MK-801 Binding Research Articles

Antidepressant-like effects of ketamine, norketamine and dehydronorketamine in forced swim test: Role of activity at NMDA receptor

Ketamine produces rapid and long-lasting antidepressant effects in patients. The involvement of ketamine metabolites in these actions has been proposed. The effects of ketamine and its metabolites norketamine and dehydronorketamine on ligand binding to 80 receptors, ion channels and transporters was investigated at a single concentration of 10 μM. The affinities of all three compounds were then assessed at NMDA receptors using [3H]MK-801 binding. The dose–response relationships of all 3 compounds in the forced swim test were also investigated in mice 30 min after IP administration. The effects of ketamine and norketamine (both 50 mg/kg) were then examined at 30 min, 3 days and 7 days post administration. Among the 80 potential targets examined, only NMDA receptors were affected with a magnitude of >50% by ketamine and norketamine at the concentration of 10 μM. The Ki values of ketamine, norketamine and dehydronorketamine at NMDA receptors were 0.119 ± 0.01, 0.97 ± 0.1 and 3.21 ± 0.3 μM, respectively. Ketamine and norketamine reduced immobility with minimum effective doses (MEDs) of 10 and 50 mg/kg, respectively; dehydronorketamine did not affect immobility at doses of up to 50 mg/kg. Neither ketamine nor norketamine reduced immobility in the forced swim test 3 and 7 days following administration. Further, oral administration of ketamine (5–50 mg/kg) did not affect immobility. We demonstrate that ketamine and norketamine but not dehydronorketamine given acutely at subanesthetic doses reduced immobility in the forced swim test. These antidepressant-like effects appear attributable to NMDA receptor inhibition.

Changes in cortical N-methyl-D-aspartate receptors and post-synaptic density protein 95 in schizophrenia, mood disorders and suicide.

In humans, depending on dose, blocking the N-methyl-D-aspartate receptor (NMDAR) with ketamine can cause psychomimetic or antidepressant effects. The overall outcome for drugs such as ketamine depends on dose and the number of its available binding sites in the central nervous system, and to understand something of the latter variable we measure NMDAR in the frontal pole, dorsolateral prefrontal, anterior cingulate and parietal cortices from people with schizophrenia, bipolar disorder, major depressive disorders and age/sex matched controls. We measured levels of NMDARs (using [(3)H]MK-801 binding) and NMDAR sub-unit mRNAs (GRINs: using in situ hybridisation) as well as post-synaptic density protein 95 (anterior cingulate cortex only; not major depressive disorders: an NMDAR post-synaptic associated protein) in bipolar disorder, schizophrenia and controls. Compared to controls, levels of NMDAR were lower in the outer laminae of the dorsolateral prefrontal cortex (-17%, p = 0.01) in people with schizophrenia. In bipolar disorder, levels of NMDAR binding (laminae IV-VI; -19%, p < 0.01) and GRIN2C mRNA (laminae I-VI; -27%, p < 0.05) were lower in the anterior cingulate cortex and NMDAR binding was lower in the outer lamina IV of the dorsolateral prefrontal cortex (-19%, p < 0.01). In major depressive disorders, levels of GRIN2D mRNA were higher in frontal pole (+22%, p < 0.05). In suicide completers, levels of GRIN2B mRNA were higher in parietal cortex (+20%, p < 0.01) but lower (-35%, p = 0.02) in dorsolateral prefrontal cortex while post-synaptic density protein 95 was higher (+26%, p < 0.05) in anterior cingulate cortex. These data suggest that differences in cortical NMDAR expression and post-synaptic density protein 95 are present in psychiatric disorders and suicide completion and may contribute to different responses to ketamine.

Src kinase as a mediator of convergent molecular abnormalities leading to NMDAR hypoactivity in schizophrenia.

Numerous investigations support decreased glutamatergic signaling as a pathogenic mechanism of schizophrenia: yet molecular underpinnings for such dysregulation are largely unknown. In the postmortem dorsolateral prefrontal cortex, we found striking decreases in tyrosine phosphorylation of N-methyl-D aspartate (NMDA) receptor subunit 2 (GluN2), which is critical for neuroplasticity. The decreased GluN2 activity in schizophrenia may not be due to downregulation of NMDA receptors since MK-801 binding and NMDA receptor complexes in the PSD were in fact increased in schizophrenia cases. At the post-receptor level, however, we found striking reductions in the protein kinase C, Pyk 2 and Src kinase activity, which in tandem can decrease GluN2 activation. Given that Src serves as a hub of various signaling mechanisms impacting GluN2 phosphorylation, we postulated that Src hypoactivity may result from convergent alterations of various schizophrenia susceptibility pathways and thus mediate their impacts on NMDA receptor signaling. Indeed, the DLPFC of schizophrenia cases exhibit increased PSD-95 and erbB4 and decreased RPTPa and dysbindin-1, each of which reduces Src activity via protein interaction with Src. To test genomic underpinnings for Src hypoactivity, we examined genome wide association study results, incorporating 13,394 cases and 34,676 controls, which yielded no significant association of individual variants of Src and its direct regulators with schizophrenia. However, a wider protein-protein interaction based network centered on Src, showed significant enrichment of gene-level associations with schizophrenia compared to other psychiatric illnesses. Our results together demonstrate striking decreases in NMDA receptor signaling at the post-receptor level and propose Src as a nodal point of convergent dysregulations impacting NMDA receptor pathway via protein-protein associations.

The major cholesterol metabolite cholestane-3β,5α,6β-triol functions as an endogenous neuroprotectant.

Overstimulation of NMDA-type glutamate receptors is believed to be responsible for neuronal death of the CNS in various disorders, including cerebral and spinal cord ischemia. However, the intrinsic and physiological mechanisms of modulation of these receptors are essentially unknown. Here we report that cholestane-3β,5α,6β-triol (triol), a major metabolite of cholesterol, is an endogenous neuroprotectant and protects against neuronal injury both in vitro and in vivo via negative modulation of NMDA receptors. Treatment of cultured neurons with triol protects against glutamate-induced neurotoxicity, and administration of triol significantly decreases neuronal injury after spinal cord ischemia in rabbits and transient focal cerebral ischemia in rats. An inducible elevation of triol is associated with ischemic preconditioning and subsequent neuroprotection in the spinal cord of rabbits. This neuroprotection is effectively abolished by preadministration of a specific inhibitor of triol synthesis. Physiological concentrations of triol attenuate [Ca(2+)]i induced by glutamate and decrease inward NMDA-mediated currents in cultured cortical neurons and HEK-293 cells transiently transfected with NR1/NR2B NMDA receptors. Saturable binding of [(3)H]triol to cerebellar granule neurons and displacement of [(3)H]MK-801 binding to NMDA receptors by triol suggest that direct blockade of NMDA receptors may underlie the neuroprotective properties. Our findings suggest that the naturally occurring oxysterol, the major cholesterol metabolite triol, functions as an endogenous neuroprotectant in vivo, which may provide novel insights into understanding and developing potential therapeutics for disorders in the CNS.

Modes of direct modulation by taurine of the glutamate NMDA receptor in rat cortex

Taurine is an endogenous brain substance with robust neuromodulatory and possible neuroprotective properties. Though other mechanisms of action have been reported, its interaction with the NMDA (N-methyl-d-aspartic acid) receptor is undocumented. We investigated taurine׳s interaction with the NMDA receptor using electrophysiological and receptor binding approaches. The effects of taurine on field potential responses in layer-5 of prelimbic cortex in rat brain slices evoked by single-pulse electrical stimulation of ventral medial cortex were determined. Picrotoxin (80µM) was present in all control and drug solutions to block the Cl− channels associated with the GABA-, taurine-, and strychnine sensitive glycine- receptors. A typical response consisted of an NBQX (2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo-[f]-quinoxaline-7-sulfonamide)-sensitive negative wave (N1) followed by a positive wave (P1) and a broad negativity (N2), both sensitive to dl-AP5 (dl-2-amino-5-phosphonopentanoic acid) inhibition. Taurine exerted a 41.5±8.3% (n=9) voltage reduction within the late phase of N2. This taurine action was prevented by 100µM AP5, but not by 10µM nifedipine, supporting a direct modulation of NMDA receptor function by taurine, without requiring the involvement of the l-type Ca2+ channel. Taurine did not alter specific [3H] MK-801 binding to rat cortical membranes in the presence of glycine or glutamate; but inhibited spermine-potentiated specific [3H] MK-801 binding to NMDA receptors by 15–20% in the presence of glycine. In addition, taurine reduced the apparent affinity of the NMDA receptor for glycine (in the presence of spermine) by 10-fold. These results show that taurine interacts directly with the NMDA receptor by multiple mechanisms.

IL-10 Gene Knockout Reduces the Expression of mGlu Receptor 1a/b and Decreases the Glutamate-Dependent Production of Nitric Oxide

IL-10 provides trophic and survival effects directly on neurons, promotes axonal outgrowth, and stimulates neuroregeneration. In this study, we analyzed the activities of arginase and nitric oxide synthase (NOS) in synaptoneurosomes derived from brain cortex of C57BL/6 IL-10 gene-knockout (KO) and wild-type (Wt) mice and determined that the synaptoneurosomes derived from KO mice present lower arginase II activity and lower spermine content than those derived from Wt mice, whereas the basal NOS activity in the KO synaptoneurosomes was higher than that observed in the control synaptoneurosomes. Moreover, our results indicate that the plasma membranes isolated from the KO mice brain exhibit significantly lower spermine-induced enhancement of [3H] MK-801 binding than the plasma membranes from the brain of Wt mice. Glutamate increases the production of nitric oxide (NO) in Wt synaptoneurosomes in a dose-dependent manner, whereas in the KO synaptoneurosomes, this amino acid does not affect the synthesis of NO. The glutamate-dependent acceleration of NO synthesis in Wt synaptoneurosomes was abrogated by LY367385, an antagonist of mGluR1a/b. The western blot analysis of the synaptoneurosomal proteins demonstrates that the expression of the subunits of NMDAR (NMDAR2A and NMDAR2B), the level of NMDAR-bound nNOS and the expression of iNOS are not changed in KO mice and that only the level of mGluR1a/b is markedly reduced in the synaptoneurosomes of KO mice. We conclude that a neuroprotective and neuroregenerative property of IL-10, in addition to its effects on polyamine metabolism and the spermine-dependent modulation of NMDAR, may involve the regulation of mGluR1a/b expression.

Neonatal olfactory bulbectomy enhances locomotor activity, exploratory behavior and binding of NMDA receptors in pre-pubertal rats

In this study, we investigated the effect of neonatal olfactory bulbectomy (nOBX) on behavioral paradigms related to olfaction such as exploratory behavior, locomotor activity in a novel environment and social interaction. We also studied the effect of nOBX on the activity of the N-methyl-d-aspartate (NMDA) subtype of glutamate receptors during development. The behavioral effects of nOBX (postnatal day 7, PD7) were investigated in pre- (PD30) and post-pubertal (PD60) Wistar rats. NMDA receptor activity was measured with [125I]MK-801 in the brain regions associated with the olfactory circuitry. A significant increase in the novelty-induced locomotion was seen in the pre-pubertal nOBX rats. Although the locomotor effect was less marked than in pre-pubertal rats, the nOBX rats tested post-pubertally failed to habituate to the novel situation as quickly as the sham- and normal- controls. Pre-pubertally, the head-dipping behavior was enhanced in nOBX rats compared with sham-operated and normal controls, while normal exploratory behavior was observed between groups in adulthood. In contrast, social interaction was increased in post-pubertal animals that underwent nOBX. Both pre- and post-pubertal nOBX rats recovered olfaction. Interestingly, pre-pubertal rats showed a significant increase in the [125I]MK-801 binding in the piriform cortex, dorsal hippocampus, inner and outer layers of the frontal cortex and outer layer of the cingulate cortex. At post-pubertal age, no significant differences in [125I]MK-801 binding were observed between groups at any of the brain regions analyzed. These results suggest that nOBX produces pre-pubertal behavioral disturbances and NMDA receptor changes that are transitory with recovery of olfaction early in adulthood.

Alterations of NMDA receptor binding in various brain regions among 6-hydroxydopamine-induced Parkinsonian rats

The N-methyl-d-aspartate (NMDA) system closely interacts with the dopaminergic system and is strongly implicated in the pathophysiological mechanisms and therapeutic paradigms of Parkinson's disease. This study aims to systematically investigate the changes of NMDA receptors in a wide range of brain structures 3 weeks after unilateral medial forebrain bundle lesion by 6-hydroxydopamine (6-OHDA). NMDA receptor distributions and alterations in the post-mortem rat brain were detected by [3H] MK-801 binding autoradiography. In the 6-OHDA-induced Parkinsonian rat model, nigrostriatal dopaminergic neuron loss significantly mediated the decreased [3H] MK-801 binding, predominantly in the hippocampus (−22.4%, p < 0.001), caudate putamen (−14.1%, p < 0.01), accumbens nucleus (−13.8%, p < 0.05), cingulate cortex (−13.4%, p < 0.001), posteromedial cortical amygdala (−14.5%, p < 0.01) and piriform cortex (−9%, p < 0.05) compared to the controls, while there was a profound reduction of tyrosine hydroxylase (TH) immunohistochemistry in the substantia nigra pars compacta. Alterations in [3H] MK-801 in the specific brain regions related to cognitive functions may indicate that cognitive dysfunctions caused by 6-OHDA lesion were via the NMDA system. The downregulation of NMDA receptor binding in the present study provides indirect evidence for plasticity in the NMDA system in the rat brain. The present study improves our understanding of the critical roles of the NMDA receptors in treating neurodegenerative disorders, and implicates NMDA receptors as a novel therapeutic target in the treatment of Parkinson's disease.

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

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

Zinc, magnesium and NMDA receptor alterations in the hippocampus of suicide victims

BackgroundThere is evidence for an association between suicidal behavior and depression. Accumulating data suggests that depression is related to a dysfunction of the brain's glutamatergic system, and that the N-methyl-d-aspartate (NMDA) receptor plays an important role in antidepressant activity. Zinc and magnesium, the potent antagonists of the NMDA receptor complex, are involved in the pathophysiology of depression and exhibit antidepressant activity. MethodsThe present study investigated the potency of Zn2+ and Mg2+ to [3H] MK-801, which binds to the NMDA receptor channel in the hippocampus of suicide victims (n=17) and sudden death controls (n=6). Moreover, the concentrations of zinc and magnesium (by flame atomic absorption spectrometry) and levels of NMDA subunits (NR2A and NR2B) and PSD-95 protein (by Western blotting) were determined. ResultsOur results revealed that there was a statistically significant decrease (by 29% and 40%) in the potency of zinc and magnesium (respectively) to inhibit [3H] MK-801 binding to NMDA receptors in the hippocampus in suicide tissue relative to the controls. These alterations were associated with increased NR2A (+68%) and decreases in both the NR2B (−46%) and PSD-95 (−35%) levels. Furthermore, lower concentrations (−9%) of magnesium (although not of zinc) were demonstrated in suicide tissue. ConclusionsOur findings indicate that alterations in the zinc, magnesium and NMDA receptor complex in the hippocampus are potentially involved in the pathophysiology of suicide-related disorders (depression), which may lead to functional NMDA receptor hyperactivity.

Comparison of the ex vivo receptor occupancy profile of ketamine to several NMDA receptor antagonists in mouse hippocampus

NMDA receptor antagonists, particularly these targeting the GluN2B subunit are of therapeutic interest for the treatment of severe mood disorders. The receptor occupancy profiles of several NMDA receptor antagonists (30mg/kg, s.c.) were compared in mouse hippocampus by ex vivo autoradiography using [3H]MK-801, a non-selective NMDA channel blocker, and [3H]ifenprodil a selective GluN2B antagonist. Subcutaneous administration of ketamine ((RS)-2-(2-Chlorophenyl)-2-(methylamino)cyclohexanone) and memantine (3,5-dimethyladamantan-1-amine) inhibited [3H]MK-801 but not [3H]ifenprodil binding in mouse hippocampus. Ketamine reached maximal occupancy of [3H]MK-801 binding sites after 15min and rapidly cleared from the brain with no significant level of occupancy measured at the 1h time point. Memantine significantly occupied [3H]MK-801 binding sites throughout the 6h time course. The selective GluN2B antagonist CP101,606 ((1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol) and Ro 25-6981 ((αR,βS)-α-(4-Hydroxyphenyl)-β-methyl-4-(phenylmethyl)-1-piperidinepropanol maleate) inhibited [3H]ifenprodil but not [3H]MK-801 binding and significant levels of occupancy (above 50%) were measured throughout the 6h time course. These data highlight the unique quick pulse target engagement profile of ketamine compared to other NMDA receptor antagonists.

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.

Exploring the Polyamine Regulatory Site of the NMDA Receptor: a Parallel Synthesis Approach

The elongated structures of polyamine inverse agonists such as 1,12-diaminododecane (N12N) and 5-(4-aminobutyl)-2-thiopheneoctanamine (N4T8N) lend themselves to a combinatorial chemistry approach to explore a potential polyamine pharmacophore at the NMDA receptor. Herein we describe more than 100 new analogues of N4T8N obtained by breaking up the long octanamine arm into a dipeptide chain of equivalent length. Solid-phase parallel synthesis based on cross-linked polystyrene and a Wang anchor allowed the low-scale preparation of four small libraries based on the combination of two amino acid residues (out of Gly, Leu, Phe, Lys, phenylglycine, Tyr, Trp, His, and Arg). The obtained compounds were tested as modulators of [(3) H]MK-801 binding to rat brain membranes and of NMDA-induced currents in cultured rat hippocampal neurons. Compounds with two aromatic residues acted as binding inhibitors (inverse agonists). Compounds with two Lys residues acted as binding stimulators (agonists) and had stimulatory and inhibitory effects on NMDA-induced currents, depending on the holding potential. High sensitivity of binding inhibition to spermine was conferred by a Tyr residue, whereas a His residue favored high potency at acidic pH.

Postmortem stability of brain GABAergic and glutamatergic receptors and enzymes under ecological conditions

Neurochemical biomarkers have emerged as useful tools for assessing the subclinical neurological impacts of environmental toxicants in birds and other wildlife. Careful consideration of biomarker stability is necessary before implementing their use on tissues from ecological studies, as receptors and enzymes in the brain may be affected by postmortem conditions. The goal of this study was to evaluate the postmortem stability of key GABAergic and glutamatergic receptors (N-methyl-d-aspartate (NMDA), gamma-aminobutyric acid (GABAA-benzodiazepine)) and enzymes (glutamine synthetase (GS), glutamic acid decarboxylase (GAD)) under environmentally relevant field and storage conditions to determine their suitability as biomarkers. We exposed chicken embryo brains to postmortem environmental and storage conditions typical for ecological studies (12, 24, and 48h at 7°C or 25°C; 1, 4, and 8 weeks at −80°C or −20°C; 1 or 2 freeze thaw cycles), and measured [3H] MK-801 binding to the NMDA receptor, [3H] flunitrazipam binding to the GABAA-benzodiazepine receptor, GS activity, and GAD activity.We found that [3H] MK-801 binding is stable under all conditions studied. GAD activity was fairly stable under each storage and environmental temperatures for all durations, but was significantly less stable when stored at −20°C than at −80°C. [3H] flunitrazipam binding and GS activity were both impacted by environmental and storage temperature and duration, and might best be utilized in studies of samples with similar histories. Our findings here demonstrate that caution is warranted when comparing samples with different collection and storage histories, but that some biomarkers are fairly stable under various conditions.

Screening of 64 Tryptamines at NMDA, 5-HT1A, and 5-HT2A Receptors: A Comparative Binding and Modeling Study

Tryptamine (T) and several T derivatives (Ts) inhibit in a voltage-dependent manner the NMDA receptor (NR). This effect is influenced by substituents at various positions, but has not yet been subjected to a detailed SAR study. Here, 64 Ts have been tested as inhibitors of [3H]MK-801 binding to NRs on rat brain membranes. For comparison, they were also tested as inhibitors of [3H]8-OHDPAT binding to 5-HT1A and of [3H]ketanserin binding to 5-HT2A receptors. Since most of these Ts have not been tested before at any of these receptors, we start with a review of the effects of Ts on 5-HT1A and 5-HT2A binding sites. NRs were inhibited with IC50s from 2 to 7 μM by Ts with alkyl or halogen at positions 2, 5, and/or 7. Inhibition by some Ts was attenuated more than 10-fold by 30 μM spermine. The most potent inhibitors at 5-HT1A receptors were 5-carboxamido-T (IC50 0.00015 μM) and serotonin (0.0016 μM), at 5-HT2A receptors 2-Me-4,7-Cl2-T (1.2 μM) and 2,7-Me2-4-Cl-T (2.0 μM). Fujita-Ban modified Free-Wilson analyses pointed to the individual significance of particular substituents. Also QSARs based on molecular operating environment descriptors resulted in sound correlations at all 4 targets. No similarities between the NR and 5-HT receptors could be found. At the NR, only L-Trp-NH2 bound 10 times better than at both 5-HT receptors studied. L-Trp-NH2 may be a structural lead to endogenous non-competitive NR antagonists.

Subunit-specific modulation of [3H]MK-801 binding to NMDA receptors mediated by dopamine receptor ligands in rodent brain

Dopamine D1 receptor (D1R) ligands may directly interact with the NMDA receptor (NMDAR), but detailed knowledge about this effect is lacking. Here we identify D1R ligands that directly modulate NMDARs and examine the contributions of NR2A and NR2B subunits to these interactions. Binding of the open channel blocker [3H]MK-801 in membrane preparations from rat- and mouse brain was used as a biochemical measure of the functional state of the NMDAR channel. We show that both D1R agonist A-68930 and dopamine receptor D2 antagonist haloperidol can decrease [3H]MK-801 binding with increased potency in membranes from the NR2A–/– mice (i.e. in membranes containing NR2B only), as compared to the inhibition obtained in wild-type membranes. Further, a wide range of D1R agonists such as A-68930, SKF-83959, SKF-83822, SKF-38393 and dihydrexidine were able to decrease [3H]MK-801 binding, all showing half maximal inhibitory concentrations ∼20μM, and with significant effects occurring at or above 1μM. With membranes from D1R–/– mice, we demonstrate that these effects occurred through a D1R-independent mechanism. Our results demonstrate that dopamine receptor ligands can selectively influence NR2B containing NMDARs, and we characterize direct inhibitory NMDAR effects by different D1R ligands.

Altered N-methyl-d-aspartate receptor function in reelin heterozygous mice: Male–female differences and comparison with dopaminergic activity

The aim of this study was to investigate the in vivo relationship between reelin and NMDA receptor function in schizophrenia. We assessed the effect of reelin deficiency in behavioral models of aspects of this illness, NMDA receptor subunit levels, and NMDA receptor, dopamine D₂ receptor, and dopamine transporter density. Male, but not female, reelin heterozygous mice showed significantly enhanced MK-801-induced locomotor hyperactivity compared to wildtype controls (7.4-fold vs. 5.2-fold effect of MK-801 over saline, respectively) but there were no genotype differences in the response to amphetamine. Both male and female reelin heterozygous mice showed enhanced effects of MK-801 on startle, but not prepulse inhibition (PPI) of startle. There were no group differences in the effect of apomorphine on startle or PPI. The levels of NMDA receptor subunits were not altered in the striatum. In the frontal cortex, male and female reelin heterozygous mice showed significant up-regulation of NR1 subunits, but down-regulation of NR2C subunits, which was associated with significantly elevated NR1/NR2A and NR1/NR2C ratios. However, there were no differences in [³H]MK-801 binding density in the nucleus accumbens or caudate nucleus, nor in the density of [³H]YM-09151 or [³H]GBR12935 in these brain regions. The enhanced effects of MK-801 in reelin heterozygous mice in this study could be reflective of the role of reelin deficiency in schizophrenia. This genotype effect was male-specific for locomotor hyperactivity, a model of psychosis, but was seen in male and female mice for startle, which could be an indication of changes in anxiety. Changes in NMDA receptor subunit levels and ratios were also seen in both male and female mice. These results suggest that the role of reelin deficiency in schizophrenia may be particularly mediated by altered NMDA receptor responses, with some of these effects being strictly sex-specific.

Deletion of the adenosine A2A receptor in mice enhances spinal cord neurochemical responses to an inflammatory nociceptive stimulus

Knockout mice lacking the adenosine A2A receptor are less sensitive to nociceptive stimuli, and this may be due to the presence of pronociceptive A2A receptors on sensory nerves. In support of this hypothesis, we have recently shown that in A2A receptor knockout mice there are marked reductions in the changes of two markers of spinal cord neuronal activity, [3H]MK801 binding to NMDA receptors and uptake of [14C]-2-deoxyglucose, in response to formalin injection. We now report that following a more prolonged inflammatory stimulus, consisting of intraplantar injections of PGE2 and paw pressure, there was in contrast an increase in [3H]MK801 binding and [14C]-2-deoxyglucose uptake in the spinal cords of the A2A receptor knockout mice which was much greater than in the wild-type mice. This increase suggests that when there is a pronounced inflammatory component to the stimulus, loss of inhibitory A2A receptors on inflammatory cells outweighs the loss of pronociceptive A2A receptors on peripheral nerves so that overall there is an increase in nociceptive signalling. This implies that although A2A antagonists have antinociceptive effects they may have only limited use as analgesics in chronic inflammatory pain.

Alterations in the emotional and memory behavioral phenotypes of transient receptor potential vanilloid type 1-deficient mice are mediated by changes in expression of 5-HT 1A, GABA A, and NMDA receptors

The transient receptor potential vanilloid type 1 channel (TRPV1) receptors are expressed in various regions of the brain. Much less is known about whether TRPV1 receptors affect higher brain functions. In the present study, we demonstrated that TRPV1-knockout (TRPV1KO) mice showed antidepressant-like behaviors in a novelty-suppressed feeding test and forced swim test when compared to wild-type (WT) mice. Additionally, TRPV1KO mice exhibited increased aggressiveness and reduced social interactions in a social dominance test and social interaction test. TRPV1KO mice showed reduced short-term memory and normal long-term memory in a novel object recognition test and passive avoidance test versus WT mice. Based on these behavioral data, we investigated changes in specific receptors related to depression, anxiety, and memory in the brains of TRPV1KO and WT mice. Binding of [ 3H]-8-OH-DPAT was significantly higher in the frontal associated cortex (FrA), nucleus accumbens (NAc), and the cingulate cortex (CC) of TRPV1KO mice than WT mice, while the expression of 5-HT 1A receptors was higher in the FrA, NAc, and cortex of TRPV1KO mice than WT mice. [ 3H]-flunitrazepam binding was also significantly higher in the FrA, striatum (CPU), and the CC of TRPV1KO versus WT mice. In contrast, [ 3H]-musicmol binding in the FrA, CPU, NAc, CC, and the dentate gyrus (DG) was significantly lower in TRPV1KO mice than WT mice. The expression of GABA Aγ 2 was higher in the NAc, CPU, and cortex of TRPV1KO versus WT mice, whereas the expression of GABA Aα 2 was lower in the FrA, CPU, NAc, and cortex in TRPV1KO mice than WT mice. Finally, [ 3H]-MK-801 binding was decreased in the CPU and CA1 of TRPV1KO versus WT mice. The expression of NR2A was lower in the hippocampus of TRPV1KO versus WT mice. These data suggest that the loss of TRPV1 results in antidepressant-like, anxiolytic, abnormal social and reduced memorial behaviors due to changes in expression of 5-HT 1A, GABA A, and NMDA receptors. This article is part of a Special Issue entitled ‘Post-Traumatic Stress Disorder’.

Levels of Ionotropic Glutamate and Muscarinic Receptors in Three Animal Models of Schizophrenia

There are well validated rodent paradigms of schizophrenia which are based on environmental manipulation (e.g. altered rearing conditions) or drug challenges. These manipulations induce behavioural changes in rodents that are thought to involve neuronal circuitry similar to the ones that are affected by the pathophysiology of the disorder. This study has investigated whether three such rodent paradigms (isolation rearing, neonatal PCP treatment or sub-chronic PCP treatment) are associated with changes in muscarinic receptors (CHRMs) or ionotropic glutamate receptors, some of which have been reported to be altered in the CNS of subjects with schizophrenia. ( 3 H)pirenzepine (CHRM1), ( 3 H)4DAMP (CHRM1/CHRM3), ( 3 H)MK801 (NMDA receptors) and ( 3 H)kainate (kainate receptors; KAR) binding were measured using in situ radioligand binding and autoradiography. Isolation rearing caused widespread decreases in ( 3 H)4DAMP (p = 0.01) and ( 3 H)kainate binding (p = 0.03). Neonatal PCP caused widespread increases in ( 3 H)4DAMP binding (p <0.0001), whereas sub-chronic PCP treatment caused widespread decreases in the binding of that radioligand (p < 0.002) and widespread increases in (3H)MK801 binding (p < 0.0001). There were no changes in ( 3 H)pirenzepine binding to CHRM1 receptors in any paradigm or no significant within region changes in the binding of any radioligand. In conclusion, in the absence of any changes in CHRM1 receptors, our ( 3 H)4DAMP and the binding of (3H)MK801 data would suggest that different rodent paradigms cause variable changes in levels of CHRM3 and KAR in the rat CNS. Our data raises the possibility that such changes may, in part, modulate the behavioural differences that have been observed after isolation rearing, neonatal PCP treatment or sub-chronic PCP treatment.