Ameliorative Effect of Cannabidiol on Topiramate-Induced Memory Loss: The Role of Hippocampal and Prefrontal Cortical NMDA Receptors and CREB/BDNF Signaling Pathways in Rats.

Cannabidiol (CBD) is a promising neurological agent with potential beneficial effects on memory and cognitive function. The combination of CBD and topiramate in the treatment of some neurological diseases has been of great interest. Since Topiramate-induced memory loss is a major drawback of its clinical application and the overall effect of the combination of CBD and topiramate on memory is still unclear, here we investigated the effect of CBD on topiramate-induced memory loss and the underlying molecular mechanisms. A one trial step-through inhibitory test was used to evaluate memory consolidation in rats. Moreover, the role of N-methyl-D-aspartate receptors (NMDARs) in the combination of CBD and topiramate in memory consolidation was evaluated through the intra-CA1 administration of MK-801 and NMDA. Western blot analysis was used to evaluate variations in brain-derived neurotrophic factor (BDNF) and phosphorylated cyclic AMP response element-binding protein (pCREB)/CREB ratio in the prefrontal cortex (PFC) and hippocampus (HPC). While the intraperitoneal (i.p.) administration of topiramate (50, 75, and 100 mg/kg) significantly reduced inhibitory time latency, the i.p. administration of CBD (20 and 40 mg/kg) could effectively reverse these effects. Similarly, the sub-effective doses of NMDA plus CBD (10 mg/kg) could improve the topiramate-induced memory loss along with an enhancement in BDNF and pCREB expression in the PFC and HPC. Contrarily, the administration of sub-effective doses of the NMDAR antagonist (MK-801) diminished the protective effects of CBD (20 mg/kg) on topiramate-induced memory loss associated with decreased BDNF and pCREB levels in the PFC and HPC. These findings suggest that CBD can improve topiramate-induced memory impairment, partially by the NMDARs of the PFC and HPC, possibly regulated by the CREB/BDNF signaling pathway.

G protein-coupled estrogen receptor 1 deficiency impairs adult hippocampal neurogenesis in mice with schizophrenia.

OBJECTIVE: This study aimed to confirm that G protein-coupled estrogen receptor 1 (GPER1) deficiency affects cognitive function by reducing hippocampal neurogenesis via the PKA/ERK/IGF-I signaling pathway in mice with schizophrenia (SZ). METHODS: Mice were divided into four groups, namely, KO Con, WT Con, KO Con, and WT SZ (n = 12 in each group). All mice were accustomed to the behavioral equipment overnight in the testing service room. The experimental conditions were consistent with those in the animal house. Forced swimming test and Y-maze test were conducted. Neuronal differentiation and maturation were detected using immunofluorescence and confocal imaging. The protein in the PKA/ERK/IGF-I signaling pathway was tested using Western blot analysis. RESULTS: GPER1 KO aggravated depression during forced swimming test and decreased cognitive ability during Y-maze test in the mouse model of dizocilpine maleate (MK-801)-induced SZ. Immunofluorescence and confocal imaging results demonstrated that GPER1 knockout reduced adult hippocampal dentate gyrus neurogenesis. Furthermore, GPER1-KO aggravated the hippocampal damage induced by MK-801 in mice through the PKA/ERK/IGF-I signaling pathway. CONCLUSIONS: GPER1 deficiency reduced adult hippocampal neurogenesis and neuron survival by regulating the PKA/ERK/IGF-I signaling pathway in the MK-801-induced mouse model of SZ.

Depletion of microglia with PLX3397 attenuates MK-801-induced hyperactivity associated with regulating inflammation-related genes in the brain.

Acute administration of MK-801 (dizocilpine), an N-methyl-D-aspartate receptor (NMDAR) antagonist, can establish animal models of psychiatric disorders. However, the roles of microglia and inflammation-related genes in these animal models of psychiatric disorders remain unknown. Here, we found rapid elimination of microglia in the prefrontal cortex (PFC) and hippocampus (HPC) of mice following administration of the dual colony-stimulating factor 1 receptor (CSF1R)/c-Kit kinase inhibitor PLX3397 (pexidartinib) in drinking water. Single administration of MK-801 induced hyperactivity in the open-field test (OFT). Importantly, PLX3397-induced depletion of microglia prevented the hyperactivity and schizophrenia-like behaviors induced by MK-801. However, neither repopulation of microglia nor inhibition of microglial activation by minocycline affected MK-801-induced hyperactivity. Importantly, microglial density in the PFC and HPC was significantly correlated with behavioral changes. In addition, common and distinct glutamate-, GABA-, and inflammation-related gene (116 genes) expression patterns were observed in the brains of PLX3397- and/or MK-801-treated mice. Moreover, 10 common inflammation-related genes ( CD68, CD163, CD206, TMEM119, CSF3R, CX3CR1, TREM2, CD11b, CSF1R, and F4/80) with very strong correlations were identified in the brain using hierarchical clustering analysis. Further correlation analysis demonstrated that the behavioral changes in the OFT were most significantly associated with the expression of inflammation-related genes ( NLRP3, CD163, CD206, F4/80, TMEM119, and TMEM176a), but not glutamate- or GABA-related genes in PLX3397- and MK-801-treated mice. Thus, our results suggest that microglial depletion via a CSF1R/c-Kit kinase inhibitor can ameliorate the hyperactivity induced by an NMDAR antagonist, which is associated with modulation of immune-related genes in the brain.

GLP-1 agonist Liraglutide prevents MK­801-induced schizophrenia­like behaviors and BDNF, CREB, p-CREB, Trk-B expressions in the hippocampus and prefrontal cortex in Balb/c mice.

Glucagon-like peptide 1 (GLP-1) agonists are among the agents that can be used to treat type 2 diabetes mellitus, and they have also been reported to have neuroprotective effects. This study examined the effects of GLP-1 agonist Liraglutide on CREB, BDNF, Trk-B expression and emotional/cognitive behaviors in an experimental schizophrenia-like behavior model induced by MK-801. MK-801 (0.25 mg/kg, 0.1 ml/kg body weight) and/or Liraglutide (300 mcg/kg) were injected intraperitoneally once a day for 7 weeks into 8-10 weeks old male Balb/c mice (n = 78). Mice were randomly divided into 5 groups: Saline+Saline, MK-801 +Saline, Liraglutide+Saline, MK-801 +Liraglutide co-treatment, and Liraglutide+MK-801 co-treatment. A Morris water maze test, an elevated plus maze test, and an open field test were performed after injection. Western blots were performed on mice' hippocampus and PFC for BDNF, Trk-B, CREB, and p-CREB expression. Our study found that MK-801 impaired emotional and cognitive functions in mice. MK-801 administration did not affect Liraglutide's positive effects on spatial learning and memory activity in the Liraglutide+MK-801 group. Liraglutide administration (Liraglutide+MK-801 group) improved the BDNF/Trk-B and p-CREB/CREB ratio in the hippocampus, and the p-CREB/CREB ratio in the PFC to the control group level. The negative effects of MK-801 on cognitive behavior were not reversed by Liraglutide in the MK-801 +Liraglutide group. In conclusion, Liraglutide does not affect NMDA receptor blockade-induced emotional and cognitive behaviors. However, it has a protective effect against cognitive impairment. Furthermore, it is possible that the GLP-1 receptors in the hippocampus and PFC are involved in the modulation of NMDA receptor activity through CREB activation/deactivation.

Nicotine pretreatment alleviates MK-801-induced behavioral and cognitive deficits in mice by regulating Pdlim5/CRTC1 in the PFC.

Increasing evidence shows that smoking-obtained nicotine is indicated to improve cognition and mitigate certain symptoms of schizophrenia. In this study, we investigated whether chronic nicotine treatment alleviated MK-801-induced schizophrenia-like symptoms and cognitive impairment in mice. Mice were injected with MK-801 (0.2 mg/kg, i.p.), and the behavioral deficits were assessed using prepulse inhibition (PPI) and T-maze tests. We showed that MK-801 caused cognitive impairment accompanied by increased expression of PDZ and LIM domain 5 (Pdlim5), an adaptor protein that is critically associated with schizophrenia, in the prefrontal cortex (PFC). Pretreatment with nicotine (0.2 mg · kg-1 · d-1, s.c., for 2 weeks) significantly ameliorated MK-801-induced schizophrenia-like symptoms and cognitive impairment by reversing the increased Pdlim5 expression levels in the PFC. In addition, pretreatment with nicotine prevented the MK-801-induced decrease in CREB-regulated transcription coactivator 1 (CRTC1), a coactivator of CREB that plays an important role in cognition. Furthermore, MK-801 neither induced schizophrenia-like behaviors nor decreased CRTC1 levels in the PFC of Pdlim5-/- mice. Overexpression of Pdlim5 in the PFC through intra-PFC infusion of an adreno-associated virus AAV-Pdlim5 induced significant schizophrenia-like symptoms and cognitive impairment. In conclusion, chronic nicotine treatment alleviates schizophrenia-induced memory deficits in mice by regulating Pdlim5 and CRTC1 expression in the PFC.

Upregulation of Connexins in the Rat Hippocampal and Cortical Neurons Following Blockade of NMDA Receptors During Postnatal Development.

BACKGROUND: Interneural gap junctional coupling represents neural development that decreases during the postnatal period. The decrease of gap junction function coincides with the main period of chemical synapse creation and increment of synaptic activity during postnatal weeks 1 to 3. METHODS: Here, we have assessed the role of chemical synapses on connexin (Cx) expression in neurons and glial cells of hippocampal and cortical neurons. We characterized the impact of NMDA receptors blockade on the expression of Cx36 and Cx43 proteins by western blot analysis in postnatal day (PND)14 and PND28. MK801 was injected subcutaneously from the first day of birth until 14 or 28 days, depending on the experimental groups. Saline was injected in the same volumes in the control group. RESULTS: Early postnatal blockade of the NMDA subtype of glutamate receptors by the non-competitive antagonist dizocilpine maleate (MK801) arrested the developmental reduction in gap junctions during the initial postnatal weeks. Expression of Cx43 declined in PND28 compared to PND14 in visual cortex (VC) neurons. Also, we found that the expression of Cx36 and Cx43 augmented in the rats' VC in PND28 following the blockade of NMDA receptors. Expression of Cx36 declined in PND28 compared to PND14 in hippocampal neurons. Also, we found that the expression of Cx36 augmented in the rats' hippocampal neurons in PND14 and PND28 following a blockade of NMDA receptors. CONCLUSION: These results suggest that the postnatal enhancement in glutamatergic synaptic activity is associated with the loss of gap junctional connections and downregulation of Cx36 and Cx43 between developing neurons and glial cells.

Inhibition of MicroRNA-182/183 Cluster Ameliorates Schizophrenia by Activating the Axon Guidance Pathway and Upregulating DCC.

Schizophrenia (SZ) is a complex disorder caused by a variety of genetic and environmental factors. Mounting evidence suggests the involvement of microRNAs (miRNAs) in the pathology of SZ. Accordingly, the current study set out to investigate the possible implication of the miR-182/183 cluster, as well as its associated mechanism in the progression of SZ. Firstly, rat models of SZ were established by intraperitoneal injection of MK-801. Moreover, rat primary hippocampal neurons were exposed to MK-801 to simulate injury of hippocampal neurons. The expression of miR-182/183 or its putative target gene DCC was manipulated to examine their effects on SZ in vitro and in vivo. It was found that miR-182 and miR-183 were both highly expressed in peripheral blood of SZ patients and hippocampal tissues of SZ rats. In addition, the miR-182/183 cluster could target DDC and downregulate the expression of DDC. On the other hand, inhibition of the miR-182/183 cluster ameliorated SZ, as evidenced by elevated serum levels of NGF and BDNF, along with reductions in spontaneous activity, serum GFAP levels, and hippocampal neuronal apoptosis. Additionally, DCC was found to activate the axon guiding pathway and influence synaptic activity in hippocampal neurons. Collectively, our findings highlighted that inhibition of the miR-182/183 cluster could potentially attenuate SZ through DCC-dependent activation of the axon guidance pathway. Furthermore, inhibition of the miR-182/183 cluster may represent a potential target for the SZ treatment.

Differentiation of neonate mouse spermatogonia on two-dimensional and three-dimensional culture systems supplemented with d-Serine and Dizocilpine (MK-801).

N-methyl-d-aspartate (NMDA) modulates the spermatogenesis process through stimulating the steroid hormone biosynthesis. The aim of this study was to evaluate the effects of NMDA receptors agonists (d-Serine) and antagonists (MK801) on spermatogonia differentiation on decellularization testicular matrix (DTM) hydrogel scaffold. Four treatment groups were planned: 2D + D-Serine, 3D + D-Serine, 2D + MK801, and 3D + MK801. Results showed that cell viability was significantly decreased after 48 h in the 3D + D-Serine group and after 24 and 48 h in the 3D + MK801 group compared to the controls. The spermatogonia proliferation after two, four, and eight weeks was significantly increased in the 3D + D-Serine culture, while it was significantly reduced in the 2D + MK801 and 3D + MK801 groups after four and eight weeks. Real-time PCR results demonstrated that pre-meiotic gene (Plzf) expression was significantly increased only in the 3D + D-Serine culture compared to the control groups after four weeks of culture. The meiotic gene (Sycp3) expression was significantly increased in the 2D + D-Serine and 3D + D-Serine compared to the 2D controls after four and eight weeks. The post-meiotic gene (Tnp1) level in the 3D + D-Serine was significantly higher than the other groups. Flow-cytometry results indicated that the protein expression of Plzf (after four and eight weeks), Sycp3 (after eight weeks), and Tnp1 (after eight weeks) in the d-Serine-treated groups was significantly increased compared with the 2D control groups. There were not any significant changes in the gene expression of spermatogenic-related markers in MK801 culture media. However, a significant decrease in the protein levels of Plzf after eight weeks and Sycp3 after four and eight weeks was observed. In conclusion, the addition of NMDARs agonists (d-Serine) could be used to regulate the differentiation of spermatogonia in the 3D culture system.

Neuronal network-based biomimetic chip for long-term detection of olfactory dysfunction model in early-stage Alzheimer's disease.

Olfactory dysfunction is an early symptom of neurodegenerative disease. Amyloid-beta oligomers (AßOs), the pathologic protein of Alzheimer's disease (AD), have been confirmed to be firstly deposited in olfactory bulb (OB), causing smell to malfunction. However, the detailed mechanisms underlying pathogenic nature of AßOs-induced olfactory neuronal degeneration in AD are not completely realized. Here, an early-stage olfactory dysfunction pathological model of AD in vitro based on biomimetic OB neuronal network chip was established for dynamic multi-site detection of neuronal electrical activity and network connection. We found both spike firing and correlation of overall neuronal network change regularly displayed gradually active state and then rapidly decay state after AßOs induction. Moreover, MK-801 and memantine were administrated at early-stage to detect alteration of OB neurons simulating nasal administration for AD treatment, which showed an almost recovery through the intermittent firing pattern. Together, this neuronal network-on-chip has revealed synaptic impairment and network neurodegeneration of olfactory dysfunction in AD, providing potential mechanisms information for early-stage progressive olfactory amyloidogenic pathology.

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 1 nM, 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 (1 nM). 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.

Effects of the N-methyl-d-aspartate receptor antagonist, MK-801, on spatial memory and influence of the route of administration.

The N-methyl-d-aspartate receptor antagonist, MK-801, is widely used to induce memory and learning impairments in preclinical studies. MK-801 is mainly injected intraperitoneally (i.p.) at doses that result in cognitive impairment and induction of motor or sensory disturbances. The aim of this study was to compare the behavioral outcomes when different administration routes (subcutaneous (s.c.) and i.p.) and MK-801 doses (0.01, 0.05, and 0.1 mg/kg) are employed in the Morris water maze (MWM) task. We also assessed the pharmacokinetics of MK-801 in rat blood plasma and its bioavailability in brain tissue. The concentrations of MK-801 in brain tissue and blood plasma were significantly higher after s.c. than i.p. administration. MK-801 administered via the s.c. route at doses of 0.1 and 0.05 mg/kg significantly impaired learning on all training days in the MWM task compared to i.p. administration at the same doses. Memory in the probe trial was significantly impaired after MK-801 administration via both routes at all doses. MK-801 also induced locomotor disturbances after i.p. and s.c. administration at the highest dose (0.1 mg/kg). Our data suggest that s.c. administration leads to higher MK-801 concentrations in brain tissue and blood plasma and evidently impairs spatial learning and memory compared to i.p. administration at the same dose. Knowledge of MK-801 concentrations in the brain and blood and the effects of the compound on memory processes and locomotor activity enable the choice of more targeted routes and doses of administration in preclinical studies.

Binding characterization of N-(2-chloro-5-thiomethylphenyl)-N'-(3-[3 H]3 methoxy phenyl)-N'-methylguanidine ([3 H]GMOM), a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist.

Labeled with carbon-11, N-(2-chloro-5-thiomethylphenyl)-N'-(3-methoxyphenyl)-N'-methylguanidine ([11 C]GMOM) is currently the only positron emission tomography (PET) tracer that has shown selectivity for the ion-channel site of N-methyl-D-aspartate (NMDA) receptors in human imaging studies. The present study reports on the selectivity profile and in vitro binding properties of GMOM. The compound was screened on a panel of 80 targets, and labeled with tritium ([3 H]GMOM). The binding properties of [3 H]GMOM were compared to those of the reference ion-channel ligand [3 H](+)-dizocilpine maleate ([3 H]MK-801), in a set of concentration-response, homologous and heterologous inhibition, and association kinetics assays, performed with repeatedly washed rat forebrain preparations. GMOM was at least 70-fold more selective for NMDA receptors compared to all other targets examined. In homologous inhibition and concentration-response assays, the binding of [3 H]GMOM was regulated by NMDA receptor agonists, albeit in a less prominent manner compared to [3 H]MK-801. Scatchard transformation of homologous inhibition data produced concave upward curves for [3 H]GMOM and [3 H]MK-801. The radioligands showed bi-exponential association kinetics in the presence of 100 µmol L-1 l-glutamate/30 µmol L-1 glycine. [3 H]GMOM (3 nmol L-1 and 10 nmol L-1 ) was inhibited with dual affinity by (+)-MK-801, (R,S)-ketamine and memantine, in both presence and absence of agonists. [3 H]MK-801 (2 nmol L-1 ) was inhibited in a monophasic manner by GMOM under baseline and combined agonist conditions, with an IC50 value of ~19 nmol L-1 . The non-linear Scatchard plots, biphasic inhibition by open channel blockers, and bi-exponential kinetics of [3 H]GMOM indicate a complex mechanism of interaction with the NMDA receptor ionophore. The implications for quantifying the PET signal of [11 C]GMOM are discussed.

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 [3 H]-AMPA binding (but not in [3 H]-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.

Cell-Specific Chemical Delivery Using a Selective Nitroreductase-Nitroaryl Pair.

The utility of small molecules to probe or perturb biological systems is limited by the lack of cell-specificity. "Masking" the activity of small molecules using a general chemical modification and "unmasking" it only within target cells overcomes this limitation. To this end, we have developed a selective enzyme-substrate pair consisting of engineered variants of E. coli nitroreductase (NTR) and a 2-nitro- N-methylimidazolyl (NM) masking group. To discover and optimize this NTR-NM system, we synthesized a series of fluorogenic substrates containing different nitroaromatic masking groups, confirmed their stability in cells, and identified the best substrate for NTR. We then engineered the enzyme for improved activity in mammalian cells, ultimately yielding an enzyme variant (enhanced NTR, or eNTR) that possesses up to 100-fold increased activity over wild-type NTR. These improved NTR enzymes combined with the optimal NM masking group enable rapid, selective unmasking of dyes, indicators, and drugs to genetically defined populations of cells.

Altered zinc sensitivity of NMDA receptors harboring clinically-relevant mutations.

Recent human genetic studies have identified a surprisingly high number of alterations in genes encoding NMDA receptor (NMDAR) subunits in several common brain diseases. Among NMDAR subunits, the widely-expressed GluN2A subunit appears particularly affected, with tens of de novo or inherited mutations associated with neurodevelopmental conditions including childhood epilepsies and cognitive deficits. Despite the increasing identification of NMDAR mutations of clinical interest, there is still little information about the effects of the mutations on receptor and network function. Here we analyze the impact on receptor expression and function of nine GluN2A missense (i.e. single-point) mutations targeting the N-terminal domain, a large regulatory region involved in subunit assembly and allosteric signaling. While several mutations produced no or little apparent effect on receptor expression, gating and pharmacology, two showed a drastic expression phenotype and two resulted in marked alterations in the sensitivity to zinc, a potent allosteric inhibitor of GluN1/GluN2A receptors and modulator of excitatory synaptic transmission. Surprisingly, both increase (GluN2A-R370W) and decrease (GluN2A-P79R) of zinc sensitivity were observed on receptors containing either one or two copies of the mutated subunits. Overexpression of the mutant subunits in cultured rat neurons confirmed the results from heterologous expression. These results, together with previously published data, indicate that disease-causing mutations in NMDARs produce a wide spectrum of receptor alterations, at least in vitro. They also point to a critical role of the zinc-NMDAR interaction in neuronal function and human health.

N'-3-(Trifluoromethyl)phenyl Derivatives of N-Aryl-N'-methylguanidines as Prospective PET Radioligands for the Open Channel of the N-Methyl-d-aspartate (NMDA) Receptor: Synthesis and Structure-Affinity Relationships.

N-Methyl-d-aspartate (NMDA) receptor dysfunction has been linked to several neuropsychiatric disorders, including Alzheimer's disease, epilepsy, drug addiction, and schizophrenia. A radioligand that could be used with PET to image and quantify human brain NMDA receptors in the activated "open channel" state would be useful for research on such disorders and for the development of novel therapies. To date, no radioligands have shown well-validated efficacy for imaging NMDA receptors in human subjects. In order to discover improved radioligands for PET imaging, we explored structure-affinity relationships in N'-3-(trifluoromethyl)phenyl derivatives of N-aryl-N'-methylguanidines, seeking high affinity and moderate lipophilicity, plus necessary amenability for labeling with a positron-emitter, either carbon-11 or fluorine-18. Among a diverse set of 80 prepared N'-3-(trifluoromethyl)phenyl derivatives, four of these compounds (13, 19, 20, and 36) displayed desirable low nanomolar affinity for inhibition of [(3)H](+)-MK801 at the PCP binding site and are of interest for candidate PET radioligand development.

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.

Syntheses and N-methyl-D-aspartate receptor antagonist pharmacology of fluorinated arylcycloheptylamines.

Selective uncompetitive antagonists of the phencyclidine (PCP) binding site of the N-methyl-D-aspartate receptor (NMDAR) are known to have therapeutic potential as anticonvulsants and neuroprotective agents. Several fluorinated molecules with each containing a cycloheptane ring were designed to probe the PCP pharmacophore and test the influence of fluorine substitution on NMDAR binding and in vivo efficacy. Syntheses and analyses of six novel compounds, 1-(4- fluorophenyl)cycloheptanamine (3), 1-(1-(4-fluorophenyl)cycloheptyl)piperidine (4), 1-(1-(4-fluorophenyl)cycloheptyl) pyrrolidine (5), 1-(3-fluorophenyl)cycloheptanamine (6), 1-(1-(3-fluorophenyl)cycloheptyl)piperidine (7), 1-(1-(3-fluorophenyl) cycloheptyl)pyrrolidine (8) and several related reference arylcyloalkylamines are described. Receptor binding was performed at the PCP site of NMDAR for each compound using [(3)H]-(+)-MK-801 displacement. Unexpectedly, the 3- fluoro- primary amine 6 had the greatest affinity of the series and these binding results support a different structure activity relationship (SAR) profile for arylcycloheptylamines when compared to arylcyclohexylamines like PCP. Five of the novel compounds have affinity (Ki) in the hundred nM (10(-7)) range. In addition, compounds 3, 5, 6, 7 and 8 were evaluated and found to exhibit neuroprotective effects from NMDA induced toxicity in vitro and compounds 6, 7 and 8 exhibited anticonvulsant activities in rats. An ED50 of 13.84 mg/kg was found for compound 6 in rat maximal electroshock (MES) test with a protective index (PI) of 3.66 against ataxia. These results support further investigation of the arylcycloheptylamine class.

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

BACKGROUND: There 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. METHODS: The present study investigated the potency of Zn(2+) and Mg(2+) to [(3)H] 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. RESULTS: Our results revealed that there was a statistically significant decrease (by 29% and 40%) in the potency of zinc and magnesium (respectively) to inhibit [(3)H] 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. CONCLUSIONS: Our 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.

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.