Selective decrease in NR1 subunit splice variant mRNA in the hippocampus of Pb2+-exposed rats: implications for synaptic targeting and cell surface expression of NMDAR complexes

We have previously shown that exposure to environmentally relevant levels of Pb(2+) during brain development decreases the expression of N-methyl-D-aspartate receptor (NMDAR) subunit 1 (NR1) and NR2A genes in the hippocampus of young adult rats and was associated with deficits in hippocampal LTP and spatial learning [Neuroscience 99 (2000) 233-242]. In the present study, we demonstrate that the lower levels of NR1 subunit mRNA expressed in the Pb(2+)-exposed hippocampus are principally due to decreased levels of the NR1-4 and NR1-2 splice variants. These changes were present in the absence of changes in GluR1, PSD-95 and alphaCaMKII gene expression. A unique characteristic of these splice variants is that they lack the C1 cassette. Further, these splice variants have been shown to impart the highest cell surface expression, PKC potentiation and calcium kinetics to NMDAR complexes. Our present findings indicate that Pb(2+)-induced changes in NR1 subunit splice variant mRNA expression in the hippocampus may provide a mechanism by which Pb(2+)-exposure can modify NMDAR-mediated calcium signaling and influence the degree of synaptic plasticity.     

Radioligand binding studies reveal agmatine is a more selective antagonist for a polyamine-site on the NMDA receptor than arcaine or ifenprodil

Ifenprodil, arcaine and agmatine have all been reported to inhibit the NMDA receptor by actions at polyamine-sites, however the specific sites with which these compounds interact is unknown. Here we used radioligand binding of [3H]MK-801 to a membrane preparation from rat cerebral cortex to investigate the interactions of these compounds with the NMDA receptor complex. In the absence of exogenous polyamines, agmatine reduced [3H]MK-801 binding only at concentrations over 500 micro M, as opposed to the putative polyamine-site antagonists arcaine and ifenprodil which directly reduce ligand binding at much lower concentrations (5 micro M) in the absence of polyamines. In our studies, all three compounds significantly reduced spermidine-potentiated [3H]MK-801 binding, however agmatine was the only compound effective at concentrations below those that produced direct inhibition of [3H]MK-801 binding. Under these conditions, agmatine had a K(i)=14.8 micro M for spermidine-potentiated [3H]MK-801 binding and displayed characteristics of a competitive antagonist. Agmatine, as well as ifenprodil and arcaine, also displaced [3H]spermidine from rat cortical membranes at concentrations similar to those that were effective at reducing spermidine-potentiated [3H]MK-801 binding. In conclusion, these data suggest that agmatine reduces the potentiating effects of polyamines by competitive antagonism at a specific site on the NMDA receptor complex, and that these actions of agmatine differ from those of ifenprodil and arcaine.     

Lead exposure during synaptogenesis alters NMDA receptor targeting via NMDA receptor inhibition

N-methyl-D-aspartate receptor (NMDAR) ontogeny and subunit expression are altered during developmental lead (Pb2+) exposure. However, it is unknown whether these changes occur at the synaptic or cellular level. Synaptic and extra-synaptic NMDARs have distinct cellular roles, thus, the effects of Pb2+ on NMDAR synaptic targeting may affect neuronal function. In this communication, we show that Pb2+ exposure during synaptogenesis in hippocampal neurons altered synaptic NMDAR composition, resulting in a decrease in NR2A-containing NMDARs at established synapses. Conversely, we observed increased targeting of the obligatory NR1 subunit of the NMDAR to the postsynaptic density (PSD) based on the increased colocalization with the postsynaptic protein PSD-95. This finding together with increased binding of the NR2B-subunit specific ligand [3H]-ifenprodil, suggests increased targeting of NR2B-NMDARs to dendritic spines as a result of Pb2+ exposure. During brain development, there is a shift of NR2B- to NR2A-containing NMDARs. Our findings suggest that Pb2+ exposure impairs or delays this developmental switch at the level of the synapse. Finally, we show that alter expression of NMDAR complexes in the dendritic spine is most likely due to NMDAR inhibition, as exposure to the NMDAR antagonist aminophosphonovaleric acid (APV) had similar effects as Pb2+ exposure. These data suggest that NMDAR inhibition by Pb2+ during synaptogensis alters NMDAR synapse development, which may have lasting consequences on downstream signaling.     

Lead exposure alters cyclic-AMP response element binding protein phosphorylation and binding activity in the developing rat brain

We examined the effect of lead (Pb(2+)) exposure during development on cyclic-AMP response element binding protein (CREB) expression and phosphorylation in cortical and hippocampal nuclear extracts at postnatal (PN) days 7, 14, 21 and 50. We also examined the binding of CREB family proteins to the cyclic-AMP response element (CRE) using a novel filter-binding assay that provides a quantitative measure of binding kinetics. In the hippocampus and cerebral cortex of control rats, CREB and phospho-CREB (pCREB; serine-133) expression is highest at PN7 and decreases steadily until PN50. Developmental Pb(2+) exposure does not affect total CREB levels but decreases pCREB levels at PN14 and PN50 in the cortex and at PN50 in the hippocampus. Using the filter-binding assay, we measured a 30% decrease in B(max) and 38% decrease in the Kd of CREB family proteins for the CRE in PN50 hippocampal nuclear fractions prepared from Pb(2+)-exposed rats. A similar, but nonsignificant, trend is observed in the cortex of PN50 lead-exposed rats. In addition, a 70% increase in the B(max) was observed in the cortex of PN14 lead-exposed rats without a significant change in the Kd. These disruptions in pCREB expression and binding activity of CREB family members during the ontogeny of the rat brain begin to decipher intracellular mechanisms of Pb(2+) neurotoxicity.     

Glutamic acid reverses Pb2+-induced reductions of NMDA receptor subunits in vitro

The objective of this study is to determine the effects of Pb2+ on N-methyl-d-aspartate receptor (NMDAR) subunits--NR1C1, NR2A and NR2B in primary cultured neuronal cells. We hypothesize that L-glutamic acid (GA) reverses Pb2+-induced NMDAR damage. Neuronal cells were isolated from the fetus brain at 18-20th day of gestation of pregnant Sprague Dawley (SD) rats. All experiments were included three independent cell preparations (N=3). The neuronal cells were exposed to Pb2+ (10(-10), 10(-9), 10(-8) and 10(-7)M) for 24 h. Neurons were pretreated with NMDAR agonist--L-glutamic acid (GA) (200 microM) and antagonists dizocipine (MK-801, 50 nM) for 1h and then exposed to 10(-7)M of Pb2+ for 24 h. Finally, GA at 2, 0.2 and 0.02 mM was incubated with neurons prior to Pb2+ exposure. Aliquots of NR1, NR2A and NR2B proteins from cell homogenate were immunoprecipitated with protein A agarose and detected by Western blotting. The addition of GA unconventionally reversed the reductions of NMDAR by Pb at protein levels, whereas MK-801 exacerbated Pb2+-induced damage. The protection by GA against Pb2+-induced reduction of NMDAR was dose-dependent. These findings suggest that the administration of GA may be a potential approach to intervene the Pb2+-induced NMDAR alterations.     

Role of neuronal NR2B subunit-containing NMDA receptor-mediated Ca2+ influx and astrocytic activation in cultured mouse cortical neurons and astrocytes

The excitatory neurotransmitter glutamate has been shown to mediate such bidirectional communication between neurons and astrocytes. In the present study, we determined the role of N-methyl-D-aspartate (NMDA) receptors on glutamate-evoked Ca(2+) influx into neurons and astrocytes. Either a nonselective NMDA receptor antagonist (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) or selective NR2B subunit-containing NMDA receptor antagonists ifenprodil and (R,S)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperid inepropanol (Ro25-6981) significantly inhibited the glutamate-evoked Ca(2+) influx into neurons, but not into astrocytes. Furthermore, we investigated whether NR2B subunit-containing NMDA receptor antagonists could suppress the astrocytic activation, as detected by glial fibrillary acidic protein (GFAP; as a specific marker of astrocyte)-like immunoreactivities in mouse cortical astrocytes. Here, we demonstrated that the increases in the level of GFAP-like immunoreactivities induced by glutamate were markedly suppressed by cotreatment with ifenprodil in cortical neuron/glia cocultures, but not in purified astrocytes. These results suggest that NR2B subunit-containing NMDA receptor plays a critical role in not only glutamate-evoked Ca(2+) influx into neurons, but also glutamate-induced astrocytic activation. Thus, glutamate-mediated pathway via NR2B subunit-containing NMDA receptor may, at least in part, contribute to neuron-to-astrocyte signaling.     

N-methyl-D-aspartate autoreceptors respond to low and high agonist concentrations by facilitating, respectively, exocytosis and carrier-mediated release of glutamate in rat hippocampus

Presynaptic NMDA autoreceptors regulating glutamate release have rarely been investigated. High-micromolar N-methyl-D-aspartate (NMDA) was reported to elicit glutamate release from hippocampal synaptosomes in a Ca(2+)-independent manner by reversal of excitatory amino acid transporters. The aim of this work was to characterize excitatory amino acid release evoked by low-micromolar NMDA from glutamatergic axon terminals. Purified rat hippocampal synaptosomes were prelabelled with [(3)H]D-aspartate ([(3)H]D-ASP) and exposed in superfusion to varying concentrations of NMDA in the presence of 1 microM glycine. The release of [(3)H]D-ASP and also that of endogenous glutamate provoked by 10 microM NMDA were external Ca(2+) dependent and sensitive to the NMDA channel blocker MK-801 but insensitive to the glutamate transporter inhibitor DL-TBOA, which, on the contrary, prevented the Ca(2+)-independent release evoked by 100 microM NMDA. The NMDA (10 microM) response was blocked by 1 nM Zn(2+) and 1 microM ifenprodil, compatible with the involvement of a NR1/NR2A/NR2B assembly, although the presence of two separate receptor populations, i.e., NR1/NR2A and NR1/NR2B, cannot be excluded. This response was strongly antagonized by submicromolar (0.01-1 microM) concentrations of kynurenic acid and was mimicked by quinolinic acid (1-100 microM) plus 1 microM glycine. Finally, the HIV-1 protein gp120 potently mimicked the NMDA co-agonists glycine and D-serine, being significantly effective at 30 pM. In conclusion, glutamatergic nerve terminals possess NMDA autoreceptors mediating different types of release when activated by different agonist concentrations: low-micromolar glutamate would potentiate glutamate exocytosis, whereas higher glutamate concentrations would also provoke carrier-mediated release.     

NMDAR-2A subunit protein expression is reduced in the hippocampus of rats exposed to Pb2+ during development

Chronic exposure to lead (Pb2+) produces deficits of learning and memory in children and spatial learning deficits in developing rats. The N-methyl-D-aspartate receptor (NMDAR) has been identified as a principal target for Pb2+-induced neurotoxicity. Age-dependent changes in NMDAR subunit gene expression were observed in hippocampi of rats chronically exposed to Pb2+ during development [T.R. Guilarte, J.L. McGlothan, Hippocampal NMDA receptor mRNA undergoes subunit specific changes during developmental lead exposure, Brain Res. 790 (1998) 98-107]. These changes were present at blood Pb2+ levels ranging from 20-60 microg/dl. Littermates were used in the present study to determine whether the changes in gene expression were reflected in protein levels. NR1, NR2A, and NR2B subunit protein levels were measured in rat hippocampus and cortex at post-natal days (PND) 7, 14, 21, and 28 by Western blot and densitometric analysis. A treatment effect was apparent for NR2A subunit protein expression in the hippocampus (F1,28=10.224, p<0.01). NR2A subunit protein was reduced by 40%, 19%, and 27% from control levels in PND14, 21, and 28 Pb2+-exposed rats, respectively. Mean comparisons indicated that rats at PND14 exhibited the most significant reduction of NR2A (p<0.001). These data concur with our previous finding of reduced NR2A mRNA found in hippocampal pyramidal and granule cells of Pb2+-exposed rats. Pb2+ exposure during development had no effect on NR1 or NR2B subunit protein expression in the hippocampus at any age. No effect was observed on any subunit in the cortex at any age. The developmental profile of the NMDAR-2A subunit protein in the hippocampus is specifically changed by chronic exposure to Pb2+. These data suggest that composition of subunits comprising NMDAR may be altered in Pb2+-exposed rats.     

NR2B-containing NMDA receptors are up-regulated in temporal cortex in schizophrenia

Saturation analyses of [3H]L-689,560, [3H]CGP 39653 and NMDA-specific [3H]ifenprodil binding revealed an equivalent increase (0.7 pmol/mg) in the number of [3H]L-689,560 and [3H]ifenprodil binding sites in superior temporal cortex (BA22) from drug-treated chronic schizophrenic patients and control subjects. No differences were observed between control and schizophrenic subjects for [3H]CGP 39653 binding in BA22, or for any of the radioligands binding to pre-motor cortex (BA6). Since [3H]L-689,560, [3H]CGP 39653 and [3H]ifenprodil label the glycine, glutamate and ifenprodil sites of the NMDA receptor complex, which are associated with NR1, NR1/NR2A and NR1/NR2B subunits respectively, our findings suggest that NR2B-containing receptors are selectively up-regulated in superior temporal cortex in schizophrenia.     

Hippocampal expression of N-methyl-D-aspartate receptor (NMDAR1) subunit splice variant mRNA is altered by developmental exposure to Pb(2+)

N-methyl-D-aspartate receptors (NMDAR) play an important role in synaptic plasticity and brain development. We have previously shown that NR1-pan mRNA is significantly increased in the hippocampus of rats chronically exposed to low levels of lead (Pb(2+)) during development [T.R. Guilarte, J.L. McGlothan, Hippocampal NMDA receptor mRNA undergoes subunit specific changes during developmental lead exposure, Brain Res., 790 (1998) 98-107]. It is not known whether this Pb(2+)-induced increase in NR1-pan mRNA is associated with changes in specific splice isoforms. To study this effect, we used in situ hybridization of oligonucleotides to probe for the NR1-a, NR1-b, NR1-1, NR1-2, and NR1-4 isoforms which are most abundantly expressed in the rat hippocampus. Developmental exposure to increasing levels of Pb(2+) resulted in significant increases in NR1-a mRNA throughout the pyramidal and granule cell layers of the rat hippocampus at postnatal day 14 (PN14). NR1-b mRNA was increased in the pyramidal cell layer of Pb(2+)-exposed rats at PN21. Splicing of the C-terminus cassettes was also regulated by developmental exposure to Pb(2+). NR1-2 mRNA was increased in CA4 pyramidal cells and in dentate granule cells of PN21 Pb(2+)-exposed rats. Notably, expression of NR1-4 mRNA in CA3 pyramidal cells was increased in Pb(2+)-exposed rats at PN14 and decreased at PN21. No significant Pb(2+) effect was measured for NR1-1 mRNA expression. These data indicate that alternative splicing of the NR1 gene shows selective anatomical and temporal regulation by Pb(2+) in the developing rat hippocampus. This study provides further support to the hypothesis that NMDARs are important targets for Pb(2+)-induced neurotoxicity.     

Autoradiographic evidence that NMDA receptor-coupled channels are located postsynaptically and not presynaptically in the perforant path-dentate granule cell system of the rat hippocampal formation

In vitro quantitative autoradiography with [3H]MK-801 was used to determine Kd and Bmax values for the NMDA receptor-coupled channel in subregions of the rat hippocampal formation. A single form of the channel with an apparent Kd in the 15-20 nM range was found for [3H]MK-801 binding in the presence of both 1 microM glutamate and 1 microM glycine. Specific binding was highest in the molecular layer of the dentate gyrus, followed by CA1 stratum radiatum and CA1 stratum oriens. Fewer binding sites were observed in the hilus of the dentate gyrus, cerebral cortex, CA1 stratum pyramidale, CA3 subregion (stratum oriens, stratum pyramidale, stratum radiatum), and thalamus. Selective destruction of dentate granule cells by colchicine microinjections reduced the amount of specific [3H]MK-801 binding by half in the molecular layer of the dentate, compared to intact tissue. [3H]MK-801 binding did not change in other hippocampal subregions as a consequence of colchicine injection. Electrolytic entorhinal cortical lesions produced no changes in regional MK-801 binding site density in any of the regions under study. To address the tissue shrinkage following entorhinal cortex lesions, detailed analysis of the binding site density per fixed (16 microns) length of granule cell dendrite, and of the aggregate density across the entire molecular layer revealed no change in the number of MK-801 binding sites per unit length of dendrite in the molecular layer of the dentate gyrus. These findings indicate that NMDA receptor-coupled channels are confined to a postsynaptic location in the perforant path-dentate granule cell system of the adult rat.     

Delayed onset of enhanced MK-801-induced motor hyperactivity after neonatal lesions of the rat ventral hippocampus.

BACKGROUND: Abnormalities in the glutamatergic system, glutamate/dopamine/gamma-aminobutyric acid interactions, and cortical development are implicated in schizophrenia. Moreover, patients with schizophrenia show symptom exacerbation in response to N-methyl-D-aspartate (NMDA) antagonist drugs. Using an animal model of schizophrenia, we compared the impact of neonatal and adult hippocampal lesions on behavioral responses to MK-801, a noncompetitive NMDA antagonist. METHODS: Neonatal rats were lesioned on postnatal day 7. Their motor activity in response to MK-801 was tested at a juvenile age, in adolescence, and in adulthood. We also measured binding of [(3)H]MK-801 and the expression of NR1 messenger RNA (mRNA) in the medial prefrontal cortex and nucleus accumbens. Adult rats received similar lesions and were tested 4 and 8 weeks after the lesion. RESULTS: As juveniles, neonatally lesioned rats did not differ from control rats in responsiveness to MK-801, whereas in adolescence and adulthood they showed more pronounced hyperactivity than control rats. The adult lesion did not alter behaviors elicited by MK-801. Neonatally lesioned rats showed no apparent changes in [(3)H]MK-801 binding or expression of the NR1 mRNA. CONCLUSIONS: These results suggest that an early lesion of the ventral hippocampus affects development of neural systems involved in MK-801 action without changes at the NMDA receptor level, and they show that the behavioral changes manifest first in early adulthood.     

Postnatal stress selectively upregulates striatal N-methyl-D-aspartate receptors in male rats

Early life events have been thought to contribute towards vulnerability to drug addiction later in life. In the present investigation, the effect of daily neonatal maternal isolation stress on NMDA channel activity was studied. [3H]MK-801 binding was measured in several brain regions from neonatally isolated (ISO) and nonhandled (NH) adult male and female rats. Maximal [3H]MK-801 binding in the caudate-putamen of male ISO rats was 58% higher compared to same sex NH rats. Unlike male rats, maximal [3H]MK-801 binding in the caudate-putamen of female ISO rats was lower than female NH rats. No other brain region showed any significant difference in maximal [3H]MK-801 binding between ISO and NH male and female rats, respectively. There was no effect of pup isolation on the binding affinity (K(d) value) in either sex. Repeated maternal isolation is associated with alterations in the NMDA channel activity in the caudate-putamen of adult rats, and may be responsible for the augmentation in the addictive behavior reported.     

The Ras inhibitor S-trans, trans-farnesylthiosalicylic acid exerts long-lasting neuroprotection in a mouse closed head injury model.

Traumatic brain injury activates N-methyl-d-aspartate receptors (NMDAR) inducing activation of the Ras protein (a key regulator of cell growth, survival, and death) and its effectors. Thus, trauma-induced increase in active Ras-GTP might contribute to traumatic brain injury pathology. Based on this hypothesis, a new concept of neuroprotection is proposed, examined here by investigating the effect of the Ras inhibitor S-trans, trans-farnesylthiosalicylic acid (FTS) in a mouse model of closed head injury (CHI). Mice subjected to CHI were treated systemically 1 h later with FTS (5 mg/kg) or vehicle. After 1 h, Ras-GTP in the contused hemisphere showed a significant (3.8-fold) increase, which was strongly inhibited by FTS (82% inhibition) or by the NMDA-receptor antagonist MK-801 (53%). Both drugs also decreased active (phosphorylated) extracellular signal-regulated kinase. FTS prevented the CHI-induced reduction in NMDAR binding in cortical, striatal, and hippocampal regions, measured by [3H]-MK-801 autoradiography, and decreased lesion size by 50%. It also reduced CHI-induced neurologic deficits, indicated by the highly significant (P < 0.0001) 60% increase in extent of recovery. Thus, FTS provided long-term neuroprotection after CHI, rescuing NMDAR binding in the contused hemisphere and profoundly reducing neurologic deficits. These findings suggest that nontoxic Ras inhibitors such as FTS may qualify as neuroprotective drugs.     

Effects of subacute lead exposure on [H]MK-801 binding in hippocampus and cerebral cortex in the adult rat

We used the NMDA receptor non-competitive antagonist, [³H]MK-801, as a ligand for an autoradiographic study to determine the effects of lead on NMDA receptor in the rat brain. Adult male rats were administered lead acetate, 100 mg/kg, or sodium acetate, 36 mg/kg (control), by i.p. for 7 days. High lead levels were detected in blood (41.1 μg/dl) and in brain (16.7–29.4 μg/g). Concentrations of lead in brain regions were not significantly different. The [³H]MK-801 binding was heterogeneously distributed throughout the rat brain with the following order of binding densities: hippocampal formation>cortex>caudate-putamen>thalamus>brainstem. Lead exposure produced a significant decrease in [³H]MK-801 binding to the NMDA receptor in the hippocampal formation including CA2 stratum radiatum, CA3 stratum radiatum, hilus dentate gyrus and presubiculum, and in the cerebral cortex including agranular insular, cingulate, entorhinal, orbital, parietal and perirhinal areas. The hippocampal formation is known as a critical neural structure for learning and memory processes, whereas, cortical and subcortical regions have been demonstrated to be involved in the modulation of complex behavioral processes. The NMDA receptor has been demonstrated to play a key role in synaptic plasticity underlying learning and memory. Lead-induced alterations of NMDA receptors in the hippocampal formation and cortical areas may play a role in lead-induced neurotoxicity.     

The novel anticonvulsant MK-801: a potent and specific ligand of the brain phencyclidine/sigma-receptor

MK-801 (5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate) is a novel anticonvulsant agent reported to antagonize certain N-methyl-D-aspartate (NMDA)-mediated effects non-competitively. The question arises of the mechanism underlying the anti-NMDA and anticonvulsant effects of MK-801. In the present study MK-801 is shown to be an extremely potent inhibitor of the binding of N-[3H] (1-[2-thienyl]cyclohexyl)piperidine ([3H]TCP) to brain phencyclidine (PCP)/sigma-receptors. Its IC50 value of 3.8 +/- 0.8 nM in this assay ranks it as the most potent known ligand of brain PCP/sigma-receptors. Addition of MK-801 altered the apparent Kd but not the apparent Bmax values for [3H]TCP binding, indicating a competitive interaction. The specificity of action of MK-801 is supported by the finding that MK-801 strongly inhibited the binding of (+)-N-[3H]allylnormetazocine ((+)-[3H]SKF 10,047) to the PCP/sigma-receptor but its effect on (+)-[3H]SKF 10,047 binding to the non-PCP, haloperidol-sensitive sigma-binding site was weaker by several orders of magnitude. Furthermore, MK-801 exerts PCP-like antagonistic effects upon NMDA-induced [3H]norepinephrine release. These findings support the concept that the anticonvulsant and anti-NMDA effects of MK-801 result from its being the most potent known ligand of PCP/sigma-receptors.     

Potential involvement of tyrosine kinase pathway in the antagonist induced upregulation of the NMDA receptor NR2B subunit in cortical neurons

The aim of this study was to study the potential mechanism(s) involved in the antagonist induced upregulation of the N-methyl-d-aspartate receptor (NMDA) NR2B subunit. The results show that chronic treatment of cortical neurons with tyrosine kinase inhibitor (genistein) resulted in downregulation of the NR2B subunit polypeptide levels, while daidzein, an inactive analog of genistein, did not alter the levels of NR2B subunit, implying that tyrosine kinases may be involved in the regulation of the NMDA NR2B subunit content. Chronic treatment of cortical neurons with the NMDA receptor antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a, d]cycloheptane-5,10-iminemaleate (MK-801) enhanced the membrane associated tyrosine kinase activity and upregulated the NR2B receptor subunit. These results suggest that MK-801 induced upregulation of NMDA (NR2B) receptor subunit might be mediated by tyrosine kinases.     

Analysis of NMDA Receptors in the Human Spinal Cord

NMDA receptors in postmortem human spinal cord were analyzed using [³H]MK-801 ligand binding and immunoblotting with NMDA receptor subunit-specific antibodies. The averageK_Dfor [³H]MK-801 binding was 1.77 nM with aB_maxof 0.103 pmol/mg. The EC₅₀for stimulation of [³H]MK-801 binding withl-glutamate was 0.34 μM. None of these parameters were affected by postmortem intervals up to 72 h. Immunoblotting of native NMDA receptors showed that NR1, NR2A, NR2C, and NR2D subunits could all be found in the human spinal cord of which NR1 was preferentially located to the dorsal half. Immunoprecipitation of solubilized receptors revealed that NR1, NR2C, and NR2D subunits coprecipitated with the NR2A subunit, indicating that native human spinal cord NMDA receptors are heteroligimeric receptors assembled by at least three different receptor subunits. These results provide a basis for the development of drugs selectively aimed at spinal cord NMDA receptors for the future treatment of spinal cord disorders.