR2A, NR1/NR2C, and NR1/NR2D receptors. When assayed over a range of glycine concentrations, ethanol exhibited both glycine-reversible and glycine-independent inhibition of NMDA receptors. In contrast, ethanol inhibition of recombinant NMDA receptors was independent of NMDA concentration. Glycine reversal of ethanol inhibition suggested that ethanol might lower the affinity of glycine for the NMDA receptor and thereby decrease response magnitude. Consistent with this hypothesis, ethanol significantly reduced glycine affinity at NR1/NR2A and NR1/NR2C receptors. Evaluation of the glycine-independent component of ethanol inhibition demonstrated that in the presence of saturating concentrations of glycine, the NR1/NR2A and NR1/NR2B receptors were more sensitive to ethanol than the NR1/NR2C and NR1/NR2D receptors. Activation of the NR1/NR2D heteromers by NMDA and low concentrations of glycine elicited responses characterized by an initial peak followed by a lower-amplitude plateau response, which is consistent with glycine-sensitive desensitization as previously described for native NMDA receptors. In addition, nondesensitizing NR1/NR2B responses elicited in the presence of subsaturating concentrations of glycine were frequently converted into desensitizing responses by the addition of ethanol, an effect that was reversed with increasing glycine concentrations. The ability of ethanol to promote glycine-sensitive desensitization further suggests an interaction between glycine and ethanol inhibition of the NMDA receptor. Taken together, the results of the present report demonstrate that ethanol inhibition of NMDA receptors has both glycine-reversible and glycine-independent components, suggesting two distinct molecular mechanisms for ethanol inhibition of NMDA receptors.

Insulin potentiates N-methyl-D-aspartate receptor activity in Xenopus oocytes and rat hippocampus.

Growth factor signal transduction pathways have recently been shown to affect voltage-gated ion channel activity. In this study we report that insulin can modulate the activity of a ligand-gated ion channel, the N-methyl-D-aspartate (NMDA) receptor. In Xenopus oocytes, brief insulin exposure rapidly potentiated NR1a/NR2A and NR1a/NR2B receptor responses 2-3 fold and weakly potentiated NR1a/NR2C and NR1a/NR2D mediated-responses. Insulin potentiation of NR1a/NR2A receptor responses was significantly blocked by staurosporine, suggesting kinase involvement in insulin action. Insulin modulation of native NMDA receptors is suggested by the observation that insulin potentiated the NMDA receptor-mediated synaptic component in hippocampal slices. Regulation of NMDA receptor activity by growth factors may account for previous observations of growth factor modulation of central nervous system excitotoxicity.

The molecular basis of NMDA receptor subtypes: native receptor diversity is predicted by subunit composition.

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.