MK-801 Binding Research Articles

The role of fatty acid-binding protein 5 and 7 on locomotor, anxiety and social behavior: Interaction with NMDA signaling

The endocannabinoid system has been shown to be a powerful mediator of anxiety, learning and memory, as well as nociception behaviors. Exogenous cannabinoids like delta-9-tetrahydrocannabinol mimic the naturally occurring endogenous cannabinoids found in the mammalian central and peripheral nervous system. The hydrophobic properties of endocannabinoids mean that these psychoactive compounds require help with cellular transport. A family of lipid intracellular carriers called fatty acid-binding proteins (FABPs) can bind to endocannabinoids. Pharmacological inhibition or genetic deletion of FABP subtypes 5 and 7 elevates whole-brain anandamide (AEA) levels, a type of endocannabinoid. This study examined locomotor behavior, anxiety-like behavior, and social behavior in FABP5-/- and FABP7-/- mice. Furthermore, we measured N-methyl-D-aspartate (NMDA) receptor levels in the brain to help identify potential underlying mechanisms related to the behavioral findings. Results showed that both male and female FABP5-/- mice exhibited significantly lower activity when compared with both FABP5/7+/+ (control) and FABP7-/-. For social behavior, male, but not female, FABP5-/- mice spent more time interacting with novel mice compared with controls (FABP5/7+/+) and FABP7-/- mice. No significant difference was found for anxiety-like behavior. Results from the NMDA autoradiography revealed [3H] MK-801 binding to be significantly increased within sub-regions of the striatum in FABP7-/- compared with control. In summary, these results show that FABP5 deficiency plays a significant role in locomotion activity, exploratory behavior, as well as social interaction. Furthermore, FABP7 deficiency is shown to play an important role in NMDA receptor expression, while FABP5 does not.

Glutamate's Effects on the N-Methyl-D-Aspartate (NMDA) Receptor Ion Channel in Alzheimer's Disease Brain: Challenges for PET Radiotracer Development for Imaging the NMDA Ion Channel.

In an effort to further understand the challenges facing in vivo imaging probe development for the N-methyl-D-aspartate (NMDA) receptor ion channel, we have evaluated the effect of glutamate on the Alzheimer's disease (AD) brain. Human post-mortem AD brain slices of the frontal cortex and anterior cingulate were incubated with [3H]MK-801 and adjacent sections were tested for Aβ and Tau. The binding of [3H]MK-801 was measured in the absence and presence of glutamate and glycine. Increased [3H]MK-801 binding in AD brains was observed at baseline and in the presence of glutamate, indicating a significant increase (>100%) in glutamate-induced NMDA ion channel activity in AD brains compared to cognitively normal brains. The glycine effect was lower, suggesting a decrease of the co-agonist effect of glutamate and glycine in the AD brain. Our preliminary findings suggest that the targeting of the NMDA ion channel as well as the glutamate site may be appropriate in the diagnosis and treatment of AD. However, the low baseline levels of [3H]MK-801 binding in the frontal cortex and anterior cingulate in the absence of glutamate and glycine indicate significant hurdles for in vivo imaging probe development and validation.

Regional binding of [3H]‐MK801 to NMDA receptors in young and aged rat brain membranes

Norepinephrine (NE) and dopamine (DA) are essential neuromodulators in the central nervous system. They regulate several crucial physiological processes, including arousal/attention, focus, neuroendocrine functions, and the sleep‐wake cycle; they also play complex roles in regulating cognition and affect; therefore, dysfunctions in catecholaminergic neurotransmission are associated with various neurological consequences. Previously, we confirmed that in brain slices from aged Fischer 344 rats, there is an age‐related decrease in glutamate‐stimulated NE release in the cerebral cortex and hippocampus (Gonzales et al., 1991 & Dezfuli et al., 2019) and a regionally specific decline in glutamate‐stimulated DA release, with a deficit in the striatum but not in the olfactory tubercle (Aljohani et al., 2021a). Importantly, our studies demonstrated that NMDA receptors mediate glutamate‐stimulated NE and DA release. We also have shown that the alpha‐2 adrenergic receptors agonist clonidine decreases this glutamate‐stimulated NE release in a concentration‐dependent manner; while in contrast, blockade of these receptors with phentolamine, an alpha‐1 and alpha‐2 adrenergic receptor blocker, or with idazoxan, a selective alpha‐2 receptor blocker, markedly potentiates glutamate‐stimulated NE release in cortical brain slices (Aljohani et al., 2021b). Since aging has heterogeneous effects on NMDA receptors in different brain areas, we have now begun to examine [3H]‐MK801 binding to NMDA receptors to compare changes in receptor density and responsiveness during aging in the cortex, hippocampus, striatum, and olfactory tubercle, as a possible mechanistic explanation for the changes in NE and DA release. The data from cortical membrane preparations suggest that young and old rats have similar NMDA receptor densities but may differ in their response to stimulation with glutamate/glycine.

Chronic oral olanzapine treatment but not haloperidol decreases [3H] MK-801 binding in the rat brain Independent of dietary conditions

Haloperidol and olanzapine are first and second-generation antipsychotic (neuroleptic) medications approved to treat schizophrenia. Glutamate signaling is known to play an important role in the manifestation of schizophrenia symptoms, as phencyclidine, a non-competitive N-methyl-D-aspartate receptor (NMDAR) antagonist, replicates and exasperates these symptoms. While initial reports show that neuroleptic treatments can impact aspects of NMDAR expression, there is little attention on the interaction between neuroleptics and dietary conditions. Thus, we examined the impact of chronic haloperidol and olanzapine treatment under both normal and high-fat dietary conditions on NMDAR expression. Adult male rats were treated for 28-days with either oral vehicle, haloperidol (1.5 mg/kg), or olanzapine (10 mg/kg), and fed either a standard control diet or a high-fat diet. In-vitro receptor autoradiography binding was performed using [3H] MK-801 as a measure of NMDAR expression. Results showed that olanzapine, irrespective of the diet, significantly decreased [3H] MK-801 binding within the cingulate cortex, substantia nigra, insular cortex, piriform cortex, ectorhinal cortex and perirhinal cortex, the forelimb region of the somatosensory cortex, and all quadrants of the caudate-putamen. In contrast, haloperidol treatment did not impact [3H] MK-801 binding, and we also report no effect of diet on [3H] MK-801 binding. These data suggest that the effects seen in olanzapine treatment are not mediated by diet, nor does a 28-day chronic high-fat diet alter [3H] MK-801 binding. Furthermore, these data also importantly support that combined consumption of a high-fat diet and pharmacological treatments are not immediately detrimental to NMDARs and contribute to the expansive literature of precision medicine.

Effects of Zinc, Mercury, or Lead on [3H]MK-801 and [3H]Fluorowillardiine Binding to Rat Synaptic Membranes.

Glutamate (Glu) is considered the most important excitatory amino acid neurotransmitter in the mammalian Central Nervous System. Zinc (Zn) is co-released with Glu during synaptic transmission and interacts with Glutamate receptors and transporters. We performed binding experiments using [3H]MK-801 (NMDA), and [3H]Fluorowillardine (AMPA) as ligands to study Zn-Glutamate interactions in rat cortical synaptic membranes. We also examined the effects of mercury and lead on NMDA or AMPA receptors. Zinc at 1nM, significantly potentiates [3H]MK-801 binding. Lead inhibits [3H]MK-801 binding at micromolar concentrations. At millimolar concentrations, Hg also has a significant inhibitory effect. These effects are not reversed by Zn (1nM). Zinc displaces the [3H]FW binding curve to the right. Lead (nM) and Hg (μM) inhibit [3H]FW binding. At certain concentrations, Zn reverses the effects of these metals on [3H]FW binding. These specific interactions serve to clarify the role of Zn, Hg, and Pb in physiological and pathological conditions.

Chronic oral methylphenidate treatment in adolescent rats promotes dose-dependent effects on NMDA receptor binding

AimExamine the effects of chronic oral Methylphenidate (MP) treatment on the N-Methyl-D-aspartic acid (NMDA) glutamate receptor binding in the rat brain using a previously established drinking paradigm that has been shown to deliver MP with similar pharmacokinetic profile as observed clinically. Main methodsBriefly, rats were divided into three treatment groups of water, low dose MP (LD; 4/10 mg/kg), or high dose MP (HD; 30/60 mg/kg). Following a 3-month treatment period, some rats were sacrificed while others went through an additional 1-month abstinence period before they were sacrificed. In vitro autoradiography (ARG) was carried out using [3H] MK801 to examine NMDA receptor binding in the brain. Key findingsThe dose-dependent effects of MP following 13 weeks of treatment on [3H] MK-801 binding were seen across the brain in the following regions: prelimbic, insular, secondary motor, primary motor, retrosplenial, rhinal, piriform, auditory, visual, dorsolateral striatum, nucleus accumbens core, hippocampus, amygdala, and thalamic regions. No differences were observed in [3H] MK-801 binding levels in animals that underwent the same treatment followed by a 4 week abstinence. SignificanceThese results demonstrate that chronic MP treatment altered NMDA receptor expression throughout the brain, which in turn may impact an individual's drug-seeking behavior, fear memory formation and overall activity. However, these effects of chronic MP were eliminated following cessation of treatment.

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

BackgroundGlutamate 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.MethodsA 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.ConclusionsLess 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.

Src deficient mice demonstrate behavioral and electrophysiological alterations relevant to psychiatric and developmental disease

Much evidence suggests that hypofunction of the N-methyl-d-aspartate glutamate receptor (NMDAR) may contribute broadly towards a subset of molecular, cognitive and behavioral abnormalities common among psychiatric and developmental diseases. However, little is known about the specific molecular changes that lead to NMDAR dysfunction. As such, personalized approaches to remediating NMDAR dysfunction based on a specific etiology remains a challenge. Sarcoma tyrosine kinase (Src) serves as a hub for multiple signaling mechanisms affecting GluN2 phosphorylation and can be disrupted by convergent alterations of various signaling pathways. We recently showed reduced Src signaling in post mortem tissue from schizophrenia patients, despite increased MK-801 binding and NMDA receptor complex expression in the postsynaptic density (PSD). These data suggest that Src dysregulation may be an important underlying mechanism responsible for reduced glutamate signaling. Despite this evidence for a central role of Src in NMDAR signaling, little is known about how reductions in Src activity might regulate phenotypic changes in cognition and behavior. As such, the current study sought to characterize behavioral and electrophysiological phenotypes in mice heterozygous for the Src Acl gene (Src+/− mice). Src+/− mice demonstrated decreased sociability and working memory relative to Src+/+ (WT) mice while no significant differences were seen on locomotive activity and anxiety-related behavior. In relation to WT mice, Src+/− mice showed decreased mid-latency P20 auditory event related potential (aERP) amplitudes, decreased mismatch negativity (MMN) and decreased evoked gamma power, which was only present in males. These data indicate that Src+/− mice are a promising new model to help understand the pathophysiology of these electrophysiological, behavioral and cognitive changes. As such, we propose that Src+/− mice can be used in the future to evaluate potential therapeutic approaches by targeting increased Src activity as a common final pathway for multiple etiologies of SCZ and other diseases characterized by reduced glutamate function.

Modulation of Na+/K+- ATPase activity by triterpene 3β, 6β, 16β-trihidroxilup-20 (29)-ene (TTHL) limits the long-term secondary degeneration after traumatic brain injury in mice

Traumatic brain injury (TBI) is a public health problem characterized by a combination of immediate mechanical dysfunction of the brain tissue, and secondary damage. Based on the hypothesis that selected targets, such as Na+ K+-ATPase are involved in the secondary damage after TBI and modulation of this enzyme activity by triterpene 3β, 6β, 16β-trihidroxilup-20 (29)-ene (TTHL) supports the ethnomedical applications of this plant, we decided to investigate whether previous TTHL treatment interrupts the progression of pathophysiology induced by TBI. Statistical analyses revealed that percussion fluid injury (FPI) increased Na+,K+-ATPase activity in all isoform (α1 and α2/3) 15 min after neuronal injury. The FPI protocol inhibited Na+,K+-ATPase activity total and α1 isoform, increased [3H]MK-801 binding but did not alter Dichloro-dihydro-fluorescein diacetate (DCFH-DA) oxidation, carbonylated proteins and free –SH groups 60 min after injury. The increase of immunoreactivity of protein PKC and state of phosphorylation of at Ser16 of Na+,K+-ATPase 60 min after FPI suggest the involvement of PKC on Na+,K+-ATPase activity oscillations characterized by inhibition of total and α1 isoform. Our experimental data also revealed that natural product rich in compounds such as triterpenes (TTHL; 30 mg/kg) attenuates [3H]MK-801 binding increase, phosphorylation of the PKC and the Na+,K+-ATPase alpha 1 subunit (Ser16) induced by FPI. The previous TTHL treatment had not effect on motor disability but protected against spatial memory deficit, BDNF, TrKB expression decrease, protein carbonylation and hippocampal cell death 7 days after FPI. These data suggest that TTHL-induced reduction on initial damage limits the long-term secondary degeneration and supports neural repair or behavioral compensation after neuronal injury.

Early antipsychotic exposure affects NMDA and GABAA receptor binding in the brains of juvenile rats

Antipsychotics were developed to treat schizophrenia in adults; however they have been increasingly prescribed in children and adolescents. The NMDA and GABAA receptors are involved in neurodevelopment and the pathophysiology of various mental disorders in children and adolescents. Male and female juvenile rats were treated orally with risperidone (0.3 mg/kg, 3 times/day), aripiprazole (1 mg/kg), olanzapine (1 mg/kg) or vehicle (control), starting from postnatal day (PD) 23 (±1 day) for 3 weeks (corresponding to the childhood-adolescent period in humans). Quantitative autoradiography was used to detect the binding density of [3H]MK-801 (an NMDA receptor antagonist) and [3H]muscimol (a selective GABAA receptor agonist). Aripiprazole elevated the [3H]MK801 binding levels in the NAcC of male rats, and the NAcS and CPu of female rats. Risperidone increased [3H]MK801 levels in the CPu of female rats, and the NAcS of male rats. Aripiprazole upregulated [3H]muscimol binding levels in the CPu and NAcC of male rats, while it elevated the [3H]muscimol levels in the PFC of female rats, compared to controls. These results suggest that early treatment with these antipsychotics modulates NMDA and GABAA neurotransmission in juveniles, which may play a role in their clinical efficacy in the control of mental disorders in children and adolescents.

Inhibition of alpha7 nicotinic receptors in the ventral hippocampus selectively attenuates reinstatement of morphine-conditioned place preference and associated changes in AMPA receptor binding.

Recurrent relapse is a major problem in treating opiate addiction. Pavlovian conditioning plays a role in recurrent relapse whereby exposure to cues learned during drug intake can precipitate relapse to drug taking. α7 nicotinic acetylcholine receptors (nAChRs) have been implicated in attentional aspects of cognition and mechanisms of learning and memory. In this study we have investigated the role of α7 nAChRs in morphine‐conditioned place preference (morphine‐CPP). CPP provides a model of associative learning that is pertinent to associative aspects of drug dependence. The α7 nAChR antagonist methyllycaconitine (MLA; 4 mg/kg s.c.) had no effect on the acquisition, maintenance, reconsolidation or extinction of morphine‐CPP but selectively attenuated morphine‐primed reinstatement of CPP, in both mice and rats. Reinstatement of morphine‐CPP in mice was accompanied by a selective increase in [3H]‐AMPA binding (but not in [3H]‐MK801 binding) in the ventral hippocampus that was prevented by prior treatment with MLA. Administration of MLA (6.7 μg) directly into the ventral hippocampus of rats prior to a systemic priming dose of morphine abolished reinstatement of morphine‐CPP, whereas MLA delivered into the dorsal hippocampus or prefrontal cortex was without effect. These results suggest that α7 nAChRs in the ventral hippocampus play a specific role in the retrieval of associative drug memories following a period of extinction, making them potential targets for the prevention of relapse.

QSAR Modeling of the NMDA Receptor Blockage by Polypharmacophoric Compounds Based on Carbazole and 1-aminoadamantane Derivatives

We investigated 14 compounds causing the NMDA receptor blockage. These polypharmacophoric compounds are conjugates of carbazole, tetrahydrocarbazole and 1-aminoadamantane derivatives. As a measure of biological activity of the compound tested, the IC50 (μM) value, reflecting 50% inhibition of [H3] MK-801 binding to the NMDA receptor, was used. The regression model with satisfactory statistical characteristics was obtained as a result of the QSAR modeling based on the Gaussian process.

Vitamin C modulates glutamate transport and NMDA receptor function in the retina

Vitamin C (in the reduced form ascorbate or in the oxidized form dehydroascorbate) is implicated in signaling events throughout the central nervous system (CNS). In the retina, a high-affinity transport system for ascorbate has been described and glutamatergic signaling has been reported to control ascorbate release. Here, we investigated the modulatory role played by vitamin C upon glutamate uptake and N-methyl-d-aspartate (NMDA) receptor activation in cultured retinal cells or in intact retinal tissue using biochemical and imaging techniques. We show that both forms of vitamin C, ascorbate or dehydroascorbate, promote an accumulation of extracellular glutamate by a mechanism involving the inhibition of glutamate uptake. This inhibition correlates with the finding that ascorbate promotes a decrease in cell surface levels of the neuronal glutamate transporter excitatory amino acid transporter 3 in retinal neuronal cultures. Interestingly, vitamin C is prone to increase the activity of NMDA receptors but also promotes a decrease in glutamate-stimulated [3 H] MK801 binding and decreases cell membrane content of NMDA receptor glutamate ionotropic receptor subunit 1 (GluN1) subunits. Both compounds were also able to increase cAMP response element-binding protein phosphorylation in neuronal nuclei in a glutamate receptor and calcium/calmodulin kinase-dependent manner. Moreover, the effect of ascorbate is not blocked by sulfinpyrazone and then does not depend on its uptake by retinal cells. Overall, these data indicate a novel molecular and functional target for vitamin C impacting on glutamate signaling in retinal neurons.

NYX-2925 Is a Novel NMDA Receptor-Specific Spirocyclic-β-Lactam That Modulates Synaptic Plasticity Processes Associated with Learning and Memory.

BackgroundN-methyl-D-aspartate receptors are one member of a family of ionotropic glutamate receptors that play a pivotal role in synaptic plasticity processes associated with learning and have become attractive therapeutic targets for diseases such as depression, anxiety, schizophrenia, and neuropathic pain. NYX-2925 ((2S, 3R)-3-hydroxy-2-((R)-5-isobutyryl-1-oxo-2,5-diazaspiro[3.4]octan-2-yl)butanamide) is one member of a spiro-β-lactam-based chemical platform that mimics some of the dipyrrolidine structural features of rapastinel (formerly GLYX-13: threonine-proline-proline-threonine) and is distinct from known N-methyl-D-aspartate receptor agonists or antagonists such as D-cycloserine, ketamine, MK-801, kynurenic acid, or ifenprodil.MethodsThe in vitro and in vivo pharmacological properties of NYX-2925 were examined.ResultsNYX-2925 has a low potential for “off-target” activity, as it did not exhibit any significant affinity for a large panel of neuroactive receptors, including hERG receptors. NYX-2925 increased MK-801 binding to human N-methyl-D-aspartate receptor NR2A-D subtypes expressed in HEK cells and enhanced N-methyl-D-aspartate receptor current and long-term potentiation (LTP) in rat hippocampal slices (100–500 nM). Single dose ex vivo studies showed increased metaplasticity in a hippocampal LTP paradigm and structural plasticity 24 hours after administration (1 mg/kg p.o.). Significant learning enhancement in both novel object recognition and positive emotional learning paradigms were observed (0.01–1 mg/kg p.o.), and these effects were blocked by the N-methyl-D-aspartate receptor antagonist CPP. NYX-2925 does not show any addictive or sedative/ataxic side effects and has a therapeutic index of >1000. NYX-2925 (1 mg/kg p.o.) has a cerebrospinal fluid half-life of 1.2 hours with a Cmax of 44 nM at 1 hour.ConclusionsNYX-2925, like rapastinel, activates an NMDA receptor-mediated synaptic plasticity process and may have therapeutic potential for a variety of NMDA receptor-mediated central nervous system disorders.

Novel conjugates of aminoadamantanes with carbazole derivatives as potential multitarget agents for AD treatment

A new group of compounds, promising for the design of original multitarget therapeutic agents for treating neurodegenerative diseases, based on conjugates of aminoadamantane and carbazole derivatives was synthesized and investigated. Compounds of these series were found to interact with a group of targets that play an important role in the development of this type of diseases. First of all, these compounds selectively inhibit butyrylcholinesterase, block NMDA receptors containing NR2B subunits while maintaining the properties of MK-801 binding site blockers, exert microtubules stabilizing properties, and possess the ability to protect nerve cells from death at the calcium overload conditions. The leading compound C-2h has been shown the most promising effects on all analyzed parameters. Thus, these compounds can be regarded as promising candidates for the design of multi-target disease-modifying drugs for treatment of AD and/or similar neuropathologies.

Сравнение фармакологических эффектов гептапептида селанка при внутрибрюшинном и интраназальном введении мышам BALB/C и C57BL/6

С использованием теста «закрытый крестообразный лабиринт» проведено сравнение влияния при внутрибрюшинном (в/б) и интраназальном (и/н) введении гептапептида селанка (300 мкг/кг/день в течение 5 дней), обладающего анксиолитическим и ноотропным свойствами, на поведение мышей инбредных линий BALB/с и C57BL/6, а также на NMDA- и ГАМКA-рецепторы мозга. Показано, что эффективность селанка при обоих путях введения выявляется лишь у мышей линии BALB/c, исходно характеризующейся сниженной по сравнению с C57BL/6 исследовательской активностью, а также более высоким уровнем тревожности. У мышей линии BALB/c при в/б введении селанк увеличивал количество мест связывания [G-3H]SR 95531 с ГАМКA-рецепторами фронтальной коры мозга на 38 %, не изменяя характеристик связывания с NMDA-рецепторами гиппокампа. Напротив, при и/н введении селанк приводил к повышению плотности мест связывания [G-3H]MK-801 на 23 % при отсутствии влияния на ГАМКA-рецепторы. По-видимому, разница в спектрах фармакологического действия пептида при разных путях введения зависит от различий фармакокинетики вещества и динамики формирования анксиолитического и ноотропного эффектов препарата.

Characterization of methadone as a β-arrestin-biased μ-opioid receptor agonist.

BackgroundMethadone is a unique µ-opioid receptor agonist. Although several researchers have insisted that the pharmacological effects of methadone are mediated through the blockade of NMDA receptor, the underlying mechanism by which methadone exerts its distinct pharmacological effects compared to those of other µ-opioid receptor agonists is still controversial. In the present study, we further investigated the pharmacological profile of methadone compared to those of fentanyl and morphine as measured mainly by the discriminative stimulus effect and in vitro assays for NMDA receptor binding, µ-opioid receptor-internalization, and µ-opioid receptor-mediated β-arrestin recruitment.ResultsWe found that fentanyl substituted for the discriminative stimulus effects of methadone, whereas a relatively high dose of morphine was required to substitute for the discriminative stimulus effects of methadone in rats. Under these conditions, the non-competitive NMDA receptor antagonist MK-801 did not substitute for the discriminative stimulus effects of methadone. In association with its discriminative stimulus effect, methadone failed to displace the receptor binding of MK801 using mouse brain membrane. Methadone and fentanyl, but not morphine, induced potent µ-opioid receptor internalization accompanied by the strong recruitment of β-arrestin-2 in µ-opioid receptor-overexpressing cells.ConclusionsThese results suggest that methadone may, at least partly, produce its pharmacological effect as a β-arrestin-biased µ-opioid receptor agonist, similar to fentanyl, and NMDA receptor blockade is not the main contributor to the pharmacological profile of methadone.

The Density of N-Methyl-D-Aspartate Receptor in Rat Striatum Varies with Differing Psychotropic Drug Treatments

Background: A number of studies in rodents suggest that antipsychotic drug treatment can either alter the levels or functionality of the N-methyl-D-aspartate receptor (NMDAR). This raises the possibility that some of the therapeutic actions or side-effects of antipsychotic drugs could be mediated through the NMDAR. Objective: To determine if treatments with combinations of psychotropic drugs change the levels of the NMDAR in rat Central Nervous System (CNS). Methods: In situ radioligand binding and autoradiography were used to quantify [3H]MK-801 binding to NMDAR in frozen CNS sections from rats treated with vehicle, haloperidol (0.1 mg/kg/day), olanzapine (1.0 mg/kg/ day), lithium (25.5 mg/kg/day) or, at the same doses, haloperidol and lithium or olanzapine and lithium for 28 days. Results: There was a significant variation of [3H]MK-801 binding with treatment in the rat striatum due to higher levels of radioligand in rats treated with haloperidol compared to those treated with olanzapine and lithium (p < 0.01; Cohen’s d = 1.47). Levels of radioligand binding did not vary with drug treatment in the cortex. Conclusion: These data argue for some effect of polypharmacy on NMDAR levels, possibly localized to the striatum. Further studies of mood stabilisers with known effects on NMDAR functionality, such as valproate and lamotrigine, with and without antipsychotic drugs could be worthwhile as they may show a greater effect on NMDAR density in the CNS. Keywords: Glutamate, haloperidol, lithium, olanzapine, NMDA, psychotropic drug.

Soaping the NMDA receptor: Various types of detergents influence differently [3H]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 [3H]MK-801 binding to NMDA receptors (NRs) was provided by the cation cetyl pyridinium (IC50 25μM) and the neutral digitonin (IC50 37μM). A 2h incubation of rat brain membranes at 24°C with 100μM of the neutral Triton X-100 resulted in about 50% reversible inhibition (without inactivation). Reversible inhibition was also effected by the anion deoxycholate (IC50 700μM), and by the zwitterions N-lauryl sulfobetaine (12-SB±, 400μM) and CHAPS (1.5mM), 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 80μM, while for deoxycholate the IC50 of inactivation was 1.2mM 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.

Inhibition of in vivo [3H]MK-801 binding by NMDA receptor open channel blockers and GluN2B antagonists in rats and mice

N-methyl-d-aspartate (NMDA) receptor antagonists, including open channel blockers and GluN2B receptor subtype selective antagonists, have been developed for the treatment of depression. The current study investigated effects of systemically administered NMDA channel blockers and GluN2B receptor antagonists on NMDA receptor activity in rodents using in vivo [3H]MK-801 binding. The receptor occupancy of GluN2B antagonists was measured using ex vivo [3H]Ro 25-6981 binding. Ketamine, a NMDA receptor channel blocker, produced a dose/exposure- and time-dependent inhibition of in vivo [3H]MK-801 binding that was maximal at ~100%. The complete inhibition of in vivo [3H]MK-801 binding was also observed with NMDA receptor channel blockers, AZD6765 (Lanicemine) and MK-801 (Dizocilpine). CP-101,606 (Traxoprodil), a GluN2B antagonist, produced a dose/exposure- and time-dependent inhibition of in vivo [3H]MK-801 binding that was maximal at ~60%. Partial inhibition was also observed with other GluN2B antagonists including MK-0657 (CERC-301), EVT-101, Ro 25-6981 and radiprodil. For all GluN2B antagonists tested, partial [3H]MK-801 binding inhibition was achieved at doses saturating GluN2B receptor occupancy. Combined treatment with ketamine (10mg/kg, i.p.) and Ro 25-6981(10mg/kg, i.p.) produced a level of inhibition of in vivo [3H]MK-801 binding that was similar to treatment with either agent alone. In conclusion, this in vivo [3H]MK-801 binding study shows that NMDA receptor activity in the rodent forebrain can be inhibited completely by channel blockers, but only partially (~60%) by GluN2B receptor antagonists. At doses effective in preclinical models of depression, ketamine may preferentially inhibit the same population of NMDA receptors as Ro 25-6981, namely those containing the GluN2B subunit.