[3H]Homoquinolinate Binds to a Subpopulation of NMDA Receptors and to a Novel Binding Site

Abstract: 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.     

Characterisation of N-methyl-D-aspartate receptor-specific [(3)H]Ifenprodil binding to recombinant human NR1a/NR2B receptors compared with native receptors in rodent brain membranes

We have performed [(3)H]ifenprodil binding experiments under NMDA receptor-specific assay conditions to provide the first detailed characterisation of the pharmacology of the ifenprodil site on NMDA NR1/NR2B receptors, using recombinant human NR1a/NR2B receptors stably expressed in L(tk-) cells, in comparison with rat cortex/hippocampus membranes. [(3)H]Ifenprodil bound to a single, saturable site on both human recombinant NR1a/NR2B receptors and native rat receptors with B:(max) values of 1.83 and 2.45 pmol/mg of protein, respectively, and K:(D) values of 33.5 and 24.8 nM:, respectively. The affinity of various ifenprodil site ligands-eliprodil, (R:(*), R:(*))-4-hydroxy-alpha-(4-hydroxyphenyl)-beta-methyl-4-pehnyl-1-pi per idineethanol [(+/-)-CP-101,606], cis-3-[4-(4-fluorophenyl)-4-hydroxy-1-piperidinyl]-3, 4-dihydro-2H:-1-benzopyran-4,7-diol [(+/-)-CP-283,097], and (R:(*), S:(*))-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperid inepropanol [(+/-)-Ro 25-6981] was very similar for inhibition of [(3)H]ifenprodil binding to recombinant human NR1a/NR2B and native rat receptors, whereas allosteric inhibition of [(3)H]ifenprodil binding by polyamine site ligands (spermine, spermidine, and arcaine) showed approximately twofold lower affinity for recombinant receptors compared with native receptors. Glutamate site ligands were less effective at modulating [(3)H]ifenprodil binding to recombinant NR1a/NR2B receptors compared with native rat receptors. The NMDA receptor-specific [(3)H]ifenprodil binding conditions described were also applied to ex vivo experiments to determine the receptor occupancy of ifenprodil site ligands [ifenprodil, (+/-)-CP-101,606, (+/-)-CP-283,097, and (+/-)-Ro 25-6981] given systemically.     

Characterization of Glutamate Binding Sites in Receptors Assembled from Transfected NMDA Receptor Subunits

Abstract: Previous studies in brain and recombinant NMDA receptors have observed heterogeneity in NMDA-sensitive glutamate binding site. We further characterized the glutamate site assembled from NR1a, NR2A, and NR2B NMDA receptor subunits using l -[³H]glutamate and [³H]CGP 39653 binding assays. In contrast to earlier reports, we demonstrate a unique pharmacology for the NR2A subunit alone, which has high affinity for agonists but low affinity for competitive antagonists compared with heteromeric combinations of NR1a + NR2A and NR1a + NR2B. Similar to previous reports, we find unequal antagonist affinity between heteromeric combinations of NR1a + NR2A and NR1a + NR2B. However, unlike earlier reports, we describe two binding components within each heteromeric transfection that more closely resemble data obtained for binding to brain membranes. In addition, we show Mg²⁺ can alter [³H]CGP 39653 binding in both the NR1a + NR2A and the NR1a + NR2B combination, thus allowing comparison of the [³H]CGP 39653-labeled site between the two heteromeric combinations. Agonist inhibition of [³H]CGP 39653 binding revealed differences between the heteromeric combinations as well as within each heteromeric combination, the latter of which more closely resembled results from brain. These results further determine components of the agonist and antagonist binding sites of the NMDA receptor as well as suggest additional possible mechanisms of heterogeneity of the glutamate site in the brain.     

Cleavage of the NR2B subunit amino terminus of N-methyl-D-aspartate (NMDA) receptor by tissue plasminogen activator: identification of the cleavage site and characterization of ifenprodil and glycine affinities on truncated NMDA receptor

Thrombolysis using tissue plasminogen activator (tPA) has been the key treatment for patients with acute ischemic stroke for the past decade. Recent studies, however, suggest that this clot-busting protease also plays various roles in brain physiological and pathophysiological glutamatergic-dependent processes, such as synaptic plasticity and neurodegeneration. In addition, increasing evidence implicates tPA as an important neuromodulator of the N-methyl-d-aspartate (NMDA) receptors. Here, we demonstrate that recombinant human tPA cleaves the NR2B subunit of NMDA receptor. Analysis of NR2B in rat brain lysates and cortical neurons treated with tPA revealed concentration- and time-dependent degradation of NR2B proteins. Peptide sequencing studies performed on the cleaved-off products obtained from the tPA treatment on a recombinant fusion protein of the amino-terminal domain of NR2B revealed that tPA-mediated cleavage occurred at arginine 67 (Arg(67)). This cleavage is tPA-specific, plasmin-independent, and removes a predicted ~4-kDa fragment (Arg(27)-Arg(67)) from the amino-terminal domain of the NR2B protein. Site-directed mutagenesis of putative cleavage site Arg(67) to Ala(67) impeded tPA-mediated degradation of recombinant protein. This analysis revealed that NR2B is a novel substrate of tPA and suggested that an Arg(27)-Arg(67)-truncated NR2B-containing NMDA receptor could be formed. Heterologous expression of NR2B with Gln(29)-Arg(67) deleted is functional but exhibits reduced ifenprodil inhibition and increased glycine EC(50) with no change in glutamate EC(50). Our results confirmed NR2B as a novel proteolytic substrate of tPA, where tPA may directly interact with NR2B subunits leading to a change in pharmacological properties of NR2B-containing NMDA receptors.     

Crystal Structure and Pharmacological Characterization of a Novel N-Methyl-d-aspartate (NMDA) Receptor Antagonist at the GluN1 Glycine Binding Site

NMDA receptors are ligand-gated ion channels that mediate excitatory neurotransmission in the brain. They are tetrameric complexes composed of glycine-binding GluN1 and GluN3 subunits together with glutamate-binding GluN2 subunits. Subunit-selective antagonists that discriminate between the glycine sites of GluN1 and GluN3 subunits would be valuable pharmacological tools for studies on the function and physiological roles of NMDA receptor subtypes. In a virtual screening for antagonists that exploit differences in the orthosteric binding site of GluN1 and GluN3 subunits, we identified a novel glycine site antagonist, 1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinoxalin-4(5H)-one (TK40). Here, we show by Schild analysis that TK40 is a potent competitive antagonist with K_b values of 21–63 nm at the GluN1 glycine-binding site of the four recombinant GluN1/N2A-D receptors. In addition, TK40 displayed >100-fold selectivity for GluN1/N2 NMDA receptors over GluN3A- and GluN3B-containing NMDA receptors and no appreciable effects at AMPA receptors. Binding experiments on rat brain membranes and the purified GluN1 ligand-binding domain using glycine site GluN1 radioligands further confirmed the competitive interaction and high potency. To delineate the binding mechanism, we have solved the crystal structure of the GluN1 ligand-binding domain in complex with TK40 and show that TK40 binds to the orthosteric binding site of the GluN1 subunit with a binding mode that was also predicted by virtual screening. Furthermore, the structure reveals that the imino acetamido group of TK40 acts as an α-amino acid bioisostere, which could be of importance in bioisosteric replacement strategies for future ligand design.     

The effects of NR2 subunit-dependent NMDA receptor kinetics on synaptic transmission and CaMKII activation

N-Methyl-D-aspartic acid (NMDA) receptors are widely expressed in the brain and are critical for many forms of synaptic plasticity. Subtypes of the NMDA receptor NR2 subunit are differentially expressed during development; in the forebrain, the NR2B receptor is dominant early in development, and later both NR2A and NR2B are expressed. In heterologous expression systems, NR2A-containing receptors open more reliably and show much faster opening and closing kinetics than do NR2B-containing receptors. However, conflicting data, showing similar open probabilities, exist for receptors expressed in neurons. Similarly, studies of synaptic plasticity have produced divergent results, with some showing that only NR2A-containing receptors can drive long-term potentiation and others showing that either subtype is capable of driving potentiation. In order to address these conflicting results as well as open questions about the number and location of functional receptors in the synapse, we constructed a Monte Carlo model of glutamate release, diffusion, and binding to NMDA receptors and of receptor opening and closing as well as a model of the activation of calcium-calmodulin kinase II, an enzyme critical for induction of synaptic plasticity, by NMDA receptor-mediated calcium influx. Our results suggest that the conflicting data concerning receptor open probabilities can be resolved, with NR2A- and NR2B-containing receptors having very different opening probabilities. They also support the conclusion that receptors containing either subtype can drive long-term potentiation. We also are able to estimate the number of functional receptors at a synapse from experimental data. Finally, in our models, the opening of NR2B-containing receptors is highly dependent on the location of the receptor relative to the site of glutamate release whereas the opening of NR2A-containing receptors is not. These results help to clarify the previous findings and suggest future experiments to address open questions concerning NMDA receptor function.     

High Level Expression of the NMDAR1 Glutamate Receptor Subunit in Electroporated COS Cells

Abstract: 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 ( K _D = 29.6 ± 6 n M ; B _max = 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-5 H -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.     

Further characterization of the molecular interaction between PSD-95 and NMDA receptors: the effect of the NR1 splice variant and evidence for modulation of channel gating

Coexpression of PSD-95(c-Myc) with NR1-1a/NR2A NMDA receptors in human embryonic kidney (HEK) 293 cells resulted in a decrease in efficacy for the glycine stimulation of [3 H]MK801 binding similar to that previously described for l-glutamate. The inhibition constants (K (I) s) for the binding of l-glutamate and glycine to NR1-1a/NR2A determined by [3 H]CGP 39653 and [3 H]MDL 105 519 displacement assays, respectively, were not significantly different between NR1-1a/NR2A receptors coexpressed +/- PSD-95(c-Myc). The increased EC(50) for l-glutamate enhancement of [3 H]MK801 binding was also found for NR1-2a/NR2A and NR1-4b/NRA receptors thus the altered EC(50) is not dependent on the N1, C1 or C2 exon of the NR1 subunit. The NR1-4b but not the NR1-1a subunit was expressed efficiently at the cell surface in the absence of NR2 subunits. Total NR1-4b and NR1-4b/NR2A expression was enhanced by PSD-95(c-Myc) but whole cell enzyme-linked immunoadsorbent assays (ELISAs) showed that this increase was not due to increased expression at the cell surface. It is suggested that PSD-95(c-Myc) has a dual effect on NMDA receptors expressed in mammalian cells, a reduction in channel gating and an enhanced expression of NMDA receptor subunits containing C-terminal E(T/S)XV PSD-95 binding motifs.     

Interleukin-2 inhibits NMDA receptor-mediated currents directly and may differentially affect subtypes

Using whole-cell patch-clamp recordings, this study investigated the effects of interleukin-2 (IL-2) on N-methyl-d-aspartate (NMDA) receptor-mediated currents (I(NMDA)) in rat cultured hippocampal neurons and human embryonic kidney (HEK) 293 cells expressing recombinant NMDA receptors. We found that IL-2 (0.01-1ng/ml) immediately and significantly decreased peak I(NMDA) in cultured neurons. Interestingly, the peak I(NMDA) induced in HEK 293 cells was also inhibited by IL-2. We also found that IL-2 differentially decreased the peak amplitudes of NR2A- and NR2B-containing NMDA receptor-mediated currents (I(NR2A) and I(NR2B)) by 54+/-5% and 30+/-4%, respectively. These results provide new evidence that IL-2 induces rapid inhibition of peak currents of NMDA receptor-mediated responses with possible NR1/NR2A and NR1/NR2B subtype-differentiation, and suggest that the inhibition is mediated by direct interaction between IL-2 and NMDA receptors.     

Inducible expression and pharmacological characterization of recombinant rat NR1a/NR2A NMDA receptors

In this study, we have established a non-neuronal cell line stably and inducibly expressing recombinant NMDA receptors (NRs) composed of rat NR1a/NR2A subunits. EcR-293 cells were transfected with rat NR1a and NR2A cDNAs using the inducible mammalian expression vector pIND. Cell colonies resistant for the selecting agents were picked and tested for NR2A mRNA as well as protein expression using quantitative RT-PCR and flow cytometry based immunocytochemistry. Clonal cells expressing functional NMDA receptors were identified by measuring NMDA-evoked ion currents, and NMDA-induced increase in cytosolic free calcium concentration in whole-cell patch-clamp and fluorimetric calcium measurements, respectively. One clone named D5/H3, which exhibited the highest response to NMDA, was chosen to examine inducibility of the expression and for pharmacological profiling of recombinant NR1a/NR2A NMDA receptors. To check inducibility, NR2A subunit expression in D5/H3 cells treated with the inducing agent muristerone A (MuA) was compared with that in non-induced cells. Both NR2A mRNA and protein expression was several folds higher in cells treated with the inducing agent. As part of the pharmacological characterization, we examined the activation of the expressed NR1a/NR2A receptors as a function of increasing concentration of NMDA. NMDA-evoked concentration-dependent increases in cytosolic [Ca2+] with an EC50 value of 41 +/- 1 microM. In addition, whereas the NMDA response was concentration-dependently inhibited by the channel blocker MK-801 (IC50 = 58 +/- 6 nM), NR2B subunit selective NMDA receptor antagonists were ineffective. Thus, this cell line, which stably and inducibly expresses recombinant NR1a/NR2A NMDA receptors, can be a useful tool for testing NMDA receptor antagonists and studying their subunit selectivity.     

Generation and Characterisation of Stable Cell Lines Expressing Recombinant Human N-Methyl-d-Aspartate Receptor Subtypes

Abstract: Transfection of mouse L(tk-) cells with human N -methyl- d -aspartate (NMDA) receptor subunit cDNAs under the control of a dexamethasone-inducible promoter has been used to generate two stable cell lines expressing NR1a/NR2A receptors and a stable cell line expressing NR1a/NR2B receptors. The cell lines have been characterised by northern and western blot analyses, and the pharmacology of the recombinant receptors determined by radioligand binding techniques. Pharmacological differences were identified between the two NMDA receptor subtypes. The glutamate site antagonist d,l -(ε)-2-[³H]amino-4-propyl-5-phosphono-3-pentanoic acid ([³H]CGP 39653) had high affinity for NR1a/NR2A receptors ( K _D = 3.93 n M ) but did not bind to NR1a/NR2B receptors. Glycine site agonists showed a 2.6–5.4-fold higher affinity for NR1a/NR2B receptors. Data from radioligand binding studies indicated that one of the cell lines, NR1a/NR2A-I, expressed a stoichiometric excess of the NR1a subunit, which may exist as homomeric assemblies. This observation has implications when interpreting data from pharmacological analysis of recombinant receptors, as well as understanding the assembly and control of expression of native NMDA receptors.     

Synthesis, radiosynthesis and in vivo evaluation of 5-[3-(4-benzylpiperidin-1-yl)prop-1-ynyl]-1,3-dihydrobenzoimidazol-2-[(11)C]one, as a potent NR(1A)/2B subtype selective NMDA PET radiotracer

Recently, a new series of potent and highly subtype-selective 1-(heteroarylalkynyl)-4-benzylpiperidine antagonists of the NMDA receptors has been described by Pfizer Laboratories. In this series, 5-[3-(4-benzylpiperidin-1-yl)prop-1-ynyl]-1,3-dihydrobenzoimidazol-2-one (1) was identified as a selective antagonist for the NR1(A)/2B subtype, displaying IC(50) values for inhibition of the NMDA responses of 5.3 nM for this subtype (compared to NR1(A)/2A: 35 microM and NR1(A)/2C>100 microM) and was active in rat at a relatively low dosage (10mg/kg po). Derivative 1 has been synthesized in four chemical steps in good overall yield and labelled with carbon-11 at its benzoimidazolone ring using [(11)C]phosgene. The pharmacological profile of [(11)C]-1 was evaluated in vivo in rats with biodistribution studies and brain radioactivity monitored with intracerebral radiosensitive beta-microprobes. The brain uptake of [(11)C]-1 was extremely low (0.07% I.D./mL on average at 30 min) and rather uniform across the different brain structures. This in vivo brain regional distribution of [(11)C]-1 did not match with autoradiographic or binding data obtained with other NR2B subtype-selective NMDA ligands. Competition studies with ifenprodil (20 mg/kg, ip, 30 min before the radiotracer injection) failed to demonstrate specific binding of the radiotracer in the brain. In view of these results, and especially considering the low brain penetration of the radiotracer, [(11)C]-1 does not have the required properties for imaging NMDA receptors using positron emission tomography.     

NMDA receptor subunit-dependent modulation by conantokin-G and Ala7-conantokin-G

The modulation of recombinant NMDA receptors by conantokin-G (con-G) and Ala7-conantokin-G (Ala7-Con-G) was investigated in Xenopus oocytes injected with capped RNA coding for NR1 splice variants and NR2 subunits using the two-electrode voltage clamp technique. Glutamate exhibited a marginally higher apparent affinity for NR2A-containing receptors than NR2B-containing receptors, regardless of the NR1 subunit present. Conantokins were bath applied to give cumulative concentration responses in the presence of 3 and 30 mum glutamate. Both contantokins exhibited biphasic concentration-response relationships at NR2A-containing NMDA receptors, producing potentiation at low conantokin concentrations and inhibition at high concentrations. These effects were stronger with glutamate concentrations near its EC50, and less marked at saturating concentrations. In contrast, the conantokin concentration-response relation was monophasic and inhibitory at NR2B-containing receptors. We conclude that the combinations of subunits that comprise the NMDA receptor complex influence conantokin and glutamate affinities and the nature of the responses to conantokins.     

Recombinant Human NMDA Homomeric NR1 Receptors Expressed in Mammalian Cells Form a High-Affinity Glycine Antagonist Binding Site

Abstract: The cDNA NMDAR1 (NR1) encodes a single polypeptide that forms a receptor-channel complex with electrophysiological and pharmacological properties characteristic of the N-methyl-d -aspartate receptor. Homomeric NR1 recombinant receptors expressed in Xenopus oocytes show functional responses with low levels of conductance. In this study we have characterized, by radioligand binding techniques, the pharmacological properties of homomeric receptors of two human NR1 isoforms (NR1a and NR1e, which differ in their C-terminal region), transiently expressed in human embryonic kidney 293 cells. The glycine site antagonist (±)-4-(trans)-2-carboxy-5,7-dichloro-4-[³H]phenylaminocarbonylamino-1,2,3,4-tetrahydroquinoline ([³H]L-689,560) bound to NR1a- and NR1e-transfected cells with high affinity (K_D = 3.29 and 1.61 nM, respectively). B_max values for NR1a- and NR1e-transfected cells were 3.82 and 1.69 pmol/mg of protein, respectively, and Hill coefficients were close to unity. K_i values for glycine site antagonists inhibiting [³H]L-689,560 binding to NR1e-transfected cells were similar to those observed with rat brain membranes. Affinity values for agonists and partial agonists were four- to 16-fold weaker, indicating that the glycine site of homomeric NR1 receptors is in an antagonist-preferring state. K_i values obtained with NR1a-transfected cells were approximately twofold lower than those obtained with NR1e-transfected cells. High-affinity binding to NR1-transfected cells was not observed with the transmitter recognition site radioligands l -[³H]glutamate and d,l -(ε)-2-[³H]amino-4-propyl-5-phosphono-3-pentanoic acid ([³H]CGP-39653) or the ion-channel radioligand [³H]dizocilpine ([³H]MK-801). These results indicate that although transfection of mammalian cells with homomeric NR1 recombinant receptors does not appear to result in functional receptors, a glycine binding site is formed that may have a physiological role if present in vivo.     

Neurosteroids allosterically modulate the ion pore of the NMDA receptor consisting of NR1/NR2B but not NR1/NR2A

Neurosteroids are endogenously derived compounds, mediating rapid effects in the central nervous system. They participate in vital processes, including memory and learning, neuroplasticity, and neuroprotection in Alzheimer’s disease. However, the mechanisms behind those effects remain to be elucidated. The neurosteroids pregnenolone sulphate (PS) and pregnanolone sulphate (3α5βS) have recently been shown to allosterically alter the NMDA receptor in nanomolar concentrations. Those studies featured ifenprodil, which is a dirty drug, with affinity to many targets. In this study we compare the NMDA receptors in the hippocampus to recombinant NMDA receptors, using [³H]-MK-801 as radioligand. The results show that neurosteroids modulate the ifenprodil binding kinetics in a narrow concentration interval, addressing it to the NR2B subunit, since no effects were recorded at recombinant NR1/NR2A receptors. The effects were also seen as changes in the manner ifenprodil displaced or induced the dissociation of [³H]-MK-801. It indicates that the neurosteroidal effects indeed alter the ion pore of the NMDA receptor, why it is reasonable to believe that these findings have physiological relevance.     

N-methyl-D-aspartate receptor antagonists enhance histamine neuron activity in rodent brain

The modulation of histamine neuron activity by various non-competitive NMDA-receptor antagonists was evaluated by changes in tele-methylhistamine (t-MeHA) levels and histidine decarboxylase (hdc) mRNA expression induced in rodent brain. The NMDA open-channel blockers phencyclidine (PCP) and MK-801 enhanced t-MeHA levels in mouse brain by 50-60%. Ifenprodil, which interacts with polyamine sites of NR2B-containing NMDA receptors, had no effect. PCP also increased hdc mRNA expression in the rat tuberomammillary nucleus. The enhancement of t-MeHA levels elicited by MK-801 (ED50 of approximately 0.1 mg/kg) was observed in the hypothalamus, cerebral cortex, striatum and hippocampus. Control t-MeHA levels and the t-MeHA response to MK-801 were not different in male and female mice. Double immunostaining for HDC and NMDA receptor subunits showed that histamine neurons of the rat tuberomammillary nucleus express NMDA receptor subunit 1 (NR1) with NMDA receptor subunit 2A (NR2A) and NMDA receptor 2B subunit (NR2B). In addition, immunoreactivity for the neuronal glutamate transporter EAAC1 was observed near most histaminergic perikarya. Hence, these findings support the existence of histamine/glutamate functional interactions in the brain. The increase in histamine neuron activity induced by NMDA receptor antagonists further suggests a role of histamine neurons in psychotic disorders. In addition, the decrease in MK-801-induced hyperlocomotion observed in mice after administration of ciproxifan further strengthens the potential interest of H3-receptor antagonist/inverse agonists for the symptomatic treatment of schizophrenia.     

Characterization of NMDA Receptor Subunit-Specific Antibodies: Distribution of NR2A and NR2B Receptor Subunits in Rat Brain and Ontogenic Profile in the Cerebellum

Abstract: Selective antisera for NMDA receptor subunits NR2A and NR2B have been developed. Each antiserum identifies a single band on an immunoblot at ∼175 kDa that appears to be the appropriate subunit of the NMDA receptor. Using these antisera the relative densities of the subunits in eight areas of adult rat brain have been determined. The NR2A subunit was found to be at its highest level in hippocampus and cerebral cortex, to be at intermediate levels in striatum, olfactory tubercle, midbrain, olfactory bulb, and cerebellum, and to be at lowest levels in the pons-medulla. The NR2B subunit was found to be expressed at its highest levels in the olfactory tubercle, hippocampus, olfactory bulb, and cerebral cortex. Intermediate levels were expressed in striatum and midbrain, and low levels were detected in the pons-medulla. No signal for NR2B was found in the cerebellum. These regional distributions were compared with that for [³H]MK-801 binding sites. It was found that although the distribution of the NR2A subunit corresponds well with radioligand binding, the distribution of the NR2B subunit does not. The ontogenic profiles of NR2A and NR2B subunits in the rat cerebellum were also determined. Just following birth [postnatal day (P) 2] NR2A subunits are undetectable, whereas NR2B subunits are expressed at amounts easily measurable. Beginning at about P12 the levels of NR2A rise rapidly to reach adult levels by P22. At the same time (P12), levels of NR2B protein begin to decline rapidly to reach undetectable levels by 22 days after birth. The results suggest that NMDA receptors are likely to be composed of different subunits in different parts of the brain and that even in the same tissue the receptors are likely to show different properties at various times during development due to alterations in the subunit composition of the receptor.     

Ethanol sensitivity of NMDA receptors

NMDA receptors are ionotropic glutamate receptors assembled of subunits of the NR1 and of the NR2 family (NR2A–NR2D). The subunit diversity largely affects the pharmacological properties of NMDA receptors and, hence, gives rise to receptor heterogeneity. As an overall result of studies on recombinant and native NMDA receptors, ethanol inhibits the function of receptors containing the subunits NR2A and/or NR2B to a greater extent than those containing NR2C or NR2D. For example, in rat cultured mesencephalic neurons, NR2C expression was developmentally increased, whereas expression of NR2A and NR2B was decreased. These changes coincided with a developmental loss of sensitivity of NMDA responses to ethanol and ifenprodil, a non-competitive NMDA receptor antagonist that shows selectivity for NR2B-containing receptors. Also in rat locus coeruleus neurons, the low ethanol sensitivity of somatic NMDA receptors could be explained by a prominent expression of NR2C. The inhibitory site of action for ethanol on the NMDA receptor is not yet known. Patch–clamp studies suggest a target site exposed to or only accessible from the extracellular environment. Apparently, amino acid residue Phe⁶³⁹, located in the TM3 domain of NR1, plays a crucial role in the inhibition of NMDA receptor function by ethanol. Since this phenylalanine site is common to all NMDA and non-NMDA receptor (AMPA/kainate receptor) subunits, this observation is consistent with accumulating evidence for a similar ethanol sensitivity of a variety of NMDA and non-NMDA receptors, but it cannot explain the differences in ethanol sensitivity observed with different NR2 subunits.