Biexponential kinetics of [3H]MK-801 binding: evidence for access to closed and open N-methyl-D-aspartate receptor channels

The phencyclidine (PCP) receptor is a site within the ion channel gated by the N-methyl-D-aspartate (NMDA)-type excitatory amino acid receptor. In the present study, kinetics of association and dissociation of the specific PCP receptor ligand [3H]MK-801 were determined in order to elucidate the mechanism of functioning of the NMDA receptor complex. Two distinct components of [3H]MK-801 association with apparent t1/2 values of approximately 10 min and 3 hr were resolved. Incubation with the NMDA receptor agonist L-glutamate increased the total steady state binding of [3H]MK-801 and increased the relative percentage of [3H]MK-801 binding that manifested fast rather than slow kinetics, without altering the observed rate constant of either the fast or slow component of association. The competitive NMDA receptor antagonist D(-)-2-amino-5-phosphonovaleric acid decreased total steady state binding of [3H]MK-801. These data support a model in which [3H]MK-801 can gain access to its binding site via two distinct paths, a fast hydrophilic path associated with a conformation of the NMDA receptor in which the channel is open and a slow hydrophobic path independent of the open channel. In the presence of L-glutamate, incubation with glycine increased the relative percentage of [3H]MK-801 binding that manifested fast rather than slow kinetics. The Hill coefficient for stimulation of specific [3H]MK-801 binding by L-glutamate was significantly greater than unity in either the absence or presence of glycine. Our data support a model of NMDA receptor functioning in which two molecules of agonist are required to convert the receptor complex to a conformation that is in equilibrium with the open conformation and in which glycine regulates the percentage of NMDA receptor complexes bound to two molecules of agonist that convert to the open configuration.     

Spermidine enhancement of [3H]MK-801 binding to the NMDA receptor complex in human cortical membranes

The effects of spermidine on the binding of [3H]MK-801 to the N-methyl-D-aspartate (NMDA) receptor complex was studied in human cerebral cortical membranes. [3H]MK-801 binding was increased from 56 +/- 5 fmol/mg protein (mean +/- S.E.M., n = 7) to 319 +/- 71 fmol/mg protein in the presence of 200 microM spermidine. The ED50 for spermidine stimulation of [3H]MK-801 binding was 89 +/- 22 microM (mean +/- S.E.M., n = 6). In the presence of glutamate (1 microM) plus glycine (1 microM) the ED50 was reduced to 5.5 +/- 0.7 microM. The increase in binding in the presence of spermidine was characterised by an increase in the rate of association of [3H]MK-801. In the presence of spermidine. [3H]MK-801 was inhibited by AP5. 7-chlorokynurenic acid and ifenprodil with IC50 values of 0.5 +/- 0.3 24 +/- 19 and 91 +/- 28 microM, respectively. None of these antagonists was a competitive inhibitor of the spermidine stimulation of [3H]MK-801 binding. Thus spermidine modulates the NMDA receptor complex in human brain, providing further evidence that the complex is similar in rat and human cortex.     

Effects of chronic lead exposure on [3H]MK-801 binding in the brain of rat

We have used quantitative autoradiographic methods to determine the effects of chronic lead exposure on N-methyl-d-aspartate (NMDA) receptors in the brain of female rat. Rats were exposed pre- and post-natally from day 4±1 post conception with 1000 ppm lead in their drinking water. This treatment continued after weaning. No effects of lead on [³H]MK-801 binding were found at PN 28. However, lead caused a significant increase in [³H]MK-801 binding in the hippocampus including CA1 and CA2, and in the occipital and temporal cortical areas at PN 56 and at PN 112. An increase in binding was also found in the entorhinal cortex and the dentate gyrus at PN 112. Because the NMDA receptor is involved in learning and memory, the lead-induced disruption of NMDA receptors in the hippocampus and cortex may be associated with the cation-induced cognition deficits.     

Polyamine and redox modulation of [3H]MK-801 binding to N-methyl-D-aspartate receptors in the spinal cord and cerebral cortex.

The pharmacology of N-methyl-D-aspartate (NMDA) receptors shows regional differences in affinity for various agonists and antagonists. We have investigated the modulatory mechanisms acting via the polyamine, redox and proton sites in the cerebral cortex and the spinal cord of adult, male rats using [3H]MK-801 binding. The affinity for glycine-independent spermine stimulation was one magnitude higher in cerebrocortical than in spinal cord membranes while the affinity for the spermine antagonist arcaine was similar. Spermine abolished the inhibiting effect of low pH in both regions. Thus, the difference in the polyamine site between the two regions seems to be restricted to agonist binding. The proportion of high affinity/total ifenprodil binding was approximately 35% both in the spinal cord and the cerebral cortex, suggesting similar relative amounts of the NMDA receptor subunit 2B. The affinity of ifenprodil to the high affinity site was however significantly higher in the cerebral cortex. Redox modulatory agents had similar effects in the two regions but spermine fully counteracted the inhibiting effect of 0.2 mM 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) in the cerebral cortex while there was only a partial effect in the spinal cord. These data show that the regional pharmacological heterogeneity involves several of the mechanisms regulating the function of the NMDA receptor. The data also indicate that the NMDA receptor subunit 2B is much more common in spinal cord than previously suggested.     

The putative anti-addictive drug ibogaine is a competitive inhibitor of [3H]MK-801 binding to the NMDA receptor complex

Ibogaine is a putative anti-addictive drug with potential efficacy for the treatment of opiate, stimulant, and alcohol abuse. We now report ibogaine is a competitive inhibitor (K_i, 1.01±0.1 µM) of [³H]MK-801 binding toN-methyl-D-aspartate (NMDA) receptor coupled cation channels. Since MK-801 can attenuate the development of tolerance to morphine and alcohol as well as sensitization to stimulants in preclinical studies, the reported ability of ibogaine to modify drug-seeking behavior in man may be attributable to a blockade of NMDA receptor coupled cation channels.P. Popik is a Fogarty Visiting Fellow on leave from Institute of Pharmacology, Polish Academy of Sciences, Kraków, Poland.     

Differential binding properties of [3H]dextrorphan and [3H]MK-801 in heterologously expressed NMDA receptors

The N-methyl-D-aspartate receptor (NMDAR) antagonists: MK-801, phencyclidine and ketamine are open-channel blockers with limited clinical value due to psychotomimetic effects. Similarly, the psychotomimetic effects of the dextrorotatory opioids, dextromethorphan and its metabolite dextrorphan, derive from their NMDAR antagonist actions. Differences in the use dependency of blockade, however, suggest that the binding sites for MK-801 and dextrorphan are distinct. In the absence of exogenous glutamate and glycine, the rate of association of [3H]MK-801 with wild-type NR1-1a/NR2A receptors was considerably slower than that for [3H]dextrorphan. Glutamate individually, and in the presence of the co-agonist glycine, had substantial effects on the specific binding of [3H]MK-801, while the binding of [3H]dextrorphan was not affected. Mutation of residues N616 and A627 in the NR1 subunit had a profound effect on [3H]MK-801 binding affinity, while that of [3H]dextrorphan was unaltered. In contrast, NR1 residues, W611 and N812, were critical for specific binding of [3H]dextrorphan to NR1-1a/NR2A complexes with no corresponding influence on that of [3H]MK-801. Thus, [3H]dextrorphan and [3H]MK-801 have distinct molecular determinants for high-affinity binding. The ability of [3H]dextrorphan to bind to a closed channel, moreover, indicates that its recognition site is shallower in the ion channel domain than that of MK-801 and may be associated with the extracellular vestibule of the NMDAR.     

Modulation of [3H]MK-801 binding to NMDA receptors in vivo and in vitro

[3H]MK-801 binding in vivo was used to determine the occupancy of NMDA receptor ligands shown to allosterically modulate binding in vitro. ED(50) values (mg/kg) were obtained for the channel blockers (+)-5-methyl-10,11-dihydro-5,4-dibenzo[a,d]cyclohepten-5,10-imine maleate ((+)-MK-801, 0.2), 1-(1-phenylcyclohexyl)piperidine (phencyclidine, PCP, 1.7) and ketamine (4.4). Antagonists at the glutamate (DL-(2-carboxypiperazine-4-yl)propyl-1-phosphonate (DL-CPP, 5.7)) and glycine site (7-Chloro-4-hydroxy-3-(3-phenoxy)-phenyl-2(H)quinolinone (L-701,324, 14.1), 3R(+)cis-4-methyl-pyrrollid-2-one (L-687,414, 15.1)) inhibited [3H]MK-801 binding in vivo to varying maximum levels (69%, 103% and 45%, respectively). NR2B subunit-selective compounds acting at the ifenprodil site inhibited [3H]MK-801 in vivo by a maximum of 52-72% and gave ED(50) values (mg/kg) of: (+/-)-(1S*, 2S*)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol ((+/-)CP-101,606), 1.9; (+/-)-(3R, 4S)-3-[4-(4-fluorophenyl)-4-hydroxypiperidin-1-yl]chroman-4,7-diol ((+/-)CP-283,097), 1.8; (+/-)-(R*, S*)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperidine propanol ((+/-)Ro 25-6981), 1.0; ifenprodil, 6.0. The glycine site agonist D-serine stimulated binding to 151% of control with an ED(50) of 1.7 mg/kg. Results show that [3H]MK-801 binding in vivo may be used to measure receptor occupancy of ligands acting not only within the ion channel but also at modulatory sites on the NMDA receptor complex.     

Effects of putative cognition enhancers on the NMDA receptor by [3H]MK801 binding

Piracetam, aniracetam, and d-cycloserine were tested for their ability to reduce inhibition of [³H]MK801 (dizocilpine) binding by 100 μM kynurenate. Piracetam (100 μM-1 mM) failed to reduce inhibition by kynurenate but stimulated [³H]MK801 binding in the absence of kynurenate. In contrast, d-cycloserine (30 μM-1 mM) and aniracetam markedly reduced this inhibition by kynurenate. Thus, cognition enhancers might function via at least some subtypes of NMDA receptors.     

In vivo labelling of the NMDA receptor channel complex by [3H]MK-801

An in vivo radioligand binding assay for the N-methyl-D-aspartate (NMDA) receptor channel complex in the mouse brain has been developed using the non-competitive NMDA receptor antagonist [3H]MK-801. In vivo binding of [3H]MK-801 was displaced by MK-801 (ED50 = 0.17 mg/kg i.p.), (-)-MK-801 (1.0 mg/kg), thienylcyclohexylpiperidine (1.8 mg/kg), etoxadrol (5.1 mg/kg) and (+)-SKF 10,047 (34.5 mg/kg). The potency of these drugs in this in vivo binding assay was highly correlated (r = 0.97) with their functional effects as antagonists of N-methyl-DL-aspartate-induced tonic convulsions.     

Biochemical and thermodynamic aspects of the binding of [3H]glycine to its strychnine-insensitive recognition site associated with the N-methyl-D-aspartate receptor complex.

The molecular mechanism of interaction between glycine and its strychnine-insensitive binding site linked to the N-methyl-D-aspartate receptor was investigated by examining on the one hand the thermodynamic properties of glycine binding, and, on the other hand, the effects of various functional group modifying agents on ligand binding. Raising the incubation temperature from 0 degrees to 37 degrees resulted in a consistent decrease of glycine binding affinity. Calculation of thermodynamic parameters from the corresponding Van't Hoff plot showed that the binding of glycine was mainly entropy-driven, the change in enthalpy contributing only little (25-30%) to the change in Gibbs free energy. Chemical modification with the sulfhydryl-directed agents p-hydroxy-mercuribenzoate and N-ethyl-maleimide showed free -SH groups to be critical for ligand binding to the receptor site. Furthermore, guanidino groups on arginyl residues, sensitive to 2,3-butanedione, were also found to participate in glycine binding. Both the -SH and the guanidino groups could be protected against their inactivation by co-incubation with glycine, indicating a direct involvement of these functional groups in the binding process. Dithiothreitol, a disulfide-reducing agent, likewise prevented [3H]glycine binding, suggesting that the glycine recognition site is stabilized by at least one disulfide bridge. It is concluded that the binding of glycine probably involves a strong ion-ion interaction between its carboxyl group and a positively charged guanidino group at the receptor site, resulting in a thermodynamically favorable increase in entropy by displacement of water molecules from the latter and a concomitant decrease in enthalpy. Furthermore, at least one free sulfhydryl group seems to participate in the binding process.     

Possible involvement of membrane phospholipids in inhibition by ferrous ions of [3H]MK-801 binding to the native N-methyl-D-aspartate channel in rat brain.

Prior treatment with ferrous chloride led to the marked inhibition of [3H](+)-5-methyl-10, 11-dihydro-5H-dibenzo[a, d]cyclohepten-5, 10-imine (MK-801) binding to an open ion channel associated with the N-methyl-D-aspartate (NMDA) receptor in a concentration-dependent manner at concentrations of higher than 1 microM in rat brain synaptic membranes. Both phospholipases A2 and C significantly prevented the inhibition when treated before the treatment with ferrous chloride, while neither superoxide dismutase nor alpha-tocopherol affected the inhibition even when treated simultaneously with ferrous chloride. Of various saturated and unsaturated fatty acids, moreover, both oleic and arachidonic acids exclusively decreased the potency of ferrous chloride to inhibit binding when membranes were treated with fatty acids, followed by a second treatment with ferrous chloride. These results suggest that membrane phospholipids may be, at least in part, responsible for the interference by ferrous ions in the opening processes of the native NMDA channel through molecular mechanisms associated with the release of unsaturated fatty acids in rat brain.     

Effects of several cerebroprotective drugs on NMDA channel function: evaluation using Xenopus oocytes and [3H]MK-801 binding.

The effects of several cerebroprotective and nootropic drugs on the function of excitatory amino acid (EAA) receptor subtypes expressed in Xenopus oocytes after injection of rodent brain poly(A)+ mRNA were investigated. The oocyte response to N-methyl-D-aspartate (NMDA) in the presence of glycine (Gly) was inhibited dose-dependently by bifemelane, indeloxazine, vinpocetine and vincamine while no effect was observed by idebenone, Ca hopantenate, aniracetam or piracetam. Bifemelane, indeloxazine and vinpocetine suppressed the maximum response of NMDA and Gly without affecting their EC50 values. Unlike Mg2+, they did not affect the current-voltage relationship of the NMDA response below 0 mV. On the non-NMDA-type responses of the injected oocytes to kainate (KA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and quisqualate (QA), no significant effects were observed by these drugs at 100 microM. On the binding of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne (MK-801) to brain membranes, the estimated IC50 values were 88 microM for bifemelane, 102 microM for indeloxazine, and 115 microM for vinpocetine. The dissociation rate of [3H]MK-801 was significantly slowed by Zn2+ and vinpocetine, but not affected by bifemelane or indeloxazine. The Kd value for [3H]MK-801 binding was increased by bifemelane and indeloxazine while Bmax was unchanged. These results suggest that the inhibition of NMDA channels by vinpocetine shows a similarity to the action of Zn2+ which closes the gate of the NMDA channel. In contrast, bifemelane and indeloxazine may affect the phencyclidine (PCP)-site in the open channels and inhibit NMDA function.     

Binding of [3H]MK-801 in subcellular fractions of Schistosoma mansoni: evidence for interaction with nicotinic receptors

Several studies have suggested that l-glutamate is a putative neurotransmitter in Schistosoma mansoni. Recently, we detected the presence of low-affinity binding sites for [(3)H]kainic acid in the heterogeneous (P(1)) subcellular fraction of S. mansoni. In an attempt to characterize N-methyl-d-aspartate (NMDA) receptors in this worm, we performed binding assays with [(3)H]MK-801, a NMDA non-competitive antagonist, in the P(1) fraction of adult S. mansoni. In competition experiments, MK-801 (IC(50) approximately 200 microM) and ketamine (IC(50) approximately 500 microM) exhibited a low affinity for the sites labeled with [(3)H]MK-801. Along with the lack of modulation of this binding by glutamatergic agonists and antagonists and the absence of stereoselectivity for MK-801 isomers, these results suggest that [(3)H]MK-801 could label a site different from the classical NMDA receptor in S. mansoni. Based on the evidences that MK-801 interacts with mammalian muscle and central nervous system nicotinic receptors as a low-affinity noncompetitive antagonist, we have investigated the effects of MK-801 on the nicotine-induced flaccid paralysis of the worm, in vivo. The motility of S. mansoni was quantified by image analysis through a measure of displacement of the worm's extremities. In the presence of (-)-nicotine (10-100 microM), we observed an immediate paralysis of the worms, that was inhibited by 1mM MK-801. Besides nicotine, choline (10-50mM) was also able to inhibit the worm's motility. As a conclusion, we suggest that [(3)H]MK-801 binds to nicotinic receptors, and not NMDA receptors, in subcellular fractions of S. mansoni.     

ACTH binds to [3H]MK-801-labelled rat hippocampal NMDA receptors.

The molecular mechanism of the clinical antiepileptic/antimyoclonic action of adrenocorticotrophic hormone (ACTH) is unknown. To explore the possible role of excitatory amino acid receptors, we studied the influence of ACTH and ACTH fragments in vitro on the binding of [3H]MK-801 to rat hippocampus, a region relevant to epilepsy. ACTH-(1-39), ACTH-(1-24), and ACTH-(1-17) displayed micromolar affinities compared to the nanomolar affinity of MK-801, whereas ACTH-(4-10) and four clinically used anticonvulsants were inactive. These findings are not specific for NMDA receptors but conform to the rank order of potency of ACTH fragments at other receptor binding sites.     

Effects of mono-, di-, and triamines on the N-methyl-D-aspartate receptor complex: a model of the polyamine recognition site.

Systematic series of monoamines, diamines, and triamines were used to define the structural requirements for interaction at the polyamine recognition site of the N-methyl-D-aspartate receptor complex. Effects of amines on binding of [3H]MK-801 to washed synaptic plasma membranes were measured in the presence of L-glutamate and glycine (100 microM each), in the absence or presence of spermine (10 microM). Linear aliphatic monoamines of methylene chain length up to 12 (dodecylamine) did not interact with the polyamine recognition site. Nonspecific inhibition of binding was observed at high concentrations of the longer monoamines. alpha,omega-Diamines of methylene chain length 2 (1,2-diaminoethane, DA2) through 12 (1,12-diaminododecane, DA12) had varying actions, depending on chain length. The shortest diamines (DA2 and DA3) acted as weak partial agonists, enhancing the binding of [3H[MK-801. Intermediate-length diamines (DA4-DA7) were selective polyamine antagonists, having little or no effect on binding of [3H]MK-801 measured in the absence of spermine but inhibiting binding measured in the presence of spermine. The longest diamines tested (DA8-DA12) acted as inverse agonists; they inhibited binding in the absence or presence of spermine, and this inhibition was blocked by the selective polyamine antagonist diethylenetriamine. Computer modeling of conformations of the diamines quantitatively documented that 1) these molecules are flexible and 2) long diamines may easily adopt conformations with inter-nitrogen distances mimicking those of short diamines. The cis and trans isomers of 1,4-diaminocyclohexane are inflexible, conformationally restricted diamines with markedly different actions. The cis isomer was a partial agonist and the trans isomer was an antagonist at the polyamine recognition site. Triamines of general structure NH2(CH2)3NH(CH2)xNH2 (TRI[3,x]), in which x = 3-12, were synthesized and tested for activity at the polyamine recognition site. Despite the large range of size, TRI[3,3] through TRI[3,9] were all fully polyamine agonists of similar potency. TRI[3,10] was a partial agonist, whereas TRI[3,12] inhibited binding of [3H]MK-801. Diethylenetriamine did not attenuate the effect of TRI[3,12]. Based on the results of the radioligand binding studies and the computer analysis, a model of the polyamine recognition site is proposed.     

Characterization, localization and pharmacological profile of a high-affinity [3H]lidocaine binding site

Electrophysiological findings support the existence of voltage-dependent, sodium channel-associated receptors for class I antiarrhythmics. We have tried to identify such receptors with tritiated lidocaine. High-affinity binding sites were discovered in heart and brain membranes, but liver and kidney particulate fractions had the highest density of sites. The dissociation constants were 75 nM in bovine heart and 29 nM in guinea-pig liver membranes. Binding was reversible (t 1/2: 102 s at 2 degrees C), optimal at pH 9-10 and was only partly destroyed by heat treatment. Subcellular fractionation experiments excluded a plasmalemmal association of the lidocaine site in heart. The competition profile of 16 antiarrhythmics indicated chemical comparability of the sites in heart and liver. These data greatly challenge the applicability of labeled lidocaine as sodium channel probe. The pharmacological significance of the site described here remains to be clarified.     

Postnatal changes in N-methyl-D-aspartate receptor binding and stimulation by glutamate and glycine of [3H]-MK-801 binding in human temporal cortex.

1. Homogenates of human infant and adult temporal cortex were used to measure [3H]-TCP and [3H]-MK-801 binding to the N-methyl-D-aspartate (NMDA)-coupled ion channel phencyclidine site. 2. Both [3H]-TCP and [3H]-MK-801 binding increased in infant cortex by > 100% between term and 26 weeks suggesting that the numbers of NMDA receptors increase during postnatal brain development. 3. [3H]-MK-801 binding was measured under non-equilibrium conditions in temporal cortex homogenates with the addition of 100 microM of L-glutamate plus a range of concentrations (0.05 microM-100 microM) of glycine. Glutamate and glycine increased [3H]-MK-801 binding by stimulating NMDA receptors and improving [3H]-MK-801 access to ion channel binding sites; maximum stimulation in adult and infant temporal cortex was achieved with 100 microM glutamate plus 5 microM glycine; a higher concentration of glycine (50 microM) reduced [3H]-MK-801 binding to below maximum. 4. The stimulation by 100 microM glutamate plus 5 microM glycine of [3H]-MK-801 binding in infant temporal cortex was affected by postnatal age. For example, although the stimulation of [3H]-MK-801 binding in 5-6 week infant cortex (236% of basal) was similar to adult cortex (230% of basal), in samples taken from infants aged 5-6 months glycine (plus glutamate) stimulation of [3H]-MK-801 binding (392% of basal) was substantially greater than that measured in adult temporal cortex. 5. The binding of [3H]-glycine to the glycine modulatory site associated with the NMDA receptor in infant cortex also increased with postnatal age by > 100% between term and 26 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)     

The discriminative stimulus effects of MK-801: Generalization to other N-methyl-D-aspartate receptor antagonists

Rats were trained to discriminate a dose of the NMDA antagonist MK-801 from saline using a standard, two-lever, operant procedure. The acquisition of this discrimination was rapid, rats reaching criterion performance in a mean of 32 sessions. The discriminative stimulus produced by MK-801 generalized to phencyclidine and (+)N-allylnormetazocine but only low levels of generalization were seen with (-)N-allylnormetazocine and with the anti- ischaemic drug ifenprodil and its derivative SL 82.0715. The MK-801 stimulus was partially antagonized by haloperidol. The results show that MK-801 can produce a discriminative stimulus which is shared by other non-competitive NMDA antagonists which have similar behavioural effects in other procedures. However, ifenprodil and SL 82.0715, which have also been shown to antagonize the effects of NMDA, do not produce similar behavioural effects or discriminative stimuli presumably because they act through a different site.     

Differential binding of [3H]MK-801 to brain regions and spinal cord of mice treated chronically with morphine

• 1.1. The effects of morphine tolerance and abstinence on the binding of [³H]MK-801, a noncompetitive antagonist of the N-methyl-D-aspartate (NMDA) receptors were determined in brain regions and spinal cord of the mouse.
• 2.2. Male Swiss-Webster mice were rendered tolerant to and physically dependent on morphine by subcutaneous implantation of a pellet containing 75 mg of morphine base for 3 days. Placebo pellet-implanted mice served as controls. In tolerant (nonabstinent) mice, the pellets were left intact at the time of sacrificing whereas, in the abstinent mice, the pellets were removed 6 hr prior to sacrificing.
• 3.3. The binding of [³H]MK-801 to membranes prepared from spinal cord and brain regions (cortex, pons-medulla, hypothalamus, hippocampus, amygdala, striatum, and midbrain) was determined by using a 5 nM concentration of the ligand in the presence of 30 μM glycine and 50 μM of glutamate.
• 4.4. In nonabstinent morphine-tolerant mice, the binding of [³H]MK-801 was decreased in pons-medulla and hypothalamus, but was increased in the spinal cord in comparison to that in placebo controls. The reduction in binding in pons-medulla was due to a decrease in the B_max value; the K_d value remained unchanged. The binding of [³H]MK-801 was increased in the hippocampus of morphine-abstinent mice.
• 5.5. These studies demonstrate that NMDA receptors of brain regions and spinal cord are differentially affected in morphine-tolerant and abstinent mice.