Less NMDA Receptor Binding in Dorsolateral Prefrontal Cortex and Anterior Cingulate Cortex Associated With Reported Early-Life Adversity but Not Suicide.

BACKGROUND: Glutamate is an excitatory neurotransmitter binding to 3 classes of receptors, including the N-methyl, D-aspartate (NMDA) receptor. NMDA receptor binding is lower in major depression disorder and suicide. NMDA receptor blocking with ketamine can have antidepressant and anti-suicide effects. Early-life adversity (ELA) may cause glutamate-mediated excitotoxicity and is more common with major depression disorder and in suicide decedents. We sought to determine whether NMDA-receptor binding is altered with suicide and ELA. METHODS: A total 52 postmortem cases were organized as 13 quadruplets of suicide and non-suicide decedents matched for age, sex, and postmortem interval, with or without reported ELA (≤16 years). Tissue blocks containing dorsal prefrontal (BA8), dorsolateral prefrontal (BA9), or anterior cingulate (BA24) cortex were collected at autopsy. Psychiatrically healthy controls and suicide decedents underwent psychological autopsy to determine psychiatric diagnoses and details of childhood adversity. NMDA receptor binding was determined by quantitative autoradiography of [3H]MK-801 binding (displaced by unlabeled MK-801) in 20-µm-thick sections. RESULTS: [3H]MK-801 binding was not associated with suicide in BA8, BA9, or BA24. However, [3H]MK-801 binding with ELA was less in BA8, BA9, and BA24 independent of suicide (P < .05). [3H]MK-801 binding was not associated with age or postmortem interval in any brain region or group. CONCLUSIONS: Less NMDA receptor binding with ELA is consistent with the hypothesis that stress can cause excitotoxicity via excessive glutamate, causing either NMDA receptor downregulation or less receptor binding due to neuron loss consequent to the excitotoxicity.

Age-dependent effects of (+)-MK801 treatment on glutamate release and metabolism in the rat medial prefrontal cortex.

NMDAR antagonist treatments in adolescent/young adult rodents are associated with augmented glutamate (Glu) release and perturbed Glu/glutamine (Gln) metabolism in the medial prefrontal cortex (mPFC) resembling those found in first-episode schizophrenia. Few studies, however, investigated NMDAR antagonist-induced changes in the adult mPFC and whether there is an age-dependence to this end. In this study, the effects of acute/repeated (+)-MK801 treatment on Glu release/metabolism were measured in the mPFC of male adolescent (postnatal day 30) and adult (14 weeks) rats. Acute (+)-MK801 treatment at 0.5 mg/kg body weight induced an approximately 4-fold increase of extracellular Glu concentration in the adolescent rats, and repeated treatment for 6 consecutive days significantly increased the levels of Glu + Gln (Glx) and glial metabolites 7 days after the last dose. Histologically (+)-MK801 treatments induced reactive astrocytosis and elevated oxidative stress in the mPFC of adolescent rats, without causing evident neuronal degeneration in the region. All (+)-MK801-induced changes observed in the mPFC of adolescent rats were not present or evident in the adult rats, suggesting that the treatments might have caused less disinhibition in the adult mPFC than in the adolescent mPFC. In conclusion, the effects of (+)-MK801 treatments on the Glu release/metabolism in the mPFC were found to be age-dependent; and the adult mPFC is likely equipped with more robust neurobiological mechanisms to preserve excitatory-inhibitory balance in response to NMDAR hypofunction.

Mechanism of NMDA receptor channel block by MK-801 and memantine.

The NMDA (N-methyl-D-aspartate) receptor transduces the binding of glutamate and glycine, coupling it to the opening of a calcium-permeable ion channel 1 . Owing to the lack of high-resolution structural studies of the NMDA receptor, the mechanism by which ion-channel blockers occlude ion permeation is not well understood. Here we show that removal of the amino-terminal domains from the GluN1-GluN2B NMDA receptor yields a functional receptor and crystals with good diffraction properties, allowing us to map the binding site of the NMDA receptor blocker, MK-801. This crystal structure, together with long-timescale molecular dynamics simulations, shows how MK-801 and memantine (a drug approved for the treatment of Alzheimer's disease) bind within the vestibule of the ion channel, promote closure of the ion channel gate and lodge between the M3-helix-bundle crossing and the M2-pore loops, physically blocking ion permeation.

Convergent Strategy to Dizocilpine MK-801 and Derivatives.

A convergent total synthesis of MK-801 has been achieved. Key synthetic transformations include a multicomponent Barbier-type reaction to construct the α-branched amine, a selective Heck α-coupling tactic to generate the exocyclic alkene skeleton, and a late-stage intramolecular hydroamination reaction between the exocyclic alkene and the secondary protected amine. The efficacy of this method was demonstrated by the synthesis of two news analogues substituted on the aromatic rings.

The synergistic effect of treatment with triptolide and MK-801 in the rat neuropathic pain model.

Triptolide (T10), an active component of Tripterygium wilfordii Hook F, is reported to have potent anti-inflammatory and analgesic effects. Additionally, MK-801, a noncompetitive N-methyl-D-aspartate receptor antagonist, can reduce glutamate toxicity and has a significant analgesic effect on chronic pain. In this study, we tested the possible synergistic analgesic ability by intrathecal administration of T10 and MK-801 for the treatment of neuropathic pain. Single T10 (3, 10, or 30 µg/kg), MK-801 (10, 30, or 90 µg/kg), or a combination of them were intrathecally administrated in rats with spinal nerve ligation. We found that single administration of T10 caused a slow-acting but long-term analgesic effect, while single administration of MK-801 caused a fast-acting but short-term effect. Administration of their combination showed obviously synergic analgesia and the 1:3 ratio of T10 to MK-801 reached the peak effect. Furthermore, application of T10 and/or MK-801 significantly inhibited the activation of microglia and astrocyte and phosphorylation of STAT3 and NR2B in the spinal dorsal horn induced by chronic neuropathic pain. Our data suggest that the combination of T10 and MK-801 may be a potentially novel strategy for treatment of neuropathic pain.

The mGluR2 positive allosteric modulator, SAR218645, improves memory and attention deficits in translational models of cognitive symptoms associated with schizophrenia.

Normalization of altered glutamate neurotransmission through activation of the mGluR2 has emerged as a new approach to treat schizophrenia. These studies describe a potent brain penetrant mGluR2 positive allosteric modulator (PAM), SAR218645. The compound behaves as a selective PAM of mGluR2 in recombinant and native receptor expression systems, increasing the affinity of glutamate at mGluR2 as inferred by competition and GTPγ35S binding assays. SAR218645 augmented the mGluR2-mediated response to glutamate in a rat recombinant mGluR2 forced-coupled Ca2+ mobilization assay. SAR218645 potentiated mGluR2 agonist-induced contralateral turning. When SAR218645 was tested in models of the positive symptoms of schizophrenia, it reduced head twitch behavior induced by DOI, but it failed to inhibit conditioned avoidance and hyperactivity using pharmacological and transgenic models. Results from experiments in models of the cognitive symptoms associated with schizophrenia showed that SAR218645 improved MK-801-induced episodic memory deficits in rats and attenuated working memory impairment in NMDA Nr1neo-/- mice. The drug reversed disrupted latent inhibition and auditory-evoked potential in mice and rats, respectively, two endophenotypes of schizophrenia. This profile positions SAR218645 as a promising candidate for the treatment of cognitive symptoms of patients with schizophrenia, in particular those with abnormal attention and sensory gating abilities.

Antidepressant Effects of (+)-MK-801 and (-)-MK-801 in the Social Defeat Stress Model.

BACKGROUND: Current data on antidepressant action of the N-methyl-D-aspartate receptor antagonist, (+)-MK-801, is inconsistent. This study was conducted to examine the effects of (+)-MK-801 and its less potent stereoisomer, (-)-MK-801, in the social defeat stress model of depression. METHODS: The antidepressant effects of (+)-MK-801 (0.1mg/kg) and (-)-MK-801 (0.1mg/kg) in the social defeat stress model were examined. RESULTS: In the tail suspension and forced swimming tests, both stereoisomers significantly attenuated increased immobility time in susceptible mice. In the sucrose preference test, (+)-MK-801, but not (-)-MK-801, significantly enhanced reduced sucrose consumption 2 or 4 days after a single dose. However, no antianhedonia effects were detected 7 days after a single dose of either stereoisomer. CONCLUSIONS: Both stereoisomers of MK-801 induced rapid antidepressant effects in the social defeat stress model, although neither produced a long-lasting effect (7 days).

The effect of the NMDA receptor-dependent signaling pathway on cell morphology and melanosome transfer in melanocytes.

BACKGROUND: The pigmentation of skin and hair in mammals is driven by the intercellular transfer of melanosome from the melanocyte to surrounding keratinocytes However, the detailed molecular mechanism is still a subject of investigation. OBJECTIVE: To investigate the effects of N-methyl-d-aspartate (NMDA) receptor-dependent signaling pathway on melanocyte morphologic change and melanosome transfer between melanocytes and keratinocytes. METHODS: The expression and the intracellular distribution of NMDA receptor in human melanocyte were analyzed by Western blot and immunofluorescence staining. Melanocytes were treated with 100µM NMDA receptor antagonist MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine maleate] and 100µM NMDA receptor agonist NMDA, after which the morphological change of melanocyte dendrites and filopodias were observed by scanning electron microscope. The ß-tubulin distribution and intracellular calcium concentration ([Ca2+]i) were observed by immunofluorescence staining and flow cytometry under the same treatment respectively. In addition, melanocytes and keratinocytes were co-cultured with or without treatment of MK-801, and the melanosome transfer efficacy were analyzed by flow cytometry. RESULTS: We show that human epidermal melanocytes expresses NMDA receptor 1, one subtype of the ionotropic glutamate receptors (iGluRs). Stimulation with agonist of NMDA receptor increased the number of melanocyte filopodia. In contrast, blockage of NMDA receptor with antagonist decreased the number of melanocyte filopodia and this morphological change was accompanied by the disorganization of ß-tubulin microfilaments in the intracellular cytoskeleton. In melanocyte-keratinocyte co-cultures, numerous melanocyte filopodia connect to keratinocyte plasma membranes; agonist of NMDA receptor exhibited an increased number of melanocyte filopodia attachments to keratinocyte, while antagonist of NMDA receptor led to a decreased. Moreover, antagonist of NMDA receptor decreased the intracellular calcium concentration in melanocytes and reduced the efficacy of melanosome transfer. CONCLUSION: Our data suggest that filopodia delivery is the major mode of melanosome transfer between melanocytes and keratinocytes. NMDA drives melanosome transfer by promoting filopodia delivery and direct morphological effects on melanocytes, while MK-801 affects the intracellular ß-tubulin redistribution and the filopodia delivery between melanocytes and keratinocytes. We hypothesize that NMDA receptor-dependent signaling is involved in melanosome transfer, which is associated with calcium influx, cytoskeleton protein redistribution, dendrites and filopodia formation. A thorough understanding of melanosome transfer is crucial for designing treatments for hyper- and hypo-pigmentary disorders of the skin.

Cortical gray matter loss in schizophrenia: Could microglia be the culprit?

Cortical gray matter loss in schizophrenia remains a great therapeutic difficulty. Each psychotic episode causes irreversible cortical gray matter loss, that causes the patients to never regain their previous state of functioning. Microglial cells are part of the innate immune system and their functions, among others, include phagocytosis and release of neurotrophic factors. They have a key impact on developmental and plasticity-induced removal of neuronal precursors, live-but-stressed neurons and synapses, while also stimulating synaptic growth and development. We hypothesize that microglia are the culprit for the cortical gray matter loss in schizophrenia through abnormal synaptic pruning, phagocytosis of stressed neurons and lacking neurotrophic factor release. Furthermore, we propose a research that could validate the hypotheses using serum samples of first-episode early-onset patients. By measuring the serum levels of milk fat globule-EGF factor 8 (MFG-E8), subcomponent in the classical pathway of complement activation (C1q), brain-derived neurotrophic factor (BDNF), interleukin-6 (IL-6) and interleukin-10 (IL-10), we could gain an insight into the state of microglial activation during various stages of the disease. If this hypothesis is valid, new targeted drugs could be developed in order to reduce the deterioration of cortical gray matter, thereby possibly improving negative symptoms and cognitive deficits.

Functional Comparison of Neuronal Cells Differentiated from Human Induced Pluripotent Stem Cell-Derived Neural Stem Cells under Different Oxygen and Medium Conditions.

Because neurons are difficult to obtain from humans, generating functional neurons from human induced pluripotent stem cells (hiPSCs) is important for establishing physiological or disease-relevant screening systems for drug discovery. To examine the culture conditions leading to efficient differentiation of functional neural cells, we investigated the effects of oxygen stress (2% or 20% O2) and differentiation medium (DMEM/F12:Neurobasal-based [DN] or commercial [PhoenixSongs Biologicals; PS]) on the expression of genes related to neural differentiation, glutamate receptor function, and the formation of networks of neurons differentiated from hiPSCs (201B7) via long-term self-renewing neuroepithelial-like stem (lt-NES) cells. Expression of genes related to neural differentiation occurred more quickly in PS and/or 2% O2 than in DN and/or 20% O2, resulting in high responsiveness of neural cells to glutamate, N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and ( S)-3,5-dihydroxyphenylglycine (an agonist for mGluR1/5), as revealed by calcium imaging assays. NMDA receptors, AMPA receptors, mGluR1, and mGluR5 were functionally validated by using the specific antagonists MK-801, NBQX, JNJ16259685, and 2-methyl-6-(phenylethynyl)-pyridine, respectively. Multielectrode array analysis showed that spontaneous firing occurred earlier in cells cultured in 2% O2 than in 20% O2. Optimization of O2 tension and culture medium for neural differentiation of hiPSCs can efficiently generate physiologically relevant cells for screening systems.

Soaping the NMDA receptor: Various types of detergents influence differently [(3)H]MK-801 binding to rat brain membranes.

Membranes prepared from rat brain were treated with increasing concentrations of cationic, neutral, anionic and zwitterionic surfactants. Potent inactivation of [(3)H]MK-801 binding to NMDA receptors (NRs) was provided by the cation cetyl pyridinium (IC50 25 µM) and the neutral digitonin (IC50 37 µM). A 2 h incubation of rat brain membranes at 24°C with 100 µM of the neutral Triton X-100 resulted in about 50% reversible inhibition (without inactivation). Reversible inhibition was also effected by the anion deoxycholate (IC50 700 µM), and by the zwitterions N-lauryl sulfobetaine (12-SB(±), 400 µM) and CHAPS (1.5 mM), with inactivation at higher concentrations. Keeping the NR cation channel in the closed state significantly protected against inactivation by cations and by 12-SB(±), but not by the other detergents. Inactivation depended differentially on the amount of the membranes, on the duration of the treatment, and on the temperature. Varying the amount of membranes by a factor 8 yielded for cetyl trimethylammonium (16-NMe3(+)) IC50s of inactivation from 10 to 80 µM, while for deoxycholate the IC50 of inactivation was 1.2 mM for all tissue quantities. Some compounds inactivated within a few min (16-NMe3(+), digitonin, CHAPS), while inactivation by others took at least half an hour (Triton X-100, deoxycholate, 12-SB(±)). These last 3 ones also exhibited the steepest temperature dependence. Knowledge about the influence of various parameters is helpful in selecting appropriate conditions allowing the treatment of brain membranes with amphiphiles without risking irreversible inactivation.

Differential influence of 7 cations on 16 non-competitive NMDA receptor blockers.

The specific binding of the NMDA receptor (NR) channel ligand [(3)H]MK-801 to rat brain membranes is sensitive to positively charged buffer ingredients as to tris(hydroxymethyl)aminomethane (Tris), to Na(+), or to protons. Here we demonstrate that 16 non-competitive NR antagonists, including 5 long-chain diamines, classical NR channel blockers and several less known compounds, differ widely in their sensitivities to cationic buffer constituents. Although chemically distinguished either as extended di-cationic or as compact mono-cationic, their sensitivities to cationic buffer ingredients did not suggest this grouping. While the di-cationic compounds are known for their sensitivity to spermine (polyamine inverse agonists), also some of the mono-cationic blockers exhibited this feature. They might share as common target a recently described negatively charged extracellular GluN1/GluN2B interface.

Pentamidine analogs as inhibitors of [(3)H]MK-801 and [(3)H]ifenprodil binding to rat brain NMDA receptors.

The anti-protozoal drug pentamidine is active against opportunistic Pneumocystis pneumonia, but in addition has several other biological targets, including the NMDA receptor (NR). Here we describe the inhibitory potencies of 76 pentamidine analogs at 2 binding sites of the NR, the channel binding site labeled with [(3)H]MK-801 and the [(3)H]ifenprodil binding site. Most analogs acted weaker at the ifenprodil than at the channel site. The spermine-sensitivity of NR inhibition by the majority of the compounds was reminiscent of other long-chain dicationic NR blockers. The potency of the parent compound as NR blocker was increased by modifying the heteroatoms in the bridge connecting the 2 benzamidine moieties and also by integrating the bridge into a seven-membered ring. Docking of the 45 most spermine-sensitive bisbenzamidines to a recently described acidic interface between the N-terminal domains of GluN1 and GluN2B mediating polyamine stimulation of the NR revealed the domain contributed by GluN1 as the most relevant target.

Targeting dopamine D3 and serotonin 5-HT1A and 5-HT2A receptors for developing effective antipsychotics: synthesis, biological characterization, and behavioral studies.

Combination of dopamine D3 antagonism, serotonin 5-HT1A partial agonism, and antagonism at 5-HT2A leads to a novel approach to potent atypical antipsychotics. Exploitation of the original structure-activity relationships resulted in the identification of safe and effective antipsychotics devoid of extrapyramidal symptoms liability, sedation, and catalepsy. The potential atypical antipsychotic 5bb was selected for further pharmacological investigation. The distribution of c-fos positive cells in the ventral striatum confirmed the atypical antipsychotic profile of 5bb in agreement with behavioral rodent studies. 5bb administered orally demonstrated a biphasic effect on the MK801-induced hyperactivity at dose levels not able to induce sedation, catalepsy, or learning impairment in passive avoidance. In microdialysis studies, 5bb increased the dopamine efflux in the medial prefrontal cortex. Thus, 5bb represents a valuable lead for the development of atypical antipsychotics endowed with a unique pharmacological profile for addressing negative symptoms and cognitive deficits in schizophrenia.

NMDA receptor structures reveal subunit arrangement and pore architecture.

N-methyl-d-aspartate (NMDA) receptors are Hebbian-like coincidence detectors, requiring binding of glycine and glutamate in combination with the relief of voltage-dependent magnesium block to open an ion conductive pore across the membrane bilayer. Despite the importance of the NMDA receptor in the development and function of the brain, a molecular structure of an intact receptor has remained elusive. Here we present X-ray crystal structures of the Xenopus laevis GluN1-GluN2B NMDA receptor with the allosteric inhibitor, Ro25-6981, partial agonists and the ion channel blocker, MK-801. Receptor subunits are arranged in a 1-2-1-2 fashion, demonstrating extensive interactions between the amino-terminal and ligand-binding domains. The transmembrane domains harbour a closed-blocked ion channel, a pyramidal central vestibule lined by residues implicated in binding ion channel blockers and magnesium, and a ∼twofold symmetric arrangement of ion channel pore loops. These structures provide new insights into the architecture, allosteric coupling and ion channel function of NMDA receptors.

In silico target fishing for the potential targets and molecular mechanisms of baicalein as an antiparkinsonian agent: discovery of the protective effects on NMDA receptor-mediated neurotoxicity.

The flavonoid baicalein has been proven effective in animal models of parkinson's disease; however, the potential biological targets and molecular mechanisms underlying the antiparkinsonian action of baicalein have not been fully clarified. In the present study, the potential targets of baicalein were predicted by in silico target fishing approaches including database mining, molecular docking, structure-based pharmacophore searching, and chemical similarity searching. A consensus scoring formula has been developed and validated to objectively rank the targets. The top two ranked targets catechol-O-methyltransferase (COMT) and monoamine oxidase B (MAO-B) have been proposed as targets of baicalein by literatures. The third-ranked one (N-methyl-d-aspartic acid receptor, NMDAR) with relatively low consensus score was further experimentally tested. Although our results suggested that baicalein significantly attenuated NMDA-induced neurotoxicity including cell death, intracellular nitric oxide (NO) and reactive oxygen species (ROS) generation, extracellular NO reduction in human SH-SY5Y neuroblastoma cells, baicalein exhibited no inhibitory effect on [(3) H]MK-801 binding study, indicating that NMDAR might not be the target of baicalein. In conclusion, the results indicate that in silico target fishing is an effective method for drug target discovery, and the protective role of baicalein against NMDA-induced neurotoxicity supports our previous research that baicalein possesses antiparkinsonian activity.

Expression of NMDA receptor and its effect on cell proliferation in the subventricular zone of neonatal rat brain.

We investigated the involvement of N-methyl D-aspartate receptor (NMDAR) in neurogenesis of rat's subventricular zone (SVZ). For this purpose, we determined expression of the NMDAR subunits NR1, NR2A, and NR2B in SVZ of the neonatal Sprague-Dawley rats using immunohistochemical techniques. All three NMDAR subunits were expressed during postnatal day (PND)-1 to PND-28 whereas each subunit showed a distinct expression pattern. We also examined the functional effect of this receptor on cell proliferation in this region and, in this regard, the animals received either intraperitoneal injection of NMDAR agonist NMDA (2 mg/kg/day) or selective non-competitive NMDAR antagonist MK-801 (10 mg/kg) or NR2B antagonist Ro25-6981 (40 mg/kg), respectively, at PND-3. A significant developmental increase of the total cell density was observed at PND-7 (P < 0.05) while proliferating cell nuclear antigen-positive cell density was significantly increased at PND-14 (P < 0.05) and at PND-28 (P < 0.05) in the SVZ after NMDA (2 mg/kg/day) injection. Our data show that the NMDAR activation promoted the cell proliferation in SVZ during the neonatal period. We, therefore, inferred that NMDAR is expressed in SVZ of the neonatal rat brain and can promote neurogenesis, as through cell proliferation process in that region, and can thus be used as a potential therapeutic target in neurodegenerative diseases.

Synthesis of a series of γ-amino alcohols comprising an N-methyl isoindoline moiety and their evaluation as NMDA receptor antagonists.

We report a series of new stereoisomeric γ-amino alcohols comprising an N-methyl isoindoline moiety as ligands for the ifenprodil binding site of the NMDA receptor. Among the four series of stereoisomers, 8a-c, 9a-c, 10a-c, and 11a-c, synthesised, the highest potencies and NMDA-NR2B subtype selectivity was found for the methyl derivative 11a and the chloro derivative 11c, both possessing the [1S,1'S] configuration. However, additional moderate potency of 11a and 11c at the hERG channel with values of 2.6 ± 2.4% and 1.6 ± 2.0%, respectively, rendered them unsuitable for medical use.

Clinical physiology and mechanism of dizocilpine (MK-801): electron transfer, radicals, redox metabolites and bioactivity.

Dizocilpine (MK-801), an extensively investigated drug possessing secondary amine and benzenoid functions, displays a wide array of biological properties, including anticonvulsant and anesthetic. There is scant discussion of biomechanism. A relevant, important finding is formation of oxidative metabolites in the hydroxylamine and phenolic categories. Analogy to cocaine metabolites suggests participation of redox entities, such as, hydroxylamine, nitroxide and nitrosonium, which can lead to electron transfer and radical formation. There is also similarity to metabolism by 3,3'-iminodipropionitrile and phencyclidine. Alternatively, the phenolic metabolites are well-known precursors of ET quinones. The review documents various physiological effects, mainly involving the central nervous system. Also of interest are the pro- and ant-oxidant properties. Considerable attention has been paid to MK-801 as an antagonist of the N-methyl-D-aspartate receptor in the glutamate category. This aspect is often associated with effects on the central nervous system. The review also provides recent literature dealing with MK-801/NMDA receptor in various areas of bioactivity. Studies were made of MK-801 involvement in working memory processing. Deficits in behavior were noted after administration of the drug. Treatment of mice with dizocilpine induced learning impairment. The influence of MK-801 on fear has been investigated. The substance is known to exert an analgesic effect in pain control. A number of reports deal with anesthetic properties.

[Ligands of glutamate and dopamine receptors evenly labeled with hydrogen isotopes].

A reaction of high-temperature solid-phase catalytic isotope exchange (HSCIE) was studied for the preparation of tritium- and deuterium-labeled ligands of glutamate and dopamine receptors. Tritium-labeled (5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclopenten-5,10-imine ([G-(3)H]MK-801) and R(+)-7-hydroxy-N,N-di-n-propyl-2-aminotetraline ([G-(3)H]-7-OH-DPAT) were obtained with a specific activity of 210 and 120 Ci/mol, respectively. The isotopomeric distribution of deuterium-labeled ligands was studied using time-of-flight mass-spectrometer MX 5310 (ESI-o-TOF) with electrospray and orthogonal ion injection. Mean deuterium incorporation per ligand molecule was 11.09 and 3.21 atoms for [G-(2)H]MK-801 and [G-(2)H]-7-OH-DPAT, respectively. The isotope label was shown to be distributed all over the ligand molecule. The radioreceptor binding of tritium-labeled ligands [G-(3)H]MK-801 and [G-(3)H]-7-OH-DPAT was analyzed using the brain structure of Vistar rats. It was demonstrated that [G-(3)H]MK-801 specifically binds to hippocampus membranes with K(d) 8.3 +/- 1.4 nM, B(max) being 3345 +/- 300 fmol/mg protein. The [G-(3)H]-7-OH-DPAT ligand specifically binds to rat striatum membranes with K(d) 10.01 +/- 0.91 nM and B(max) 125 +/- 4.5 fmol/mg protein. It was concluded that the HSCIE reaction can be used for the preparation of highly tritium-labeled (+)-MK-801 and 7-OH-DPAT with retention of their physiological activities.