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The novel mGlu2/3 receptor antagonist, LY3020371, has been shown to produce antidepressant-like effects comparable to that of the clinically-effective antidepressant ketamine. In the present study, we investigated whether LY3020371 would be predicted to be free of the side-effects and safety pharmacology issues associated with ketamine. In contrast to ketamine, LY3020371 produced small increases in locomotion and did not impair motor performance on an inverted screen. Ketamine, but not LY3020371, increased dopamine efflux in the nucleus accumbens of rats. Ketamine also produced cognitively-impairing effects in rats in a T-maze and in a psychomotor vigilance task and altered theta synchrony between the hippocampus and mPFC, whereas LY3020371 had either no significant impact or lesser effects in these assays. In mice, ketamine, but not LY3020371, negatively affected spontaneous alternation in a Y-maze. Rats were trained to discriminate LY3020371 from vehicle where 30mg/kg produced 100% drug-appropriate responding and the ED50 for LY3020371 was 9.4mg/kg, i.p. In rats discriminating LY3020371, neither d-amphetamine nor phencyclidine fully substituted for LY3020371 (35-45%) and the mGlu2/3 receptor agonist LY354740 partially attenuated the discriminative stimulus effects of LY3020371. These are the first data to demonstrate the discriminative stimulus effects of an mGlu2/3 receptor antagonist. Some alterations were suggested to occur in the density of mGlu2/3 receptor binding sites in the drug discrimination rats relative to their age-matched non-drug-exposed controls. In preclinical toxicology studies of 14day dosing of doses up to 1000mg/kg, i.v. in rats and up to 500m/kg, i.v. in Cynomologous monkeys, LY3020371 produced uM plasma exposures without producing critical toxicological findings. It is concluded that LY3020371 does not recapitulate the motor, cognitive, subjective, neurochemical, electrophysiological, or toxicological findings reported with ketamine. Thus, LY3020371 possesses both the efficacy signatures of a rapidly-acting antidepressant and a safety profile enabling proof of concept studies in patients.

Group II metabotropic glutamate (mGlu) receptor agonists were efficacious in randomized clinical research trials for schizophrenia and generalized anxiety disorder. The regional quantification of mGlu(2) and mGlu(3) receptors remains unknown. A selective and structurally novel mGlu(2/3) receptor agonist, 2-amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY459477) was tritiated and the distribution of mGlu(2) and mGlu(3) receptors was studied in transgenic mice lacking either mGlu(2), mGlu(3) or both receptors. LY459477 is an agonist with 1-2 nM potency for rodent and human mGlu(2) and mGlu(3) receptors. The functional selectivity of LY459477 was demonstrated by over 640-fold selectivity and the displacement binding selectivity was greater than 320-fold for all glutamate receptors except mGlu(6) (∼230-fold). More than 1000-fold selectivity was demonstrated for all non-glutamate receptors known to be targeted by antipsychotic drugs. Like atypical antipsychotic drugs, LY459477 reversed in vitro electrophysiological effects of a serotonergic hallucinogen and behavioral effects of phencyclidine or amphetamine. There was virtually no binding of [(3)H]LY459477 to any brain region in mice with a deletion of both mGlu(2) and mGlu(3) receptors. Regions enriched in mGlu(2) receptors included the medial prefrontal cortex, select hippocampal regions, the medial mammillary nucleus, the medial habenula, and the cerebellar granular cell layer. Regions enriched in mGlu(3) receptors were the dorsolateral entorhinal cortex, the hippocampal CA1 field, the piriform cortex, the substantia nigra, the thalamic reticular nucleus, and primary sensory thalamic nuclei. These findings suggest [(3)H]LY459477 should be a useful tool to further define the role of mGlu(2) and mGlu(3) receptors throughout the brain with respect to major neuropsychiatric syndromes. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'.

The synthesis of a series of C3'-cis-substituted carboxycyclopropyl glycines bearing a wide variety of functional groups is described, and the structure-activity relationship for this series as agonists of group II metabotropic glutamate receptors is reported.

A series of novel group I metabotropic glutamate receptor (mGlu) antagonists have been designed on the basis of the 4-carboxyphenylglycine pharmacophore. The compounds are either mGlu1 receptor selective or equipotent for both mGlu1 and mGlu5 receptors and have IC 50 values ranging from 1 to 30 μM determined by phosphoinositide hydrolysis (PI) assay in vitro. All the compounds produced dose-dependent inhibition of group I mGlu receptor agonist ( RS )-3,5-dihydroxyphenylglycine (DHPG)-induced limbic seizure responses in mice with ED 50 values ranging from 9 nmol for LY393053 to 138 nmol for LY339840 after intracerebroventricular injection and were more potent than the mGlu1 receptor antagonist 1-aminoindan-1,5-dicarboxylic acid (ED 50 =477 nmol). Further antagonist actions were also demonstrated in a model of ( RS )-DHPG-induced PI hydrolysis in vivo such that LY367385 and the active cis isomer of LY393053 produced dose-dependent inhibition of PI responses in both cerebellum and hippocampus. Cis LY393053 also inhibited hippocampal PI responses when administered intraperitoneally at a dose of 30 mg/kg. These compounds define a new series of group I mGlu receptor antagonists which may serve as useful experimental tools.

Previous work has shown that mGlu2/3 receptor agonists such as LY379268 inhibit motor responses to acutely administered phencyclidine (PCP) in rats. However, it has not been determined whether mGlu2/3 receptor agonists will reverse the enhanced effects of repeatedly administered PCP (so called PCP sensitization). In these studies, rats were administered daily PCP and monitored for the number of ambulations, fine movements, time at rest and rears using an automated activity system. At Day 10, when compared the first (Day 1) response, PCP-treated animals showed enhanced responses to all measures tested. Augmentations of these PCP-induced behaviors generally peaked between the third and tenth day after PCP administration had begun. Acute administration of LY379268 effectively suppressed PCP-evoked motor behaviors in rats sensitized to PCP. However, daily administrations of LY379268 (for 9 days), along with PCP, did not prevent the expression of the enhanced PCP response on Day 10. Thus, LY379268 administration can suppress PCP responses after either acute or chronic exposure to PCP. However, the underlying plasticity that leads to PCP sensitization was not affected by this treatment.

Pre-synaptic metabotropic glutamate (mGlu) receptors modulate neuronal excitability by controlling glutamate and gamma-aminobutyric acid (GABA) release. The mGlu8 receptor is predominantly found in pre-synaptic terminals and its expression is highly restricted. To study the role of this receptor, mGlu8 receptor-deficient mice were generated. Here we report that naïve mGlu8 receptor-deficient mice showed increased anxiety-related behavior in the elevated plus maze in low illumination conditions (red light). Open arm avoidance and risk assessment behavior were both significantly increased in mutant mice. Increased stressfulness of the testing conditions abolished this behavioral difference. Fluorescent light or prior restraint stress decreased the open arm activity of wild-type mice, while the open arm activity of mutant mice was essentially unaffected, leading to similar values in both strains. The total number of arm entries or closed arm entries was not significantly different between strains, indicating that the lack of mGlu8 receptor does not affect locomotor activity. No gross behavioral changes, or changes in the function of the autonomic nervous system or somatomotor systems were observed in mutant mice. Moreover, no significant differences in seizure susceptibility were detected between strains. Our results suggest that mGlu8 receptor may play a role in responses to novel stressful environment.

(2S,4S)-2-Amino-4-(4,4-diphenylbut-1-yl)-pentane-1,5-dioic acid 1m, is a novel metabotropic glutamate receptor (mGluR) antagonist with insignificant ionotropic affinity. It is selective antagonist of negatively-coupled cAMP-linked mGluRs with no effect on phosphoinositide coupled mGluRs. A series of 4-substituted glutamic acid analogues were prepared and it was found that compound 1k is tenfold more potent than 1m. Compound 1k has neither significant affinity for ionotropic glutamate receptors nor group 1 and 3 metabotropic receptors.

The in vitro pharmacology of a structurally novel compound, LY341495, was investigated at human recombinant metabotropic glutamate (mGlu) receptor subtypes expressed in non-neuronal (RGT, rat glutamate transporter) cells. LY341495 was a nanomolar potent antagonist of 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD)-induced inhibition of forskolin-stimulated cAMP formation at mGlu2 and mGlu3 receptors (respective IC50S of 0.021 and 0.014 microM). At group I mGlu receptor expressing cells, LY341495 was micromolar potent in antagonizing quisqualate-induced phosphoinositide (PI) hydrolysis, with IC50 values of 7.8 and 8.2 microM for mGlu1a and mGlu5a receptors, respectively. Among the human group III mGlu receptors, the most potent inhibition of L-2-amino-4-phosphonobutyric acid (L-AP4) responses was seen for LY341495 at mGlu8, with an IC50 of 0.17 microM. LY341495 was less potent at mGlu7 (IC50 = 0.99 microM) and least potent at mGlu4 (IC50 = 22 microM). Binding studies in rat brain membranes also demonstrated nanomolar potent group II mGlu receptor affinity for LY341495, with no appreciable displacement of ionotropic glutamate receptor ligand binding. Thus, LY341495 has a unique range of selectivity across the mGlu receptor subtypes with a potency order of mGlu3 > or = mGlu2 > mGlu8 > mGlu7 >> mGlu1a = mGlu5a > mGlu4. In particular, LY341495 is the most potent antagonist yet reported at mGlu2, 3 and 8 receptors. Thus, it represents a novel pharmacological agent for elucidating the function of mGlu receptors in experimental systems.

The novel compound LY354740 is a conformationally constrained analog of glutamate, which was designed for interaction at metabotropic glutamate (mGlu) receptors. In this paper the selectivity of LY354740 for recombinant human mGlu receptor subtypes expressed in non-neuronal (RGT) cells is described. At human mGlu2 receptors, LY354740 produced >90% suppression of forskolin-stimulated cAMP formation with an ec50 of 5.1 ± 0.3 nM. LY354740 was six-fold less potent in activating human mGlu3 receptors (ec50 = 24.3 ± 0.5 nM). LY354740 inhibition of forskolin-stimulated cAMP formation in human mGlu2 receptor-expressing cells was blocked by competitive mGlu receptor antagonists, including (+)-α-methyl-4-carboxyphenylglycine (MCPG) and LY307452 ((2S,4S0-2-amino-4-(4,4-diphenylbut-1-yl)-pentane-1,5-dioic acid). LY354740 had no agonist or antagonist activities at cells expressing human mGlu4 or mGlu7 (group III mGlu receptors) (ec50s > 100 000 nM). When tested at group I phosphoinositide-coupled human mGlu receptors (mGlu1a and mGlu5a), LY354740 did not activate or inhibit mGlu receptor agonist-evoked phosphoinositide hydrolysis at up to 100 000 nM. Electrophysiological experiments also demonstrated that LY354740 also had no appreciable activity in cells expressing human recombinant AMPA (GluR4) and kainate (GluR6) receptors. Thus, LY354740 is a highly potent, efficacious and selective group II (mGlu2/3) receptor agonist, useful to explore the functions of these receptors in situ. © 1997 Elsevier Science Ltd. All rights reserved.

Metabotropic glutamate receptors (mGluRs) are a heterogeneous family of G-protein coupled receptors that are linked to multiple second messengers in the rat hippocampus. The compound 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) has been widely used to activate this class of receptors and study their functions in situ. However, 1S,3R-ACPD acts on multiple mGluR subtypes to produce multiple alterations in second messengers. We report here that the aza-substituted analog of 1S,3R-ACPD, 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC), is a highly selective agonist for negatively-coupled cAMP-linked mGluRs in the rat hippocampus, with similar potency in mGluR2 expressing cells. 1S,3R-ACPD decreases forskolin-stimulated cAMP formation, increases basal cAMP formation, and increases phosphoinositide hydrolysis in the rat hippocampus. However, 2R,4R-APDC inhibited forskolin-stimulated cAMP, but had none of the other activities of 1S,3R-ACPD. Furthermore, 2R,4R-APDC had no measurable ionotropic glutamate receptor affinity in rat hippocampus, as indicated by lack of effects on basal and glutamate agonist-evoked [3H]norepinephrine release. 2R,4R-APDC also inhibited forskolin-stimulated cAMP formation in human mGluR2 expressing cells with about three-fold greater potency than 1S,3R-ACPD, but unlike 1S,3R-ACPD, showed no appreciable activation of phosphoinostide hydrolysis in human mGluR1 alpha expressing cells. Thus, 2R,4R-APDC should be a useful pharmacological agent to explore the functions of mGluRs coupled to inhibition of adenylate cyclase.

The pharmacology and cellular mechanism by which metabotropic glutamate receptor (mGluR) activation modulates cAMP formation was studied in cross-chopped hippocampal slices from neonatal (7 day old) rats. The selective mGluR agonist 1S,3R-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD), and other non-selective mGluR agonists produced concentration-related stimulation of basal cAMP formation in this tissue. The relative agonist potency order was 1S,3R- ACPD = quisqualate > ibotenate ⪢ 1R,3S-ACPD. 1S,3R-ACPD stimulated cAMP accumulation was antagonized in a stereoselective manner by l -2-amino-3-phosphonopropionate ( l -AP3), but not by higher chain homologues such as l -2-amino-4-phosphonobutyrate ( l -AP4) and 2-amino-5-phosphonopentanoate (AP5). 1S,3R-ACPD-enhanced cAMP formation was greatly inhibited by incubation with adenosine deaminase. In the adult rat hippocampus, 1S,3R-ACPD did not appreciably increase basal cAMP, but inhibited forskolin-stimulated cAMP formation, and this effect was observed with or without adenosine deaminase. In the presence of the adenosine receptor antagonist and cAMP phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX), 1S,3R-ACPD did not enhance cAMP formation in the neonatal hippocampus, but inhibited forskolin-stimulated cAMP (like in the adult tissue). These results demonstrate that mGluRs that increase cAMP in the neonatal hippocampus have a unique pharmacology when compared to mGluRs that decrease cAMP accumulation and increase phosphoinositide hydrolysis. 1S,3R-ACPD stimulation of cAMP in the neonatal rat hippocampal slice involves potentiation of responses to endogenous adenosine. Negatively coupled cAMP linked mGluRs are also present in the neonatal tissue, but are masked by the predominence of the positively coupled mGluR cAMP response.

Phosphoinositide-linked metabotropic glutamate receptors have been well characterized in a variety of CNS tissues. In this study the pharmacology of metabotropic glutamate receptors negatively coupled to cAMP formation was investigated in cross-chopped slices of the adult rat hippocampus. Excitatory amino acid agonists and antagonists were examined for effects on forskolin (30 microM)-simulated cAMP formation. The selective metabotropic glutamate agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) and various nonselective metabotropic/ionotropic agonists were found to inhibit forskolin-stimulated cAMP formation. Inhibition of cAMP formation was observed using 1S,3R-ACPD (57% at 100 microM), quisqualate (92% at 500 microM), ibotenate (44% at 500 microM), L-glutamate (41% at 1000 microM), and L-aspartate (59% at 1000 microM). Inhibition of forskolin-stimulated cAMP formation induced by these agonists was observed even in the presence of the ionotropic antagonists MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione. Up to 500 microM of the ionotropic agonists N-methyl-D-aspartate, AMPA, and kainate did not inhibit forskolin-stimulated cAMP formation. L-2-amino-3-phosphonopropionate (500 microM) greatly inhibited the stimulatory effect of 1S,3R-ACPD on phosphoinositide hydrolysis, even in the presence of forskolin. However when measuring cAMP formation, L-2-amino-3-phosphonoproprionate (500 microM) mimicked the effect of 1S,3R-ACPD, producing 64% inhibition of forskolin-stimulated cAMP. These studies show that in the adult rat hippocampus metabotropic glutamate receptors that are negatively linked to cAMP formation have a pharmacology that is distinct from ionotropic glutamate receptors and phosphoinositide-linked metabotropic glutamate receptors.

In previous work we found that 2-amino-3-phosphonopropionic acid, the β-phosphono-substituted analog of aspartic acid, is a selective in vitro inhibitor of the excitatory amino acid agonist [ibotenate, quisqualate, trans(±)-1-amino-1,3-cyclopentanedicarboxylic acid (trans-ACPD)]-stimulated phosphoinositide hydrolysis. Here we investigated if the inhibitory effects of 2-amino-3-phosphonopropionic acid on metabotropic excitatory amino acid receptors could be observed in the ex vivo brain slice following in vivo administration to neonatal rats. In vitro addition of 2-amino-3-phosphonopropionic acid noncompetitively antagonized trans-ACPD-stimulated [3H]phosphoinositide hydrolysis in hippocampal slices from 9-day-old rats. Intraperitoneal (i.p.) administration of 2-amino-3-phosphonopropionic acid (250, 500 and 750 mg/kg) for two consecutive days (at 7 and 8 days of age) also produced dose-related inhibition of trans-ACPD-stimulated [3H]phosphomositide hydrolysis in hippocampal slices from 9-day-old rats. 2-Amino-3-phosphonopropionic acid (500 mg/kg × 2) inhibited quisqualate, ibotenate, and glutamate activations of [3H]phosphoinositide hydrolysis, but had no effect on carbachol or norepinephrine stimulations. Thus, multiple in vivo dosing with 2-amino-3-phosphonopropionic acid produces selective inhibition of excitatory amino acid, but not nonexcitatory amino acid receptors coupled to phosphoinositide hydrolysis. 2-Amino-3-phosphonopropionic acid dosing could be used to inhibit the metabotropic excitatory amino acid receptor in vivo to study its function in the developing brain.

The relative roles of protein kinase C in regulating excitatory amino acid-, cholinoceptor-, and adrenoceptor-stimulated phosphoinositide hydrolysis were studied. Slices of rat hippocampus were prelabeled with [3H]-myo-inositol, and agonist-induced [3H]-phosphoinositide hydrolysis was measured by the formation of [3H]-inositol monophosphate ([3H]-IP) in the presence of lithium ion. Activation of protein kinase C with phorbol 12,13-dibutyrate (PDB) (10(-6) M) completely inhibited ibotenate (IBO) (10(-3) M)-induced [3H]-phosphoinositide hydrolysis. Half-maximal inhibition was observed at about 10(-7) M PDB. Higher concentrations of PDB were required to inhibit stimulation of [3H]-IP by either carbachol (CARB) (10(-3) M) or norepinephrine (NE) (10(-4) M, and only partial inhibition could be attained. Preincubation with staurosporine (STAURO) (10(-5) M) or 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) (10(-4) M), inhibitors of protein kinase C, potentiated IBO- but not CARB- or NE-induced stimulation of [3H]-IP. PDB inhibition of IBO- or NE-stimulated [3H]-phosphoinositide hydrolysis was reversed by co-addition of STAURO or H-7. In the case of IBO + STAURO, this reversal was to the potentiated level observed with STAURO alone. Enhanced agonist stimulation and reversal of PDB inhibition were also produced by STAURO when [3H]-phosphoinositide hydrolysis was stimulated by either L-glutamate or quisqualate. These experiments show that direct activation of protein kinase C by PDB leads to inhibition of phosphoinositide hydrolysis mediated by excitatory amino acid receptors, cholinoceptors, or adrenoceptors. However, the enhanced agonist-stimulated phosphoinositide hydrolysis elicited by inhibitors of protein kinase C suggests that, when protein kinase C is indirectly activated, only excitatory amino acids rapidly inhibit further receptor-coupling.