Incubation of cocaine seeking following brief cocaine experience in mice is enhanced by mGluR1 blockade.

The incubation of cocaine craving describes the time-dependent augmentation of cue-induced cocaine seeking during withdrawal from prolonged cocaine self-administration and requires time-dependent changes in neuroplasticity at the level of glutamatergic synapses in the nucleus accumbens (NAc). In contrast to most studies that use multiple cocaine-cue conditioning sessions, the present study tested mice with limited cocaine experience (i.e., a single conditioning session) in the incubation of cue-mediated cocaine seeking and its associated changes in the glutamate system. Mice that self-administered cocaine during a single session exhibited a time-dependent increase in their response for the drug-associated cue as compared to mice that self-administered saline. This behavior was associated with changes in AMPA and NMDA receptor binding characteristics. Furthermore, Group I metabotropic glutamate receptor (mGluR1) mRNA levels were altered in several brain regions, including the NAc. Because of the pivotal role of mGluR1 in the control of cocaine-induced plasticity, we investigated the role of mGluR1 in the formation of drug cue-mediated cocaine seeking. After prolonged withdrawal, mice in which an mGluR1 antagonist was administered following cocaine self-administration displayed increased cocaine seeking compared to vehicle-treated mice. These results suggest that limited cocaine experience is sufficient to induce neurobiological changes that enable an initially neutral cue to acquire motivational value that increases over time, an effect that likely involves glutamate signaling through mGluR1.

Effect of pomaglumetad methionil on the QT interval in subjects with schizophrenia.

OBJECTIVE: This thorough QT/QTc (TQT) study assessed the effects of a supratherapeutic dose of pomaglumetad methionil, a potential treatment for schizophrenia, compared to placebo on the QT interval in subjects with schizophrenia. METHODS: This double-blind, 3-period crossover study enrolled 86 subjects aged 22 - 63 years, who met Diagnostic and Statistical Manual, Fourth Edition, Test Revision (DSM-IV-TR) criteria for schizophrenia; 78 subjects completed the study. Subjects were randomly assigned to sequences of 3 treatment periods of single oral doses of pomaglumetad methionil 400 mg, moxifloxacin 400 mg, and placebo. Quadruplicate electrocardiograms (ECGs) were extracted from 2 hours predose to 12 hours postdose and were overread by a blinded central reader. Time-matched pharmacokinetic (PK) parameters were assessed. RESULTS: At all-time points, the upper bound of the 90% 2-sided confidence interval (CI) for the least squares (LS) mean difference in changes from baseline in Fridericia's corrected QT interval (ΔQTcF) between pomaglumetad methionil and placebo was < 10 milliseconds (msec). Sufficient assay sensitivity was not achieved, likely due to food effect; although the maximum observed drug concentration (Cmax) with moxifloxacin (1,410 ng/mL) was lower than expected, the slope of the regression line of moxifloxacin plasma concentrations versus placebo-subtracted ΔQTcF was similar to that reported in the literature. CONCLUSIONS: A single supratherapeutic dose of 400 mg pomaglumetad methionil did not prolong QTcF to a clinically significant degree and, importantly, did not result in any absolute QTcF > 450 msec or increase in QTcF from predose > 30 msec.

Avermectins differentially affect ethanol intake and receptor function: implications for developing new therapeutics for alcohol use disorders.

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.

Quantification of the glycine transporter 1 in rhesus monkey brain using [18F]MK-6577 and a model-based input function.

BACKGROUND: Glycine transporter 1 (GlyT1) inhibitors have emerged as potential treatments for schizophrenia due to their potentiation of NMDA receptor activity by modulating the local concentrations of the NMDA co-agonist glycine. [18F]MK-6577 is a potent and selective GlyT1 inhibitor PET tracer. Although differences in ligand kinetics can be expected between non-human primates and humans, the tracer pre-clinical evaluation can provide valuable information supporting protocol design and quantification in the clinical space. The main objective of this work was to evaluate the in vivo kinetics of [18F]MK-6577 in rhesus monkey brain. Additionally, a method for estimating the tracer input function from the tracer brain tissue kinetics and venous sampling was validated. This technique was applied for determination of the dose-occupancy relationship of a GlyT1 inhibitor in monkey brain. METHODS: Compartmental and Logan graphical analysis were utilized for quantification of the [18F]MK-6577 binding using the measured tracer arterial input function. The stability of the tracer volume of distribution relative to scan length was assessed. The proposed model-based input function method takes advantage of the agreement between the tracer concentration in arterial and venous plasma from ~5 min. The approach estimates the initial peak of the input curve by adding a gamma like function term to the measured venous curve. The parameters of the model function were estimated by simultaneously fitting several brain time activity curves to a compartmental model. RESULTS: Good agreement was found between the model-based and the measured arterial plasma curve and the corresponding distribution volumes. The Logan analysis was the preferred method of analysis providing reliable and stable volume of distribution and occupancy results using a 90 and possibly 60 min scan length. CONCLUSION: The model-based input function method and Logan analysis are well suited for quantification of [18F]MK-6577 binding and GlyT1 occupancy in monkey brain.

NMDA-mediated Ca(2+) influx drives aberrant ryanodine receptor activation in dendrites of young Alzheimer's disease mice.

Deficits in synaptic function, particularly through NMDA receptors (NMDARs), are linked to late-stage cognitive impairments in Alzheimer's disease (AD). At earlier disease stages, however, there is evidence for altered endoplasmic reticulum (ER) calcium signaling in human cases and in neurons from AD mouse models. Despite the fundamental importance of calcium to synaptic function, neither the extent of ER calcium dysregulation in dendrites nor its interaction with synaptic function in AD pathophysiology is known. Identifying the mechanisms underlying early synaptic calcium dysregulation in AD pathogenesis is likely a key component to understanding, and thereby preventing, the synapse loss and downstream cognitive impairments. Using two-photon calcium imaging, flash photolysis of caged glutamate, and patch-clamp electrophysiology in cortical brain slices, we examined interactions between synaptically and ER-evoked calcium release at glutamatergic synapses in young AD transgenic mice. We found increased ryanodine receptor-evoked calcium signals within dendritic spine heads, dendritic processes, and the soma of pyramidal neurons from 3xTg-AD and TAS/TPM AD mice relative to NonTg controls. In addition, synaptically evoked postsynaptic calcium responses were larger in the AD strains, as were calcium signals generated from NMDAR activation. However, calcium responses triggered by back-propagating action potentials were not different. Concurrent activation of ryanodine receptors (RyRs) with either synaptic or NMDAR stimulation generated a supra-additive calcium response in the AD strains, suggesting an aberrant calcium-induced calcium release (CICR) effect within spines and dendrites. We propose that presenilin-linked disruptions in RyR signaling and subsequent CICR via NMDAR-mediated calcium influx alters synaptic function and serves as an early pathogenic factor in AD.

Fragment-Based Optimization of Dihydropyrazino-Benzimidazolones as Metabotropic Glutamate Receptor-2 Positive Allosteric Modulators against Migraine.

Our previous scaffold-hopping attempts resulted in dihydropyrazino-benzimidazoles as metabotropic glutamate receptor-2 (mGluR2) positive allosteric modulators (PAMs) with suboptimal drug-like profiles. Here, we report an alternative fragment-based optimization strategy applied on the new dihydropyrazino-benzimidazolone scaffold. Analyzing published high-affinity mGluR2 PAMs, we used a pharmacophore-guided approach to identify suitable growing vectors and optimize the scaffold in these directions. This strategy resulted in a new fragment like lead (34) with improved druglike properties that were translated to sufficient pharmacokinetics and validated proof-of-concept studies in migraine. Gratifyingly, compound 34 showed reasonable activity in the partial infraorbital nerve ligation, a migraine disease model that might open this indication for mGluR2 PAMs.

Transepithelial transport of the group II metabotropic glutamate 2/3 receptor agonist (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate (LY354740) and its prodrug (1S,2S,5R,6S)-2-[(2'S)-(2'-amino)propionyl]aminobicyclo[3.1.0]hexane-2,6-dicarboxylate (LY544344).

The limited oral bioavailability of the potent and selective group II metabotropic glutamate (mGlu) 2/3 receptor agonist, (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate (LY354740), was shown to be improved by its peptidyl prodrug, (1S,2S,5R,6S)-2-[(2'S)-(2'-amino)propionyl]aminobicyclo[3.1.0]hexane-2,6-dicarboxylate (LY544344). The purpose of this study was to elucidate the mechanisms of intestinal absorption of LY354740 and its prodrug LY544344. Transepithelial transport and accumulation studies were performed in Caco-2 cell monolayers; the involvement of the peptide transporter 1 (PEPT1) transporter was also examined. In absorptive transport studies, the peptidyl prodrug LY544344 partially hydrolyzed to release LY354740 intracellularly, and both compounds appeared in the basolateral compartment. The absorptive transport rate of LY544344, basolateral appearance rate of LY354740, and their cellular accumulation after incubation with LY544344 were concentration-dependent. PEPT1 inhibition reduced transepithelial transport and cellular accumulation of LY544344 to 22 and 1.1% of control, respectively. LY354740 showed concentration-independent absorptive transport with negligible cellular accumulation. Efflux and trans-stimulation studies revealed predominantly apical efflux and the existence of specific transporters for LY544344 and intracellularly released LY354740 on the apical and basolateral membranes. LY544344 efflux was also trans-stimulated at the apical side by glycyl-glutamate but not by glycyl-sarcosine. Transport of neither compound was affected by P-glycoprotein-mediated efflux, as shown in transport and uptake inhibition studies in Madin-Darby canine kidney multidrug resistance 1-transfected cell line and inverted membrane vesicles. In conclusion, LY354740 is mainly transported by the paracellular pathway, whereas intestinal absorption of LY544344 is mediated by PEPT1. However, the absorptive transport of LY544344 seems to be modulated by an apical efflux transporter and a rate-limiting transport step across the basolateral membrane.

Synthesis and Pharmacological Characterization of C4ß-Amide-Substituted 2-Aminobicyclo[3.1.0]hexane-2,6-dicarboxylates. Identification of (1 S,2 S,4 S,5 R,6 S)-2-Amino-4-[(3-methoxybenzoyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylic Acid (LY2794193), a Highly Potent and Selective mGlu3 Receptor Agonist.

Multiple therapeutic opportunities have been suggested for compounds capable of selective activation of metabotropic glutamate 3 (mGlu3) receptors, but small molecule tools are lacking. As part of our ongoing efforts to identify potent, selective, and systemically bioavailable agonists for mGlu2 and mGlu3 receptor subtypes, a series of C4ß-N-linked variants of (1 S,2 S,5 R,6 S)-2-amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 1 (LY354740) were prepared and evaluated for both mGlu2 and mGlu3 receptor binding affinity and functional cellular responses. From this investigation we identified (1 S,2 S,4 S,5 R,6 S)-2-amino-4-[(3-methoxybenzoyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 8p (LY2794193), a molecule that demonstrates remarkable mGlu3 receptor selectivity. Crystallization of 8p with the amino terminal domain of hmGlu3 revealed critical binding interactions for this ligand with residues adjacent to the glutamate binding site, while pharmacokinetic assessment of 8p combined with its effect in an mGlu2 receptor-dependent behavioral model provides estimates for doses of this compound that would be expected to selectively engage and activate central mGlu3 receptors in vivo.

Metabotropic glutamate receptor/phospholipase C system in female rat heart.

Glutamate is the main excitatory neurotransmitter in the central nervous system. This amino acid mediates learning and memory processes acting through ionotropic and metabotropic receptor binding. Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors that stimulate phospholipase C (PLC) or inhibit adenylyl cyclase (AC). MGluRs have been widely described in CNS. However, little is known about these receptors in peripheral system. The present work describes the mGluR/PLC pathway in membranes from pregnant and non-pregnant rat heart by radioligand binding, Western-blot assays and PLC activity determination. Furthermore, mRNA coding mGluR1, mGluR5, alphaGq/11 and PLCbeta1 was identified by RT-PCR. Binding assays indicated total mGlu receptor numbers of 4.7+/-0.2 pmol/mg protein and 4.2+/-1.0 pmol/mg protein in non-pregnant and pregnant rats respectively, and their corresponding KD values were 545.3+/-85.6 nM and 1062.8+/-393.6 nM. Western blots revealed bands corresponding to mGluR1 and mGluR5 receptors, confirming that these receptors are expressed in heart. The beta1 isoform of PLC, which mediates group I mGluRs (mGluR I) response, was also expressed in rat heart. Moreover, PLC activity was modulated by calcium in a dose-dependent manner. Finally, specific agonists for mGluRs increased the PLC activity and the increase was prevented by specific mGluR antagonists. These results demonstrate the presence of group I mGlu receptors and their functional coupling to the PLC stimulation in female rat heart, suggesting a possible role of mGluR/PLC pathway in this tissue.

Changes of metabolite profile in kainic acid induced hippocampal injury in rats measured by HRMAS NMR.

The solid-state high resolution magic angle spinning nuclear magnetic resonance (HRMAS NMR) technique was applied in this work to characterize and quantify the neurochemical changes in the rat hippocampus (CA1 or CA3) after local administration of kainic acid (KA). Intact tissue samples obtained from the KA treated and control brain samples were analyzed using HRMAS NMR. Metabolite profiles from NMR spectra of KA treated and control samples revealed the statistical significant decrease of N-acetylaspartate (NAA) and an increase of choline derivatives in the CA1 and CA3 directly receiving KA injection. Less extensive KA-induced metabolic changes were found in the hippocampi sample from the area contralateral to the site receiving KA administration. These results provided quantitative metabolic information on KA-induced neuronal loss and cell breakdown. In addition, the present study also revealed increased level of gamma-aminobutyric acid (GABA) and glutamate after KA treatment, suggesting that the cellular release of inhibitory and excitatory amino acids can be quantified using this method. KA induced microglia activation was evidenced by increased level of myo-insitol (myo-I). This study demonstrates that ex vivo HRMAS NMR is a useful tool for analyzing and quantifying changes of neurochemistry and cerebral metabolism in the intact brain.

Developmental lead (Pb) exposure reduces the ability of the NMDA antagonist MK-801 to suppress long-term potentiation (LTP) in the rat dentate gyrus, in vivo.

Chronic developmental lead (Pb) exposure increases the threshold and enhances decay of long-term potentiation (LTP) in the dentate gyrus of the hippocampal formation. MK-801 and other antagonists of the N-methyl-D-aspartate (NMDA) glutamate receptor subtype impair induction of LTP. In addition, Pb exposure reduces presynaptic glutamate release and is associated with alterations in NMDA receptor expression. This study examined LTP in Pb-exposed animals challenged with a low dose of MK-801 to assess the sensitivity of this receptor to inhibition. Pregnant rats received 0.2% Pb acetate in the drinking water beginning on gestational day 16, and this regimen was continued through lactation. Adult male offspring maintained on this solution from weaning were prepared with indwelling electrodes in the perforant path and dentate gyrus. Several weeks later, input/output (I/O) functions were collected in awake animals before and after saline or MK-801 administration (0.05 mg/kg, s.c.). LTP was induced using suprathreshold train stimuli 60 min post-drug. Post-train I/O functions were reassessed 1 and 24 h after train delivery. Upon full decay of any induced LTP, drug conditions were reversed such that each animal was tested under saline and MK-801. I/O functions measured 1 and 24 h after train induction as well as immediate post-train responses revealed significant LTP of comparable magnitude that was induced in both control and Pb-exposed animals tested under saline conditions. In contrast, MK-801 reduced LTP in control but not in Pb-exposed animals. The broadening of the excitatory postsynaptic potential evident in responses evoked by train stimuli is NMDA-dependent. Pb exposure attenuated the MK-801-induced reduction in area of this NMDA component by approximately 50%. These findings are consistent with other neurochemical and behavioural observations and suggest that up-regulation of postsynaptic NMDA receptors produces subsensitivity to the inhibitory effects of MK-801 on hippocampal LTP following chronic developmental Pb exposure.

Cortical glutamatergic markers in schizophrenia.

Post-mortem studies have yet to produce consistent findings on cortical glutamatergic markers in schizophrenia; therefore, it is not possible to fully understand the role of abnormal glutamatergic function in the pathology of the disorder. To better understand the changes in cortical glutamatergic markers in schizophrenia, we measured the binding of radioligands to the ionotropic glutamate receptors (N-methyl D-aspartate, [3H]CGP39653, [3H]MK-801), amino-3-hydroxy-5-methyl-4-isoxazole ([3H]AMPA), kainate ([3H]kainate), and the high-affinity glutamate uptake site ([3H]aspartate) using in situ radioligand binding with autoradiography and levels of mRNA for kainate receptors using in situ hybridization in the dorsolateral prefrontal cortex from 20 subjects with schizophrenia and 20 controls matched for age and sex. Levels of [3H]kainate binding were significantly decreased in cortical laminae I-II (p = 0.01), III-IV (p < 0.05), and V-VI (p < 0.01) from subjects with schizophrenia. By contrast, levels of [3H]MK-801, [3H]AMPA, [3H]aspartate, or [3H]CGP39653 binding did not differ between the diagnostic cohorts. Levels of mRNA for the GluR5 subunit were decreased overall (p < 0.05), with no changes in levels of mRNA for GluR6, GluR7, KA1, or KA2 in tissue from subjects with schizophrenia. These data indicate that the decreased number of kainate receptors in the dorsolateral prefrontal cortex in schizophrenia may result, in part, from reduced expression of the GluR5 receptor subunits.

Positive allosteric modulators of the metabotropic glutamate receptor subtype 2 (mGluR2).

This article describes recent medicinal chemistry progress toward selective potentiators of the metabotropic glutamate receptor 2 (mGluR2). Groups at Lilly and Merck have identified new classes of potentiators that exhibit selectivity for mGluR2 over the seven other subtypes of mGluRs. Structure-activity relationships as well as pharmacokinetic properties and in vivo activity are reviewed.

4-Alkylated homoibotenic acid (HIBO) analogues: versatile pharmacological agents with diverse selectivity profiles towards metabotropic and ionotropic glutamate receptor subtypes.

4-Alkylated analogues of homoibotenic acid (HIBO) have previously shown high potency and selectivity at ionotropic and metabotropic glutamic acid receptor (iGluR and mGluR) subtypes. Compounds with different selectivity profiles are valuable pharmacological tools for neuropharmacological studies, and the series of 4-alkyl-HIBO analogues have been extended in this paper in the search for versatile agents. Pharmacological characterization of five new analogues, branched and unbranched 4-alkyl-HIBO analogues, have been carried out. The present compounds are all weak antagonists at Group I mGluRs (mGluR1 and 5) presenting only small differences in potencies (Ki values ranging from 89 to 670 microM). Affinities were studied at native and cloned iGluRs, and the compounds described show preference for the AMPA receptor subtypes GluR1 and 2 over GluR3 and 4. However, compared to previous 4-alkyl-HIBO analogues, these compounds show a remarkably high affinity for the Kain preferring subtype GluR5. The observed GluR5 affinities were either similar or higher compared to their GluR1 and 2 affinity. Isopropyl-HIBO showed the highest affinity for GluR5 (Ki=0.16 microM), and represents a unique compound with high affinity towards the three subtypes GluR1, 2 and 5. In general, these compounds represent new selectivity profiles compared to previously reported Glu receptor analogues.

Ionotropic glutamate receptor binding in the posterior cingulate cortex in schizophrenia patients.

Using quantitative autoradiography, the present study examined ionotropic glutamatergic receptor binding sites using [3H]dizocilpine, [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate, and [3H]kainate in the posterior cingulate cortex of schizophrenia patients and matched controls. We found a significant increase in [3H]dizocilpine binding in the superficial layers (41%, p<0.001) and deep layers (30%, p=0.004) of the posterior cingulate cortex in the schizophrenia group compared with controls. No significant differences were observed in [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate and [3H]kainate binding. In summary, the present study has for the first time demonstrated that the glutamatergic system is affected in the posterior cingulate cortex in schizophrenia patients. The fact that only the N-methyl-D-aspartate receptor densities are significantly altered suggests that this is unlikely to be caused by a simple decrease in glutamatergic transmission.

[3H]muscimol binding to gamma-aminobutyric acid(A) receptors is upregulated in CA1 neurons of the gerbil hippocampus in the ischemia-tolerant state.

BACKGROUND AND PURPOSE: Excitotoxic activation of glutamate receptors is currently thought to play a pivotal role in delayed neuronal death (DND) of highly vulnerable CA1 neurons in the gerbil hippocampus after transient global ischemia. Postischemic degeneration of these neurons can be prevented by "preconditioning" with a short sublethal ischemic stimulus. The present study was designed to test whether ischemic preconditioning is associated with specific alterations of ligand binding to excitatory glutamate and/or inhibitory gamma-aminobutyric acid (GABA)A receptors compared with ischemia severe enough to induce DND. METHODS: With the use of quantitative receptor autoradiography, postischemic ligand binding of [3H]MK-801 and [3H]alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) to excitatory N-methyl-D-aspartate (NMDA) and AMPA receptors as well as [3H]muscimol to inhibitory GABA(A) receptors in hippocampal subfields CA1, CA3, and the dentate gyrus were analyzed in 2 experimental paradigms. Gerbils were subjected to (1) a 5-minute ischemic period resulting in DND of CA1 neurons and (2) a 2.5-minute period of ischemia mediating tolerance induction. RESULTS: [3H]MK-801 and [3H]AMPA binding values to excitatory NMDA and AMPA receptors showed a delayed decrease in relatively ischemia-resistant CA3 and dentate gyrus despite maintained neuronal cell density. [3H]Muscimol binding to GABA(A) receptors in CA1 neurons was transiently but significantly increased after preconditioning but not after global ischemia with consecutive neuronal death. CONCLUSIONS: Downregulation of ligand binding to glutamate receptors in relatively ischemia-resistant CA3 and dentate gyrus neurons destined to survive suggests marked synaptic reorganization processes despite maintained structural integrity. More importantly, upregulation of binding to inhibitory GABA(A) receptors in the hippocampus indicates a relative shift between inhibitory and excitatory neurotransmission that we suggest may participate in endogenous postischemic neuroprotection.

[3H]ATPA: a high affinity ligand for GluR5 kainate receptors.

The pharmacological properties of [3H]ATPA ((RS)-2-amino-3(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid) are described. ATPA is a tert-butyl analogue of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid) that has been shown to possess high affinity for the GluR5 subunit of kainate receptors. [3H]ATPA exhibits saturable, high affinity binding to membranes expressing human GluR5 (GluR5) kainate receptors (Kd approximately 13 nM). No specific binding was observed in membranes expressing GluR2 and GluR6 receptors. Several compounds known to interact with the GluR5 kainate receptor inhibited [3H]ATPA binding with potencies similar to those obtained for competition of [3H]kainate binding to GluR5. Saturable, high affinity [3H]ATPA binding (Kd approximately 4 nM) was also observed in DRG neuron (DRG) membranes isolated from neonatal rats. The rank order potency of compounds to inhibit [3H]ATPA binding in rat DRG and GluR5 membranes were in agreement. These finding demonstrate that [3H]ATPA can be used as a radioligand to examine the pharmacological properties of GluR5 containing kainate receptors.

[3H]CGP 61594, the first photoaffinity ligand for the glycine site of NMDA receptors.

Activation of NMDA receptors requires the presence of glycine as a coagonist which binds to a site that is allosterically linked to the glutamate binding site. To identify the protein constituents of the glycine binding site in situ the photoaffinity label [3H]CGP 61594 was synthesized. In reversible binding assays using crude rat brain membranes, [3H]CGP 61594 labeled with high affinity (K(D) = 23 nM) the glycine site of the NMDA receptor. This was evident from the Scatchard analysis, the displacing potencies of various glycine site ligands and the allosteric modulation of [3H]CGP 61594 binding by ligands of the glutamate and polyamine sites. Electrophysiological experiments in a neocortical slice preparation identified CGP 61594 as a glycine antagonist. Upon UV-irradiation, a protein band of 115 kDa was specifically photolabeled by [3H]CGP 61594 in brain membrane preparations. The photolabeled protein was identified as the NR1 subunit of the NMDA receptor by NR1 subunit-specific immunoaffinity chromatography. Thus, [3H]CGP 61594 is the first photoaffinity label for the glycine site of NMDA receptors. It will serve as a tool for the identification of structural elements that are involved in the formation of the glycine binding domain of NMDA receptors in situ and will thereby complement the mutational analysis of recombinant receptors.

Inhibition of uptake and release of a novel mGluR agonist (L-F2CCG-I) by anion transport blockers in the rat spinal cord.

A new metabotropic glutamate receptor (mGluR) agonist, (2S,1'S,2'S)-2-(2-carboxy-3,3-difluorocyclopropyl)glycine (L-F2CCG-I), induces a priming effect on (RS)-alpha-aminopimelate in the isolated spinal cord of newborn rats. Similar to (RS)-alpha-aminopimelate, L-glutamate (30-100 microM) neither affected spinal reflexes nor the resting membrane potentials of motoneurones, but preferentially potentiated the depression of monosynaptic excitation caused by L-F2CCG-I (0.4 microM). Following L-F2CCG-I treatment (1-2 microM), L-glutamate decreased the monosynaptic spinal reflexes in a concentration dependent manner, indicating a priming' effect of L-F2CCG-I. Thus L-glutamate is completely compatible with (RS)-alpha-aminopimelate in revealing the priming effect. An anion transport blocker, 4,4'-dinitrostilbene-2,2'-disulphonic acid (DNDS) (100 microM), markedly inhibited both the response to (RS)-alpha-aminopimelate and the induction of the L-F2CCG-I priming effect. The data suggest that L-F2CCG-I is Cl- -dependently incorporated into certain stores, and that (RS)-alpha-aminopimelate or L-glutamate must stimulate the release of L-F2CCG-I from the storage site. There were pharmacological similarities between the quisqualate and L-F2CCG-I priming effect. The physiological significance of the quisqualate or L-F2CCG-I priming is not yet established. L-F2CCG-I would be expected to be a useful pharmacological probe for elucidating the mechanism of the priming.

Newer anticonvulsant drugs: role of pharmacology, drug interactions and adverse reactions in drug choice.

In the last few years a number of new anticonvulsants have been introduced into clinical practice mainly as add-on therapy in patients who do not become seizure-free while receiving established anticonvulsants. Up to now, no single drug has been shown to be more effective at controlling seizures of a particular type than another, so other factors such as mechanism of action, pharmacokinetics, dosage regimens or the spectrum of adverse drug reactions and interactions are used when making a choice between one agent and another. The mechanism of action of tiagabine and vigabatrin is very specific; both agents increase gamma-aminobutyric acid (GABA) levels through inhibition of reuptake and catabolism respectively. However, the mechanism of action of gabapentin is unknown and those of felbamate, lamotrigine and topiramate are not sufficiently clarified as yet, and may be multiple. Great advances have been made in improving the pharmacokinetic characteristics of these newer anticonvulsants. Gabapentin and vigabatrin exhibit relatively ideal pharmacokinetic properties as they are not bound to proteins, are excreted mostly unchanged in the urine and show linear pharmacokinetics. Lamotrigine possesses a highly variable elimination half-life depending on the co-medication. Tiagabine is highly protein bound and zonisamide shows nonlinear pharmacokinetics; both these drugs are extensively metabolised. Problematic drug interactions between newer anticonvulsants and other drugs in general occur rarely when these agents are given concomitantly. However, in common with most new drugs, there are very few data on the use of the newer anticonvulsants in women of childbearing age. Studies done so far on interactions with oral contraceptives used low anticonvulsant dosages for a very short time. The newer anticonvulsants elicit adverse reactions that, while not being unique, are particularly associated with that drug. For example, felbamate may cause aplastic anaemia and fulminant liver failure, lamotrigine is prone to cause skin rash, and oxcarbazepine may cause symptomatic hyponatraemia. Topiramate and zonisamide cause kidney stones, and vigabatrin may induce psychiatric syndromes. Although highly diverse in structure and activity, these newer drugs offer new possibilities for treating refractory epilepsy. However, since no single factor can dictate the choice of drug nor predict the success of treatment, prescribing of these rather expensive drugs has to depend upon careful consideration of the aims of treatment, the characteristics of the drug and the needs of the individual patient.