The Rapidly Acting Antidepressant Ketamine and the mGlu2/3 Receptor Antagonist LY341495 Rapidly Engage Dopaminergic Mood Circuits.

Ketamine is a rapidly acting antidepressant in patients with treatment-resistant depression (TRD). Although the mechanisms underlying these effects are not fully established, inquiry to date has focused on the triggering of synaptogenesis transduction pathways via glutamatergic mechanisms. Preclinical data suggest that blockade of metabotropic glutamate (mGlu2/3) receptors shares many overlapping features and mechanisms with ketamine and may also provide rapid efficacy for TRD patients. Central dopamine circuitry is recognized as an end target for mood regulation and hedonic valuation and yet has been largely neglected in mechanistic studies of antidepressant-relevant effects of ketamine. Herein, we evaluated the changes in dopaminergic neurotransmission after acute administration of ketamine and the mGlu2/3 receptor antagonist LY341495 [(2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid ] in preclinical models using electrophysiologic, neurochemical, and behavioral endpoints. When given acutely, both ketamine and LY341495, but not the selective serotonin reuptake inhibitor (SSRI) citalopram, increased the number of spontaneously active dopamine neurons in the ventral tegmental area (VTA), increased extracellular levels of dopamine in the nucleus accumbens and prefrontal cortex, and enhanced the locomotor stimulatory effects of the dopamine D2/3 receptor agonist quinpirole. Further, both ketamine and LY341495 reduced immobility time in the tail-suspension assay in CD1 mice, which are relatively resistant to SSRI antidepressants. Both the VTA neuronal activation and the antidepressant phenotype induced by ketamine and LY341495 were attenuated by the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo- (9CI)-benzo[f]quinoxaline-7-sulfonamide, indicating AMPA-dependent effects. These findings provide another overlapping mechanism of action of ketamine and mGlu2/3 receptor antagonism that differentiates them from conventional antidepressants and thus support the potential rapidly acting antidepressant actions of mGlu2/3 receptor antagonism in patients.

Metabotropic glutamate receptors in brain function and pathology.

Metabotropic glutamate receptors (mGluRs) are a novel family of recently cloned G protein-coupled receptors. These receptors are heterogeneous and coupled to multiple second messenger systems that include increases in phosphoinositide hydrolysis, activation of phospholipase D, decreases in cAMP formation, increases in cAMP formation, and changes in ion channel function. Using the selective mGluR agonist 1-aminocyclopentane-1,3-dicarboxylic acid (1s,3R-ACPD), considerable progress has been made towards understanding the role of this glutamate receptor class in the central nervous system. This article reviews the molecular aspects and pharmacology of mGluRs, and recent studies elucidating their role in brain function and pathology.

Comparison of c-Fos induction in the brain by the mGlu2/3 receptor antagonist LY341495 and agonist LY354740: evidence for widespread endogenous tone at brain mGlu2/3 receptors in vivo.

LY341495 and LY354740 are potent and selective antagonist and agonist, respectively, for Group II metabotropic glutamate (mGlu2/3) receptors. Here we demonstrate that LY341495 (3 mg/kg) significantly increased c-Fos expression in almost all brain regions analyzed (44 out of 52 regions) in animals that were prehandled and kept in home-cage environment to minimize stress. Robust c-Fos induction was observed in all cortical regions, hippocampal CA1 and CA3 subregions, amygdala and several other subcortical nuclei. In contrast to LY341495, changes in c-Fos expression following LY354740 were more modest and not generally widespread (decreased in 1 region, dentate gyrus; and increased in 13 out of 52 regions). Interestingly, although LY354740 is anxiolytic in animals, LY341495 did not increase c-Fos expression in the paraventricular nucleus of the hypothalamus which is usually activated by stress/fear and several anxiogenic compounds. To further investigate the behavioral consequences of mGlu2/3 receptor antagonism, LY341495 was administered to prehandled animals that were placed in the elevated plus maze test under low light (low stress) conditions. Here LY341495 increased mouse elevated plus maze (EPM)-anxiety in a dose-dependent manner, significantly decreasing the time spent in open arms, but not affecting total ambulations. The behavioral consequences and associated widespread pattern of brain neuronal activations following blockade of mGlu2/3 receptors suggest that there is considerable endogenous glutamate tone throughout the brain at negative feedback peri-synaptic mGlu2/3 receptors, even under low stress conditions where synaptic glutamate release spillover would be expected to be minimized.

Anxiolytic-like activity of the mGLU2/3 receptor agonist LY354740 in the elevated plus maze test is disrupted in metabotropic glutamate receptor 2 and 3 knock-out mice.

RATIONALE: (1S,2S,5R,6S)-2-Aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY354740) is a potent and selective agonist for group II metabotropic glutamate (mGlu2 and mGlu3) receptors, with anxiolytic-like activity in animal and human models, and efficacy in anxiety patients. However, the lack of mGlu2 or mGlu3 receptor specific agonists has prevented in vivo characterization of individual functions of these two receptors in mediating the anxiolytic-like effects of LY354740. OBJECTIVE: To utilize mGlu2 receptor and mGlu3 receptor knockout animals and the mGlu2/3 selective antagonist (2S,1'S,2'S)-2-(9-xanthylmethyl)-2-(2'-carboxycyclopropyl)glycine (LY341495) to further investigate the roles of mGlu2 and mGlu3 receptors in mediating the anxiolytic-like actions of LY354740 in a mouse model of anxiety [elevated plus maze (EPM) test]. METHODS: To confirm that mGlu2/3 receptors are responsible for anxiolytic-like activity in the EPM under these test conditions, mice were pretreated with LY341495 at 30 min prior to s.c. administered LY354740. Subsequently, saline vehicle or LY354740 was administered (s.c.) 30 min before the EPM testing in wild-type, mGlu2 receptor knockout, and mGlu3 receptor knockout mice. RESULTS: LY354740 reduced in a dose-dependent manner anxiety-related behavior on the EPM in wild-type mice with a maximally effective dose of 10--20 mg/kg s.c. Pretreatment with LY341495 potently prevented the anxiolytic-like effects of LY354740 (20 mg/kg, s.c.) in mice. Although the mGlu2 receptor knockout and mGlu3 receptor knockout mice were grossly normal, the anxiolytic-like activity of LY354740 (20 mg/kg, s.c.) was not evident in either mGlu2 or mGlu3 receptor knockout mice, when compared to their wild-type controls. CONCLUSIONS: The activation of both mGlu2 and mGlu3 receptors by LY354740 appears to be required for anxiolytic-like activity in the EPM test in mice. These studies serve as a foundation for additional studies on underlying circuits, brain structures, and receptor subtypes involved in the anxiolytic-like actions of mGlu receptor active agents, and the design of future drugs for anxiety disorders in humans.

Thyrotropin-releasing hormone (protirelin) inhibits potassium-stimulated glutamate and aspartate release from hippocampal slices in vitro.

Excess excitatory amino acid release is involved in pathways associated with seizures and neurodegeneration. Thyrotropin-releasing hormone (TRH; protirelin), a brain-derived tripeptide, has shown efficacy in the treatment of such disorders, yet its mechanism of neuroprotection is poorly understood. Using superfused hippocampal slices, we tested the hypothesis that TRH could inhibit evoked glutamate/aspartate release in vitro. Rat hippocampal slices were first equilibrated in oxygenated Krebs buffer (KRB) (120 min) then superfused for 10 min with KRB (control), or KRB containing 0.1, 1, or 10 microM TRH respectively, prior to and during 5 min depolarization with high potassium KRB (50 mM [K(+)] +/- TRH). Fractions (1 min) were collected during the 5 min stimulation and for an additional 10 min thereafter and analyzed for glutamate and aspartate by HPLC. TRH had no effect on baseline glutamate/aspartate release, while all three TRH doses significantly (P < 0.05) inhibited peak 50 mM [K(+)]-stimulated glutamate/aspartate release, and glutamate remained below control (P < 0.05) at 15 min post stimulation. A 5 min pulse of TRH (10 microM) had no affect on basal glutamate/aspartate release, whereas the TRH pre-pulsed slices failed to release glutamate/aspartate by [K(+)]-stimulation given 15 min later. These results are the first to show a potent and prolonged inhibitory effect of TRH on evoked glutamate/aspartate release in vitro. These initial studies suggest that exogenous and/or endogenous TRH may function, in part, to modulate excess glutamate release in specific CNS loci. Additional studies are in progress to fully understand the mechanism of this potent effect of TRH and its implication in various CNS disorders.

Group II selective metabotropic glutamate receptor agonists and local cerebral glucose use in the rat.

The novel mGluR agonist LY354740 and a related analogue LY379268 are selective for mGluR2/3 receptors and are centrally active after systemic administration. In this study, rates of local cerebral glucose use were measured using the [14C]2-deoxyglucose autoradiographic technique to examine the functional consequences of their systemic administration in the conscious rat. Both LY354740 (0.3, 3.0, 30 mg/kg) and LY379268 (0.1, 1.0, 10 mg/kg) produced dose-dependent changes in glucose use. After LY354740 (3.0mg/kg), 4 of the 42 regions measured showed statistically significant changes from vehicle-treated controls: red nuclei (-16%), mammillary body (-25%), anterior thalamus (-29%), and the superficial layer of the superior colliculus (+50%). An additional 15 regions displayed significant reductions in function-related glucose use (P < .05) in animals treated with LY354740 (30 mg/ kg). LY379268 (0.1, 1.0, 10 mg/kg) produced changes in glucose metabolism in 20% of the brain regions analyzed. Significant increases (P < .05) in glucose use were evident in the following: the superficial layer of the superior colliculus (+81%), locus coeruleus (+57%), genu of the corpus callosum (+31%), cochlear nucleus (+26%), inferior colliculus (+20%), and the molecular layer of the hippocampus (+14%). Three regions displayed significant decreases: mammillary body (-34%), anteroventral thalamic nucleus (-28%), and the lateral habenular nucleus (-24%). These results show the important functional involvement of the limbic system together with the participation of components of different sensory systems in response to the activation of mGluR2 and mGluR3 with LY354740 and LY379268.

Anxiolytic activity of the MGLU2/3 receptor agonist LY354740 on the elevated plus maze is associated with the suppression of stress-induced c-Fos in the hippocampus and increases in c-Fos induction in several other stress-sensitive brain regions.

LY354740 is a potent and selective agonist for group II metabotropic glutamate (mGlu) receptors, mGlu2 and mGlu3 receptors, with anxiolytic activity in several animal models of anxiety, including the elevated plus maze (EPM) test. Here, we studied neuronal activation in mouse brain after EPM exposure in saline- and LY354740-treated mice using c-Fos immunoreactivity as a marker. The effect of LY354740 on c-Fos expression was also studied in cage control (no EPM) mice. Pretreatment with LY354740 (20 mg/kg, s.c.) produced robust anxiolytic behavior on the EPM. LY354740 administration decreased EPM-induced increases in c-Fos expression in the CA3 of the hippocampus, while having no significant effects on basal c-Fos expression in the hippocampus. LY354740 administration significantly increased c-Fos expression in specific limbic regions, including the lateral division of the central nucleus of the amygdala (CeL), lateral parabrachial nucleus, locus coeruleus, and Edinger-Westphal nucleus, whether or not animals were exposed to the EPM. Moreover, LY354740 administration per se significantly increased c-Fos expression in regions processing sensory information, including the paraventricular and lateral geniculate nucleus of the thalamus as well as the nucleus of the optic tract and superior colliculus. In particular, the suppression of fear-evoked neuronal activity in the hippocampus and drug-induced increases in neuronal activation in the CeL have been previously linked to the anxiolytic effects of clinically effective drugs such as benzodiazepines, and thus may contribute to anxiolytic actions of LY354740 in animal models and human anxiety patients.

Metabotropic glutamate receptor modulation of cAMP accumulation in the neonatal rat hippocampus.

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 predominance of the positively coupled mGluR cAMP response.

Induction or protection of limbic seizures in mice by mGluR subtype selective agonists.

The behavioral consequences of metabotropic glutamate receptor (mGluR) activation were investigated following intracerebral administration of the mGluR selective agonists (RS)3,5-dihydroxyphenyl-glycine (3,5-DHPG), (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (1S,3R-ACPD), (1R,3S)-1-aminocyclopentane-1,3-dicarboxylate (1R,3S-ACPD), L-2-amino-4-phosphonobutyrate (L-AP4), L-serine-O-phosphate (L-SOP) and (2S,3S,4S)alpha-(carboxycyclopropyl)glycine (L-CCGI) into the thalamus in mice. Injections of 3,5-DHPG, 1S,3R-ACPD and L-CCGI produced dose-dependent increases in limbic seizures with a potency order of 3,5-DHPG = 1S,3R-ACPD > L-CCGI. This effect of 1S,3R-ACPD was stereoselective, since the inactive isomer (1R,3S-ACPD) did not elicit seizure activity. Limbic seizures induced by the phosphoinositide-coupled mGluR subtype selective agonist 3,5-DHPG were attenuated by the mGluR antagonist L-2-amino-3-phosphonopropanoic acid (L-AP3) and dantrolene, inhibitors of mGluR-mediated intracellular calcium mobilization. Interestingly, L-AP4, L-SOP and low doses of L-CCGI also protected against 3,5-DHPG seizures. These data indicate that mGluR agonist-induced limbic seizures in mice are mediated by activation of phosphoinositide-coupled mGluRs. Furthermore, these seizures can be protected against by activation of mGluRs that are negatively-linked to cAMP formation.

Pharmacological agents acting at subtypes of metabotropic glutamate receptors.

Metabotropic (G-protein-coupled) glutamate (mGlu) receptors have now emerged as a recognized, but still relatively new area of excitatory amino acid research. Current understanding of the roles and involvement of mGlu receptor subtypes in physiological/pathophysiological functions of the central nervous system has been recently propelled by the emergence of various structurally novel, potent, and mGlu receptor selective pharmacological agents. This article reviews the evolution of pharmacological agents that have been reported to target mGlu receptors, with a focus on the known receptor subtype selectivities of current agents.

LY354740: a metabotropic glutamate receptor agonist which ameliorates symptoms of nicotine withdrawal in rats.

LY354740 is a conformationally constrained analog of glutamate with high selectivity and nanomolar agonist activity at Group II metabotropic glutamate receptors (mGluRs). This orally active compound is a new drug candidate which is being developed for the treatment of anxiety. In this study, LY354740 was investigated in a model of nicotine withdrawal using the acoustic startle reflex (sensorimotor reactivity) in rats. Nicotine (6 mg/kg/day) was administered for 12 days subcutaneously by osmotic minipumps. After 12 days the pumps were removed and the animals were allowed to go through spontaneous withdrawal. Cessation of chronic nicotine exposure led to increased startle responding for 4 days following withdrawal. Treatment with LY354740 (0.0001-0.1 mg/kg, i.p.; 0.03-3 mg/kg, oral) produced a dose-dependent attenuation of the enhanced auditory startle responding following withdrawal of nicotine with intraperitoneal and oral ED50 values of 0.003 mg/kg and 0.7 mg/kg, respectively. These effects were stereoselective since the (-)-enantiomer of LY354740, LY366563, was without effect in this model. LY354740 produced no changes in the sensorimotor reactivity of rats not exposed to nicotine at oral doses up to 10 mg/kg. These data support the functional role of mGluR agonists in nicotine withdrawal and indicate that LY354740 may be efficacious in reducing the symptoms associated with nicotine withdrawal during smoking cessation in humans.

1-BCP, a memory-enhancing agent, selectively potentiates AMPA-induced [3H]norepinephrine release in rat hippocampal slices.

It is now clear that the AMPA subtype of ionotropic glutamate receptors (iGluRs) undergoes a rapid desensitization in response to activation by AMPA receptor agonists. This desensitization is inhibited by compounds such as aniracetam and cyclothiazide, which act at a distinct site on the AMPA receptor complex. In particular, cyclothiazide greatly potentiates AMPA receptor-mediated depolarizing responses in the hippocampus. We have recently shown cyclothiazide also increases AMPA-induced release of [3H]norepinephrine ([3H]NE). More, recently, a benzamide compound, 1-(1,3-benzodioxol-5-ylcarbonyl)-piperidine (1-BCP), has been reported to enhance AMPA-induced currents and to facilitate memory retention in rats in a number of memory tasks. In this study, the effects of 1-BCP on excitatory amino acid agonist-induced [3H]NE release in rat hippocampal slices were determined. We report that 1-BCP, like cyclothiazide, selectively potentiates AMPA-induced [3H]NE release. However, cyclothiazide was more potent and efficacious than 1-BCP. Nevertheless, these data suggest a role for AMPA receptor-mediated enhancement of norepinephrine release as a mechanism of action for nootropic compounds such as 1-BCP.

Acute increases in monoamine release in the rat prefrontal cortex by the mGlu2/3 agonist LY379268 are similar in profile to risperidone, not locally mediated, and can be elicited in the presence of uptake blockade.

Our recent work (Cartmell et al., Journal of Neurochemistry, 75 (2000) 1147-1154) demonstrated that systemic injection of the potent, selective mGlu2/3 receptor agonist, LY379268, acutely increased extracellular levels of dopamine, its metabolites DOPAC and HVA, and the 5-HT metabolite, 5-HIAA, in rat medial prefrontal cortex (mPFC). Here, we compared the acute effects of LY379268 with those of clozapine and risperidone (atypical antipsychotics) on extracellular levels of both dopamine and 5-HT in the mPFC of freely-moving rats. Uptake blockers were included to minimize metabolism of monoamines near the probe area. One hour after injection, LY379268 (10 mg/kg s.c.), clozapine (10 mg/kg s.c.) or risperidone (1 mg/kg s.c.) maximally increased dopamine by 224, 257 and 234% of basal levels. These effects were followed by maximal increases in DOPAC and HVA levels 2 to 3.5 hours after administration. LY379268, at 3 and 10 mg/kg s.c., and risperidone (1 mg/kg s.c.) also increased dialysate 5-HT to 169, 179 and 140% of basal levels and 5-HIAA to 144, 154 and 121% of basal levels, respectively. These neurochemical changes in the mPFC could not be mimicked when LY379268 (3 or 30 microM) was administered locally via the microdialysis probe. These data demonstrate that increases in extracellular monoamines in the rat prefrontal cortex evoked acutely by the mGlu2/3 agonist, LY379268, are similar in profile to risperidone, not locally mediated, and can be elicited in the presence of uptake blockade.

The novel metabotropic glutamate receptor agonist 2R,4R-APDC potentiates stimulation of phosphoinositide hydrolysis in the rat hippocampus by 3,5-dihydroxyphenylglycine: evidence for a synergistic interaction between group 1 and group 2 receptors.

The mGlu receptor subtypes and second messenger pathways that mediate 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) responses in brain tissues are not fully understood. 1S,3R-ACPD differs from 3,5-dihydroxyphenylglycine (DHPG) or quisqualate in that 1S,3R-ACPD also activates group 2 mGlu receptors (mGlu2 and mGlu3) that are negatively linked to cAMP formation. To investigate the contribution of group 2 mGlu receptor activity of 1S,3R-ACPD to the phosphoinositide response in the rat hippocampus, we examined the effects of the novel group 2 mGlu receptor agonist 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC). 2R,4R-APDC did not activate or inhibit group 1 mGlu receptors (human mGlu1 alpha and mGlu5a) or group 3 mGlu receptors (human mGlu4 and mGlu7), but potently decreased forskolin-stimulated cAMP formation in human mGlu2- and mGlu3-expressing cells. In slices of the adult rat hippocampus 2R,4R-APDC had no effect on basal phosphoinositide hydrolysis; however, it was found to greatly enhance phosphoinositide hydrolysis to DHPG or quisqualate. In the neonatal rat hippocampus, 2R,4R-APDC enhanced the potency of DHPG, while not affecting the maximal response to group 1 mGlu receptor agonists. Thus, the phosphoinositide response in the rat hippocampus to 1S,3R-ACPD is mediated by a synergistic interaction between group 1 and group 2 mGlu receptors.

LY341495 is a nanomolar potent and selective antagonist of group II metabotropic glutamate 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.

(S)-3,4-DCPG, a potent and selective mGlu8a receptor agonist, activates metabotropic glutamate receptors on primary afferent terminals in the neonatal rat spinal cord.

(S)-3,4-Dicarboxyphenylglycine (DCPG) has been tested on cloned human mGlu1-8 receptors individually expressed in AV12-664 cells co-expressing a rat glutamate/aspartate transporter and shown to be a potent and selective mGlu8a receptor agonist (EC(50) value 31+/-2 nM, n=3) with weaker effects on the other cloned mGlu receptors (EC(50) or IC(50) values >3.5 microM on mGlu1-7). Electrophysiological characterisation on the neonatal rat spinal cord preparation revealed that (S)-3,4-DCPG depressed the fast component of the dorsal root-evoked ventral root potential (fDR-VRP) giving a biphasic concentration-response curve showing EC(50) values of 1.3+/-0.2 microM (n=17) and 391+/-81 microM (n=17) for the higher and lower affinity components, respectively. The receptor mediating the high-affinity component was antagonised by 200 microM (S)-alpha-methyl-2-amino-4-phosphonobutyrate (MAP4, K(D) value 5.4+/-1.5 microM (n=3)), a group III metabotropic glutamate (mGlu) receptor antagonist. The alpha-methyl substituted analogue of (S)-3,4-DCPG, (RS)-3,4-MDCPG (100 microM), antagonised the effects of (S)-3,4-DCPG (K(D) value 5.0+/-0.4 microM, n=3) in a similar manner to MAP4. (S)-3,4-DCPG-induced depressions of the fDR-VRP in the low-affinity range of the concentration-response curve were potentiated by 200 microM (S)-alpha-ethylglutamate (EGLU), a group II mGlu receptor antagonist, and were relatively unaffected by MAP4 (200 microM). However, depressions of the fDR-VRP mediated by the AMPA selective antagonist (R)-3,4-DCPG were not potentiated by EGLU, suggesting that the low-affinity component of the concentration-response curve for (S)-3,4-DCPG is not due to antagonism of postsynaptic AMPA receptors. It is suggested that the receptor responsible for mediating the high-affinity component is mGlu8. The receptor responsible for mediating the low-affinity effect of (S)-3,4-DCPG has yet to be identified but it is unlikely to be one of the known mGlu receptors present on primary afferent terminals or an ionotropic glutamate receptor of the AMPA or NMDA subtype.

Selective inhibition of forskolin-stimulated cyclic AMP formation in rat hippocampus by a novel mGluR agonist, 2R,4R-4-aminopyrrolidine-2,4- dicarboxylate.

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.

In vitro and in vivo antagonism of AMPA receptor activation by (3S, 4aR, 6R, 8aR)-6-[2-(1(2)H-tetrazole-5-yl) ethyl] decahydroisoquinoline-3-carboxylic acid.

The in vitro and in vivo pharmacology of a structurally novel competitive antagonist for the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) subtype of excitatory amino acid receptors is described. LY215490, (+/-)(6-(2-(1-H-tetrazol-5-yl)ethyl) decahydroisoquinoline-3-carboxylic acid), was shown to displace selectively 3H-AMPA and 3H-6-cyano-7-nitro- quinoxaline-2,3-dione (3H-CNQX) binding to rat brain membranes. LY215490 potently antagonized quisqualate-and AMPA-induced depolarizations of rat cortical slices in a competitive manner, while requiring higher concentrations to antagonize the effects of N-methyl-D-aspartate (NMDA) and kainate. In slices of rat hippocampus, LY215490 also selectively antagonized AMPA-evoked release of 3H-norepinephrine. These AMPA receptor activities were due to the (-) isomer of the compound. (3S,4aR,6R, 8aR)-6-[2-(1(2)H-tetrazole-5-yl)ethyl] decahydroisoquinoline-3-carboxylic acid (LY293558). LY215490 was centrally active following parenteral administration in mice as demonstrated by protection versus maximal electroshock seizures and decreases in spontaneous motor activity. LY215490 (its active isomer being LY293558) represents a novel pharmacological agent for in vitro and in vivo studies of AMPA receptor function in the CNS.

[3H]-LY341495 as a novel antagonist radioligand for group II metabotropic glutamate (mGlu) receptors: characterization of binding to membranes of mGlu receptor subtype expressing cells.

Metabotropic glutamate (mGlu) receptors are a family of eight known subtypes termed mGlu1-8. Currently, few ligands are available to study the pharmacology of mGlu receptor subtypes. In functional assays, we previously described LY341495 as a highly potent and selective mGlu2 and mGlu3 receptor antagonist. In this study, radiolabeled [3H]-LY341495 was used to investigate the characteristics of receptor binding to membranes from cells expressing human mGlu receptor subtypes. Using membranes from cells expressing human mGlu2 and mGlu3 receptors, [3H]-LY341495 (1 nM) specific binding was > 90% of total binding. At an approximate K(D) concentration for [3H]-LY341495 binding to human mGlu2 and mGlu3 receptors (1 nM), no appreciable specific binding of [3H-]LY341495 was found in membranes of cells expressing human mGlu1a, mGlu5a, mGlu4a, mGlu6, or mGlu7a receptors. However, modest (approximately 20% of mGlu2/3) specific [3H]-LY341495 (1 nM) binding was observed in human mGlu8 expressing cells. [3H]-LY341495 bound to membranes expressing human mGlu2 and mGlu3 receptors in a reversible and saturable manner with relatively high affinities (Bmax 20.5 +/- 5.4 and 32.0 +/- 7.0 pmol/mg protein; and K(D) = 1.67 +/- 0.20 and 0.75 +/- 0.43 nM, respectively). The pharmacology of [3H]-LY341495 binding in mGlu2 and mGlu3 expressing cells was consistent with that previously described for LY341495 in functional assays. [3H]-LY341495 binding provides a useful way to further investigate regulation of receptor expression and pharmacological properties of mGlu2 and mGlu3 receptor subtypes in recombinant systems.

LY354740 is a potent and highly selective group II metabotropic glutamate receptor agonist in cells expressing human glutamate receptors.

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 (+)-alpha-methyl-4-carboxyphenylglycine (MCPG) and LY307452 ((2S,4S)-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) (EC50 > 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.