Search

Background: Currently, the most dynamic areas in the glutamate receptor system neurobiology are the identification and development of positive allosteric modulators (PAMs) of glutamate ionotropic receptors. PAM-based drugs are of great interest as promising candidates for the treatment of neurological diseases, such as epilepsy, Alzheimer's disease, schizophrenia, etc. Understanding the molecular mechanisms underlying the biological action of natural and synthetic PAMs is a key point for modifying the original chemical compounds as well as for new drug design., Objective: We are trying to elaborate a system of molecular functional screening of ionotropic glutamate receptor probable PAMs., Methods: The system will be based on the radioligand - receptor method of analysis and will allow rapid quantification of new AMPAR probable PAMs molecular activity. We plan to use a tritiumlabeled analogue of recently elaborated ionotropic GluR probable PAM ([3H]PAM-43) as the main radioligand., Results: Here, we characterized the specific binding of the ligand and its ability to potentiate ionotropic GluR currents. The existence of at least two different sites of [3H]PAM-43 specific binding has been shown. One of the above sites is glutamate-dependent and is characterized by higher affinity. "Patchclamp" technique showed the ability of PAM-43 to potentiate ionotropic GluR currents in rat cerebellar Purkinje neurons in a concentration-dependent manner., Conclusion: The possibility of using PAM-43 as a model compound to study different allosteric effects of potential regulatory drugs (AMPAR allosteric regulators) was shown. [3H]PAM-43 based screening system will allow rapid selection of new AMPAR probable PAM structures and quantification of their molecular activity., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors produce postsynaptic current by transmitting an agonist-induced structural change in the ligand-binding domain (LBD) to the transmembrane channel. Receptors carrying T686S/A substitutions in their LBDs produce weaker glutamate-evoked currents than wild-type (WT) receptors. However, the substitutions induce little differences in the crystal structures of their LBDs. To understand the structural mechanism underlying reduced activities of these AMPAR variants, we analyzed the structural dynamics of WT, T686S, and T686A variants of LBD using nuclear magnetic resonance. The HD exchange studies of the LBDs showed that the kinetic step where the ligand-binding cleft closes was changed by the substitutions, and the substitution-induced population shift from cleft-closed to cleft-open structures is responsible for the reduced activities of the variants. The chemical shift analyses revealed another structural equilibrium between cleft-locked and cleft-partially-open conformations. The substitution-induced population shift in this equilibrium may be related to slower desensitization observed for these variants., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The metabotropic glutamate 7 (mGlu 7 ) receptor belongs to the group III of mGlu receptors. Since the mGlu 7 receptor can control excitatory neurotransmission in the hippocampus and cortex, modulation of the receptor may have therapeutic benefit in several CNS diseases. However, mGlu 7 remains relatively unexplored among the eight known mGlu receptors partly because of the limited availability of tool compounds to interrogate its potential therapeutic utility. Here we report the discovery of a new class of mGlu 7 allosteric agonists. Hits originating from virtual screening were followed up with further analogue searching and screening, leading to a novel series of mGlu 7 allosteric agonists. Guided by docking into a structural model of the mGlu 7 receptor the initial hit 5 was successfully optimized to analogues with comparable potencies and more attractive drug-like attributes than AMN082.

α-amino-3-hydroxy-5-methyl-4-isoaxazolepropionic acid (AMPA) ionotropic glutamate receptors mediate fast excitatory neurotransmission in the central nervous system, and their dysfunction is associated with neurological diseases. Glutamate binding to ligand-binding domains (LBDs) of AMPA receptors induces channel opening in the transmembrane domains of the receptors. The T686A mutation reduces glutamate efficacy so that the glutamate behaves as a partial agonist. The crystal structures of wild-type and mutant LBDs are very similar and cannot account for the observed behavior. To elucidate the molecular mechanism inducing partial agonism of the T686A mutant, we computed the free-energy landscapes governing GluA2 LBD closure using replica-exchange umbrella sampling simulations. A semiclosed state, not observed in crystal structures, appears in the mutant during simulation. In this state, the LBD cleft opens slightly because of breaking of interlobe hydrogen bonds, reducing the efficiency of channel opening. The energy difference between the LBD closed and semiclosed states is small, and transitions between the two states would occur by thermal fluctuations. Evidently, glutamate binding to the T686A mutant induces a population shift from a closed to a semiclosed state, explaining the partial agonism in the AMPA receptor., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Positive modulators of NMDA receptors are important candidates for therapeutic development to treat psychiatric disorders including autism and schizophrenia. Sulfated neurosteroids have been studied as positive allosteric modulators of NMDA receptors for years, but we understand little about the cellular fate of these compounds, an important consideration for drug development. Here we focus on a visualizable sulfated neurosteroid analogue, KK-169. As expected of a pregnenolone sulfate analogue, the compound strongly potentiates NMDA receptor function, is an antagonist of GABA A receptors, exhibits occlusion with pregnenolone sulfate potentiation, and requires receptor domains important for pregnenolone sulfate potentiation. KK-169 exhibits somewhat higher potency than the natural parent, pregnenolone sulfate. The analogue contains a side-chain alkyne group, which we exploited for retrospective click labeling of neurons. Although the anionic sulfate group is expected to hinder cell entry, we detected significant accumulation of KK-169 in neurons with even brief incubations. Adding a photolabile diazirine group revealed that the expected plasma membrane localization of KK-169 is likely lost during fixation. Overall, our studies reveal new facets of the structure-activity relationship of neurosteroids at NMDA receptors, and their intracellular distribution suggests that sulfated neurosteroids could have unappreciated targets in addition to plasma membrane receptors., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Metabotropic glutamate receptors belong to class C G-protein-coupled receptors and consist of eight subtypes that are ubiquitously expressed throughout the central nervous system. In recent years, the metabotropic glutamate receptor subtype 5 (mGlu 5 ) has emerged as a promising target for a broad range of psychiatric and neurological disorders. Drug discovery programs targetting mGlu 5 are primarily focused on development of allosteric modulators that interact with sites distinct from the endogenous agonist glutamate. Significant efforts have seen mGlu 5 allosteric modulators progress into clinical trials; however, recent failures due to lack of efficacy or adverse effects indicate a need for a better understanding of the functional consequences of mGlu 5 allosteric modulation. Biased agonism is an interrelated phenomenon to allosterism, describing how different ligands acting through the same receptor can differentially influence signaling to distinct transducers and pathways. Emerging evidence demonstrates that allosteric modulators can induce biased pharmacology at the level of intrinsic agonism as well as through differential modulation of orthosteric agonist-signaling pathways. Here, we present key considerations in the discovery and development of mGlu 5 allosteric modulators and the opportunities and pitfalls offered by biased agonism and modulation., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Receptors of glutamic acid are known for over 30 years for their action and for about 20 years for their structure. Presence of at least three classes of ionotropic receptors was confirmed at the beginning of 80'. Recognition of the sequence and first cloning were done at the beginning of 90'. In 1994 ligand binding site was recognized at the junction of two subunits S1-S2 in the ligand-binding domain. Since then, many subtypes of ionotropic and metabotropic glutamate receptors were recognized, together with their localization and functions. In the meantime numerous orthosteric ligands, both agonists and antagonists were developed especially for NMDA ion channels. Their usefulness as drugs was rather low, due to the involvement in the excitatory tract. More interest was focused on metabotropic receptors, which are GPSR's and can be modulated both by orthosteric and allosteric modulators. It seems like allosterism could be considered as promising future for glutamate receptors and ion channels, especially when first allosteric negative modulators of the mGluR2 went close into the clinical trial., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

The cyanobacterial toxin β-N-methylamino-L-alanine (BMAA) now appears to be a cause of Guamanian amyotrophic lateral sclerosis/parkinsonism dementia complex (ALS/PDC). Its production by cyanobacteria throughout the world combined with multiple mechanisms of BMAA neurotoxicity, particularly to vulnerable subpopulations of motor neurons, has significantly increased interest in investigating exposure to this non-protein amino acid as a possible risk factor for other forms of neurodegenerative illness. We here provide a brief overview of BMAA studies and provide an introduction to this collection of scientific manuscripts in this special issue on BMAA.

AMPA receptors mediate fast excitatory neurotransmission in the mammalian brain and transduce the binding of presynaptically released glutamate to the opening of a transmembrane cation channel. Within the postsynaptic density, however, AMPA receptors coassemble with transmembrane AMPA receptor regulatory proteins (TARPs), yielding a receptor complex with altered gating kinetics, pharmacology, and pore properties. Here, we elucidate structures of the GluA2-TARP γ2 complex in the presence of the partial agonist kainate or the full agonist quisqualate together with a positive allosteric modulator or with quisqualate alone. We show how TARPs sculpt the ligand-binding domain gating ring, enhancing kainate potency and diminishing the ensemble of desensitized states. TARPs encircle the receptor ion channel, stabilizing M2 helices and pore loops, illustrating how TARPs alter receptor pore properties. Structural and computational analysis suggests the full agonist and modulator complex harbors an ion-permeable channel gate, providing the first view of an activated AMPA receptor., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Positive allosteric modulators of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are small molecules that decrease deactivation of AMPARs via an allosteric site. These molecules keep the receptor in an active state. Interestingly, this type of modulator has been proposed for treating cognitive decline in ageing, dementias, and Alzheimer's disease (AD). S 47445 (8-cyclopropyl-3-[2-(3-fluorophenyl)ethyl]-7,8-dihydro-3H-[1,3]oxazino[6,5-g][1,2,3]benzotriazine-4,9-dione) is a novel AMPAR positive allosteric modulator (AMPA-PAM). Here, the mechanisms by which S 47445 could improve synaptic strength and connectivity were studied and compared between young and old mice. A single oral administration of S 47445 at 10 mg/kg significantly increased long-term potentiation (LTP) in CA3-CA1 hippocampal synapses in alert young mice in comparison to control mice. Moreover, chronic treatment with S 47445 at 10 mg/kg in old alert animals significantly counteracted the deficit of LTP due to age. Accordingly, chronic treatment with S 47445 at 10 mg/kg seems to preserve synaptic cytoarchitecture in old mice as compared with young control mice. It was shown that the significant decreases in number and size of pre-synaptic buttons stained for VGlut1, and post-synaptic dendritic spines stained for spinophilin, observed in old mice were significantly prevented after chronic treatment with 10 mg/kg of S 47445. Altogether, by its different effects on LTP, VGlut1-positive particles, and spinophilin, S 47445 is able to modulate both the structure and function of hippocampal excitatory synapses known to be involved in learning and memory processes. These results open a new window for the treatment of specific age-dependent cognitive decline and dementias such as AD., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

A series of analogues based on serine as lead structure were designed, and their agonist activities were evaluated at recombinant NMDA receptor subtypes (GluN1/2A-D) using two-electrode voltage-clamp (TEVC) electrophysiology. Pronounced variation in subunit-selectivity, potency, and agonist efficacy was observed in a manner that was dependent on the GluN2 subunit in the NMDA receptor. In particular, compounds 15a and 16a are potent GluN2C-specific superagonists at the GluN1 subunit with agonist efficacies of 398% and 308% compared to glycine. This study demonstrates that subunit-selectivity among glycine site NMDA receptor agonists can be achieved and suggests that glycine-site agonists can be developed as pharmacological tool compounds to study GluN2C-specific effects in NMDA receptor-mediated neurotransmission.

A new algorithm for comparison of protein dynamics is presented. Compared protein structures are superposed and their modes of motions are calculated using the anisotropic network model. The obtained modes are aligned using the dynamic programming algorithm of Needleman and Wunsch, commonly used for sequence alignment. Dynamical comparison of hemoglobin in the T and R2 states reveals that the dynamics of the allosteric effector 2,3-bisphosphoglycerate binding site is different in the two states. These differences can contribute to the selectivity of the effector to the T state. Similar comparison of the ionotropic glutamate receptor in the kainate+(R,R)-2b and ZK bound states reveals that the kainate+(R,R)-2b bound states slow modes describe upward motions of ligand binding domain and the transmembrane domain regions. Such motions may lead to the opening of the receptor. The upper lobes of the LBDs of the ZK bound state have a smaller interface with the amino terminal domains above them and have a better ability to move together. The present study exemplifies the use of dynamics comparison as a tool to study protein function. Proteins 2017; 85:1507-1517. © 2014 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Kainate receptors (KARs) consist of a class of ionotropic glutamate receptors, which exert diverse pre- and postsynaptic functions through complex signaling regulating the activity of neural circuits. Whereas numerous small-molecule positive allosteric modulators of the ligand-binding domain of ( S )-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propanoic acid (AMPA) receptors have been reported, no such ligands are available for KARs. In this study, we investigated the ability of three benzothiadiazine-based modulators to potentiate glutamate-evoked currents at recombinantly expressed KARs. 4-cyclopropyl-7-fluoro-3,4-dihydro-2 H -1,2,4-benzothiadiazine 1,1-dioxide (BPAM344) potentiated glutamate-evoked currents of GluK2a 21-fold at the highest concentration tested (200 μ M), with an EC 50 of 79 μ M. BPAM344 markedly decreased desensitization kinetics (from 5.5 to 775 ms), whereas it only had a minor effect on deactivation kinetics. 4-cyclopropyl-7-hydroxy-3,4-dihydro-2 H -1,2,4-benzothiadiazine 1,1-dioxide (BPAM521) potentiated the recorded peak current amplitude of GluK2a 12-fold at a concentration of 300 μ M with an EC 50 value of 159 μ M, whereas no potentiation of the glutamate-evoked response was observed for 7-chloro-4-(2-fluoroethyl)-3,4-dihydro-2 H -1,2,4-benzothiadiazine 1,1-dioxide (BPAM121) at the highest concentration of modulator tested (300 μ M). BPAM344 (100 μ M) also potentiated the peak current amplitude of KAR subunits GluK3a (59-fold), GluK2a (15-fold), GluK1b (5-fold), as well as the AMPA receptor subunit GluA1 i (5-fold). X-ray structures of the three modulators in the GluK1 ligand-binding domain were determined, locating two modulator-binding sites at the GluK1 dimer interface. In conclusion, this study may enable the design of new positive allosteric modulators selective for KARs, which will be of great interest for further investigation of the function of KARs in vivo and may prove useful for pharmacologically controlling the activity of neuronal networks., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

The metabotropic glutamate receptor 2 (mGlu 2 ) plays an important role in the presynaptic control of glutamate release and several mGlu 2 positive allosteric modulators (PAMs) have been under assessment for their potential as antipsychotics. The binding mode of mGlu 2 PAMs is better characterized in functional terms while few data are available on the relationship between allosteric and orthosteric binding sites. Pharmacological studies characterizing binding and effects of two different chemical series of mGlu 2 PAMs are therefore carried out here using the radiolabeled mGlu 2 agonist 3 [H]-LY354740 and mGlu 2 PAM 3 [H]-2,2,2-TEMPS. A multidimensional approach to the PAM mechanism of action shows that mGlu 2 PAMs increase the affinity of 3 [H]-LY354740 for the orthosteric site of mGlu2 as well as the number of 3 [H]-LY354740 binding sites. 3 [H]-2,2,2-TEMPS binding is also enhanced by the presence of LY354740. New residues in the allosteric rat mGlu2 binding pocket are identified to be crucial for the PAMs ligand binding, among these Tyr 3.40 and Asn 5.46 . Also of remark, in the described experimental conditions S731A (Ser 5.42 ) residue is important only for the mGlu 2 PAM LY487379 and not for the compound PAM-1: an example of the structural differences among these mGlu 2 PAMs. This study provides a summary of the information generated in the past decade on mGlu 2 PAMs adding a detailed molecular investigation of PAM binding mode. Differences among mGlu 2 PAM compounds are discussed as well as the mGlu2 regions interacting with mGlu 2 PAM and NAM agents and residues driving mGlu2 PAM selectivity. This article is part of the Special Issue entitled 'Metabotropic Glutamate Receptors, 5 years on'., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Memory loss observed as a consequence of aging is paralleled by a down-regulation of AMPA-type glutamate receptors (AMPARs) that mediate fast excitatory synaptic transmission. Activation of these receptors enhances long-term potentiation (LTP), a neuronal process demonstrated to be crucial for memory storage and thought to be a cellular substrate of learning and memory. In the present studies, we determined that LTP was reduced in aged rats when compared to young rats and that acute treatment with CX1846, a novel AMPAR positive allosteric modulator, fifteen minutes prior to tetanic stimulation completely reversed the significant deficit in LTP observed in aged rats. These results suggest that CX1846 might be useful for the treatment of age-related memory impairments., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

The controlled activation of proteins in living cells is an important goal in protein-design research, but to introduce an artificial activation switch into membrane proteins through rational design is a significant challenge because of the structural and functional complexity of such proteins. Here we report the allosteric activation of two types of membrane-bound neurotransmitter receptors, the ion-channel type and the G-protein-coupled glutamate receptors, using coordination chemistry in living cells. The high programmability of coordination chemistry enabled two His mutations, which act as an artificial allosteric site, to be semirationally incorporated in the vicinity of the ligand-binding pockets. Binding of Pd(2,2'-bipyridine) at the allosteric site enabled the active conformations of the glutamate receptors to be stabilized. Using this approach, we were able to activate selectively a mutant glutamate receptor in live neurons, which initiated a subsequent signal-transduction pathway.

Allosteric modulation of metabotropic glutamate (mGlu) receptors offers a promising pharmacological approach to normalize neural circuit dysfunction associated with various psychiatric and neurological disorders. As mGlu receptor allosteric modulators progress through discovery and clinical development, both technical advances and novel tool compounds are providing opportunities to better understand mGlu receptor pharmacology and neurobiology. Recent advances in structural biology are elucidating the structural determinants of mGlu receptor-negative allosteric modulation and supplying the means to resolve active, allosteric modulator-bound mGlu receptors. The discovery and characterization of allosteric modulators with novel pharmacological profiles is uncovering the biological significance of their intrinsic agonist activity, biased mGlu receptor modulation, and novel mGlu receptor heterodimers. The development and exploitation of optogenetic and optopharmacological tools is permitting a refined spatial and temporal understanding of both mGlu receptor functions and their allosteric modulation in intact brain circuits. Together, these lines of research promise to provide a more refined understanding of mGlu receptors and their allosteric modulation that will inform the development of mGlu receptor allosteric modulators as neurotherapeutics in the years to come., (© The Author 2015. Published by Oxford University Press on behalf of CINP.)

5-Arylbenzothiadiazine type compounds acting as positive allosteric modulators of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA-PAMs) have received particular attention in the past decade for their nootropic activity and lack of the excitotoxic side effects of direct agonists. Recently, our research group has published the synthesis and biological activity of 7-chloro-5-(3-furanyl)-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide (1), one of the most active benzothiadiazine-derived AMPA-PAMs in vitro to date. However, 1 exists as two stereolabile enantiomers, which rapidly racemize in physiological conditions, and only one isomer is responsible for the pharmacological activity. In the present work, experiments carried out with rat liver microsomes show that 1 is converted by hepatic cytochrome P450 to the corresponding unsaturated derivative 2 and to the corresponding pharmacologically inactive benzenesulfonamide 3. Surprisingly, patch-clamp experiments reveal that 2 displays an activity comparable to that of the parent compound. Molecular modeling studies were performed to rationalize these results. Furthermore, mice cerebral microdialysis studies suggest that 2 is able to cross the blood-brain barrier and increases acetylcholine and serotonin levels in the hippocampus. The experimental data disclose that the achiral hepatic metabolite 2 possesses the same pharmacological activity of its parent compound 1 but with an enhanced chemical and stereochemical stability, as well as an improved pharmacokinetic profile compared with 1.

Understanding the thermodynamics of binding of a lead compound to a receptor can provide valuable information for drug design. The binding of compounds, particularly partial agonists, to subtypes of the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor is, in some cases, driven by increases in entropy. Using a series of partial agonists based on the structure of the natural product, willardiine, we show that the charged state of the ligand determines the enthalpic contribution to binding. Willardiines have uracil rings with pKa values ranging from 5.5 to 10. The binding of the charged form is largely driven by enthalpy, while that of the uncharged form is largely driven by entropy. This is due at least in part to changes in the hydrogen bonding network within the binding site involving one water molecule. This work illustrates the importance of charge to the thermodynamics of binding of agonists and antagonists to AMPA receptors and provides clues for further drug discovery.

Our laboratory is investigating ivermectin (IVM) and other members of the avermectin family as new pharmaco-therapeutics to prevent and/or treat alcohol use disorders (AUDs). Earlier work found that IVM significantly reduced ethanol intake in mice and that this effect likely reflects IVM's ability to modulate ligand-gated ion channels. We hypothesized that structural modifications that enhance IVM's effects on key receptors and/or increase its brain concentration should improve its anti-alcohol efficacy. We tested this hypothesis by comparing the abilities of IVM and two other avermectins, abamectin (ABM) and selamectin (SEL), to reduce ethanol intake in mice, to alter modulation of GABAARs and P2X4Rs expressed in Xenopus oocytes and to increase their ability to penetrate the brain. IVM and ABM significantly reduced ethanol intake and antagonized the inhibitory effects of ethanol on P2X4R function. In contrast, SEL did not affect either measure, despite achieving higher brain concentrations than IVM and ABM. All three potentiated GABAAR function. These findings suggest that chemical structure and effects on receptor function play key roles in the ability of avermectins to reduce ethanol intake and that these factors are more important than brain penetration alone. The direct relationship between the effect of these avermectins on P2X4R function and ethanol intake suggest that the ability to antagonize ethanol-mediated inhibition of P2X4R function may be a good predictor of the potential of an avermectin to reduce ethanol intake and support the use of avermectins as a platform for developing novel drugs to prevent and/or treat AUDs.

Accurate calculation of the binding affinity of small molecules to proteins has the potential to become an important tool in rational drug design. In this study, we use the free energy perturbation (FEP) method with restraints to calculate the standard binding free energy of five ligands (ACPA, AMPA, CNQX, DNQX, and glutamate) to the glutamate receptor GluA2, which plays an essential role in synaptic transmission. To deal with the convergence problem in FEP calculations with charged ligands, we use a protocol where the ligand is coupled in the binding site while it is decoupled in bulk solution simultaneously. The contributions from the conformational, rotational, and translational entropies to the standard binding free energy are determined by applying/releasing respective restraints to the ligand in bulk/binding site. We also employ the confine-and-release approach, which helps to resolve convergence problems in FEP calculations. Our results are in good agreement with the experimental values for all five ligands, including the charged ones which are often problematic in FEP calculations. We also analyze the different contributions to the binding free energy of each ligand to GluA2 and discuss the nature of these interactions.

Gustatory cortex (GC), an assemblage of taste-responsive neurons in insular cortex, is widely regarded as integral to conditioned taste aversion (CTA) retention, a link that has been primarily established using lesion approaches in rats. In contrast to this prevailing view, we found that even the most complete bilateral damage to GC produced by ibotenic acid was insufficient to disrupt postsurgical expression of a presurgical CTA; nor were such lesions sufficient to disrupt postsurgical acquisition and initial expression of a second CTA. However, some rats with lesions were significantly impaired on these tests. Further examination of all conditioned rats with lesions, regardless of the lesion topography, revealed a significant positive association between damage in the posterior portion of GC and especially within adjacent posterior regions of insular cortex. Accordingly, we developed a high-resolution lesion-mapping program that permitted the overlay of the individual lesion maps from rats with CTA impairments to produce a groupwise aggregate lesion map. Comparison of this map with one derived from the unimpaired counterparts indicated a specific lesion "hot spot" associated with CTA deficits that included the most posterior end of GC and overlying granular layer and encompassed an area provisionally referred to in the literature as visceral cortex. Thus, the detailed mapping of the lesion in behaviorally defined subgroups of rats allowed us to exploit the variability in performance to uncloak an important potential component of the functional topography of insular cortex; such an approach could have general applicability to other brain structure-function endeavors as well.

Optical probing and manipulation of cellular signaling has revolutionized biological studies ranging from isolated cells to intact tissues in the live animal. A promising avenue of optical manipulation is Chemical Optogenetics (or Optogenetic Pharmacology), an approach for engineering specific proteins to be rapidly and reversibly switched on and off with light. The approach employs synthetic photoswitched ligands, which can be reversibly photo-isomerized to toggle back and forth between two conformations in response to two wavelengths of light. We focus here on the photoswitched tethered ligand (PTL) approach in which the PTL is covalently attached in a site-directed manner to a signaling protein. For this a ligand anchoring site is introduced at a location which allows the ligand to dock only in one of the light-controlled conformations, thus enabling liganding to be rapidly switched. The ligand can be an agonist, antagonist or an active site (or pore) blocker. In principle, orthogonal chemistries of attachment would make PTL anchoring completely unique. However, extremely high specificity of remote control is also obtained by cysteine attachment because of the ligand specificity and precise geometric requirements for liganding. We describe here the design of light-gated ionotropic and metabotropic glutamate receptors, the selection of a site for cysteine placement, the method for PTL attachment, and a detailed protocol of photoswitching experiments in cultured cells. These descriptions can guide applications of Chemical Optogenetics to other receptors and serve as a starting point for use in more complex preparations.

Metabotropic glutamate (mGlu) receptors play important roles in regulating CNS function and are known to function as obligatory dimers. Although recent studies have suggested heterodimeric assembly of mGlu receptors in vitro, the demonstration that distinct mGlu receptor proteins can form heterodimers or hetero-complexes with other mGlu subunits in native tissues, such as neurons, has not been shown. Using biochemical and pharmacological approaches, we demonstrate here that mGlu2 and mGlu4 form a hetero-complex in native rat and mouse tissues which exhibits a distinct pharmacological profile. These data greatly extend our current understanding of mGlu receptor interaction and function and provide compelling evidence that mGlu receptors can function as heteromers in intact brain circuits.

Total syntheses of structurally and biologically intriguing natural products relying on new synthetic methodologies are described. This article features cinchona alkaloid-catalyzed asymmetric Morita-Baylis-Hillman reactions, heterocycle syntheses based on rhodium-catalyzed C-H amination and indium-catalyzed Conia-ene reactions, and their utilization for the syntheses of the phoslactomycin family of antibiotics, glutamate receptor agonists and antagonists, and alkaloids with characteristic highly substituted pyrrolidinone core structures.

On the basis of the results obtained in previous series of AMPA potentiators belonging to 3,4-dihydro-2H-benzo- and 3,4-dihydro-2H-pyrido-1,2,4-thiadiazine 1,1-dioxides, the present work focuses on the design of original isosteric 3,4-dihydro-2H-thieno-1,2,4-thiadiazine 1,1-dioxides. Owing to the sulfur position, three series of compounds were developed and their activity as AMPA potentiators was characterized. In each of the developed series, potent compounds were discovered. After screening the selected active compounds on a safety in vivo test, 6-chloro-4-ethyl-3,4-dihydro-2H-thieno[2,3-e]-1,2,4-thiadiazine 1,1-dioxide (24) appeared as the most promising compound and was further evaluated. Its effects on long-term potentiation in vivo and on AMPA-mediated noradrenaline release were measured to predict its potential cognitive enhancing properties. Finally, an object recognition test performed in mice revealed that 24 was able to significantly enhance cognition, after oral administration, at doses as low as 0.3 mg/kg. This study validates the interest of the isosteric replacement of the benzene or pyridine nuclei by the thiophene nucleus in the ring-fused thiadiazine dioxides class of AMPA potentiators.

Ionotropic glutamate receptors (iGluRs) are ligand-gated cation channels that mediate neurotransmission in animal nervous systems. Homologous proteins in plants have been implicated in root development, ion transport, and several metabolic and signaling pathways. AtGLR3.4, a plant iGluR homolog from Arabidopsis thaliana, has ion channel activity and is gated by asparagine, serine, and glycine. Using heterologous expression in Xenopus oocytes, we found that another Arabidopsis iGluR homolog, AtGLR1.4, functioned as a ligand-gated, nonselective, Ca(2+)-permeable cation channel that responded to an even broader range of amino acids, none of which are agonists of animal iGluRs. Seven of the 20 standard amino acids--mainly hydrophobic ones--acted as agonists, with methionine being most effective and most potent. Nine amino acids were antagonists, and four, including glutamate and glycine, had no effect on channel activity. We constructed a model of this previously uncharacterized ligand specificity and used knockout mutants to show that AtGLR1.4 accounts for methionine-induced membrane depolarization in Arabidopsis leaves.

Positive allosteric modulators of α-amino-3-hydroxy-5-methyl-isoxazole-propionic acid (AMPA) receptors facilitate synaptic plasticity and can improve various forms of learning and memory. These modulators show promise as therapeutic agents for the treatment of neurological disorders such as schizophrenia, ADHD, and mental depression. Three classes of positive modulator, the benzamides, the thiadiazides, and the biarylsulfonamides differentially occupy a solvent accessible binding pocket at the interface between the two subunits that form the AMPA receptor ligand-binding pocket. Here, we describe the electrophysiological properties of a new chemotype derived from a structure-based drug design strategy (SBDD), which makes similar receptor interactions compared to previously reported classes of modulator. This pyrazole amide derivative, JAMI1001A, with a promising developability profile, efficaciously modulates AMPA receptor deactivation and desensitization of both flip and flop receptor isoforms. This article is part of a Special Issue entitled 'Cognitive Enhancers'., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Introduction: The NMDA receptor is a ligand-gated ion channel that plays a critical role in higher level brain processes and has been implicated in a range of neurological and psychiatric conditions. Although initial studies for the use of NMDA receptor antagonists in neuroprotection were unsuccessful, more recently, NMDA receptor antagonists have shown clinical promise in other indications such as Alzheimer's disease, Parkinson's disease, pain and depression. Based on the clinical observations and more recent insights into receptor pharmacology, new modulatory approaches are beginning to emerge, with potential therapeutic benefit., Areas Covered: The article covers the known pharmacology and important features regarding NMDA receptors and their function. A discussion of pre-clinical and clinical relevance is included, as well. The subsequent patent literature review highlights the current state of the art targeting the receptor since the last review in 2010., Expert Opinion: The complex nature of the NMDA receptor structure and function is becoming better understood. As knowledge about this receptor increases, it opens up new opportunities for targeting the receptor for many therapeutic indications. New strategies and advances in older technologies will need to be further developed before clinical success can be achieved. First-in-class potentiators and subunit-selective agents form the basis for most new strategies, complemented by efforts to limit off-target liability and fine-tune on-target properties.

Introduction: Metabotropic glutamate receptor 4 (mGlu(4)) is a group III GPCR and has been demonstrated to play a major role in a number of therapeutic areas within the CNS. As the orthosteric site of all glutamate receptors is highly conserved, modulating mGlu(4) via allosteric modulation has emerged as a very attractive mode-of-action and has been validated preclinically in a number of animal models for Parkinson's disease, anxiety, pain, and neuroinflammation., Areas Covered: In this review, the patent literature for mGlu(4)-positive allosteric modulators over the past 4 years will be provided. Patents from all companies are discussed and an overview of the chemical matter and relevant biological properties will be given., Expert Opinion: Although there has yet to be an mGlu(4)-positive allosteric modulator progressed into clinical trials, there is a wealth of preclinical data from the primary literature that shows the promise of this emerging target. A number of academic and industry laboratories have recently published exciting patent data covering a multitude of chemical matter. Positive allosteric modulation of mGlu(4) remains one of the more attractive non-dopaminergic therapies for Parkinson's disease, as well as emerging data for other indications such as pain, neuroinflammation, schizophrenia and diabetes, which could potentially make mGlu(4) a significant therapeutic target going forward.

Neurons in layer 4 of the primary auditory cortex receive convergent glutamatergic inputs from thalamic and cortical projections that activate different groups of postsynaptic glutamate receptors. Of particular interest in layer 4 neurons are the Group II metabotropic glutamate receptors (mGluRs), which hyperpolarize neurons postsynaptically via the downstream opening of GIRK channels. This pronounced effect on membrane conductance could influence the neuronal processing of synaptic inputs, such as those from the thalamus, essentially modulating information flow through the thalamocortical pathway. To examine how Group II mGluRs affect thalamocortical transmission, we used an in vitro slice preparation of the auditory thalamocortical pathways in the mouse to examine synaptic transmission under conditions where Group II mGluRs were activated. We found that both pre- and post-synaptic Group II mGluRs are involved in the attenuation of thalamocortical EPSP/Cs. Thus, thalamocortical synaptic transmission is suppressed via the presynaptic reduction of thalamocortical neurotransmitter release and the postsynaptic inhibition of the layer 4 thalamorecipient neurons. This could enable the thalamocortical pathway to autoregulate transmission, via either a gating or gain control mechanism, or both., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Metabotropic glutamate (mGlu) receptors are promising targets to treat numerous brain disorders. So far, allosteric modulators are the only subtype selective ligands, but pure agonists still have strong therapeutic potential. Here, we aimed at investigating the possibility of developing subtype-selective agonists by extending the glutamate-like structure to hit a nonconsensus binding area. We report the properties of the first mGlu4-selective orthosteric agonist, derived from a virtual screening hit, LSP4-2022 using cell-based assays with recombinant mGlu receptors [EC(50): 0.11 ± 0.02, 11.6 ± 1.9, 29.2 ± 4.2 μM (n>19) in calcium assays on mGlu4, mGlu7, and mGlu8 receptors, respectively, with no activity at the group I and -II mGlu receptors at 100 μM]. LSP4-2022 inhibits neurotransmission in cerebellar slices from wild-type but not mGlu4 receptor-knockout mice. In vivo, it possesses antiparkinsonian properties after central or systemic administration in a haloperidol-induced catalepsy test, revealing its ability to cross the blood-brain barrier. Site-directed mutagenesis and molecular modeling was used to identify the LSP4-2022 binding site, revealing interaction with both the glutamate binding site and a variable pocket responsible for selectivity. These data reveal new approaches for developing selective, hydrophilic, and brain-penetrant mGlu receptor agonists, offering new possibilities to design original bioactive compounds with therapeutic potential.

Excessive excitability or hyperexcitability of glutamate-containing neurons in the brain has been proposed as a possible explanation for anxiety, stress-induced disorders, epilepsy, and some neurodegenerative diseases. However, direct measurement of glutamate on a rapid time scale has proven to be difficult. Here we adapted enzyme-based microelectrode arrays (MEA) capable of detecting glutamate in vivo, to assess the effectiveness of hyperexcitability modulators on glutamate release in brain slices of the rat neocortex. Using glutamate oxidase coated ceramic MEAs coupled with constant voltage amperometry, we measured resting glutamate levels and synaptic overflow of glutamate after K(+) stimulation in brain slices. MEAs reproducibly detected glutamate on a second-by-second time scale in the brain slice preparation after depolarization with high K(+) to evoke glutamate release. This stimulus-evoked glutamate release was robust, reproducible, and calcium dependent. The K(+)-evoked glutamate release was modulated by ligands to the α(2)δ subunit of voltage sensitive calcium channels (PD-0332334 and PD-0200390). Meanwhile, agonists to Group II metabotropic glutamate (mGlu) receptors (LY379268 and LY354740), which are known to alter hyperexcitability of glutamate neurons, attenuated K(+)-evoked glutamate release but did not alter resting glutamate levels. This new MEA technology provides a means of directly measuring the chemical messengers involved in glutamate neurotransmission and thereby helping to reveal the role multiple glutamatergic system components have on glutamate signaling., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate most excitatory synaptic transmission in the central nervous system. The free energy of neurotransmitter binding to the ligand-binding domains (LBDs) of iGluRs is converted into useful work to drive receptor activation. We have computed the principal thermodynamic contributions from ligand docking and ligand-induced closure of LBDs for nine ligands of GluA2 using all-atom molecular dynamics free energy simulations. We have validated the results by comparison with experimentally measured apparent affinities to the isolated LBD. Features in the free energy landscapes that govern closure of LBDs are key determinants of binding free energies. An analysis of accessible LBD conformations transposed into the context of an intact GluA2 receptor revealed that the relative displacement of specific diagonal subunits in the tetrameric structure may be key to the action of partial agonists.

The Japanese beetle (JB), Popillia japonica, exhibits rapid paralysis after consuming flower petals of zonal geranium, Pelargonium x hortorum. Activity-guided fractionations were conducted with polar flower petal extracts from P. x hortorum cv. Nittany Lion Red, which led to the isolation of a paralysis-inducing compound. High-resolution-MS and NMR ((1)H, (13)C, COSY, heteronuclear sequential quantum correlation, heteronuclear multiple bond correlation) analysis identified the paralytic compound as quisqualic acid (C(5)H(7)N(3)O(5)), a known but rare agonist of excitatory amino acid receptors. Optical rotation measurements and chiral HPLC analysis determined an L-configuration. Geranium-derived and synthetic L-quisqualic acid demonstrated the same positive paralytic dose-response. Isolation of a neurotoxic, excitatory amino acid from zonal geranium establishes the phytochemical basis for induced paralysis of the JB, which had remained uncharacterized since the phenomenon was first described in 1920.

Subunit-selective ligands for glutamate receptors remains an area of interest as glutamate is the major excitatory neurotransmitter in the brain and involved in a number of diseased states in the central nervous system (CNS). Few subtype-selective ligands are known, especially among the N-methyl-D-aspartic acid (NMDA) receptor class. Development of these ligands seems to be a difficult task because of the conserved region in the binding site of the NMDA receptor subunits. A few scaffolds have been developed showing potential to differentiate between the NMDA receptors.

A series of 1,5-disubstituted pyridones was identified as positive allosteric modulators (PAMs) of the metabotropic glutamate receptor 2 (mGluR2) via high throughput screening (HTS). Subsequent SAR exploration led to the identification of several compounds with improved in vitro activity. Lead compound 8 was further profiled and found to attenuate the increase in PCP induced locomotor activity in mice.

The extracellular amino-terminal domains (ATDs) of the ionotropic glutamate receptor subunits form a semiautonomous component of all glutamate receptors that resides distal to the membrane and controls a surprisingly diverse set of receptor functions. These functions include subunit assembly, receptor trafficking, channel gating, agonist potency, and allosteric modulation. The many divergent features of the different ionotropic glutamate receptor classes and different subunits within a class may stem from differential regulation by the amino-terminal domains. The emerging knowledge of the structure and function of the amino-terminal domains reviewed here may enable targeting of this region for the therapeutic modulation of glutamatergic signaling. Toward this end, NMDA receptor antagonists that interact with the GluN2B ATD show promise in animal models of ischemia, neuropathic pain, and Parkinson's disease.

Working memory impairments are a core aspect of schizophrenia, yet current medicines do not address such cognitive dysfunction. We have developed a model of these working memory deficits by acutely disrupting glutamatergic synaptic transmission by administration of the N-methyl-d-aspartate (NMDA) antagonist ketamine in the nonhuman primate. The current studies evaluated the effect of positive allosteric modulators ("potentiators") of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors on the working memory and behavioral effects of ketamine. AMPA receptors mediate fast excitatory synaptic transmission throughout the brain and play a critical role in the activity-dependent regulation of NMDA receptors. We find that positive modulation of AMPA receptors with LY451646 (0.1-1.0mg/kg, SC) and structurally distinct PF-4778574 (0.01mg/kg, SC) robustly ameliorates ketamine-induced working memory impairments without altering behavioral effects of acute ketamine we consider related to positive- and negative-like symptoms. These results support AMPA receptor potentiators as a potential adjunctive treatment for cognitive impairment associated with schizophrenia (CIAS)., (Copyright 2010 Elsevier B.V. All rights reserved.)

Metabotropic glutamate receptors (mGlu receptors), with their unique signaling systems and pharmacological characteristics, have emerged as a new topic in excitatory amino acid research. Among them, the unique distribution of group II mGlu receptors, such as mGlu(2) and mGlu(3) receptors, and the involvement of these receptors in the regulation of neurotransmission are particularly interesting. Recently, potent agonists for mGlu(2/3) receptor have been synthesized, and their pharmacological roles have been intensively investigated using animal models. mGlu(2/3) receptors clearly have crucial roles in the central nervous system, and accumulating evidence in both rodents and human studies has suggested that agonists for mGlu(2/3) receptors may be beneficial for the treatment of psychiatric disorders such as schizophrenia. Possible neuronal circuits through which mGlu(2/3) receptor agonists exert their pharmacological effects have also been investigated., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

In order to investigate the 4-hydroxy-1,2,5-oxadiazol-3-yl moiety as a carboxylic acid bioisoster at ionotropic glutamate receptors (iGluRs), a series of acidic alpha-aminocarboxylic acids in which the distal carboxy group was replaced by the 4-hydroxy-1,2,5-oxadiazol-3-yl group was synthesized. Ionization constants were determined. All target compounds, except the Asp analogue 12, were resolved using chiral HPLC. Whereas 12 showed good affinity exclusively at NMDA receptors, the Glu analogue, (+)-10, was an unselective, though potent AMPA receptor preferring agonist (EC(50) = 10 microM at iGluR2) showing only low stereoselectivity. The two higher Glu homologues, (+)-15 and (+)-18, turned out to be weak agonists at iGluR2 as well as weak antagonists at NR1/NR2A, whereas the corresponding (-)-isomers were selective NR1/NR2A antagonists with somewhat higher potency. The results proved the 4-hydroxy-1,2,5-oxadiazol-3-yl moiety to be a useful bioisoster at all three classes of iGluRs, capable of being integrated into agonists as well as antagonists.

The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) is a subtype of the ionotropic glutamate receptors that plays a prominent role in neurotransmission and is widespread throughout the CNS. Because of this, its malfunction can result in a multitude of nervous system diseases. This review looks at compounds that are able to modulate AMPAR function by binding to one of several sites on the receptor that either downregulate its function (competitive, noncompetitive and uncompetitive antagonists) or upregulate its function (positive modulators). It will also give an account of the various diseases that have implicated AMPAR dysfunction and how specific types of AMPAR modulator may be beneficial in their treatment. The AMPAR remains an unexploited but important therapeutic target.

AMPA receptors (AMPARs) are tetrameric ligand-gated ion channels that couple the energy of glutamate binding to the opening of a transmembrane channel. Crystallographic and electrophysiological analysis of AMPARs has suggested a coupling between (1) cleft closure in the bilobate ligand-binding domain (LBD), (2) the resulting separation of transmembrane helix attachment points across subunit dimers, and (3) agonist efficacy. In general, more efficacious agonists induce greater degrees of cleft closure and transmembrane separation than partial agonists. Several apparent violations of the cleft-closure/efficacy paradigm have emerged, although in all cases, intradimer separation remains as the driving force for channel opening. Here, we examine the structural basis of partial agonism in GluA4 AMPARs. We find that the L651V substitution enhances the relative efficacy of kainate without increasing either LBD cleft closure or transmembrane separation. Instead, the conformational change relative to the wild-type:kainate complex involves a twisting motion with the efficacy contribution opposite from that expected based on previous analyses. As a result, channel opening may involve transmembrane rearrangements with a significant rotational component. Furthermore, a two-dimensional analysis of agonist-induced GluA2 LBD motions suggests that efficacy is not a linearly varying function of lobe 2 displacement vectors, but is rather determined by specific conformational requirements of the transmembrane domains.

The optimum geometries and binding energies of the complexes formed by AMPA and Kainic acid, as well as their anions with tyrosine, proline and some tripeptides are investigated with quantum chemical calculations (HF/6-31G**). It was found that receptors featuring the Tyr-Ala-Pro sequence exhibit stronger binding energies to the substrates than the Tyr-Ser-Pro and Tyr-Ser-Ser. As expected, the anions are more bound than the neutral species. This work can lead to investigations on the effect of AMPA receptors mutations on the brain functions, possibly related to criminal tendencies.

The design, synthesis, and pharmacological characterization of a highly potent and selective glutamate GluR5 agonist is reported. (S)-2-Amino-3-((RS)-3-hydroxy-8-methyl-7,8-dihydro-6H-cyclohepta[d]isoxazol-4-yl)propionic acid (5) is the 8-methyl analogue of (S)-2-amino-3-(3-hydroxy-7,8-dihydro-6H-cyclohepta[d]isoxazol-4-yl)propionic acid ((S)-4-AHCP, 4). Compound 5 displays an improved selectivity profile compared to 4. A versatile stereoselective synthetic route for this class of compounds is presented along with the characterization of the binding affinity of 5 to ionotropic glutamate receptors (iGluRs). Functional characterization of 5 at cloned iGluRs using a calcium imaging assay and voltage-clamp recordings show a different activation of GluR5 compared to (S)-glutamic acid (Glu), kainic acid (KA, 1), and (S)-2-amino-3-(3-hydroxy-5-tert-butyl-4-isoxazolyl)propionic acid ((S)-ATPA, 3) as previously demonstrated for 4. An X-ray crystallographic analysis of 4 and computational analyses of 4 and 5 bound to the GluR5 agonist binding domain (ABD) are presented, including a watermap analysis, which suggests that water molecules in the agonist binding site are important selectivity determinants.

The design and synthesis of four pyrrolidine scaffolds that are structurally related to the known ionotropic glutamate receptor antagonist, (-)-kaitocephalin, is described. Additionally, preliminary results of the biological evaluation of these compounds are disclosed.

Kainate receptors (KARs) are involved in both NMDA receptor-independent long-term potentiation (LTP) and synaptic facilitation at mossy fibre synapses in the CA3 region of the hippocampus. However, the identity of the KAR subtypes involved remains controversial. Here we used a highly potent and selective GluK1 (formerly GluR5) antagonist (ACET) to elucidate roles of GluK1-containing KARs in these synaptic processes. We confirmed that ACET is an extremely potent GluK1 antagonist, with a Kb value of 1.4+/-0.2 nM. In contrast, ACET was ineffective at GluK2 (formerly GluR6) receptors at all concentrations tested (up to 100 microM) and had no effect at GluK3 (formerly GluR7) when tested at 1 microM. The X-ray crystal structure of ACET bound to the ligand binding core of GluK1 was similar to the UBP310-GluK1 complex. In the CA1 region of hippocampal slices, ACET was effective at blocking the depression of both fEPSPs and monosynaptically evoked GABAergic transmission induced by ATPA, a GluK1 selective agonist. In the CA3 region of the hippocampus, ACET blocked the induction of NMDA receptor-independent mossy fibre LTP. To directly investigate the role of pre-synaptic GluK1-containing KARs we combined patch-clamp electrophysiology and 2-photon microscopy to image Ca2+ dynamics in individual giant mossy fibre boutons. ACET consistently reduced short-term facilitation of pre-synaptic calcium transients induced by 5 action potentials evoked at 20-25Hz. Taken together our data provide further evidence for a physiological role of GluK1-containing KARs in synaptic facilitation and LTP induction at mossy fibre-CA3 synapses.

Recent studies suggest that subtype specific activators of metabotropic glutamate receptors (mGluRs) have exciting potential for the development of novel treatment strategies for numerous psychiatric and neurological disorders. A number of positive allosteric modulators (PAMs) have been identified that are highly selective for mGluR1, including the compounds Ro 01-6128, Ro 67-4853, and Ro 67-7476. These PAMs have been previously found to interact with a site distinct from that of negative allosteric modulators (NAMs), typified by R214127. These mGluR1 PAMs do not have an effect on baseline calcium levels but induce leftward shifts in the concentration-response of mGluR1 to agonists. However, their effects on a variety of signaling pathways and their mechanism of action have not been fully explored and are of critical importance for further development of mGluR1 allosteric modulators as novel drugs. In baby hamster kidney (BHK) cells, mGluR1 activates calcium mobilization, cAMP production, and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation; signaling cascades which are distinct and differentially regulated. In contrast to their effects on calcium mobilization, these compounds were found to activate ERK1/2 phosphorylation in the absence of exogenously added agonist, an effect that was fully blocked by both orthosteric (LY341495) and allosteric (R214127) mGluR1 antagonists. The mGluR1 PAMs were also found to activate cAMP production in the absence of agonist. Thus, these mGluR1 PAMs have qualitatively different effects on a variety of mGluR1-mediated signal transduction cascades. Together, these data provide further evidence that allosteric compounds can differentially modulate the coupling of a single receptor to independent signaling pathways or act in a system-dependent manner.

We developed a simple and effective analysis procedure that includes pretreatment and determination methods for beta-N-methylamino-l-alanine (BMAA), a cyanobacterial neurotoxin. BMAA may be produced by all known groups of cyanobacteria living in freshwater as well as marine environments. In this paper, we report a novel determination method for BMAA. A cation-exchange resin was effective for the selective concentration of BMAA from cyanobacterial extracts and yielded a high recovery rate. Moreover, liquid chromatography (LC)-electrospray ionization mass spectrometry with a hydrophilic LC column was effective for determining BMAA levels. The quantitation limit for BMAA based on selected ion monitoring (SIM) was determined as 0.5 ng at a signal/noise ratio of 5.

The fate of glyphosate and its degradation product aminomethylphosphonic acid (AMPA) was studied in soil. Labeled glyphosate was used to be able to distinguish the measured quantities of glyphosate and AMPA from the background values since the soil was sampled in a field where glyphosate had been used formerly. After addition of labeled glyphosate, the disappearance of glyphosate and the formation and disappearance of AMPA were monitored. The resulting curves were fitted according to a new EU guideline. The best fit of the glyphosate degradation data was obtained using a first-order multi compartment (FOMC) model. DT(50) values of 9 days (glyphosate) and 32 days (AMPA) indicated relatively rapid degradation. After an aging period of 6 months, the leaching risk of each residue was determined by treating the soil with pure water or a phosphate solution (pH 6), to simulate rain over a non-fertilized or fertilized field, respectively. Significantly larger (p < 0.05) amounts of aged glyphosate and AMPA were extracted from the soil when phosphate solution was used as an extraction agent, compared with pure water. This indicates that the risk of leaching of aged glyphosate and AMPA residues from soil is greater in fertilized soil. The blank soil, to which 252 g glyphosate/ha was applied 21 months before this study, contained 0.81 ng glyphosate/g dry soil and 10.46 ng AMPA/g dry soil at the start of the study. Blank soil samples were used as controls without glyphosate addition. After incubation of the blank soil samples for 6 months, a significantly larger amount of AMPA was extracted from the soil treated with phosphate solution than from that treated with pure water. To determine the degree of uptake of aged glyphosate residues by crops growing in the soil, (14)C-labeled glyphosate was applied to soil 6.5 months prior to sowing rape and barley seeds. After 41 days, 0.006 +/- 0.002% and 0.005 +/- 0.001% of the applied radioactivity was measured in rape and barley, respectively.