Abstracts of the 7th International Meeting on Metabotropic Glutamate ...

Current Neuropharmacology, 2011, 9, 1-70

Abstracts of the 7th International Meeting on Metabotropic Glutamate Receptors (Taormina, Italy, October 2-7, 2011) 1.New Orthosteric Agonists of Group-III Metabotropic Glutamate Receptors F.C. Acher1, T. Courtiol1, I. Brabet2, C. Selvam1, N. Oueslati2, D. Rigault1, I.A. Lemasson1, S. Cesarini1, Bertrand H.-O3, C. Goudet2 and J.-P. Pin2 1

Université Paris Descartes, UMR8601-CNRS, 45 rue des SaintsPères, Paris 06, France; 2Institut de Génomique Fonctionnelle, CNRS UMR5203, INSERM U661, Universités Montpellier 1 & 2, 141 rue de la Cardonille, 34094 Montpellier, France; 3Accelrys, Parc Club Orsay Université, 20 rue Jean Rostand, 91893 Orsay, France Among the 8 subtypes of metabotropic glutamate receptors (mGluRs), mGlu4 receptors have been identified as clearly promising therapeutic targets for the treatment of Parkinson’s disease, pain and anxiety. In the recent years, several positive allosteric modulators (PAM) were disclosed revealing beneficial effects. On the other hand, we showed that using mGlu4R preferential agonist LSP1-2111 in animal models of Parkinson’s disease or anxiety, a positive response was observed [1,2]. Although PAMs are usually preferred to orthosteric agonists because of better brain penetration and better mGluR subtype selectivity, orthosteric agonists may also display advantageous properties. Notably they are water soluble, little metabolized because of their polar structure, they may penetrate the brain by active transport and show fewer off target effects. The major hurdle to overcome with orthosteric ligands is subtype selectivity. However in a virtual high throughput screening campaign, we discovered PCEP that disclosed a new binding pocket adjacent to the glutamate binding site and lined with subtype variable residues [3,4]. Taking advantage of that new pocket, we discovered LSP42022 that indeed displays some selectivity among the group III receptors (EC50’s of 0.11, 4.2, 11.6, 29.2 M at mGlu4, 6, 7, 8 receptors). Derivatives with various R1 - R3 substituents were synthesized and display notable potency and selectivity. R1

NO2 O H2N HO2C

P OH

O CO2H

(+)-(3S)-PCEP

H2N HO2C

P HO OH LSP1-2111

OH OCH3

O H2N HO2C

P HO OH

R2 R3

LSP4-2022 R1=R3=H R2= OCH2CO2H

[1] Beurrier, C, et al, FASEB J. 2009, 23, 3619-3628. [2] Wieronska, J. M. et al. Neuropharmacology, 2010, 59, 627-634. [3] Selvam, C., et al. J. Med. Chem., 2010, 53, 2797-2813. [4] Acher, F. C., et al. Neuropharmacology, 2011, 60, 102-107.

2.Control of Ethanol Consumption and Preference by Metabotropic Glutamate Receptor Subtype 7 Expressed in Striatum L. Adelfinger1, A. Dillinger1, A. Bahi1, K. Fizia1, M. Dietz1, F. Gasparini2 and P.J. Flor1 1

University of Regensburg, Faculty of Biology and Preclinical Medicine, Regensburg, Germany; 2Novartis Pharma AG, Neuroscience Discovery, Basel, Switzerland Growing evidence supports a role for the central nervous system (CNS) neurotransmitter L-glutamate and its metabotropic receptors (mGluRs) in drug addiction in general and alcohol-use disorders in particular. Alcohol dependence, for instance, has a genetic component, and the recent discovery that variations in the gene 1570-159X/11 $58.00+.00

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coding for mGluR7 modulate alcohol consumption further validates involvement of the L-glutamate system. Consequently, increasing interest emerges in developing L-glutamatergic therapies for the treatment of alcohol abuse and dependence. To this end, we performed a detailed behavioral pharmacology study to investigate the regulation of alcohol consumption and preference following administration of the mGluR7-selective drugs N,N'-dibenzyhydrylethane-1,2-diamine dihydrochloride (AMN082) and 6-(4methoxyphenyl)-5-methyl-3-pyridin-4-ylisoxazonolo[4,5-c]pyridin4(5H)-one (MMPIP). Upon administration of the allosteric agonist AMN082 (10 mg/kg, i.p.) in rats, there was a significant decrease in ethanol consumption and preference, without affecting ethanol blood metabolism. In contrast, mGluR7 blockade with MMPIP (10 mg/kg, i.p.) showed an increase in alcohol intake and reversed AMN082‘s effect on ethanol consumption and preference. Moreover, mice carrying a genetic deletion of mGluR7 showed increased alcohol drinking and preference, confirming our pharmacological findings. Recently, we started to use intra-cerebral injections of adeno-associated viruses expressing small interfering RNAs that knock-down mGluR7 in rats. Using this approach we were able to identify striatal nuclei that mediate mGluR7’s regulatory role on ethanol intake. In conclusion, our findings support a specific regulatory role for mGluR7 on alcohol drinking and preference and provide evidence for the use of mGluR7-directed drugs as potential new treatments for alcohol-use disorders in man.

3.Antidepressant-like Effect of a Metabotropic Glutamate 2/3 Receptor Antagonist Y. Ago, S. Chaki, K. Takuma and T. Matsuda Laboratory of Medicinal Pharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan Preclinical and clinical evidence supports the hypothesis that brain glutamatergic system plays a crucial role in the pathophysiology of major depression and the mechanism of action of antidepressants. The metabotropic glutamate 2/3 receptor (mGluR2/3) is abundantly localized within forebrain regions, and its antagonists have an antidepressant-like potential in experimental animal models [1, 2]. Recently, the upregulation of mGluR2/3 expression was shown in the prefrontal cortex of patients with major depression [3], indicating its etiological contribution via glutamatergic dysfunction. We have previously reported that chronic corticosterone treatment and social isolation-rearing increased immobility time of mice in the forced swim and tail suspension tests, suggesting the reliable models of depression for studying its neurobiological mechanisms [4]. Thus, we examined the binding of the mGluR2/3 antagonist [3H]LY341495 to mGluR2/3 in the brain by in vitro autoradiography and the effects of administration of the mGluR2/3 antagonists MGS0039 and LY341495 in these two models. Isolation-rearing increased [3H]LY341495 binding in mouse prefrontal cortex, cortical layers I–III, and hippocampus (due to an increase in Bmax), while chronic corticosterone did not. MGS0039 and LY341495 decreased the immobility time in the forced swim test in both corticosterone-treated and isolation-reared mice [5]. Moreover, chronic corticosterone and isolation-rearing markedly increased high K+-induced dopamine release, but not glutamate and serotonin release, in mouse prefrontal cortex, and this enhanced release of dopamine was reversed by the mGluR2/3 antagonist. These findings suggest that mGluR2/3 antagonists exhibit an antidepressant-like effect irrespective of the expression of mGluR2/3, probably via an inhibition of enhanced prefrontal dopaminergic neurotransmission, and imply that mGluR2/3 is a promising target for the treatment of depression. [1] Chaki, S, et al. Neuropharmacology, 2004, 46, 457-467. [2] Yoshimizu, T, et al. Psychopharmacology (Berl) 2006, 186 , 587-593. ©2011 Bentham Science Publishers

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[3] Feyissa, A. M, et al. Prog. Neuropsychopharmacol. Biol. Psychiatry 2010, 34, 279-283. [4] Ago, Y, et al. Neuropharmacology 2008, 55, 1355-1363. [5] Kawasaki, T, et al. Neuropharmacology, 60, 2011, 397-404.

4.Genetic Dissection of mGluR1 Function in Cerebellar Purkinje Cells A. Aiba Laboratory of Animal Resources, Center for Disease Biology and Integrative Medicine, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan The subtype 1 of mGluR (mGluR1) is a Gq-coupled receptor and expressed strongly in Purkinje cells of cerebellum. We previously showed that mGluR1 knockout (KO) mice have characteristic cerebellar symptoms such as ataxic gait and motor discoordination. mGluR1 KO mice also exhibited deficient long-term depression at parallel fiber-Purkinje cell synapses and impaired developmental transition from multiple to mono-innervation of Purkinje cells by climbing fibers [1, 2]. These phenotypes were rescued by introduction of an mGluR1a transgene with Purkinje cell-specific L7 promoter (mGluR1-rescue mice) [3]. However, roles of mGluR1 in the adult brain remain elusive, mainly due to lack of conventional and reproducible method to block mGluR1 expression at a certain developmental stage. We established mGluR1 conditional knockout (cKO) mice using the tetracycline-controlled gene expression system [4]. The mGluR1 cKO mice express mGluR1 only in Purkinje cells and its expression can be turned off with oral administration of a tetracycline analog, doxycycline. The mGluR1 cKO mice showed normal motor coordination without doxycyline administration. Blockade of expression of mGluR1 in the adult mGluR1 cKO mice led to impaired motor coordination, suggesting that mGluR1 is essential for cerebellar function in mice not only during postnatal development but also in adulthood. Our previous studies using mGluR1 KO mice have revealed that mGluR1 in Purkinje cell is required for delay paradigm of eyeblink conditioning [1, 5]. By using mGluR1 cKO mice, we also investigated the role of the mGluR1 in each learning process of eyeblink conditioning. Current progress will be presented. [1] Aiba, et al. Cell, 1994, 79, 377. [2] Kano, et al. Neuron, 1997, 18, 71. [3] Ichise, et al. Science. 2000, 288, 1832 . [4] Nakao, et al. Neurosci. Res., 2007, 57, 538. [5] Kishimoto, et al. Eur. J. Neurosci., 2002, 16, 2416 .

5.Neuron-astrocyte Communication Mediated by Endocannabinoid/ mGluR Signaling at Tripartite Synapses A. Araque Cajal Institute (CSIC), Doctor Arce 37, Madrid 28002, Spain Accumulating evidence indicates the existence of bidirectional communication between astrocytes and neurons, which has led to the establishment of the Tripartite Synapse concept, in which astrocytes actively exchange information with the synaptic elements, responding to synaptically released neurotransmitters and releasing gliotransmitters that regulate synaptic transmission [1]. I will present data showing the existence of neuron-astrocyte signaling mediated by endocannabinoids (ECBs) in mice hippocampal slices. Hippocampal astrocytes express functional cannabinoid receptors (CB1Rs) that upon activation increase the intracellular calcium through Gq/11- and phospholipase C-dependent release of calcium from internal stores. Depolarization of hippocampal pyramidal neurons stimulates the release of ECBs, which activate CB1Rs in adjacent astrocytes, elevating their calcium levels. These endocannabinoid-induced calcium elevations stimulate glutamate

Conference Proceedings

release from astrocytes and evoke slow inward currents in adjacent neurons through activation of NMDARs [2]. I will also present more recent data showing the consequences of the ECB-mediated neuron-astrocyte signaling on synaptic transmission. Endocannabinoids released by hippocampal pyramidal neurons transiently increase the probability of transmitter release at single CA3-CA1 hippocampal synapses. This synaptic potentiation is due to CB1Rinduced calcium elevations in astrocytes, which stimulate the release of glutamate that activates presynaptic group I metabotropic glutamate receptors (mGluRs). While endocannabinoids induce Gi/o protein-mediated synaptic depression of homoneuronal synapses by direct activation of presynaptic CB1Rs, they lead to synaptic potentiation of heteroneuronal synapses by activation of CB1Rs in astrocytes. Therefore, ECB signaling may lead to mGluR-dependent potentiation of synaptic transmission through activation of astrocytes [3]. I will finally present our more recent results showing that the previously found ECB/mGluR signaling at tripartite synapses may lead to long-lasting hippocampal synaptic plasticity, because the temporal coincidence of the astrocyte-mediated ECB/mGluR signaling and a postsynaptic signal evoked by mild (to -30 mV) neuronal depolarization induces the long-term potentiation of the transmitter release at CA3-CA1 hippocampal synapses. [1] Perea, G, et al. Trends Neurosci., 2009, 32, 421-431. [2] Navarrete, M., Araque, A. Neuron, 2008, 57, 883-893. [3] Navarrete, M., Araque, A. Neuron, 2010, 68, 113-126. Supported by: MICINN (BFU2010-15832), Cajal Blue Brain, and European Union (Health- F2-2007-202167).

6.Mirror Symmetric Alterations in Synaptic Plasticity in the Mouse Models of Fragile X Syndrome and Tuberous Sclerosis B.D. Auerbach, E. Osterweil and M.F. Bear HHMI/MIT, Picower Institute for Learning and memory, Cambridge, MA; MIT Brain and Cognitive Sciences, Cambridge, MA Autism spectrum disorder (ASD) is a highly pervasive neurodevelopmental condition and finding effective therapies for it is of the utmost importance. Understanding disease pathophysiology is crucial for development of novel therapies. Despite its strong heritability, the genetic heterogeneity of autism makes it difficult to study systematically and currently there is no unifying mechanism that accounts for the diverse constellation of symptoms associated with ASD. However, recent progress using animal models suggests that ASD may arise from altered function and plasticity of synapses in the brain. Tuberous sclerosis (TSC) and Fragile X syndrome (FXS) are single gene disorders associated with autism and intellectual impairment, and genetically engineered animal models permit investigation of whether altered synaptic function is a common disruption in these disorders as well as other forms of ASD. Here we tested the hypothesis that protein synthesisdependent long-term depression (LTD) induced by activation of group 1 metabotropic glutamate receptors (mGluR1 and mGluR5) is exaggerated in the hippocampus of the Tsc2+/- mouse, similar to the Fmr1-/y model of FXS. Unexpectedly, we discovered a selective impairment in mGluR-dependent LTD in Tsc2+/- mice that could be corrected acutely by the mTOR inhibitor rapamycin. Consistent with this deficit in synaptic plasticity, we also found that basal protein synthesis rates are diminished in Tsc2+/- mice, which was similarly rescued by rapamycin. Both the LTD and protein synthesis impairments were also reversed by a positive modulator of metabotropic glutamate receptor 5 (mGluR5 PAM), and in vivo administration of the mGluR5 PAM corrected a memory deficit in the Tsc2+/- mice. These results suggest, first, that excessive mTOR signaling suppresses the protein synthesis required for mGluRLTD, second, that mGluR5 PAMs have therapeutic utility in TSC and, third, that the synaptic pathophysiology of TSC and FXS, at least in the hippocampus, may be mirror images of one another.

Abstracts of the 7th International Meeting on Metabotropic Glutamate Receptors

While Tsc2+/- and Fmr1-/y mice have opposite alterations in mGluRLTD and protein synthesis we found they share a behavioral deficit in context discrimination, suggesting that opposing synaptic deviations may result in similar cognitive impairments. To test this idea of mirror symmetry we crossed Tsc2+/- mice with Fmr1-/y mice. Remarkably, both mGluR-LTD and context discrimination are restored to wildtype levels in the Tsc2+/ x Fmr1-/y double mutant mice. Our findings demonstrate that bidirectional changes in mGluR mediated protein synthesis and plasticity can manifest as similar behavioral impairments and may be a common pathophysiology in ASD. This discovery has important implications for the design of treatments for TSC, FXS, and autism of unknown etiology.

7.Selective mGlu2/3 Receptor NAM Alleviate Pharmacological or Age-Related Cognitive Impairment in Rodents and Monkeys T. M. Ballard, F. Knoflach, T. J. Woltering, E. Goetschi, R. Moog, J. Huwyler, M. Schmitt, J. Wichmann and S. Gatti F. Hoffmann-La Roche Ltd., pRED,Basel,Grenzacherstrasse 124, CH-4070 Basel, Switzerland We have synthesised brain penetrant, potent and selective negative allosteric modulators (NAM) of metabotropic glutamate (mGlu) 2/3 receptors, such as compounds 7am and 7i [1]. Morphological, behavioural and electrophysiological studies have indicated a role for mGlu2/3 receptors in cognitive processes dependent on medial temporal lobe activity [2]. To explore this hypothesis further we tested the mGlu2/3 NAM on predominantly hippocampal-dependent tests of cognition, such as an operant conditioning delayed match to position (DMTP) task and the Morris water maze. In both the DMTP task and the water maze, mGlu2/3 NAM dose-dependently attenuated the cognitive impairment induced by either the mGlu2/3 selective agonist LY354740 or the muscarinic antagonist scopolamine. Combination of sub-threshold doses of compound 7am and donepezil, a cholinesterase inhibitor, significantly reversed scopolamine-induced impairment of working memory in the DMTP task. To further profile the effects of mGlu2/3 NAM on cognition, compound 7i (3-10 mg/kg p.o.) was tested in aged Fischer-344 rats (22-24 months) and in PS2APP transgenic mice, a mouse model of Alzheimer’s disease [3]. After four days of treatment compound 7i significantly improved spatial acquisition in aged rats. Ten days treatment with compound 7i completely attenuated the spatial learning deficit observed in 12 month old PS2APP transgenic mice. Compound 7i (3-30 mg/kg p.o.) was further evaluated on a neurological test battery, locomotor activity and a chain pull test, which assesses motor and/or motivational performance. The mGlu2/3 NAM did not disrupt behaviour at doses up to and above the cognition-enhancing dose-range. Finally, compound 7i (3-10 mg/kg p.o.) was shown to improve choice accuracy in a delayed match to sample (DMTS) task in aged rhesus monkeys (23-30 years). In summary, the current study demonstrates that negative modulation of mGlu2/3 receptors results in enhanced performance of hippocampal-dependent cognitive tasks in rodents without nonspecific behavioural effects. Furthermore, there is evidence that this class of compounds improves performance of a working memory task in non-human primates. [1] Woltering, T. J, et al. Bioorg. Med. Chem. Lett., 2010, 20(23), 6969. [2] Higgins, G.A, et al. Neuropharmacology, 2004, 26, 907. [3] Richards, J.G. J. Neurosci., 2003, 23, 8989.

8.An Assessment of Brain Metabotropic Glutamate Receptor 5 (mGluR5) Occupancy for STX107 in Nonhuman Primates O. Barret, G. Tamagnan, R. Rush‡, D. Lanzetta‡, N. Uddin‡ and J. Seibyl

Current Neuropharmacology, 2011, Vol. 9, Suppl. 1

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‡

Seaside Therapeutics, Inc., 840 Memorial Drive, Cambridge, MA 02139, USA and Molecular NeuroImaging, LLC, 60 Temple Street, Suite 8A, New Haven, CT 06510, USA

Preclinical and early clinical evaluation of metabotropic glutamate receptor 5 (mGluR5) occupancy and displacement by candidate drugs is an important part of in vivo characterization during the clinical development of potential new treatments for neurological disorders including autism and other neurodevelopmental disorders, neurodegenerative diseases, pain, and anxiety. The aim of this study was to use positron emission tomography (PET) with [18F]fluoropyridineethynylbenzonitrile ([18F]-FPEB) to interrogate brain mGluR5 receptor occupancy of the proprietary mGluR5 antagonist STX107 in the non-human primate (Macaca mulatta). [18F]-FPEB1 is a specific high affinity ligand that binds to the allosteric modulator site on the mGluR5 receptor. STX1071 is a small molecule, negative allosteric modulator of the mGluR5 receptor which is under development at Seaside Therapeutics for the treatment of Fragile X Syndrome and autism. In an open label, blockade protocol, dose occupancy curves and pharmacokinetic measurements were acquired in non-human primates over a 3 hour post-dose period for four intravenous dose levels of STX107 (0.08, 0.25, 0.8 & 2.5 mg/kg; n=2/dose). Occupancy across several brain regions was assessed by comparison of the regional volume of distribution measured at baseline and following STX107 administration. A strong dose response relationship with mGluR5 occupancy ranging from 29 to 89% was established. The dose of STX107 that provided 50% occupancy at the mGluR5 receptor was calculated to be 0.21 mg/kg. This dose was associated with a Cmax and C120 total plasma concentration of 1.4 and 0.14 M respectively. These results illustrate the brain penetration and mGluR5 modulation potential of STX107 and support its evaluation for the treatment of Fragile X Syndrome and autism. 1[18F]-FPEB and STX107 were discovered by Merck Research Laboratories (MRL); we gratefully acknowledge the support and helpful discussions of MRL in this work.

9.Group-II mGluRs as Drug Targets in the Treatment of Amyotrophic Lateral Sclerosis Giuseppe Battaglia1, Barbara Riozzi1, Gemma Molinaro1, Domenico Bucci1, Marianna Storto1, Simone Pallottino1,2, Ferdinando Nicoletti1 and Valeria Bruno1,2 1

I.N.M. Neuromed, Pozzilli, Italy; 2Department of Physiology and Pharmacology, University “Sapienza”, Rome

Amyotrophic Lateral Sclerosis (ALS), a chronic neurodegenerative disorder characterized by a progressive degeneration of motor neurons in the cerebral cortex and ventral horns of the spinal cord, is uncurable at present. A growing body of evidence suggests that glial cell line-derived neurotrophic factor (GDNF) has survivalpromoting and restorative effects in neurons, and attenuates neurodegeneration in mouse models of ALS. We have demonstrated that activation of mGlu3 receptors mediates neuroprotection through the production of neurotrophic factors, such asGDNF itself and transforming growth factor- (TGF-), which is required for the neurotrophic action of GDNF in a variety of models. Here we show that in primary cultures of mouse spinal cord cells exposed to kainic acid (25 M for 24 hours), application of the orthosteric mGlu2/3 receptor agonist, LY379268 (10 M), significantly reduced the death of motor neurons, as assessed by immunocytochemistry for SMI-32. LY379268 also increased GDNF levels in cultures of spinal cord cells. We then treated C57Black mice with LY379268 (0.5, 1 and 5 mg/kg per day) by


means of subcutaneously implanted osmotic minipumps releasing the drug for 28 days. This treatment led to increases in GDNF levels in the cerebral cortex and spinal cord. Double immunohistochemical showed that astrocytes were the cellular source of the increase in GDNF. Increases in GDNF levels were also seen in G93A ALS mutant mice, which over-express a mutated form of superoxide dismutase-1 (SOD1). Additional groups of G93A mice were treated with LY379268 (0.5, 1 and 5 mg/kg/day) until death. We examined neurological signs and motor behavior by the rotarod test at different times. Treatment with LY379268 induced a substantial improvement of motor behavior and neurological score at the doses of 1 and 5 mg/kg/day. No effect was observed in the survival rate. We conclude that mGlu2/3 receptor agonists may be beneficial in the treatment of ALS.

10.Metabotropic Glutamate Receptors and Autism: Insights from Animal Models of Syndromic Disorders Mark F. Bear, Benjamin D. Auerbach and Emily K. Osterweil HHMI, The Picower Institute for Learning and Memory, Department of Brain and Cognitive Sciences, MIT, Cambridge, MA 02139 USA Fragile X syndrome (FXS) and tuberous sclerosis complex (TSC) are single-gene disorders of brain development that often manifest, in part, as autism and intellectual disability. Genetically engineered mouse models permit detailed investigation of the synaptic pathophysiology of these disorders. Previously we discovered excessive synaptic protein synthesis downstream of activation of mGluR5 in the Fmr1 KO mouse model of FXS. One way this excess is observed functionally is exaggerated mGluR-dependent long-term depression (LTD) in the hippocampus. The “mGluR theory” of fragile X proposes that multiple disease phenotypes emerge as a consequence of hypersensitivity to mGluR5 activation, and predicts amelioration of FXS by inhibitors of mGluR5 signaling. These predictions have been confirmed in numerous animal studies and preliminary human studies appear promising. The intracellular signaling network that couples mGluR5 to local synaptic mRNA translation remains to be fully elucidated, but it has been proposed to include the Akt-mTOR pathway which is negatively regulated by the TSC1/2 complex. Heterozygous loss of function of either the TSC1 or TSC2 genes is the cause of tuberous sclerosis complex, and excessive mTOR signaling is believed to be pathogenic in this disease. We used the Tsc2+/- mouse to test the hypothesis that one consequence of excessive mTOR activity is increased mGluR5-dependent protein synthesis and LTD. Contrary to our hypothesis, however, we find impaired protein synthesis and LTD in the Tsc2+/- mice, which can be rescued by a positive allosteric modulator (PAM) of mGluR5. Moreover, treatment of the mutant mice with the PAM reversed a deficit in context discrimination learning, comparable to what has been observed using mTOR inhibitors. These results suggest that normal synaptic function occurs within a defined range of mGluR5-dependent protein synthesis regulation, and that deviations in either direction can manifest as autism and intellectual disability.

11.Synergy between L-DOPA and a Novel Positive Allosteric Modulator of Metabotropic Glutamate Receptor 4: Implications for Parkinson's Disease Treatment K.-E. Bennouar1, M.A. Uberti2, M.D. Bacolod2, M. Cajina2, H.N. Jimenez2, C. Melon1, D. Doller2, L. Kerkerian-Le Goff1 and P. Gubellini1

Conference Proceedings 1

Institut de Biologie du Développement de Marseille-Luminy (IBDML), UMR6216 (CNRS/Aix-Marseille Université), Marseille, France; 2 Lundbeck Research USA, Paramus, NJ, USA

The research of symptomatic treatments for Parkinson's disease (PD) has led to identify group III metabotropic glutamate (mGlu) receptors as possible targets to modulate synapses becoming overactive due to dopamine loss into the basal ganglia [1]. We have recently shown that mGlu4 receptor agonists are able to reduce akinesia when injected in striatum [2] or globus pallidus [3] in rat PD models. Here we present a novel mGlu4 receptor positive allosteric modulator (PAM) efficiently passing the blood-brain barrier (Lu AF21934). In vitro electrophysiological recordings show that AF21934 inhibits dose-dependently corticostriatal synaptic transmission and enhances the inhibitory effect of the orthosteric mGlu4 receptor agonist LSP1-2111 [3]. When injected intraperitoneally in hemiparkinsonian rats (6-hydroxydopamine lesion of the substantia nigra pars compacta), AF21934 (10 and 30 mg/kg) had no effect per se on akinesia assessed by the cylinder test. On the other hand, AF21934 acted in synergy with L-DOPA when the two compounds were co-injected: in particular, this combined treatment significantly improved akinesia with doses of L-DOPA that were ineffective alone (1 and 5 mg/kg). Interestingly, this provided the bonus of significantly reducing the number of rats developing L-DOPA-induced dyskinesia (LID). However, at LDOPA doses systematically inducing LID (25 mg/kg), AF21934 did not further affect this motor side-effect. These data suggest that a combined therapy with L-DOPA and an mGlu4 receptor PAM might allow reducing L-DOPA doses while maintaining the same benefit on PD motor troubles, and at the same time minimizing the induction of LID. [1] Duty, S. Br. J. Pharmacol., 2010, 61, 271-287. [2] Cuomo, D, et al. J. Neurochem., 2009, 109, 1096-1105. [3] Beurrier, C, et al. FASEB J, 2009, 23, 3619-3628.

12.Ceftriaxone Attenuates post-Surgical Human Pain and Acts Synergistically with mGlu5 Receptor Blockade in Causing Analgesia in Mice Matteo Bernabucci1,2, Angela Macaluso3, Ferdinando Nicoletti4,1 and Saul Collini3 1

I.N.M. Neuromed, Pozzilli; 2Dept. of Neuroscience University of Rome Sapienza; 3Department of Anaesthesiology and Intensive Care,

University of Rome 'La Sapienza', Faculty of Medicine and Psychology, 4 Sant'Andrea Hospital, Rome, Italy; Dept. of Physiology and Pharma-

cology University of Rome Sapienza The glial glutamate transporter, GLT-1 (EAAT2), has recently been considered as a target in the treatment of chronic pain, and, interestingly, the antibiotic ceftriaxone causes analgesia by enhancing GLT-1 expression in spinal cord astrocytes [1] GLT-1 limits the amount of glutamate that spreads to the sides of the synapses, thus restraining the activation of mGlu5 receptors in the peripheral portion of the dendritic spines. Knowing that mGlu5 receptor antagonists cause analgesia, we decided to examine whether ceftriaxone and mGlu5 receptor antagonists could act synergistically in relieving inflammatory pain. As a model, we used mice unilaterally injected with formalin in the hindpaw. In the formalin test, nocifensive behaviour shows an early phase, which reflects the stimulation of peripheral nociceptors, and a second phase, that instead reflects the development of central sensitization in the dorsal horns of the spinal cord. Both ceftriaxone (200 mg/kg, i.p.) and the mGlu5 receptor antagonists MPEP (30 mg/kg, i.p.) reduced nocifensive behaviour in the second phase of the formalin test. The combination between ceftriaxone and mGlu5 receptor antagonists produced a remarkable analgesic effect, which was


greater than that predicted if the effect of two drugs was additive. Cefazoline, an antibiotic structurally related to ceftriaxone that fails to induce GLT-1, did not cause analgesia and showed no influence on the analgesic effect of MPEP. We tested for the first time the analgesic action of ceftriaxone on postsurgical pain in humans. Forty-three patients with carpal tunnel syndrome underwent surgical release of the carpal tunnel ligament. Two additional patients underwent surgery for ulnar neuropathy. Two groups of fifteen patients received i.m. injection of ceftriaxone (2 g) and cefazoline (1 g), respectively. An additional group of patients served as control. Mechanical allodynia was assessed with the Von Frey filaments 6 hours after drug injection. Allodynia was significantly reduced in the group of patients receiving ceftriaxone as compared to control patients or to patients receiving cefazoline. We speculate that a combination between ceftriaxone and mGlu5 receptor antagonists might be beneficial in the treatment of chronic pain in humans. [1] Ramos, et al., Neuroscience, 2010, 169.

13.Beyond GABAB Receptor Heteromers: Insights Into the Role of Auxiliary Subunits Bernhard Bettler Department of Biomedicine, Institute of Physiology, University of Basel, Klingelberstr. 50/70, CH-4056 Basel, Switzerland Molecular cloning revealed that functional GABAB receptors are formed by the heteromeric assembly of GABAB1 with GABAB2 subunits. However, cloned GABAB(1,2) receptors failed to reproduce the functional diversity observed with native GABAB receptors. Using functional proteomics we recently could demonstrate that GABAB receptors in the brain are high molecular weight complexes of GABAB1, GABAB2 and members of a subfamily of the 'potassium channel tetramerization domain-containing' (KCTD) proteins [1] Co-assembly with KCTD proteins changes kinetic and pharmacological properties of the GABAB(1,2) core receptor in a KCTD subtype-specific manner. I will provide an update of the effects of the KCTD proteins on recombinant and native GABAB responses. Work in my laboratory is supported by grants of the Swiss Science Foundation (31003A-133124, Sinergia CRSII3_136210/1), the National Center of Competences in Research (NCCR) “Synapsy, Synaptic Bases of Mental Diseases” and the European Community’s Seventh Framework Program (FP7/2007-2013) under Grant Agreement 201714. [1] Schwenk, et al., 2010, Nature, 465, 231-235.

14.mGluR1 Negatively Signaling in Melanoma

Regulates

Wnt/-Catenin

Travis L. Biechele1,2, Nathan D. Camp1,2, Daniel M. Fass3,4, Rima M. Kulikauskas2,5, Nick C. Robin1,2, Bryan D. White1,2, Corinne M. Taraska1,5, Erin C. Moore1,5, Jeanot Muster1,2, Rakesh Karmacharya3,6, Stephen J. Haggarty3,4, Andy J. Chien2,5 and Randall T. Moon1,2 1 Howard Hughes Medical Institute, Department of Pharmacology, and Institute for Stem Cell and Regenerative Medicine, University of Washington School of Medicine, Box 357370, Seattle, WA 98195, USA; 2Institute for Stem Cell and Regenerative Medicine, University of Washington School of Medicine, Box 357370, Seattle, WA 98195, USA; 3The Broad Institute of Harvard University and Massachusetts Institute of Technology, 7 Cambridge St., Cambridge, MA 02141, USA; 4Current address: Center for Human Genetic Research, Mass General Hospital, 185 Cambridge St., Boston, MA 02114 USA; 5Department of Medicine, Division of


5

Dermatology University of Washington, Seattle, Washington 98195-7280; 6Schizophrenia and Bipolar Disorder Program, McLean Hospital, Harvard Medical School, 115 Mill Street, Belmont, MA 02478 To identify new protein and pharmacological regulators of Wnt/ßcatenin signaling we used a cell-based reporter assay to screen a collection of 1857 human-experienced compounds for their ability to enhance activation of the ß-catenin reporter by a low concentration of WNT3A. This identified 44 unique compounds, including the FDA-approved drug riluzole, which is presently in clinical trials for treating melanoma. We found that treating melanoma cells with riluzole in vitro enhances the ability of WNT3A to regulate gene expression, to promote pigmentation, and to decrease cell proliferation. Furthermore riluzole, like WNT3A, decreases metastases in a mouse melanoma model. Interestingly, siRNAs targeting the metabotropic glutamate receptor, GRM1, a reported indirect target of riluzole, enhance ß-catenin signaling. The unexpected regulation of ß-catenin signaling by both riluzole and GRM1 has implications for the future uses of this drug.

15. In vitro and in vivo Characterization of two mGlu2/3 Negative Allosteric Modulators I. Biesmansa, A. Megensa, J.A. Bouwknechta, R. Woutersb, X. Langloisa, P. Te Rielea, A.A. Trabancoc, C.M. Wassvikc, J.R. Atacka and H. Lavreysena a

Dept. Neuroscience a and C.R.E.A.Te b, Janssen Research & Development, A Division of Janssen Pharmaceutica NV, Turnhoutseweg 30, 2340 Beerse, Belgium; Neuroscience Medicinal Chemistry c, Janssen Research & Development, A Division of Janssen Cilag S. A., c/ Jarama 75-45007 Toledo, Spain Changing glutamate activity by modulators of Group II metabotropic (i.e., mGlu2 and mGlu3) receptors is believed to have potential for the treatment of neuropsychiatric disorders. In the present work, we describe the in vitro and in vivo properties of two structurally-related mGlu2/3 negative allosteric modulators (NAMs): RO4491533 and an analogue JNJ-42112265. Both compounds antagonized the glutamate-induced modulation of [35S]GTPS binding and induced a rightward shift of the glutamate concentration-response curve in membranes from cells expressing recombinant human and rat mGlu2 receptors (IC50 in both species: 1-3 nM). Both compounds showed activity at human mGlu3 receptors but were selective over other mGlu receptor subtypes. Consistent with in vitro binding data, both compounds occupied the rat mGlu2 receptor in an ex vivo occupancy study using a tritiated mGlu2 positive allosteric modulator with an ED50 of 2.3 mg/kg (p.o.) for RO4491533 and 3.8 mg/kg (s.c.) for JNJ-42112265. Using a variety of other models, we confirmed in vivo mGlu2 target engagement. For example, both NAMs were able to antagonize the mGlu2/3 agonist LY404039 (10 mg/kg s.c.) -induced blockade of conditioned avoidance responding in rats (ED50: 8.8 mg/kg s.c. for JNJ-42112265 and 8.7 mg/kg p.o. for RO4491533, corresponding to approximately 75% receptor occupancy). Similarly, the increase in c-fos expression in the parietal cortex following LY404039 treatment in rats (10 mg/kg s.c.) was abolished when pretreated with RO4491533 and JNJ-42112265. While both compounds did not affect spontaneous locomotion, they decreased immobility time in the tail suspension test, which suggests a potential antidepression-like effect.

16.Characterisation of mGlu5 PAMs in Behavioural Assays for Schizophrenia, Anxiety, and Cognition B. Biemans, L. Aubert, C. Fischer, C. Diener, R. Gasser, W. Spooren, G. Jaeschke and L. Lindemann


CNS Discovery, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, CH-4070 Basel, Switzerland The group I metabotropic glutamate receptor subtype 5 (mGlu5) is under investigation as target for neuropsychiatric illnesses. Drug development efforts focus on allosteric binding sites located in the transmembrane domain of the mGlu5 receptor. Negative allosteric modulators of mGlu5 with demonstrated preclinical anxiolytic and antidepressant activity are under clinical investigation for several indications, including depression and Fragile X syndrome. Positive allosteric modulation of mGlu5 on the other hand may offer a novel approach for improving positive and negative symptoms of schizophrenia, as well as cognitive impairments. Positive allosteric modulators (PAMs) can enhance the endogenous glutamatetriggered receptor activity while avoiding the risk of receptor desensitisation described for agonists. Three selective mGlu5 PAMs, i.e. CDPPB, DFB, and ADX47273 (ADX) were characterised in vitro and in vivo. In vitro, these compounds potentiate human mGlu5 receptor activation by glutamate with submicromolar potencies (ADX > CDPPB > DFB), and bind mGlu5 with micromolar potency in a [3H]-MPEP binding assay. In vivo, compounds were examined in the rat for their ability to reverse mGlu5 NAM–induced anxiolytic effects in the Vogel conflict drinking test. Furthermore, their antipsychotic and pro-cognitive potential was assessed in the rat amphetamine-induced hyperactivity paradigm and in the scopolamine-disrupted spontaneous alternation test in mice, respectively. Consistent with the low potency in the [3H]-MPEP binding assay, all three compounds did not reverse the mGlu5 NAM-induced increase in drinking time in the Vogel test. ADX was not active in the amphetamine-induced hyperactivity test but reversed the scopolamine-induced deficit in working memory at 1 mg/kg. The in vitro and in vivo properties of the three mGlu5 PAMs will be discussed.

17.Drug Metabolism and Disposition of a Novel N-4(2,5-dioxopyrrolidin-1-yl)phenylpicolinamide Series of Positive Allosteric Modulators of Metabotropic Glutamate Receptor 4: Identification of ML182 as an Orally Efficacious mGluR4-PAM Anna L. Blobaum†‡, Ryan D. Morrison†‡, Satyawan Jadhav†‡, Darren W. Engers‡, Stacey R. Lindsley‡, Ya Zhou‡, Rocco D. Gogliotti‡, Carrie K. Jones‡, Colleen M. Niswender‡, P. Jeffrey Conn‡, Craig W. Lindsley‡, Corey R. Hopkins‡ and J. Scott Daniels.†‡ †

Drug Metabolism & Pharmacokinetics, ‡Vanderbilt Center for Neuroscience Drug Discovery, Department of Pharmacology, Vanderbilt Specialized Chemistry Center for Accelerated Probe Development (MLPCN), Vanderbilt University Medical Center, Nashville, Tennessee, 37232 A chorus of pharmacology data has established metabotropic glutamate receptor 4 (mGluR4) as a viable target for therapeutic intervention in Parkinson’s disease (PD), specifically through the activation of this receptor with small molecule positive allosteric modulators (PAMs). Towards the identification of PAMs bearing drug metabolism & pharmacokinetic (DMPK) attributes of an orally active CNS therapeutic, our group implemented an approach utilizing predictive high throughput (HT) in vitro tools (metabolic stability, protein binding and P450 inhibition) and in vivo rodent PK to establish the structure-activity relationships (SAR) influencing the bioavailability, disposition and pharmacodynamics (PD) of small molecule mGluR4 PAMs. Our initial appraisal of a new mGluR4 selective scaffold led to ML128 (1), a compound possessing excellent CNS exposure but poor metabolic stability and P450 inhibition liabilities. In an attempt to improve upon


the properties of ML128, a series of N-(4-(1,1,3,3tetraoxidobenzo[d][1,3,2]dithiazol-yl) phenyl)carboxamides (e.g. 2) was screened and demonstrated an improved P450 inhibition profile (>30μM), predicted hepatic clearance (3 mL/min/kg in rat), and plasma protein binding (fu, 0.04). However, in vivo PK analysis indicated a scaffold that possessed poor CNS permeability (brain:plasma ratio, B:P, < 0.1). To enable an iterative medicinal chemistry effort around 2, we employed HT in vitro stability screens and a single dose-time point rodent PK design to identify the resulting N-4-(2,5-dioxopyrrolidin-1-yl)-phenylpicolinamide series (e.g., 3, ML182) with marked improvements in DMPK properties. Although microsomal predicted hepatic clearance was moderate-high (50 mL/min/kg), ML182 demonstrated other favorable attributes, such as improved protein binding (plasma and brain fu > 0.03), and safety characteristics (>30μM IC50, P450 enzymes). Coupled with an attractive in vivo PK and tissue distribution profile (CLP = 37 mL/min/kg; %F = 80; B:P = 1), a limited in vivo P450-interaction potential and efficacy in acute models of PD, the lead phenyl-substituted picolinamide, 3, represents a successful outcome of rational drug design and a means to assess Parkinson’s disease-modification via mGluR4 activation. This work was supported by the National Institutes of Health, the Michael J. Fox Foundation for Parkinson’s Research (MJFF) and the Molecular Libraries Probe Production Centers Network (MLPCN).

18.Blockade of in vivo Accumulation of [3H]ABP688 by mGluR5 NAMs: MPEP and MTEP, Relationship with Anxiolytic Activity A. Bobok, B. Kiss, K. Sághy, I. Gyertyán and K. Gál Gedeon Richter Plc.; H-1103 Budapest, Gyömri út 19-2 Hungary [3H]ABP688 is a brain-penetrable high affinity radioligand selective for the allosteric site of metabotropic glutamate 5 receptors (mGluR5). The aim of this study was to compare the in vitro binding affinities of MPEP and MTEP to their in vivo blocking effect on the cerebral accumulation of [3H]ABP688. Furthermore, we wished to explore the relationship between blockade of [3H]ABP688 accumulation by MTEP and MPEP and their in vivo anxiolytic effect established in the Vogel assay. In vitro MPEP and MTEP concentration-dependently displaced [3H]ABP688 binding using rat cortical membrane preparation with Ki values of 11.0 and 17.5 nM, respectively. For the determination of in vivo accumulation of [3H]ABP688, the radioligand was administered to restrained male Wistar rats intravenously through tail vein for baseline determination. Thirty minutes after [3H]ABP688 administration (5 Ci/animal) the highest accumulation of radioactivity was found in the striatum followed by hippocampus, cortical areas, olfactory tubercle and the amygdala. The cerebellum exhibited the lowest accumulation in line with the known low level of expression of mGluR5 in this brain region. In ”blocking” experiments, both MPEP (3-10-30 mg/kg,) and MTEP (1-3-10-30 mg/kg,) were administered orally 30 min before iv. injection of [3H]ABP688. MTEP and MPEP caused a dosedependent reduction in the radioactivity in the mGluR5-rich brain regions. The inhibition of [3H]ABP688 accumulation by MPEP and MTEP was significant at anxiolytic doses determined in the Vogel assay (MPEP MED= 3 mg/kg, MTEP MED= 10 mg/kg).


19.Targeting Group I Metabotropic Receptors in Amyotrophic Lateral Sclerosis

Glutamate


20.Searching for a New Potential mGluR4 Positive Allosteric Modulators

T. Bonifacino1, F.Giribaldi1, A Puliti2, I. Musante2, M. Melone3, M. Milanese1, P. Rossi3, F. Conti3, A. Pittaluga1 and G. Bonanno1

P. Braski1, A. Bojarski2, G. Burnat1, P. Chorobik1, R. Bugno2, R. Kurczab2, J. Staro2, B. Chrucicka1, A. Palucha-Poniewiera1 and A. Pilc1

1

1

Dept. of Experimental Medicine and Centre of Excellence for Biomedical Research, Univ. of Genova, Italy; 2Dept. of Pediatric Sciences, Univ. of Genova, and Mol. Genetics Unit, Gaslini Inst. Genova, Italy; 3Dept. of Neuroscience, Univ. of Marche, Ancona, Italy Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease characterized by muscle wasting, weakness and spasticity, reflecting a degeneration of upper and lower motor-neurons (MNs). The mechanisms of neuronal death in ALS are still largely obscure, thus impairing the establishment of efficacious therapies. Glutamate(Glu)-mediated excitotoxicity plays a major role in the degeneration of MNs and reduced astrocytary transport, which in turn increases the synaptic availability of Glu, was suggested as a cause. On the basis of our studies, we proposed that abnormal release of Glu may well be another source of excessive Glu [1-3]. Recently, mGlu1 and mGlu5 Glu-releasing autoreceptors were described in rat cerebral cortex nerve terminals, whose activation produced potentiation of Glu release [4]. In the present work we investigated on the presence of similar autoreceptors in the spinal cord of SOD1/G93A mice, a widely used animal model of human ALS. Exposure of spinal cord synaptosomes to increasing concentrations of the mGluR1/5 agonist 3,5-DHPG produced distinct effects in SOD1/G93A mice and controls: concentration above 0.3 μM stimulated the basal release of [3H]D-Aspartate, used to label the endogenous pools of Glu, both in control and SOD1/G93A mice. At variance, concentrations of 3,5-DHPG equal to or lower than 0.31 μM increased [3H]D-Asp release in SOD1/G93A mice, only. Experiments with selective mGluR1 or mGluR5 antagonists indicated that the both high and low affinity effects of 3,5-DHPG involved mGluR1 and mGluR5 activation. Low 3,5-DHPG concentrations induced increase of IP3 in SOD1/G93A but not in control mice; whereas, high 3,5-DHPG induced IP3 formation in both mouse strains. Release experiments confirmed that 3,5-DHPG produced exocytotic release of [3H]DAspartate, involving intra-terminal Ca2+ release through IP3sensitive channels. Protein and mRNA determination pointed towards a more elevated expression of mGluR5 in SOD1/G93A mice. We generated mice carrying half expression of mGlu1 receptors in the SOD1/G93A background, by crossing ALS mutant mice with Grm1+/crv4 mouse, lacking mGluR1 because of a spontaneous recessive mutation [5], thus providing a genetic tool to evaluate the role of mGluR1 in ALS. Double mutants showed prolonged survival probability respect to single mutant SOD1/G93A mice. Accordingly, slower disease progression and improved motor performances were observed. Histological studies showed higher number of ChAT-positive MNs in spinal cord, reduced axonal degeneration and mitochondrial damage, reduced astrocyte and microglia activation in double mutant compared to SOD1/G93A mice. Our results demonstrate the existence of an abnormal mGluR1/5-mediated increase of Glu release in SOD1/G93A mice, which may represent a cause of excessive Glu and of neurodegeneration. mGluR1 deletion has a significant impact in-vivo on survival and motor performances of SOD1/G93A mice, thus supporting this assumption. [1] Raiteri, et al. Neuropharmacology, 2004. [2] Milanese, et al. J. Neurochem., 2010. [3] Milanese, et al. J. Neurochem., 2011. [4] Musante, et al. Neuropharmacology, 2008. [5] Conti, et al. Int. J. Mol. Med., 2006, 18 (4), 593-600. Supported by MURST (PRIN project 2006058401).

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Department of Neurobiology,Institute of Pharmacology PAS, 12 Smtna St. Kraków, Poland; 2Department of Medicinal Chemistry, Institute of Pharmacology PAS, 12 Smtna St. Kraków, Poland Background: Glutamate, the most abundant excitatory neurotransmitter in the brain, regulates neuronal firing via ionotropic and eight subtypes of metabotropic glutamate receptors (mGluR). Among mGluRs, mGlu4 primarily found presynaptically in several brain areas andfunction as an autoreceptors or heteroreceptors: mGlu4 receptor activation inhibits GABA and glutamate neurotransmission. Intervention in glutamatergic neurotransmission through mGlu4 receptor has been pursued intensively for the treatment of vast number of neurological and psychiatric disorders such as anxiety, schizophrenia, epilepsy Parkinson disease, and addiction. Aim: Identification novel chemical scaffold possessing mGlu4 positive allosteric modulation activityby interaction with transmembrane region of mGlu4 receptor. Methods: GRM4 was cloned into genome of HEK-293 cells contained T-Rex expression system (Invitrogen). Expression of the receptor was analyzed by means of qRT-PCR and Western blot in both: cells before and after stable transfection with GRM4. For the functional characterization of mGlu4 was used cAMP accumulation assay. The screening study and activity of potential PAM was determined using forskolin-induced cAMP accumulation, in a HEK-293 T-Rex cell line stably expressing mGlu4 or mock transfected HEK-293 T-Rex cell line. Cells were labeled [3H] adenine. [3H] cAMP was then purified by sequential chromatography over Dowex resin and aluminum oxide columns. Activity of potential PAM was determined using [35S]-GTPS assay, too. Results: HEK-293 T-Rex cell line stably expressing mGlu4 has been characterized. Endogenous mGlu4orthosteric agonists LGlutamic acid (L-Glu) decreased the forskolin-induced cAMP production in cells, with EC50 value of 10,2 μM. Non selective mGluR4 orthosteric agonists L-AP4, L-SOP, ACPT-I and DCPG decreased the forskolin-induced cAMP production in cells, with EC50 values of 1,3 μM, 1,2 μM, 3,3 μM and 4,1 μM, respectively. We developed chemo–informatics tools and screened in silico compound collections from external source. We designed and synthetized compound collection, too. Via in vitro screening of compound collection we have identified chemical scaffolds possessing mGluR4 potential PAM activity. Twelve active compounds have been identified. Active compounds: (i) induce a leftward-shift of the glutamate concentration-response curve. (ii) None of compounds do interact with mGluR8 receptor up to 30 μM. One of identified compounds it is not an mGluR4positive allosteric modulator. This compound inhibit adenylyl cyclase activity and mimic mGluR4 PAM activity. Conclusions: We have now identified, and characterized new potential mGluR4 PAMs. This study is supported by project UDA-POIG.01.03.010-12-100/08-00 co-financed by European Union from the European Fund of Regional Development (EFRD) http://modall.pl.

21.Chemogenomic Discovery of the First Selective Allosteric Antagonists at the GPRC6A Receptor H. Bräuner-Osborne, V.K. Bhatia, B. Christiansen, C. Clemmensen, K.B. Hansen, D.E. Gloriam, L.D. Johansen, D.S. Pedersen, S. Smajilovic and P. Wellendorph Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Fruebjergvej 3, 2100 Copenhagen, Denmark. * E-mail: [email protected]


In 2004 we reported the cloning and tissue expression of a novel human family C G protein-coupled receptor, termed GPRC6A [1]. Measurement of Ca2+-dependent chloride currents in Xenopus laevis oocytes facilitated the deorphanization of GPRC6A and identification of L-alpha-amino acids as agonists. The most active agonists were basic L-alpha-amino acids, L-Arg, L-Lys and Lornithine, suggesting that these may function as endogenous signaling molecules [2]. Cloning, cell surface expression and deorphanization of the mouse and rat orthologues further reinforces the assignment of the agonist preferences of human GPRC6A [3]. We have developed efficient assays based on measurement of intracellular calcium and inositol phosphate levels in mammalian cells, which have been used to characterize a range of commercially available L-Arg and L-Lys analogs and allosteric modulation by divalent cations [4]. In order to study the physiological function of the receptor we have developed a GPRC6A knock-out mouse, which has so far been used to show that the GPRC6A receptor does not have a function in bone under normal physiological conditions [5]. Most recently, we have also developed a GPRC6A specific antagonist using a chemogenomic approach, which will likewise be used to elucidate the function of the receptor. Here we will present this compound series and mutational analyses of the binding site which has been shown that the compounds bind in the 7TM domain. [1] Wellendorph, and Bräuner-Osborne. Gene, 2004,335,37-46. [2] Wellendorph , et al. Mol. Pharmacol., 2005, 67,589-597. [3] Wellendorph, et al. Gene, 2007, 396, 257-267. [4] Christiansen, et al. Br. J. Pharmacol., 150, 798-807. [5] Wellendorph, et al. J. Mol. Endocrinol., 2009, 42, 215-223.

22.Novel Metabotropic Glutamate Receptor 4 (mGluR4) Allosteric Potentiators for the Treatment of Parkinson’s Disease and Anxiety Brice Campo Addex Pharmaceuticals SA 12 Chemin des Aulx, CH-1228, Plan Les Ouates Geneva, Switzerland. E-mail: [email protected] Parkinson's Disease (PD) is characterized by depletion of dopamine in Substantia Nigra neurons which, in turn, causes hyperactivity in GABAergic and glutamatergic transmission, which contribute to the movement disorders associated with PD. Metabotropic glutamate receptor type 4 (mGluR4) is a Family C GPCR, that is primarily found presynaptically in basal ganglia circuits that control movement. Data suggest mGluR4 activation inhibits GABA and glutamate neurotransmission at these synapses. Thus, drugs that activate mGluR4 may normalize neurotransmission in the basal ganglia thereby treating movement disorders in PD. So far, the exploration of this hypothesis has been challenging due to the lack of subtype selective mGluR4 molecules with drug-like characteristics. Allosteric modulators have shown greater selectivity and better modulatory control compared to orthosteric agonists and/or antagonists of various receptors, including most mGluRs. The first mGluR4 positive allosteric modulator (PAM), (-)-PHCCC, was described by Marino et al. in 2003 and it was reported to improve motor fluctuation in rodent models of PD [1]. In 2007, Merck & Co., Inc. entered into an exclusive research collaboration and license agreement with Addex Pharmaceuticals in order to discover and develop orally available mGluR4 PAM drugs for the treatment of Parkinson's disease and other undisclosed therapeutic indications. The presentation will highlight the discovery of a novel chemical class of mGluR4 PAMs including some structure-activity relationship and in vitro ADME profile. In vitro pharmacological characterization of lead compounds, as well as pharmacokinetic properties [2] and in vivo efficacy in preclinical rodent models of PD and anxiety will be discussed. [1] Marino, et al. Proc. Natl. Acad. Sci. U.S.A., 2003, 100, 13668. (b) Maj, et al. Neuropharmacology, 2003, 45, 895.

Conference Proceedings [2] Campo, B. Allosteric Modulator Drug Discovery Conference, San Diego, Nov. 2010.

23.Early Changes in the Transcript of mGlu4 Receptors in Mice Treated with Haloperidol or 1-Methyl-4-Phenyl1,2,3,6,-Tetrahydropyridine Cannella Milena1, Motolese Marta1, Bucci Domenico1, Molinaro Gemma1, Bruno Valeria1,2, Nicoletti Ferdinando1,2 and Battaglia Giuseppe1 1

I.N.M. Neuromed, Pozzilli, Italy; 2Department of Physiology and Pharmacology, University “Sapienza”, Rome The neostriatum (caudate nucleus and putamen), the primary input region of the basal ganglia, is connected to output nuclei (internal globus pallidus and substantia nigra pars reticulata) by a “direct” inhibitory GABAergic pathway, and by an “indirect” pathway, which includes a striatal GABAergic projection to the external globus pallidus, an additional GABAergic projection from the external globus pallidus to the subthalamic nucleus, and an excitatory glutamatergic projection from the subthalamic nucleus to the output nuclei. Dopamine (DA) released from the nigro-striatal pathway stimulates the direct pathway acting at D1 receptors and inhibits the indirect pathway acting at D2 receptors. The progressive loss of nigro-striatal neurons associated with PD leads to a decreased activity of the direct pathway and an increased activity of the indirect pathway, which lead to inhibition of thalamocortical neurons and motor dysfunction (bradykinesia, rigidity, and resting tremor). It is now believed that during the early “compensated” phase of PD a number of mechanisms reduce the activity of the indirect pathway. These include an increased production of endogenous opioids, which suppress synaptic transmission in the external globus pallidus by activating presynaptic μ opioid receptors. Whether expression of mGlu receptors undergoes plastic modifications in response to dopaminergic denervation or dopamine receptor blockade is unknown. We addressed this issue using mice treated with the parkinsonian toxin, MPTP, or with the DA receptor blockade, haloperidol. We investigated gene expression variation of different genes that are involved in direct or indirect pathway. MPTP was used at a dose (30 mg/kg, i.p., single injection) that caused the death of about 40-50% of nigral dopaminergic neurons. MPTP treatment led to an increased expression of the transcripts of mGlu4 receptors, CB1 receptors, and pre-proenkephalin-A (PPE) in the striatum at 3 days. At 7 days, the three transcripts returned back to control levels, whereas mGlu4 and CB1 (but not PPE) mRNA levels were reduced at 14 days following MPTP injection. A single haloperidol injection (1 mg/kg, i.p.) led to a transient up-regulation of mGlu4 receptor and PPE (but not CB1 receptor) mRNA levels after 1 day. Levels were not different from controls at 3 and 5 days. The transcripts of mGlu5 receptors, A2A adenosine receptors, and the potassium/chloride co-transporter, KCC2, did not change at any time in the striatum of mice treated with MPTP or haloperidol. We conclude that mGlu4 receptors and PPE, which are selective markers of the indirect pathway, show neuroadaptive modifications in response of DA denervation or DA receptor blockade. Supported by a grant from the Era-net NEURON program (Neurodegeneration 2008: mGluRpatho).

24.Discovery and Characterization of Novel Metabotropic Glutamate Receptor 4 (mGluR4) Allosteric Potentiators S. Célanirea, C. Boléaa, S. Brücknera, N. Livertonb, D. Charvina, F. Hessb, S. Polia, B. Bournique a, M. Fonsia, S. Polsky-Fisherb, C. Gibsonb, F. Girarda, S. Browneb,


A. Dilellab, E. Lisb, B. Luob, E. Le Poula, I. Reynoldsb and B. Campoa a

Addex Pharmaceuticals SA, 12 Chemin des Aulx, CH-1228, Plan Les Ouates Geneva, Switzerland ; b Merck Research Laboratories, Merck & Co., Inc. West Point, PA, 19486 Metabotropic glutamate receptor type 4 (mGluR4) is a member of Group III mGluRs of Family C GPCR, that is primarily found presynaptically in several brain areas. Structurally diverse mGluR4 ligands have been identified in the past 10 years, such as LAP-4 and LSP1-2111 (agonists), VU0155041 (allosteric agonist) and the first allosteric modulator (-)-PHCC discovered at Merck&Co, demonstratring in vivo activity in rodent models of Parkinson’s disease, anxiety, epilepsy or neuropathic pain. Allosteric modulators are an emerging class of orally available small molecule therapeutic agents that may offer a competitive advantage over classical orthosteric drugs. This potential stems from their ability to offer greater selectivity and better modulatory control at disease mediating receptors. In 2007, Addex Pharmaceuticals and Merck & Co., Inc. entered into an exclusive research collaboration and license agreement with the goal of developing orally available mGluR4 PAMs for the treatment of Parkinson's disease and other therapeutic indications. Recently, we have reported the in vivo pharmacological activity of a novel proprietary mGluR4 PAM in rodent models of Parkinson’s Disease and anxiety. [1] We reporting here the discovery of this novel chemical class of selective, brain penetrant, orally active mGluR4 PAMs for the treatment CNS disorders. Synthesis, structure-activity/property relationship, in vitro pharmacological characterization as well as pharmacokinetic properties of lead compounds will be described. [1] Reynolds, I.J. At Drug Discovery Summit, Targeting Parkinson’s Disease, Philadelphia, June 2011. [2] Campo, B. At Allosteric Modulator Drug Discovery Conference, San Diego, Nov. 2010.

25.mGluR5 as a Target for the Treatment of L-DOPAInduced Dyskinesia M. Angela Cenci Lund University, Dept. Experimental Medical Science, Basal Ganglia Pathophysiology Unit, BMC F11, 221 84 Lund (Sweden). E-mail: [email protected] L-DOPA-induced dyskinesia in Parkinson´s disease has been linked to altered dopamine and glutamate transmission within the basal ganglia. We have compared compounds targeting specific subtypes of glutamate receptors or calcium channels for their ability to attenuate L-DOPA-induced dyskinesia and the associated changes in striatal nuclear signalling and gene expression in rats with unilateral 6-hydroxydopamine (6-OHDA) lesions [1,2]. Rats were treated with L-DOPA in combination with antagonists of metabotropic glutamate receptor (mGluR) type 5 and type 1 (MTEP and EMQMCM, respectively), one agonist of group II mGluR (LY379268), one NR2B-selective NMDA receptor antagonist (Ro256981), and one antagonist of L-type calcium channels (isradipine). Of all the compounds tested, only MTEP significantly reduced L-DOPA-induced dyskinesia at doses that did not have any negative effect on the animals´ physiological motor performance. At these doses, the compound also inhibited the upregulation of phosphorylated ERK1/2 and prodynorphin mRNA induced by LDOPA in striatal neurons. These effects of MTEP were confirmed in 6-OHDA-lesioned mice. In another study, we evaluated fenobam, a noncompetitive mGluR5 antagonist already tested in humans, using both rodent and nonhuman primate models of LDOPA-induced dyskinesia [3]. In both animal models, acute


9

administration of fenobam significantly attenuated peak-dose LDOPA-induced dyskinesia. Chronic administration of fenobam to previously drug-naïve animals (de novo treatment) attenuated the development of peak-dose dyskinesia without compromising the anti-parkinsonian effect of L-DOPA. Our studies have offered a rationale for the clinical trials that are currently evaluating the antidyskinetic efficacy of mGluR5 antagonist compounds in people with Parkinson´s disease [4]. [1] Mela, F, et al. J. Neurochem., 2007, 101, 483-497. [2]. Rylander, D, et al. J. Pharmacol. Exp. Ther., 2009, 227-235. [3]. Rylander, D, et al. Neurobiol. Dis., 2010, 39, 352-361. [4]. Berg, D, et al. Mov. Disord., 2011, 26, 1243-1250.

26.A Wistar Rat Strain Lacking mGlu2 Receptors: Initial Phenotyping L. Ceolin1, G.R.I Barker1, E.C. Warburton1, Z.A. Bortolotto1, G.L. Collingridge1 and D. Lodge1 1

MRC Centre for Synaptic Plasticity, School of Physiology and Pharmacology, Medical School, University of Bristol, Bristol

The important role of glutamate in brain function is well established [1]. Hyperactivity of glutamatergic transmission found in the cortex, thalamus, striatum, amygdala and hippocampus has been associated with the pathogenesis of anxiety [2]. Therefore, pharmacological manipulations to decrease glutamate activity in these areas were hypothesised to be effective in the treatment of anxiety disorders [3]. The modulatory effects on neuronal excitability, synaptic transmission and synaptic plasticity of the Gprotein coupled mGluRs offer such a possibility [4, 5]. In particular, the brain distribution, the presynaptic localization, their ability to modulate glutamatergic activity and their inhibition of adenyl cyclase and cAMP formation make group II receptors, (mGlu2 and mGlu3), an attractive target for studying in models of psychiatric disorders [5]. We have shown reproducible evidence, using LY395756 (10 M), a mixed mGluR2 agonist (EC50 = 0.397 ± 0.04 M) /mGluR3 antagonist (IC50 = 2.94 ± 0.20 M) [6], of heterogeneous expression of mGluR2. Thus, on the basis of the inhibition induced by this mGluR2 agonist of the entorhinal input into CA1-stratum lacunosum moleculare, rats could be divided into those that were ‘sensitive’ (>65% inhibition of the fEPSP) or ‘insensitive’(4 million structures) to high potency shape-based allosteric modulator pharmacophores developed using SAR from receptor specific, advanced allosteric modulator lead series.

36.Awake Rat Pharmacological MRI as a Translational Pharmacodynamic Biomarker: mGluR2/3 Agonist and PAM Modulation of Ketamine-induced BOLD Signals Mark Day, Chih-Liang Chin, Jaymin Upadhyay, Gerard J. Marek , Scott J. Baker, Min Zhang , Mario Mezler, Gerard B. Fox and Gerard Marek Neuroimaging techniques have been exploited to characterize the effect of N-methyl-d-aspartate (NMDA) receptor antagonists on brain activation in humans and animals. However, most preclinical imaging studies were conducted in anesthetized animals and could be confounded by potential drug-anesthetic interactions as well as

Fig. (1). Flexible alignment of mGlu4 PAMs with SurflexSim to generate shape-based hypotheses for virtual screening. [1] Jain, A.N. J. Med. Chem., 2004, 47, 947-961. [2] Irwin, J.J. and Shoichet, B.K. J. Chem. Inf. Model., 2005, 45(1), 177182. [3] Mueller, R, et al. ACS Chem. Neurosci., 20101, 288-305.

Funding sources for this work include NIH, Michael J. Fox Foundation and Johnson and Johnson Pharmaceuticals. Vanderbilt



is a Specialized Chemistry Center within the Molecular Libraries Probe Centers Network (MLPCN).

Department of Neuroscience, Karolinska Institutet, 171 77 Stockholm, Sweden

38.Addressing the Functional Cross-Talk of mGlu 2 and 5-HT2A by Measurement of Intracellular Signaling Pathways

[1] Marek, et al. J. Pharmacol. Exp. Ther., 2000, 292(1), 76-87. [2] González-Maeso, et al. Nature, 2008, 452(7183), 93-7.

Group I metabotropic glutamate receptors (mGluRs), which include the mGluR type 1 and type 5, are abundantly expressed in the medium spiny neurons (MSNs) of the striatum and are generally regarded as an important target in the treatment of Parkinson’s disease and L-DOPA-induced motor complications. mGluR1 and mGluR5 are known to be primarily coupled to Gaq proteindependent activation of phospholipase C, which leads to increased phosphoinositide turnover and stimulation of protein kinase C (PKC). Previous work showed that activation of group I mGluRs increases the state of phosphorylation of the dopamine- and cAMPregulated phosphoprotein of 32 kDa (DARPP-32), an important mediator of cAMP signaling, highly expressed in striatal MSNs. Here, we have used striatal slices obtained from wild type and transgenic mice to investigate the effects produced by DHPG, a group I mGluR agonist, on the GluR1 subunit of the AMPA glutamate receptor. We found that incubation of striatal slices with DHPG increased the state of phosphorylation of GluR1 at Ser845, a site specifically regulated by cAMP-dependent protein kinase (PKA). In contrast, we did not observe any change in phosphorylation at Ser831, a site regulated by Ca2+/calmodulin protein kinase II and PKC. The effect of DHPG on Ser845 phosphorylation was concentration- and time-dependent. Incubation for 10 min in the presence of 25 M DHPG resulted in a 200% increase in p-Ser845-GluR1. DHPG-induced phosphorylation of Ser845 was prevented by the mGluR5 inhibitor MPEP, but not by the mGluR1 inhibitor, LY367385. Adenosine A2A receptors (A2ARs) and dopamine D1 receptors (D1Rs) are selectively expressed in the MSNs of the striatopallidal and striatonigral pathway, where they act by stimulating cAMP signaling. We found that the effect of DHPG was blocked in the presence of the A2AR antagonist, ZM241385, but not in the presence of the D1R antagonist, SCH23390. These data suggested that the action of DHPG was limited to a specific population of striatal neurons, corresponding to the A2AR-expressing MSNs of the striatopallidal pathway. This idea was supported by the observation that activation of dopamine D2 receptors, which are coupled to inhibition of cAMP signaling in striatopallidal neurons, also abolished the increase in GluR1 phosphorylation produced by DHPG. We also examined the involvement of DARPP-32 in mGluR5-mediated regulation of GluR1. In agreement with previous work, we found that DHPG increased the PKA-dependent phosphorylation of DARPP-32 at Thr34, thereby converting DARPP-32 into an inhibitor of protein phosphatase-1. The effect of DHPG on Ser845 phosphorylation was absent in knock-in mice, in which DARPP-32 had been inactivated by replacing the PKA phosphorylation site (i.e. Thr34) with an Ala. A similar blockade of the effect of DHPG was observed in mice in which DARPP-32 was specifically inactivated in striatopallidal MSNs. In contrast, deletion of DARPP-32 in striatonigral MSNs did not affect DHPG-induced GluR1 phosphorylation. Taken together, these observations indicate that, in the striatum, activation of mGluR5 leads to A2ARdependent activation of cAMP/DARPP-32 signaling and phosphorylation of GluR1 at Ser845. This effect is likely to promote glutamatergic transmission at striatopallidal MSNs and may help to elucidate the antiparkinsonian properties of drugs acting at mGluR5 and their ability to counteract L-DOPA-induced dyskinesia.

39.mGluR5-Mediated Regulation of cAMP/DARPP-32 Signaling and AMPA Receptor Phosphorylation in Striatopallidal Medium Spiny Neurons

40.Orthosteric Group III mGlu Receptor Agonists for Parkinson’s Disease Treatment: Symptomatic and Neurochemical Action on Pallidal Synaptic Transmission

M.T. Dell’Anno, S. Pallottino and G. Fisone

Thierry Deltheil, Nathalie Turle-Lorenzo and Marianne Amalric

H.K. Delille, J. Becker, S. Burkhardt, B. Bleher, L. Unger, G.C. Terstappen, H. Schoemaker, G.J. Marek and M. Mezler Neuroscience Discovery, Global Pharmaceutical Research & Development, Abbott, Knollstraße 50, 67061 Ludwigshafen, Germany Schizophrenia is a complex mental disorder and changes in several neurotransmitter systems or in their interplay seem to be part of the etiology. In this regard, the glutamatergic and serotonergic systems are thought to be altered and both systems are (potential) targets of antipsychotic drugs. Previously, a functional cross-talk/antagonism between the serotonin receptor 5-HT2A and the metabotropic glutamate receptor mGlu2 has been described [1], and a reciprocal influence on receptor binding and signaling has been demonstrated [2]. In addition to the activation of Gq/11 subtype G proteins, hallucinogenic 5-HT2A agonists also activate Gi/o proteins resulting in specific cellular responses like the induction of egr-2. This signaling through Gi/o proteins could be influenced by activation of mGlu2. Furthermore, a physical interaction between mGlu2 and 5HT2A has been demonstrated recently [2]. Here, the functional cross-talk between mGlu2 and 5-HT2A was studied employing an inducible mGlu2 expression system on a stable 5-HT2A background in Hek293 cells and several functional signaling readouts. First, the formation of a 5-HT2A/mGlu2 complex was confirmed using classical FRET and htr-FRET methods. Additionally, dimer formation of mGlu2 with 5-HT2B and mGlu5 but not with 5-HT2C was observed, indicating promiscuity of the mGlu2 receptor with respect to its potential physical interaction partners. Interestingly, we did not observe an effect of 5-HT2A coexpression on the pharmacology of mGlu2, measuring the negative Gi/o-mediated effect on cellular cAMP levels with mGlu2/3-selective agonists, or mGlu2-selective positive allosteric modulators (PAMs). Vice versa, the mobilization of intracellular Ca2+ by 5-HT2A was not fundamentally altered by co-expression of mGlu2, neither with nonhallucinogenic 5-HT2A agonists nor with hallucinogenic drugs like DOI and mescaline. Also, antagonist response for both receptors was not changed by the co-expression of the other receptor in our recombinant expression system and, surprisingly, we could not replicate the activation of Gi/o proteins with the hallucinogenic compounds in this system. In addition, in rat cerebral cortex membrane preparations we did not find an altered response of LY354740 binding in the presence of DOI. In conclusion, our data suggest that mGlu2 and 5-HT2A indeed physically interact and form dimeric or oligomeric receptor forms. However, a direct functional cross-talk of mGlu2 and 5-HT2A can not be observed by measurement of the classical signaling pathways and a fundamental difference between hallucinogenic and non-hallucinogenic substances could not be confirmed assessing calcium or cAMP signaling. While these data do not question the physiological crosstalk of the two receptor types, they do not fully support a biological relevance of the physical receptor dimers.


Lab. Neurobiologie de la Cognition, UMR6155 CNRS Université de Provence, Marseille (France) Developing nondopaminergic palliative treatments for Parkinson’s disease represents a major challenge to avoid the debilitating side effects produced by L-DOPA therapy. This is due to the progressive degeneration of substantia nigra pars compacta dopaminergic neurons. This dopamine loss leads to an imbalance in basal ganglia network activity with a noticeable subthalamic nucleus overactivation. A strong research effort has been made to identify a target whose modulation could lower subthalamic nucleus activity. Metabotropic glutamate receptor 4 (mGlu4) appears to be a relevant target because it is expressed in the basal ganglia as an autoreceptor on glutamatergic and heteroreceptor on GABAergic nerve endings. Indeed, several agonists of this receptor have antiparkinsonian effects in either pharmacological or lesional models of the disease. Nevertheless the mechanisms involved in their antiparkinsonian action have still to be understood. To achieve this goal, we have used intracerebral in vivo microdialysis to monitor glutamate and GABA extracellular concentrations in the globus pallidus (GP) of either sham or unilaterally 6-OHDA lesioned awake rats. GP is the primary output structure of the striatum which receives both GABA striatopallidal and glutamate subthalamopallidal inputs. Consequences on GABA and glutamate extracellular concentrations of both systemic and local (through reverse dialysis) administration of a group III orthosteric mGlu receptor agonist LSP1-2111, were studied. LSP1-2111 (15 mg/kg i.p.) induces a decrease in glutamate and GABA pallidal concentrations in 6-OHDA lesioned animals and no effect in sham animals. To confirm the involvement of pallidal mGluR4 receptors in the previous effect we locally applied LSP1-2111 through reverse dialysis. In sham animals, LSP1-2111 (500 to 1000 M) induces a decrease of glutamate extracellular concentration, whereas it has no clear cut effect on GABA extracellular concentration. In contrast, LSP1-2111 at a dose of 1000 M decreases both GABA and Glutamate extracellular concentrations in 6-OHDA lesioned rats. Antiparkinsonian effects of LSP1-2111 could thus be mediated by the modulation of both GABAergic and glutamatergic activites in the GP. These results point to a critical role of the GP in Parkinsonian conditions and reveal a specific action of mGlu4 receptors in this structure to reduce motor symptoms of the disease. (supported by EranetNeuron, ANR).

41.Reduced Metabotropic Glutamate Receptor 5 Density in Major Depression Determined by [11C]ABP688 Positron Emission Tomography and Postmortem Study


15

site with high specificity in 11 unmedicated subjects with major depression and 11 matched healthy controls; the amount of mGluR5 protein was investigated using Western blot method in brain samples of 15 depressed subjects and 15 matched controls (postmortem study). Results: The PET study revealed decreased regional mGluR5 binding in the prefrontal cortex, the cingulate cortex, the insula, the thalamus and the hippocampus of the depressed individuals (uncorrected p1000-fold selective versus mGluR1) mGluR5 NAM. GRN-529 had dose dependent efficacy across a relevant battery of animal models, addressing both the core and the co-morbid symptoms overrepresented in TRD. Acute administration of GRN-529 was effective in antidepressant (tail suspension, forced swim) anxiolytic (stress induced hyperthermia, four plate, Vogel) and analgesic models (sciatic nerve ligation, inflammatory hyperalgesia). Measurement of receptor occupancy revealed a strong correlation between target engagement, concentration of compound in the brain and efficacy across behavioral endpoints. This relationship supports the translational value of PET imaging to dose selection in patients. Collectively the broad spectrum of efficacy of GRN-529 supports the hypothesis that negative allosteric modulation of mGluR5 could represent an innovative therapeutic approach to the treatment of TRD.

83.Regulation of Recombinantly Expressed mGlu7 Receptors by G-Protein Coupled Receptor Kinases L. Iacovelli1, A. De Blasi2 and F. Nicoletti1,3 1 2

Department of Physiology and Pharmacology “V. Erspamer” and Department of Molecular Medicine, University of Rome


“Sapienza”, P.le Aldo Moro 5, 00185 Roma, Italy; Neuromed, Località Camerelle, Pozzilli, Italy

Conference Proceedings 3

I.N.M.

Group III metabotropic glutamate receptors (mGlu4, mGlu6, mGlu7 and mGlu8 receptors) are potential drug targets for a variety of neurological and psychiatric disorders, including Parkinson’s disease, anxiety, neuroinflammation and pain [1]. Within this family, the mGlu7 receptor shows the highest evolutionary conservation and plays a key role in the regulation of neurotransmitter release [2]. The mGlu7 receptor acts as a presynaptic autoreceptor, inhibiting neurotransmitter release at both glutamatergic and GABAergic terminals. The mGlu7 receptor has a relatively low affinity for glutamate, suggesting that it remains inactive during normal neurotransmission, being activated in the case of excessive glutamate release, to inhibit further release [1]. The role of mGlu7 receptor in the regulation of synaptic plasticity and synaptic transmission has been elucidated by several studies converging on the importance of mGlu7 and PICK1 interaction [3]. Mice and rats in which this interaction is disrupted show a phenotype characteristic of the absence epilepsy. In spite of the increasing knowledge on the pathophysiology of mGlu7 receptors, no data are yet available on the molecular mechanisms that regulate the signalling of this receptor subtype. We have addressed this issue in HEK293 cells transiently transfected with mGlu7 receptor cDNA. In these cells, the group III receptor agonist L-AP4 reduced in a dose-dependent manner forskolin-stimulated cAMP formation. This effect was prevented by pre-incubation with pertussis toxin (10 nM), supporting the coupling of mGlu7 receptor to Gi protein. In cells co-expressing mGlu7 receptor and different G coupled receptor kinases (GRK2 or GRK4 or GRK6), only GRK4 was effective in reducing the inhibition of cAMP formation by L-AP4. The stimulation of mGlu7 receptors by L-AP4 also stimulated MAPK kinase activation and this effect was prevented by pertussis toxin pre-treatment. The stimulation of MAPK pathway by L-AP4 was reduced when the receptor was co-expressed in HEK 293 cells with GRK2, but not with GRK4. -arrestin proteins have many functions in G protein-coupled receptor signalling (GPCR). They participate to the endocytic complex internalizing GPCR, mostly in clatrin coated pits and, on the other hand, -arrestins function as adaptor proteins, ensuring the formation of signalling protein complexes upon GPCR activation [4]. In HEK 293 cells, mGlu7mediated MAPK activation was increased by -arrestin1 coexpression, suggesting its role as adaptor protein in MAPK signalling. We conclude that adenylyl cyclase inhibition and MAPK activation, the two transduction pathways linked to mGlu7 receptor activation, are selectively desensitized by different GRKs. As GRK2 is ubiquitously expressed, while the expression of GRK4 has been reported only found in selected brain regions (such as hippocampus and cerebellum), the picture of mGlu7 receptor regulation could be influenced by the cell context. Our data also support a role for -arrestin1 as a scaffold protein, mediating mGlu7-induced MAPK activation.

84.18F-FPEB PET Imaging Targeting the mGluR5 Receptor in Neurodegenerative Disorders D. Jennings, J. Seibyl, O. Barret, G. Tamagnan, J. Batis, D. Alagille, R. Baldwin, G. Zubal, D. Russell, K. Marek Objective: To utilize 18F-FPEB PET imaging to target the metabotrophic glutamate receptor Type 5 (mGluR5) imaging in patients with early Parkinson disease, Huntington disease (HD) Alzheimer disease and Fragile X syndrome in compared with healthy controls (HC). Background: mGluR5 is highly expressed in neocortical structures and striatal neurons. A reduction in mGluR5 expression may occur as a result of neurodegeneration of the

striatum due to PD or HD. For example, evidence from PD animal models suggests levodopa induced dyskinesia (LID) is associated with enhanced expression of mGluR5. mGluR5 antagonists have been shown to attenuate LID in PD subjects and possibly ameliorate symptoms in fragile X patients. 18F-FPEB binds mGluR5 with high affinity and specificity and good test retest reliability. Methods: Early PD (n=8), PD with LID (n=5), and HC (n=7), HD (2) underwent 18F-FPEB PET imaging. Subjects received 5 mCi 18F-FPEB and serial dynamic PET projection data was acquired over 2 hrs. Images were realigned in PMOD, normalized using custom FPEB template. After initial kinetic modeling studies demonstrated correlation with non-invasive analysis methods subsequent studies used a target to cerebellar reference region as the primary imaging outcome. Activity displayed for striatal, neocortical, thalamic,and cerebellar regions as a function of time. Results: Mean age for HC 46 yrs, early PD 60.5 yrs, PD with LID 65.6 yrs, HD 51 yrs. Mean UPDRS for early PD was 23 and PD with LID 39. Duration from diagnosis was 0.9 yrs, for early PD, 10.4 yrs. For HD subjects, mean UHDRS motor score was 16, duration of diagnosis 4.4yrs. The mean putamenal: cerebellar ratio (SD) was: early PD 3.32 (0.37, PD with LID 3.53 (0.37), HD 2.5 (0.17), HC 3.29 (0.27). Conclusions: 18F-FPEB is a promising PET radiotracer for evaluating mGluR5 with excellent characteristics for human imaging. Preliminary data from this ongoing study indicates that 18F-FPEB PET shows reductions in striatal mGluR5 in HD subjects and a possible increase in PD with LID subjects. The results obtained across these neurodegenerative diseases are consistent with the proposed mGluR5 pathophysiology for these conditions.

85.Activation of Group II Metabotropic Glutamate Receptors Induces Long-Term depression of Excitatory Synaptic Transmission in the Substantia Nigra Pars Reticulate K.A. Johnson, C.M. Niswender, P.J. Conn and Z. Xiang a

Department of Pharmacology, Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University, Nashville, TN 37232 USA

Several subtypes of metabotropic glutamate receptors (mGlus) have been implicated as therapeutic targets for treating the motor symptoms and progressive degeneration of substantia nigra pars compacta (SNc) dopamine neurons observed in Parkinson’s disease (PD). Agonists of group II mGlus (mGlu2 or mGlu3) reduce excitatory transmission at multiple synapses in the basal ganglia and may have antiparkinsonian effects by reducing excessive excitatory transmission in the output nuclei of the basal ganglia, including the substantia nigra pars reticulata (SNr). While previous studies have found that brief application of a group II mGlu agonist to rat midbrain slices produces a reversible depression of excitatory postsynaptic currents (EPSCs) recorded from GABAergic SNr neurons, we have found that a more prolonged bath application of the group II mGlu agonist LY379268 (100 nM, 10 minutes) induces a striking long-term reduction in EPSC amplitude. This novel form of drug-induced long-term depression (LTD) lasts at least 45 minutes after drug washout, is concentration-dependent, and is completely blocked by pretreatment of slices with the group II mGlu-preferring antagonist LY341495 (500 nM). To determine the relative contributions of mGlu2 and mGlu3 to the LTD induced by LY379268, we tested the ability of LY379268 (100 nM) to induce LTD in wild type mice and mice lacking mGlu2 or mGlu3. LY379268 induced similar LTD in wild type mice and mGlu3 knockout mice, whereas LTD was absent in mGlu2 knockout mice, indicating that mGlu2 activation is necessary for the induction of LTD in the mouse SNr. Surprisingly, the mGlu2-selective positive allosteric modulator biphenyl indanone-A (BINA, 3 μM) was unable to potentiate the EPSC depression induced by a subthreshold


concentration of LY379268 in rat brain slices, and LTD was not mimicked by the mGlu2-selective agonist LY541850 (10 μM). These studies suggest a novel role for group II mGlus in the longterm regulation of excitatory transmission in the SNr and invite further exploration of mGlu2 as a therapeutic target for treating the motor symptoms of PD. This work was supported by NIH grants NS48334, NS67737, and MH74953. Vanderbilt is a Specialized Chemistry Center within the Molecular Libraries Probe Centers Network.

86.Preclinical Characterization of LY2300559, a Dual Acting mGlu2 Receptor Potentiator and Leukotriene CysLT1 Receptor Antagonist, for Potential Use in Migraine M.P. Johnson, J. Schkeryantz, S. Swanson, A.N. Perkins, and K.W. Johnson Neuroscience Discovery, Eli Lilly and Company, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Indiana, 46033, U.S.A The metabotropic glutamate 2 (mGlu2) receptor is a presynaptic receptor on central and peripheral nerve endings that negatively regulates neurotransmitter release. The mGlu2 receptor is localized within the leptomeninges in rat and human. The cysteinyl leukotriene receptor 1 (CysLT1) receptor mediates the proinflammatory leukotriene D4 (LTD4), located on circulating leukocytes, macrophages and resident mast cells. Mast cells are localized in association with Substance P (SP) positive nerve endings innervating meningeal arteries. Activation of the CysLT1 receptor induces release of numerous proinflammatory mediators. In a dural plasma protein extravasation (PPE) model with trigeminal ganglia electrical stimulation, agents that were either selective mGlu2 potentiators or CysLT1 antagonists showed efficacy, and in combination an apparent synergistic response was found. While investigating various chemotypes with positive allosteric modulatory activity at the mGlu2 receptor, a series of molecules that also processed antagonist activity at CysLT1 were recognized. One such molecule is LY2300559, (3-{[4-(4-acetyl3-hydroxy-2-propylphenoxymethyl)-phenyl]-(S)-hydroxymethyl} benzoic acid). LY2300559 is a potent potentiator of the mGlu2 receptor (EC50 = 50 nM in an intracellular calcium based functional assay) and an antagonist of the CysLT1 receptor (IC50= 727 nM in an intracellular calcium based functional assay, Kb= 22 nM). No activity as either an agonist, antagonist, or as an allosteric modulator of the mGlu1/3/4/5/8 receptors at concentrations of 10 M was found. Similarly, no activity in a diverse panel of over 80 targets (CEREP panel) was observed at 10 M, indicating LY2300559 appears to be very selective for the mGlu2 and CysLT1 receptors. The site of allosteric interaction with the mGlu2 receptor was confirmed to be within the transmembrane (TM) domain with selectivity over mGlu3 driven largely by an asparagine to aspartate difference within TM5. As with previously characterized potentiators of the mGlu2 receptor, while a small increase in GTPS35 autoradiograph binding was seen with LY2300559 alone, in combination with an orthosteric agonist LY379268 the GTPS35 was greatly enhanced. The brain region pattern of these signals overlapped with the known distribution of mGlu2 receptors and no effect was seen in brain slices from mGlu2 -/- mice. LY2300559’s bioavailability in rats and dogs was 41% and 76% with clearance values of 2.1 and 3.8 mg/min/kg in rat and dog, respectively. Volumes of distributions were low and the brain to plasma ratio in rat was < 0.01, possibly due to a high degree of plasma protein binding ( 99%). Moreover, LY2300559 showed no appreciable inhibitory activity (> 5 M) when tested in a panel of seven CYP enzymes and was devoid of hERG activity. When tested in the PPE model LY2300559 was a potent (full effect at 0.01 mg/kg po)


31

inhibitor of dural extravasation, with a long duration of effect (18 hours). LY2300559 showed no indications of tolerance with repeat dosing. In pilot toxicology studies, daily administration of LY2300559 to rats (15 d) and dogs (7 d) resulted in no-observed adverse effect levels (NOAELs) of 500 mg/kg. Exposures (AUC and Cmax) at the NOAELs were >500-fold higher than those following the effective dose in the PPE model. In conclusion, LY2300559 is a potent and selective potentiator of the mGlu2 receptor and antagonist of the CysLT1 receptor that is efficacious in an animal model of migraine, and appears to have a favorable safety profile in preclinical animal testing.

87.Novel mGluR5 NAMs: Potential Utility to Treatment in Fragile X Syndrome and Affective Disorders C. K. Jonesa,b, A. L. Rodrigueza,b, J. M. Rookea,b, C. M. Niswendera,b, K. A. Emmittea,b,c, A. S. Feltsa,b, K. P. Lawson, B. S. Batesa,b, A. L. Blobauma,b, R.D. Morrisona,b, F. W. Byersa,b, J.S. Danielsa,b, R.L. Carpenterd, A. Healyd, M. Shumwaya, C. W. Lindsleya,b,c and P.J. Conna,b a

Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232, USA; bVanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, Nashville, TN 37232, USA; cDepartment of Chemistry, Vanderbilt University, Nashville, TN 37232, USA; dSeaside Therapeutics, Cambridge , MA 02139, USA Selective negative allosteric modulators (NAMs) of the metabotropic glutamate receptor subtype 5 (mGlu5) represent anexcitingnovel approach for the treatment of multiple neurologic and psychiatric disorders, including Fragile X syndrome, autism, anxiety, and major depressive disorder. We have made excellent progress in the discovery and optimization of novel classes of mGlu5 NAMsand in performing detailedpharmacologic characterization of a number of different novel chemical series. These include full and partial mGlu5 NAMs with suitable physiochemical properties and PK profiles for in vivo studies. Examples includethe 3-Fluoro-5-(3-(pyridine-2-yl)-1,2,4-oxadiazol-5-yl)benzonitrile (VU0285683) and the 1,3 substituted aryl ether benzamide (VU0409106). In recombinant systems, these mGluR5 NAMs exhibit low nM potency for the inhibition of mGluR5-mediated responses and in many cases high affinity for the 2-methyl-6(phenylethynyl)-pyridine (MPEP) binding site. Using the MPEP site PET ligand [18F]-FPEB, in vivo occupancy at central mGluR5 was determined for VU0409106in ratswith a calculated ED50 of approximately 5.0 mg/kg, which was consistent with the potency of VU0409106 in a number ofrodent preclinical models of neurologic disorders. Moreover, these novel chemical series of mGluR5 NAMs, such asVU0285683, exhibit efficacy in a dose range that does not appear to mimic or potentiate the effects of N-methyl Daspartate receptor antagonistssuggesting that a favorable therapeutic index may be achieved with mGluR5 NAMs in vivo. This project was supported through funding from the NIH and Seaside Therapeutics. Vanderbilt is a Specialized Chemistry Center within the Molecular Libraries Probe Centers Network.

88.mGluR Heteromers: Function and Pharmacology Paul J. Kammermeier University of Rochester Medical Center, Department of Pharmacology and Physiology, Rochester, NY 14642, USA Nearly a decade of biochemical and structural data has demonstrated that metabotropic glutamate receptors (mGluRs) form stable, covalently linked homodimers. While the functional


consequences of dimerization are beginning to be elucidated [1, 2], recent evidence is beginning to show that, contrary to established dogma, some mGluRs can form heteromeric receptors [3]. In some cases, such as heteromeric mGluR2/4, the interactions likely take the form of heterodimers, but the possibility of higher order multimers forming among other subtypes cannot be ruled out. Further, the function and pharmacological consequences of heteromerization have not been explored. Using heterologous mGluR expression in rat sympathetic neurons from the superior cervical ganglion (SCG) and using natively expressed ion channel modulation as an assay for receptor signaling, the possibility of functional interactions between mGluR1 and mGluR5 was explored. By combining a glutamate binding-pocket mutant of mGluR1 (Y74A) with the wild type mGluR5 to render glutamate a highly selective mGluR5 agonist, it was found that the efficacy and potency of the combined mGluR1YA/5 receptors was significantly different than either receptor alone, while mGluR1YA and mGluR2 functioned independently. Further, the dose-response curve of the combined receptors showed that mGluR1YA and mGluR5 seemed to interact in a fundamentally different way than mGluR1YA with mGluR1, suggesting that the interaction may not be dimeric in nature. Interestingly, while application of the selective mGluR5 antagonist MPEP to cells expressing the mGluR1YA/5 combination produced predictable results, selective mGluR1 antagonists strongly antagonized glutamate responses at all concentrations, indicating that heteromeric mGluR1/5 receptors may not respond to known pharmacological compounds as predicted by their constituent subunits. The heteromeric interaction profile described above was reproduced in HeLa cells using glutamate-induced rises in intracellular calcium as an assay for receptor function, indicating that the functional interaction profile is a general phenomenon, not restricted to heterologous expression in sympathetic neurons. These data led us to propose a model in which mGluR1 and 5 are hypothesized to form tetrameric receptors (dimers of homodimers). The implications of this model were further tested using selective pharmacological tools and examining coupling to native N-type calcium channels and M-type potassium channels in SCG neurons. [1] Kammermeier, P. J. and Yun, J. JPET, 2005, 312(2), 502-508. [2] Kniazeff, J., et al., Nat. Struct. Mol. Biol., 2004, 11, 706-713. [3] Doumazane, E., et al., 2011, 25(1), 66-77.


absent in the cerebellum, hippocampus and striatum of DGL knockout mice, whereas they were intact in DGL knockout brains. Depolarization-induced retrograde synaptic suppression was also abolished in DGL knockout mice but was intact in DGL knockout brains. Expression of the molecules requires for 2-AG production (mGluR1 and phospholipase C4 (PLC4) in the cerebellum, and mGluR5, M1–mAChR and PLC1 in the hippocampus and striatum) and CB1 receptor were normal. The basal 2-AG content was markedly reduced and stimulus-induced elevation of 2-AG was absent in DGL knockout brains, whereas the 2-AG content was normal in DGL knockout brains. These results indicate that 2-AG produced by DGL mediates retrograde synaptic suppression. The second topic is the finding that proteaseactivated receptor 1 (PAR1), a member of the Gq/11-coupled receptors that are proteolytically activated by serine proteases, can trigger 2-AG production and causes retrograde suppression of synaptic transmission in the hippocampus [2]. In rat cultured hippocampal neurons, PAR1 activation by thrombin or PAR1specific peptide agonists transiently suppressed inhibitory transmission at cannabinoid-sensitive, but not cannabinoid-insensitive, synapses. The PAR1-induced suppression of synaptic transmission was accompanied by an increase in paired-pulse ratio, and was blocked by a CB1 receptor antagonist. The PAR1-induced suppression was absent in DGL knockout mice. The PAR1induced IPSC suppression remained intact under the blockade of group I mGluRs, and was largely resistant to the treatment that blocked Ca2+ elevation in glial cells following PAR1 activation, which exclude the major contribution of glial PAR1 in IPSC suppression. We conclude that activation of neuronal PAR1 triggers retrograde signaling mediated by 2-AG, which activates presynaptic CB1 receptors and suppresses transmitter release at hippocampal inhibitory synapses. [1] Tanimura, et al. Neuron, 2010, 65, 320-327. [2] Hashimotodani, et al. J. Neurosci., 2011, 31, 3104-3109.

90.LY2140023 Monohydrate: Preclinical and Clinical Update on the Development of an mGluR2/3 Agonist Treatment for Schizophrenia B. J. Kinon

89.Endocannabinoid Release and Retrograde Synaptic Suppression Mediated by Group I mGluR and other Gq/11-Coupled Receptors

Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN, 46285

M. Kano

While dopamine has been viewed as the key neurotransmitter mediating the symptoms of schizophrenia over the last 5 decades, there is accumulating evidence implicating dysregulation of the glutamatergic system as a prominent contributor to the pathophysiology of the disease. Understanding the role of glutamate in schizophrenia may provide a new direction for the development of innovative and potentially more effective drug therapies for schizophrenia. As an overview of our research, I will summarize data from several preclinical animal models of schizophrenia identifying metabotropic glutamate (mGlu) 2/3 receptors as potential antipsychotic targets, and present data from several recent clinical trials assessing the role of the mGlu 2/3 receptor agonist LY2140023 monohydrate, an oral prodrug of LY404039, as a possible therapeutic agent in the treatment of schizophrenia. The results of Study HBBD (a proof-of-concept clinical study of 40 mg twice daily LY2140023), and Study HBBI (a dose-ranging clinical study of 4 doses of LY2140023 vs. olanzapine or placebo given for 4 weeks) in adult patients with schizophrenia will be briefly reviewed. In addition, the results of Study HBBR will be presented. Study HBBR was a multicenter, randomized, Phase 2, openlabel study comparing the safety and tolerability of LY2140023 with standard-of-care (SOC; investigator’s choice of atypical antipsychotics: olanzapine, risperidone, or aripiprazole) for schizophrenia. Patients had moderate symptomatology, with

Department of Neurophysiology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Endocannabinoids are produced in activity-dependent manners and released from postsynaptic neurons. The released endocannabinoids travel backward across the synapse, activate presynaptic CB1 cannabinoid receptors, and cause transient or persistent suppression of neurotransmitter release. Endocannabinoid release is triggered by strong depolarization of postsynaptic neurons and the resultant elevation of calcium concentration, strong activation of Gq/11coupled receptors such as group I metabotropic glutamate receptors (mGluRs) and M1/M3 muscarinic acetylcholine receptors (M1/M3– mAChRs) at basal intracellular calcium level, or simultaneous calcium elevation and Gq/11-coupled receptor activation. The first topic of my talk is the identification of the endocannabinoid that mediates retrograde signaling at central synapses [1]. To determine which of the two major endocannabinoids, anandamide or 2arachidonoylglycerol (2-AG), mediates retrograde signaling, we generated and analyzed mutant mice lacking either of the two 2-AG synthesizing enzymes diacylglycerol lipase (DGL) and (DGL). We found that retrograde synaptic suppression triggered by activation of group I mGluRs or M1/M3–mAChRs were totally


prominent negative symptoms and evidence of functional impairment. Those who met entry criteria were randomized to open-label treatment with either LY2140023 monohydrate (target dose: 40 mg twice daily; n=130) or SOC (n=131). Analysis of the primary objective showed LY2140023 monohydrate to be comparable to SOC in time to discontinuation due to lack of tolerability (i.e., adverse events) (p=.184). The incidence of serious adverse events was comparable between groups. LY2140023 monohydrate-treated patients had a significantly greater incidence of the treatment-emergent adverse events of insomnia, vomiting, agitation, and dyspepsia, while SOC-treated patients reported significantly more akathisia and weight gain. SOC was associated with greater increases in parkinsonian symptoms, akathisia, and prolactin. One patient had a seizure during the placebo lead-in phase of the study before receiving any study medication. A second patient had 2 seizures 1 day after discontinuing 3 weeks of LY2140023 monohydrate treatment and initiating treatment with a conventional antipsychotic. The patient spontaneously recovered without any residual effects. All-cause discontinuation and discontinuation due to lack of efficacy were significantly greater in the LY2140023 monohydrate group compared with the SOC group. Initial improvement on PANSS total and positive scores over the 6– 8 weeks of acute treatment did not differ between treatments. Improvement at the 24-week endpoint was significantly greater in the SOC group. LY2140023 monohydrate and SOC had comparable negative symptom improvement over the 24 weeks. These data provide further evidence that the potential antipsychotic LY2140023 monohydrate, with a glutamatergic mechanism of action, may have a unique tolerability profile characterized by a low association with adverse events that characterize currently available dopaminergic antipsychotics.

91.Selective Mglur2 Negative Allosteric Modulators for the Treatment of Cognitive Deficits Associated with Alzheimer’s Disease Nathalie Lambeng Addex Pharmaceuticals SA, 12 Chemin des Aulx, CH-1228, Plan Les Ouates, Geneva, Switzerland. E-mail: [email protected] Group II metabotropic glutamate receptor (mGluR) orthosteric agonists have been reported to affect working and spatial memory in rodents and monkeys [1,2]. As shown in mGluR2 KO mice, this effect is specific to mGluR2 receptor activation. In contrast, orthosteric mGluR2/3 antagonists improved acquisition of shortterm working memory, spatial learning and social memory [1, 3]. In addition, some non-competitive group II antagonists improved cognitive performance in scopolamine impaired normal rats, in aged rats and in PS2APP mice, a model of Alzheimer’s disease [4,5]. Finally, group II mGluR stimulation was recently shown to induce Amyloid (42) accumulation and the group II antagonist LY341495 blocked this effect [6]. Altogether, these results highlight the interest of mGluR2 blockade as a therapeutic approach for treating memory deficits in Alzheimer’s disease. Interestingly, all those studies have been performed using non-selective mGluR2 molecules which retain affinity for either mGluR3 or group I and/or III mGluRs, which could lead to toxic effects such as neurotoxicity in the case of blockade of mGluR3 receptors [7,8]. Therefore, our aim was to identify novel, selective and brain penetrant mGluR2 Negative Allosteric Modulators (NAM), and to evaluate their ability in reversing scopolamine- or mGluR2/3 agonist- induced memory deficit in the novel object recognition test in rats. We identified several compounds as mGluR2 NAMs active in fullyblocking the glutamate-induced calcium mobilization in a human recombinant system. Their allosteric mode of action was confirmed using Schild-plot, reversibility and radioligand binding competition


33

experiments. These compounds were inactive on human mGluR3 and all other mGluR subtypes. Their in vitro pharmacological characterization will be presented, together with the pharmacokinetic properties and in vivo activity in preclinical rodent models of memory deficits of the best compound. [1] Higgins, et al. Neuropharmacology, 2004, 46, 907-917. [2] Spinelli, et al. Psychopharmacology, 2005, 179, 292-302. [3] Shimazaki, et al. Eur. J. Pharmacol., 2007, 575(1-3), 94-97. [4] Knoflach, et al. Neuropharmacology, 2005, 49(S1), 1-288. [5] Woltering, et al. Bioorg. Med. Chem. Lett., 2010, 20(23), 6969-74. [6] Kim, et al. J. Neurosci., 2010, 30(11), 3870-5. [7] Corti, et al. J. Neurosci., 2007, 27(31), 8297-8308. [8] Caraci, et al. Mol. Pharmacol., 2011, 79(3), 618-626.

92.mGlu1 Receptors and Endocannabinoids: Neuroprotection Studies in an in vitro Model of Cerebral Ischemia E. Landucci, T. Scartabelli, E. Gerace, F. Moroni, G. Mannaioni and D.E. Pellegrini-Giampietro Department of Preclinical and Clinical Pharmacology University of Florence, Viale Pieraccini 6, 50139 Florence, Italy Antagonists of mGlu1 receptors attenuate CA1 pyramidal cell death in models of ischemia by a mechanism that involves the release of GABA [1]. Stimulation of mGlu1 postsynaptic receptors in CA1 pyramidal cells promote the release of endogenous cannabinoids [2], that may act as retrograde transmitters to suppress the output of GABA via activation of CB1 receptors located on interneuron presynaptic terminals [3]. The effects of synthetic and endogenous CBs were evaluated in rat organotypic hippocampal slices exposed to 20 min oxygen-glucose deprivation (OGD). When present in the incubation medium, the synthetic CB agonists WIN 55212-2 and CP 55940 (1-30 M) and the CB1 agonist ACEA exacerbated CA1 injury induced by OGD, whereas the CB1 receptor antagonists AM 251 and LY 320135 were neuroprotective with maximal activity at 1 M. The endocannabinoid 2-arachidonoylglycerol (2-AG) reduced OGD injury in hippocampal slices at 0.1-1 M, whereas anandamide (AEA) was neurotoxic at the same concentrations. The effects of WIN 55212-2, AEA and 2-AG in slices were all dependent on the activation of CB1 but not CB2 receptors, except for the toxic effects of AEA that were also dependent on vanilloid TRPV1 receptors. In the same model of rat organotypic hippocampal slices we investigated the cross-talk between mGlu1 and CB1 receptors. WIN 55212-2, but not AM251, significantly reverted the neuroprotective effects of LY367385 and 3-MATIDA in 30 min of OGD. The mGlu1/5 agonist DHPG (100 M) exacerbated CA1 injury induced by 20 min OGD, but this effect was not synergic with that of WIN 55212-2. AM251, but not WIN 55212-2, was able to revert the neurotoxic effects of the mGlu1/5 agonist DHPG. We then determined by LC-MS/MS the CA1 contents of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in control and OGD-treated hippocampal slices. AEA, and to a lesser extent 2-AG, increased significantly immediately after OGD and returned to basal levels 3 h later; LY367385 was able to prevent the increase induced by OGD. Our results suggest that endocannabinoids and mGlu1 receptors may play a cooperative role in the mechanisms of post-ischemic neuronal death. [1] Pellegrini-Giampietro, D.E. Trends Pharmacol. Sci., 2003, 24, 461-470. [2] Varma, et al. J. Neurosci., 2001, 21, 1-5, [3] Alger, B.E. Prog. Neurobiol., 2002, 68, 247-286.

93.Neurochemical Characterisation of Group II Metabotropic Receptor Knockout Mice T. Lane1, L. Lyon1, D. Bannerman2, T. Sharp3, J. Kew4, C. Corti5 and P. Harrison1

34 Current Neuropharmacology, 2011, Vol. 9, Suppl. 1 1 Department of Psychiatry, University of Oxford, Warneford Hospital, Oxford, UK. 2Department of Experimental Psychology, University of Oxford, Oxford, UK 3Departmnet of pharmacology, University of Oxford, Oxford, UK 4Neurosciences CEDD, Glaxo SmithKline, New Frontiers Science Park, Harlow, U.K 5Neurosciences CEDD, GlaxoSmithKline, Via Fleming 4, Verona, Italy

The group II metabotropic glutamate receptors (mglur) play a role in cognition as well being implicated in the pathophysiology of a number of psychiatric disorders such as anxiety, addiction and schizophrenia. The ability of group II agonists to reverse spatial working memory deficits induced by phencyclidine in rats [1] and ketamine in humans [2] highlighted the possibility that these drugs may help to improve the cognitive dysfunction observed in schizophrenia patients. Recently an agonist for the group II mglurs has shown promise as an anti-psychotic agent [3]. Behavioural studies of mice lacking group II mgluRs have shown an interesting phenotype, with a spatial memory deficit which is present in appetitively motivated but not aversively motivated tasks [4]. The mice have normal function in other appetitive tasks, suggesting that the greater arousal involved in aversive tasks is able to overcome the memory deficit. The group II double knockout mice (dko) show reduced locomotor activity in general and in response to amphetamine. Given that group II agonists and antagonists are known to modulate dopamine, noradrenalin and serotonin [5,6] all of which are implicated in different aspects of attention, stress and arousal, as well as reward and cognitive flexibility [7,8]. We carried out an HPLC study to examine the basal tissue levels of monoamines in the cortex, hippocampus and striatum of group II dko and wild type mice. 16 wild type and 16 dko mice (maintained as two separate wild type and dko colonies) were used. Frontal cortex, hippocampus and striatum were dissected out, snap frozen and stored at -80oC. Dopamine, noradrenalin, serotonin and metabolites were measured in tissue homogenates by HPLC using electrochemical detection. Dopamine and its metabolites DOPAC and HVA were significantly reduced in the striatum of dko mice. Cortical and hippocampal dopamine and its metabolites were unaffected. Cortical levels of the noradrenalin metabolite MHPG were significantly increased in the cortex of dko mice while the levels of noradrenalin were not altered, suggesting greater turnover of noradrenalin. Cortical 5-HT was increased in male but not female knockout animals. Decreased striatal DA is consistent with the reduced hyperlocomotor activity in response to amphetamine in these animals and with the regulation of striatal DA in the literature [6,9]. The degree to which the present findings are reflective of neurotransmitter release will need to be further investigated by microdialysis. Work supported by a pilot industry award from the medial research council [1] Moghaddam, B, et al. Science, 1998, 281, 1349-1352. [2] Krystal, J.H, et al. Psychopharmacology, 2005, 179, 303-309. [3] Patil, S.T, et al. Nat. Med., 2007, 13, 1102-1107. [4] Lyon, L, et al. Neuropsychopharmacology, 2011 (in press). [5] Cartmell, J, et al. Brain Res., 2000, 887, 378-384. [6] Lorrain, D, et al. Neuropharmacology, 2005, 48, 927-935. [7] Nikiforuk, A., et al , J. Pharmacol. Exp. Ther., 2010, 335, 665-673. [8] Hu, G, et al. J. Pharmacol. Exp. Ther., 1999, 289, 412-416. [9] Pherson, A.L, et al. Psychopharmacology, 2010, 211, 443-455.

94.The Development of mGlu2 Positive Allosteric Modulators: In vitro and in vivo Characterization of JNJ40068782 H. Lavreysena, J. Cida, A.A. Trabancoa, W. Drinkenburga, A. Ahnaoua, A. Megensa, I. Biesmansa, I. Van der Lindena, L. Peetersa, X. Langloisa, P. Te Rielea, F. Dautzenberg, J.R. Atacka, G. Duveyb, R. Lütjensb, E. Le Poul, and G. Macdonalda

Conference Proceedings a

Neuroscience, Janssen Research and Development, A division of Janssen Pharmaceutica NV, Turnhoutseweg 30, 2340 Beerse, Belgium; bAddex Pharmaceuticals SA, Chemin des Aulx 12, 1228 Plan-les-Ouates, Geneva, Switzerland

Stimulation of the mGlu2 receptor reduces glutamatergic transmission in brain regions where excess glutamate signaling may be implicated in the pathophysiology of disorders such as anxiety and schizophrenia. Hence, activation of mGlu2 receptors is being pursued as a novel therapeutic approach for the treatment of these diseases. To date, several mGlu2/3 receptor agonists have shown activity in preclinical animal models of anxiety and schizophrenia, while recent clinical trials have demonstrated beneficial effects of the mechanism in patients with generalized anxiety disorder or schizophrenia. mGlu2 positive allosteric modulators (PAMs) have been shown to reproduce aspects of the pharmacological effects of direct mGlu2/3 agonists in animal models and are therefore considered as an alternative way for modulating mGlu2 receptor function and treating CNS disorders. We will present the pharmacological properties of the 3-cyano-1,4-disubstituted pyridone JNJ-40068782, a new selective mGlu2 receptor PAM. In human and rat mGlu2 receptor over-expressing cell lines, JNJ-40068782 potentiated the receptor response to glutamate with EC50 values of 150 and 300 nM, respectively. The effects of JNJ-40068782 result from binding to a site on the mGlu2 receptor distinct from the agonist binding site, and thereby increasing the binding affinity of agonists. Conversely, the affinity of novel mGlu2 PAM radioligands is increased by agonists, highlighting the reciprocal allosteric interaction between agonists and PAMs. Using an in vivo occupancy assay, it was shown that JNJ-40068782 readily enters the brain and occupies the rat mGlu2 receptor with an ED50 of 4.2 mg/kg p.o., corresponding to a plasma concentration of 400 ng/mL. Profiling in a sleep-wake EEG assay showed that JNJ40068782 decreased REM sleep in rats, consistent with changes in sleep-wake architecture associated with mGlu2 receptor activation, with a minimum effective dose corresponding to a plasma concentration of 300 ng/mL. Finally, in the mouse, JNJ-40068782 was able to attenuate PCP-induced hyperlocomotion to a similar extent as obtained with the mGlu2/3 receptor agonist LY404039, suggesting a potential anti-psychotic activity of JNJ-40068782.

95.Interactions Between mGlu5 Receptors and other GPCRs: Relevance to Drug-Seeking A.J. Lawrence1 , R.M. Brown1, C.L. Adams1, M. Stagnitti1, J. Duncan1 and C. Ledent2 1

Florey Neuroscience Institutes, University of Melbourne, Parkville, Vic, 3010; 2Institut de Recherche Interdisciplinaire, Faculte de Medecine, Universite de Bruxelles, Brussels, Belgium

Adenosine A2A receptors and metabotropic glutamate type 5 (mGlu5) receptors are co-localised both presynaptically and postsynaptically in the striatum and have been shown to functionally interact to regulate transmitter release, signal transduction and behaviour. We have pursued this interaction in relation to drug-seeking and have documented a powerful synergy between A2A receptor antagonists and mGlu5 negative allosteric modulators (NAMs). For example, individually sub-threshold doses of SCH58261 (A2A receptor antagonist) and MTEP (mGlu5 NAM), when given in combination reduce alcohol self-administration in iP rats by >50%. Moreover, this combination treatment also prevented a cue-induced reinstatement of alcohol-seeking, without impacting upon locomotor activity [1]. Specificity of this interaction was confirmed with similar studies examining combination treatments including mGlu5-CB1, mGlu5-A1, A2A-CB1 and A2A-A1 receptor antagonism [1, 2]. To examine this interaction further, and extend


studies beyond alcohol, we have used mice with a genetic deletion of A2A receptors (A2A KO) and assessed the response to MTEP in these versus WT littermates. The conditioned rewarding and locomotor activating properties of cocaine were evaluated using the conditioned place preference (CPP) paradigm. Adenosine A2A KO and WT mice were subjected to alternating daily conditioning injections of cocaine (20mg/kg, i.p.) or saline. Twenty min prior to cocaine administration mice were pre-treated with either MTEP or vehicle. CPP was assessed following 8 days of conditioning. During each session the time spent in each compartment as well as locomotor activity was measured. Vehicle-treated mice of both genotypes expressed a CPP to cocaine. MTEP pretreatment abolished cocaine CPP in WT, but not A2A KO mice. In contrast, MTEP attenuated the acute locomotor activating properties of cocaine similarly in both genotypes. These data provide evidence for a functional interaction between adenosine A2A and mGlu5 receptors in mediating the conditioned effects of cocaine (either directly or via modulation of incentive learning) but not direct cocaine-induced hyperactivity. This functional interaction is further supported by modulation of [125I]ZM241385 binding to the A2A receptor by MTEP. Thus, preincubation of mouse striatal slices with MTEP prior to [125I]ZM241385 resulted in a reduction in specific binding to A2A receptors. Collectively, these findings demonstrate a functionally relevant interaction between mGlu5 and adenosine A2A receptors, that has relevance to drug-seeking and possibly mechanisms of incentive learning. [1] Adams, C.L. et al. Int. J. Neuropsychopharmacol., 2008, 11, 229-241. [2] Adams, C.L. et al. Br. J. Pharmacol., 2010, 159, 534-542.

96.Metabotropic Glutamate Receptors in Thalamus in Relationship to Epilepsy C. Lee and J. Huguenard Department of Neurology and Neurological Sciences Stanford University School of Medicine, Stanford, CA 94305 USA mGluRs modulate neuronal excitability through pre- and postsynaptic mechanisms. Group I receptors generally have postsynaptic actions, while Group II and III receptors suppress neurotransmission through presynaptic mechanisms. Group III but not II receptors are localized to active zones of both symmetric and asymmetric synapses. Group III mGluRs have been implicated in absence epilepsy. mGluR4 knockout mice are resistant to seizures induced by low dose GABAA receptor antagonists, and infusion of mGluR antagonists into nRt conferred a similar resistance, while mGluR7 knockout mice exhibited spontaneous seizures and enhanced seizure susceptibility. Knock-in mice lacking the PDZligand motif of mGluR7a also showed increased susceptibility to seizures. mGluR7 is located presynaptically within excitatory synapses where it can serve as a low affinity (EC50 = 1mM) autoreceptor. Recent studies have documented a key role for PICK1 (protein interacting with C kinase 1) in trafficking and function of mGluR7. Of two mGluR7 splice variants, only mGluR7a is expressed in the thalamus, especially in cell bodies and fibers in nRt. Recently, it was shown that in vivo uncoupling of mGluR7 from its PDZ-domain-interacting ligand, PICK1, induced absence seizures indicating mGluR7 might prevent excessive excitation in the thalamocortical circuit. We adopted the protein transduction domain approach of Bertaso (2008) to deliver TAT-R7-LVI and its inactive control TAT-R7-AAA, which should not interfere with PICK1-mGlur7A interactions, in vivo and examine their effects on thalamic networks in vitro. We find that the peptide is taken up into the brain and is detectable in neurons for at least an hour following IV injection. We find that the LVI form of the protein enhances


35

in vitro thalamic oscillations, increasing their duration by more than twofold. The effects in vitro lasted for at least 90 minutes, indicating that effects of in vivo injection persist in slices for sufficient time to allow detailed in vitro synaptic and circuit analysis. To test for effects of the peptide on synaptic transmission in the thalamic circuit we first injected into the cortex of 4 week old animals an adeno-associated viral construct (rAAV5/CamKIIChR2 (H134R)-EYFP) for a fusion protein containing an increased function channelrhodopsin along with yellow fluorescent protein (EYFP) driven by the Cam Kinase 2 promoter that drives expression in principal cells. 4 weeks later brain slices were made and voltage clamp recordings in nRt cells were obtained. Cortical afferents were activated by stimulation of the internal capsule via a 488 nm laser. Pairs of optical stimuli were applied at 10 Hz and repeated every 5 seconds. In control slices (with AAA peptide) paired pulse facilitation was observed, which is consistent with the low release probabilities of cortical afferent terminals. In contrast, in slices from LVI peptide treated animals, EPSCs showed little facilitation, suggesting that inactivation of mGluR7a/PICK1 interactions increases release probability. These results show that by using optogenetics and TAT mediated delivery of intracellular peptides it will be possible to discern epilepsy related circuit disruptions in thalamus related to epilepsy. Supported by the NINDS.

97.Discovery of Orally Active 5,6,7,8-Tetrahydroquinazolin-5-yl Amide Derivatives as mGlu5 Receptor Negative Allosteric Modulators G. Li, M. April, J. Austin, H. Zhou, X. Pu, N. J. Boyle, M. A. Uberti, R. M. Brodbeck, M. D. Bacolod, D. G. Smith and D. Doller Chemical & Pharmacokinetic Sciences Department, Lundbeck Research USA. 215 College Road. Paramus, NJ 07652, USA A series of 5,6,7,8-tetrahydroquinazolin-5-yl amide derivatives was designed and synthesized as novel mGlu5 receptor negative allosteric modulators (NAMs) lacking an alkyne moiety. Structureactivity and structure-property relationships within this chemotype were explored, leading to Lu AE88928, characterized by hmGlu5 IC50 of 10 nM, excellent selectivity across a broad panel of GPCRs and ion channels, fair physicochemical properties, good safety profile, and excellent PK properties in rodents. A close analog of Lu AE88928 demonstrated anxiolytic-like activity in the GellerSeifter conflict test at 30 mg/kg, PO. Results of our SAR and optimization will be discussed. O

Chiral N N

N N

Lu AE88928 hmGlu5 IC50 10 nM rat B/P 4.5 rat F 100% absolute stereochemistry arbitrarily assigned

98.Heteroarylcyclohexylamides Negative Allosteric Modulators

as

mGlu5

Receptor

G. Li, M. April, H. Zhou, X. Pu, M. A. Uberti, R.M. Brodbeck, M. Cajina, G. Chandrasena and D. Doller


Chemical & Pharmacokinetic Sciences Department, Lundbeck Research USA. 215 College Road. Paramus, NJ 07652, USA A number of mGlu5 receptor negative allosteric modulators (mGlu5 NAMs) have been evaluated in the clinic for anxiety, and several other indications. Several of these development compounds, including STX107, AFQ056, ADX10059 and ADX48621, contain an alkyne moiety in their structure. The discontinuation of ADX10059 due to liver toxicity concerns suggests a strong need to identify structurally diverse non-alkyne mGlu5 NAMs. Structural modification of a lead generated from computational modeling afforded a novel series of trans-heteroarylcyclohexylamides (I) as mGlu5 NAMs. Structure-activity and structure-property relationship studies were conducted, leading to select compounds showing good potency in a functional mGlu5 receptor FLIPR assay and good brain penetration in rodents.

99.Molecular Mechanisms Underlying a Late Phase of Cerebellar Long-Term Synaptic Depression David J. Linden The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School Of Medicine, USA Cerebellar LTD of parallel fiber–Purkinje synapses is a form of neuronal information storage that may be studied in a cell culture model system. It is induced postsynaptically through an mGluR1/Ca influx/protein kinase C (PKC) cascade and it’s initial expression requires phosphorylation of ser-880 in the carboxy-terminal PDZ-ligand region of GluA2 AMPA receptors and consequent binding of PICK1. This triggers clathrin/dynamin – dependent endocytosis of GluA2–containing surface AMPA receptors. Cerebellar LTD also has a distinct late phase beginning 45–60 min after induction that is blocked by transcription or translation inhibitors. It has been suggested that gene expression and protein synthesis are required for both long–term memory consolidation and late phases of LTP and LTD. What has been lacking is identification of the necessary genes and specific transcription factor binding sites in their promoters. We show that inhibition of the transcription factor SRF or its cofactor MAL, blocks the late–phase of LTD in cultured cerebellar Purkinje cells as does deletion of the immediate–early gene Arc. Using neuronal BAC transfection, we show that, in Arc–/– cells transfected with a wildtype Arc BAC, late phase LTD is rescued. However, mutation of one SRF–binding site in the Arc promoter (SRE 6.9) blocks this rescue. Co–transfection of SRF engineered to bind mutated SRE 6.9 can restore late–phase LTD in Arc–/–, SRE 6.9 mutant BAC cells. Thus, SRF binding to SRE 6.9 in the Arc promoter is required for the late phase of cerebellar LTD. We have sought to determine the expression mechanism of the late phase of LTD by applying various drugs and peptides after the protein synthesis-dependent late phase is well-established. Neither bath application of mGluR1 antagonists (JNJ 16259685 or LY 456236) nor the PKC inhibitor GF 109203X starting 60-70 min after LTD induction attenuated the late phase. Similarly, achieving the whole-cell configuration with a second pipette loaded with the peptide PKC inhibitor PKC(19-36) starting 60 min post-induction also failed to alter the late phase. Late internal perfusion with peptides designed to disrupt PICK1GLUA2 interaction (phospho-SVKI, PICK1139-166) also failed to impact late phase LTD expression. However, late internal perfusion with two different blockers of dynamin, the drug dynasore and a dynamin inhibitory peptide (QVPSRPNRAP) produced a rapid and complete reversal of cerebellar LTD


expression. These findings suggest that the protein synthesisdependent late phase of LTD requires persistent dynamin-mediated endocytosis of AMPA receptors, but not persistent PICK1-GluA2 binding or its upstream mGluR1/PKC signaling cascade.

100.‘Molecular Switches’ on mGluR Allosteric Ligands that Modulate Modes of Pharmacology and/or Subtype Selectivity Craig W. Lindsley Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center Department of Pharmacology, Vanderbilt University Department of Chemistry, Nashville, TN 37212 G-Protein-coupled receptors (GPCRs) represent the largest class of drug targets, accounting for over 40% of marketed drugs; however, discovery efforts for many GPCRs have failed to provide viable drug candidates. Historically, drug discovery efforts have focused on developing ligands that act at the orthosteric site of the endogenous agonist. Recently, efforts have focused on functional assay paradigms and the discovery of ligands that act at allosteric sites to modulate receptor function in either a positive, negative or neutral manner. Allosteric modulators have numerous advantages over orthosteric ligands including high subtype selectivity, the ability to mimic physiological conditions, lack of densensitization/ down regulation/internalization and reduced side effects. Despite these virtues, challenging issues have now arisen for allosteric modulators of metabotropic glutamate receptors (mGluRs): shallow SAR, ligand-directed trafficking and the identification of subtle ‘molecular switches’ that modulate the modes of pharmacology (from PAM to NAM or SAM), as well as mGluR subtype selectivity. Here, we will discuss the impact of modest structural changes to multiple mGluR allosteric ligands scaffolds that unexpectedly modulate pharmacology and/or subtype selectivity, as well as a concept of ‘molecular locks’, to describe series free from ‘molecular switches’. Finally, this talk will convey lessons learned, and raise concerns over metabolism and the pharmacology of metabolites.

101.Cinnabarinic acid: A Novel Endogenous Agonist of mGlu4 Receptors L. Lionetto3,*, F. Fazio1,2, G. Molinaro1 , R. T. Ngomba1, S. Notartomaso1,4, M. Curini5, O. Rosati5, P. Scarselli1, M. Simmaco3, F. Mastroiacovo1, R. Di Marco1,4, G. Battaglia1, V. Bruno1,2, H. Bertrand6, F. C. Acher7, J.-P. Pin8,9, F. Nicoletti1,2 and C. Goudet8,9 1 Neuromed I.R.C.C.S., Pozzilli, Italy; 2Departments of Physiol. and Pharmacol. and 3DESMOS, Univ.of Rome Sapienza, Italy; 4Dept. of Hlth. Sci., Univ. of Molise, Campobasso, Italy; 5Dept. of Chem. and Pharmaceut. Technology, Organic Chemistry Unit, Univ. of Perugia, Perugia, Italy; 6Accelrys Inc., Parc-Club Orsay Univ., Orsay, France; 7Lab. de Chimie et de Biochimie Pharmacologiques et Toxicologiques, CNRS, Univ. Paris Descartes, Paris, France; 8 Inst. de Génomique Fonctionnelle, Univ. de Montpellier, Montpellier, France; 9U661, INSERM, Montpellier, France

Cinnabarinic acid (CA), a metabolite of the kynurenine pathway, derives from the condensation of two molecules of 3hydroxyanthranilic acid, and meets the structural requirements to interact with glutamate receptors. CA was able to selectively activate recombinant mGlu4 receptors. Mutagenesis studies and molecular modeling indicated that CA behaved as a partial orthosteric agonist of mGlu4 receptors. CA also activated native mGlu4 receptors in cultured cerebellar granule cells and mixed


mouse cortical cells, where its actions (inhibition of cAMP formation and neuroprotection, respectively) were attenuated by the genetic deletion of mGlu4 receptors. In addition, CA mimicked the protective activity of the mGlu4 receptor enhancer, PHCCC, against MPTP toxicity when locally infused in the mouse external globus pallidus. HPLC-MS/MS analysis showed the presence of endogenous CA in different organs. In the brain, CA could be detected under inflammatory conditions resulting from systemic injection of the bacterial endotoxin, LPS. We conclude that CA is a putative endogenous ligand of mGlu4 receptors.

102.Toward Differentiating the Roles of mGlu2 and mGlu3 Receptors in the Rat CNS. D. Lodge, L. Ceolin, L. Hanna, S.J. Lucas, Z.A. Bortolotto and G.L. Collingridge MRC Centre for Synaptic Plasticity, School of Physiology and Pharmacology, Medical School, University of Bristol, Bristol BS8 1TD, United Kingdom There is a considerable body of evidence from experimental work on rats supporting the important role of Group II metabotropic glutamate (mGluR) receptors in synaptic function and plasticity in the CNS and in behaviours which pertain to human psychiatric and neurological diseases [1]. The contribution, however, of the individual mGlu2 and mGlu3 receptors is not well understood. This is a particularly important matter as compounds acting at these receptors are being progressed toward the clinic. To date the most advanced compounds appear to be mixed mGlu2/mGlu3 orthosteric receptor agonists. The potential advantages and disadvantages of molecules combining both activities are unclear. Two main approaches have been taken to solving this issue. Firstly, useful evidence is being published on allosteric compounds active at one or other of the two receptors but direct comparisons with orthosteric compounds cannot be made. Secondly, transgenic mice not expressing one or both of the two receptors are also providing useful evidence but mice are not rats and their behaviours are not necessarily comparable. We have made use of a largely uncited discovery by Eli Lilly chemists, that LY395756 and its active enantiomer, LY541850, are selective orthosteric agonists at the mGlu2 receptor and at the same time orthosteric antagonists at the mGlu3 receptor [2]. With this compound, we have interrogated inter alia the inhibitory effects of group II receptors on entorhinal inputs into CA1-stratum lacunosum moleculare and into the dentate gyrus and on cortical and intra-nuclear inputs into the lateral amygdale in brain slices from both rats and mice. In the latter, initial studies suggest the presence of an mGlu3 receptor dependent long term depression. Using DCG-IV as a typical mixed mGlu2/mGlu3 agonist, most of its effect could be mimicked by the novel mGlu2 agonist. But there are a couple of interesting anomalies. Firstly in the amygdale, there appears to be a substantial inhibition mediated by mGlu3 receptors. Secondly, while studying the entorhinal input into CA1, rats were found that did not respond to the mGlu2 agonist; in the same rats, DCG-IV produced a substantial inhibition which was reversed by LY395756. In other rats this novel mGlu2 agonist induced inhibition comparable with that of DCG-IV. This anomaly was traced to strain differences within the Wistar rats [3]. Lack of expressed mGlu2 receptors throughout the brain was confirmed by Western blots [3]. Furthermore, the submicromolar agonist and antagonist properties of LY395756 and LY541850 was confirmed in mGlu3 and mGlu2 knock-out mice respectively. Thus in the medial perforant path, the action of DCG-IV (0.3 -1.0 μM) was mimicked by or antagonised by LY541850 (0.1 -1.0 μM) in the mGlu3 and mGlu2 knock-out mice respectively. A start has been made on behavioural studies exploiting the difference in expression of mGlu2 in two rat strains. The rats lacking mGlu2 receptors show an anxious phenotype in


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elevated plus maze and open field assays [3]. Other studies exploring the cognitive, psychotic and other behavioural domains are on-going. [1] Nicoletti, F, et al. Neuropharmacology, 2010, 60, 1-25. [2] Dominguez, C. et al. J. Med. Chem., 2005, 48, 3605-3612. [3] Ceolin, L, et al. J. Neurosci., 2011, 31(18), 6721-31. We would like to thank Eli Lilly & Co. for providing us with such a useful pharmacological tool, knock-out mice and general support for this project and the MRC for financial support.

103.Effects of a Novel mGlu2/3 Receptor Agonist Prodrug, LY2140023 Monohydrate, on central Dopaminergic and Serotonergic Turnover as Determined in Human and Rat Cerebrospinal Fluid S. Lowe1, R. Dean2, B. Ackermann3, K. Jackson4, F. Natanegara2, S. Anderson2, J. Eckstein3, E. Yuen4, M. Ayan-Oshodi2, M. Ho1, D. McKinzie2, K. Perry2 and K. Svensson2 1

Lilly-NUS Centre for Clinical Pharmacology, National University of Singapore, Singapore; 2Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, 46285 IN, USA; 3Eli Lilly and Company, Greenfield Laboratories, Greenfield, Indiana, USA 4Lilly Research Laboratories, Windlesham, Surrey, UK Accumulating evidence suggests that the primary symptoms of schizophrenia may be associated with altered central glutamate transmission. LY2140023 monohydrate is the methionine prodrug of the selective mGlu2/3 receptor agonist LY404039, and is currently being assessed for the treatment of schizophrenia. The objectives of the present studies were to evaluate the central pharmacological activity of LY2140023 monohydrate in preclinical and clinical studies. Effects on neurotransmitter/metabolite concentrations were assessed in male rats following single oral doses of LY2140023 monohydrate (microdiasylates from the prefrontal cortex), single intraperitoneal injection of LY404039 (cerebrospinal fluid [CSF]), or LY2140023 monohydrate dosed once-daily for 7 days (CSF). A clinical study in 16 healthy subjects assessed effects of LY2140023 monohydrate 40 mg orally twicedaily for 14 days in lumbar CSF. In the rat Studies, acute dosing with LY2140023 monohydrate resulted in significant dosedependent increases in extracellular concentrations of dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) but not 5-hydroxyindoleacetic acid (5-HIAA) in the prefrontal cortex. LY2140023 monohydrate dosing for 7 days elevated concentrations of HVA and 5-HIAA in CSF, while acutely dosed LY404039 increased concentrations of DOPAC, HVA, and methoxyhydroxyphenylglycol (MHPG), but not 5-HIAA. In the clinical study, significant increases were seen for DOPAC, HVA, 5hydroxytryptophan, 5-HIAA, and MHPG in CSF of subjects receiving LY2140023 monohydrate but not placebo. In conclusion, LY2140023 monohydrate and/or LY404039 dosing potently affected dopamine turnover and also significantly affected serotonin turnover in the human and rat central nervous systems. The measurement of biogenic amine metabolites such as DOPAC and HVA may serve as useful biomarkers of LY2140023 monohydrate and/or LY404039 central pharmacodynamic activity.

104.Structures of Apo and Agonist Bound Human mGluR2 Extracellular Domain F. Lu1, J. Monn2, M. Kearins1, S. Antonysamy1, D. Clawson2, A. Zhang1, T. Gheyi1, M. Russell1 and S. Atwell1 Lilly Research Laboratories, Eli Lilly and Company, 1San Diego, CA, USA, 2Indianapolis, IN, USA


Metabotropic glutamate receptors (mGluRs) are G-protein-coupled receptors (GPCRs) that transmit excitatory signals in the central nervous system. We report here the first x-ray crystal structures of the extracellular portion of human mGluR2: in an apo, open form as well as in a closed, agonist-bound form with either LY354740 or LY404040. Crystallization of the closed, agonist bound form utilized protein lacking the cysteine rich domain and incorporating a C234S mutation to eliminate the resulting unpaired surface cysteine. The open form crystallizes with the N-terminal lobes in close contact. This contact juxtaposes disordered 108-133 loops, responsible for a disulfide linkage through Cys121. This disulfide linkage functions as the hinge around which the remaining ligand binding domain pivots upon agonist binding, bringing the entire ligand binding domains and, by inference, the cysteine rich domains together.

105.Pharmacological and Molecular Characterization of Metabotropic Glutamate Receptor 2/3 Negative Allosteric Modulators L. Lundström, C.Bissantz, J. Beck, J. Messer, T.J. Woltering, J.G Wettstein, E. Goetschi , J. Wichmann, F. Knoflach and S. Gatti F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland The unique features of the allosteric ligands for both mGlu2 and mGlu3 receptors are described using several affinity and functional assays and different radioligands for both the orthosteric and the allosteric binding sites. In particular two new potent mGlu2/3 NAMs, RO4988546 and RO5488608, are described in this study and the allosteric binding site in the transmembrane (TM) domain of mGlu2 is characterized in details. Site directed mutagenesis, functional measurements and 2- adrenergic-based modeling of mGlu2 were employed to identify important molecular determinants of potent mGlu2/3 NAMs. RO4988546 and RO5488608 affect both 3[H]-LY354740 agonist binding at the orthosteric site and the binding of a tritiated positive allosteric modulator (3[H]-PAM), indicating that NAMs and PAMs could have overlapping binding sites in the mGlu2 TM domain. We identified eight residues in the allosteric binding pocket that are crucial for non-competitive antagonism of agonist-dependent activation of mGlu2 and directly interact with the NAMs: Arg3.28, Arg3.29, Phe3.36, HisE2.52, Leu5.43, Trp6.48, Phe6.55, and Val7.43. The mGlu2 specific residue HisE2.52 is likely to be involved in selectivity and residues located in the outer part of the binding pocket are more important for 3[H]-LY354740 agonist binding inhibition which is independent of the highly conserved Trp6.48 residue. This is the first complete molecular investigation of the allosteric binding pocket of mGlu2 and Group II mGluRs and provides new information on what determines mGlu2 NAMs selective interactions and effects on both agonist ligand binding and signal transduction.

106.Molecular Determinants of the Selective Interaction of Positive Allosteric Modulators with the Transmembrane Region of Metabotropic Glutamate Receptor 2 L. Lundström1, C. Bissantz2, J Beck1, J.G. Wettstein1, T.J.Woltering2, J.Wichmann2 and S. Gatti1 1 Neuroscience Research; 2Discovery Chemistry, F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland

Metabotropic glutamate receptor 2 (mGlu2) plays an important role on the presynaptic control of glutamate release and positive allosteric modulators (mGlu2 PAMs) are under assessment for their potential as antipsychotics. Several ligands for the allosteric binding


site in the transmembrane mGlu2 (TM) domain were generated in recent year and it is now possible to use them to map the allosteric binding site and understand at the molecular level the selective changes in receptor function induced by the presence of mGlu2 PAM. Pharmacological studies were carried out to characterize the cellular effects of several mGlu2 PAM (also radioligands) in cells expressing rat mGlu2.Site directed mutagenesis studies, affinity and functional measurements in recombinant cell systems and 3Dmodeling of mGlu2 were then employed to identify important molecular determinants of two structurally different mGlu2 PAMs. The results show that the presence of mGlu2 PAMs increases the affinity of 3[H]-LY354740 (mGlu2 agonist) for the orthosteric site of mGlu2 as well as the number of binding sites. The 3[H]-2,2,2TEMPS (3[H]-mGlu2 PAM) radioligand selective interaction with the allosteric site of mGlu2 is completely displaced by either mGlu2 PAMs or mGlu2 Negative allosteric modulators (NAMs). Moreover the 3[H]-mGlu2 PAM binding is enhanced by the presence of mGlu2 agonists, confirming a modulatory interaction between ligands for the orthosteric and the allosteric binding sites of mGlu2. In mutagenesis studies five residues, Phe3.36, Tyr3.40, Leu5.43, Asn5.46 and Trp6.48 were identified in the allosteric mGlu2 binding pocket to be crucial for both PAM interaction and potentiation of agonist induced activity; Ser5.42 residue is important for receptor potentiation only for LY487379. This study provides a complete molecular investigation of the mGlu2 PAM binding site and function. Binding differences between mGlu2 PAMs and NAMs are discussed as well as the determinants of mGlu2 PAM selectivity.

107.Role of Metabotropic Glutamate Receptors in the Basolateral Amygdala-Driven Prefrontal Cortical Deactivation or Over-activation in Models of Inflammatory or Neuropathic Pain in the Rat Sabatino Maione, Enza Palazzo, Livio Luongo, Francesco Rossi and Vito de Novellis Department of Experimental Medicine, Pharmacology Division, The Second University of Naples, Itay The medial prefrontal cortex (mPFC) and the basolateral nucleus (BLA) or the central nucleus (CeA) of the amygdala are anatomically and functionally interconnected and cooperate for the processing and integration of emotionally salient sensory input. Specifically, the unpleasantness and affective component of chronic pain are critically driven by prelimbic and infralimbic (PL/IL) divisions of the mPFC and amygdala reciprocal connections. On this subject, emerging evidence shows that supraspinal brain regions can be profoundly affected by persistent pain generated by peripheral nerve injury or chronic inflammation in rodents, as well as in patients. Accordingly, we show in this study that nociceptive responding BLAmPFC pyramidal neurons undergo deactivation related to carrageenan-induce inflammation together with downregulation of the endovanilloid/glutamate synaptic machinery (while GABAergic tone was enhanced) that can participate for impairing pain behaviour mediated by BLA–mPFC circuits. In particular, it has been found here that, conversely to neuropathic pain where excitatory synaptic transmission was increased in layer II/III pyramidal cells of rats [1, 2], the same pyramidal cells are inhibited by intra-plantar injection of carrageenan. We show unprecedented in vivo evidence of a neural mechanism by which selective mGluR1 up-regulation in the BLA division of the amygdala may impair mPFC function in inflammatory pain conditions. These contrasting results between neuropathic and inflammatory pain may suggest that, even if the amygdala hyperactivity can be observed in different pain models, differential changes in functionally distinct subsets of mPFC neurons in inflammatory versus neuropathic pain can be observed. In


conclusion, we believe that the present data will help us understand the cross-talk between the amygdala and mPFC, and the roles of mGluR1 in the BLA for the functional changes of amygdalocortical interactions within the framework of the pain-related neuronal affective mechanisms. [1] Metz, A.E, et al. Proc. Natl. Acad. Sci. U.S.A., 2009, 106(7),2423-8. [2] de Novellis, V, et al. Mol. Pain, 2011, 17, 7.

108.Increased Trait Anxiety in Infancy Caused by Maternal Stress J. Mairesse1,2,*, C. Laloux1,2,*, G. Van Camp1, M.L. Reynaert1, J. Marrocco1, H. Bouwalerh1, A. Giovine2, I. Branchi3, S.G. Bouret4,5, S. Morley-Fletcher1, G.E. Bergonzelli6, M. Malagodi1, R. Gradini2,7, F. Nicoletti2,7, M. Darnaudéry8,# and S. Maccari1,# 1

Neuroplasticity Team, UMR 8576 CNRS, North University of Lille, Villeneuve d’Ascq, France; 2.Department of Human Physiology and Pharmacology, University of Rome “La Sapienza”, Rome, Italy; 3 Dept. Cell Biology and Neuroscience, Istituto Superiore di Sanità, Rome, Italy; 4Inserm, U837, Jean-Pierre Aubert Research Center, Development and plasticity of the postnatal brain, Lille, France; 5 The Saban Research Institute, Neuroscience Program, Childrens Hospital of Los Angeles, University of Southern California, Los Angeles, California, USA; 6Nutrition and Health, Nestlé Research Center, Lausanne, Switzerland 7Istituto Neurologico Mediterraneo Neuromed, Pozzilli, Italy; 8INRA UMR 1286 and CNRS UMR 5226, University of Bordeaux, Bordeaux, France Background: Rats exposed to prenatal restraint stress (“PRS rats”) represent a valuable model for the study of the interplay between environmental triggers and neurodevelopment in the pathogenesis of anxious/depressive disorders. It is unclear when the pathological phenotype becomes expressed during ontogenesis of PRS rats. Methods: Repeated episodes of restraint stress were delivered to the dams during pregnancy. Ultrasonic vocalization (USV) was assessed under different behavioural paradigms, i.e. isolation, “maternal potentiation”, and exposure to an unfamiliar male odour (UMO). Anxiety in mothers was measured by conventional tests. Maternal behaviour was scored in the first 8 post-partum days (PPDs). Expression of GABAA receptor subunits and metabotropic glutamate (mGlu) receptors was assessed by immunoblotting. Results: PRS reduced maternal behaviour in the first 6 PPDs. The offspring of stressed dams emitted more USVs in response to isolation, and did not show the phenomenon of “maternal potentiation”, i.e. the increase in USV in response to maternal reunion after isolation. USV suppression in response to UMO persisted beyond postnatal day-10 (PND10) only in PRS rats. Infant PRS rats also showed a reduced expression of the 2 subunit of GABAA receptors and an increased expression of mGlu5 receptors in the amygdala, and lacked the developmental peak in blood leptin levels at PND14. Conclusions: These data offer a clear-cut demonstration that the epigenetic programming triggered by PRS is already translated into an “anxious” phenotype during early postnatal life, thus supporting the causal relationship between gestational stress and emotional liability in infants.

109.One Step Towards Understanding Metabotropic Glutamate Receptor Kinetics P. Marcaggi1,2, H. Mutoh2, D. Dimitrov2, M. Beato1 and T. Knöpfel2 1 Dept of Neuroscience, Physiology and Pharmacology, University College London, Gower Street, London, WC1E 6BT, UK; 2 Laboratory for Neuronal Circuit Dynamics, Brain Science Institute, RIKEN, Saitama, Japan


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Metabotropic glutamate receptors (mGluRs) can be expressed in the vicinity of excitatory synapses where extracellular glutamate transients follow sub-millisecond temporal patterns. The dependency of mGluR activation on the temporal properties of glutamate exposure will depend on mGluR kinetics. The effects of mGluR activation, downstream of G-protein mediated transduction, are delayed and amplified, impairing mGluR kinetics analysis. Here we report an attempt to determine mGluR kinetics after insertion of fluorescent proteins enabling real-time monitoring of conformational changes by fluorescence energy transfer (FRET). We focused on type 1 mGluR which is expressed at synapses and mediate synaptic plasticity. Structural studies have predicted a rearrangement of mGluR dimers following glutamate binding. Assuming that the rearranged dimer equates the active state of the receptor, we set up to monitor this rearrangement in real time. Cyan and yellow fluorescent proteins (CFP and YFP) were inserted in the second intracellular loop of mGluR1 subunits. When expressed in PC12 cells, these constructs showed clear expression at the cell membrane and FRET was observed as expected from dimers containing both CFP and YFP. Consistent with a conformational rearrangement of mGluR1 dimer upon glutamate binding, transient glutamate exposure produced a transient FRET increase, which had an EC50 of 7.5μM, was mimicked by the agonist DHPG, abolished by the competitive antagonist MCPG and partially inhibited by mGluR1-selective allosteric modulators. Thus, we hypothesize that the FRET increase reflects the conformational change to the active state of the receptor. When glutamate was applied at increasing concentrations up to 2mM, the activation time course decreased to a minimum of ~10ms, a time course similar to the solution exchange achieved at the cell membrane, and faster than previously expected. In contrast, the deactivation time course remained constant at ~50ms. During long lasting applications, no desensitization was observed. Instead, a sensitization developed over ~400ms. Activation, deactivation and sensitization time courses and amplitudes were used to derive a kinetic scheme and rate constants, from which we inferred the EC50 and frequency dependence of mGluR1 activation in non steady-state conditions, as occurs during synaptic transmission. This work was supported by RIKEN intramural grants (TK), a MRC Career Development Award and a Royal Society travel grant (PM).

110.mGluR2/3 Activation Decreases Drug Seeking and Taking by Impacting Primarily the Conditioned Behavioral and Neurochemical Effects of Drugs of Abuse A. Markou1, M. D’Souza1, S. Semenova1, M. Liechti1, D.J. Sheffler2, P.J. Conn2, R. Kuczenski1, and N.D.P. Cosford3 1

Department of Psychiatry, School of Medicine, University of California San Diego, La Jolla, CA 92093, U.S.A.; 2Department of Pharmacology and Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, Nashville, TN 37232, U.S.A.; 3Program on Apoptosis and Cell Death Research, The Sanford-Burnham Institute for Medical Research, La Jolla, CA 92037, U.S.A The motivation to maintain drug seeking, self-administration, and dependence involves alterations in glutamatergic neurotransmission. Metabotropic glutamate receptors 2/3 (mGlu2/3) regulate glutamate and dopamine release in the ventral tegmental area (VTA) and the nucleus accumbens (NAc) shell, two brain areas critically involved in reward and motivation. Acute systemic, as well as intra-VTA or intra-NAc, administration of the mGlu2/3 agonist LY379268 decreased nicotine, but not food, self-administration in rats [1]. In addition, nicotine self-administration downregulated mGlu2/3 receptor function in corticolimbic rat brain sites including the VTA and the NAc, demonstrated by decreased coupling of mGlu2/3


receptors to G proteins in the GTP35S binding assay [1]. Interestingly, LY379268 administration decreased both cue-induced reinstatement of nicotine and food seeking [1], indicating that mGlu2/3 receptors modulate conditioned behavioral responses to reinforcer-associated stimuli independent of whether the reinforcer was a drug or food. To further understand the role mGlu2/3 receptors on the primary rewarding and conditioned effects of drugs of abuse, we compared the effects of the relatively selective mGlu2 positive allosteric modulator (PAM) potassium 30-([2-cyclopentyl-6-7dimethyl-1-oxo-2,3-dihydro-1H-inden-5yloxy]methyl)biphenyll-4 carboxylate (BINA) and the mGlu2/3 agonist LY379268 on cocaine self-administration and seeking in rats [2]. BINA decreased cocaine self-administration with no effect on food self-administration, while LY379268 non-selectively decreased both cocaine and food selfadministration. Further, BINA decreased cue-induced reinstatement of cocaine seeking with no effect on food seeking. The higher selectivity of BINA, compared to LY379268, for drug- versus foodmotivated behaviors suggests that positive allosteric modulation of mGlu2 selectively influences the increased glutamate transmission induced by drugs of abuse and their conditioned effects and not the effects of natural reinforcers that are likely to induce only small increases in glutamate transmission. To understand the neurochemical mechanisms that mediate the effects of mGlu2/3 activation on drug-seeking and taking, the effects of LY379268 on nicotine-induced increases in the NAc shell dopamine were assessed using in vivo microdialysis [3]. Interestingly, LY379268 pretreatment blocked nicotine-induced increases in NAc shell dopamine only when tested in the presence of a nicotine-associated context. By contrast, LY379268 did not block nicotine-induced increases in NAc shell dopamine in nicotine-naive or nicotineexperienced rats tested in the absence of a nicotine-associated context. These intriguing findings suggest an important role for mGlu2/3 receptors in regulating the combined effects of nicotine and nicotine-associated contexts/cues on NAc dopamine. Taken together, these findings indicate an important role for mGlu2/3 receptors in limbic areas, such as the VTA and the NAc shell, in modulating the direct pharmacological effects of psychostimulant drugs, such as nicotine and cocaine, as well as the conditioned behavioral and neurochemical effects of contexts/stimuli associated with drugs of abuse. [1] Liechti, M.E, et al. J. Neurosci., 2007, 27, 9077-9085. [2] Jin, X, et al. Neuropsychopharmacology, 2010, 235, 2021-2036. [3] D’Souza, M, et al. Neuropsychopharmacology, 2011, In press.

111.Neuroadaptation Induced by Early Life Stress in the Nigrostriatal Motor Circuit: Prenatal Restraint Stress as a Model of “Compensated” Parkinsonism J. Marrocco1, T. Cassano2, G. Van Camp1, J. Mairesse1, M-L. Reynaert1, H. Bouwalerh1, J-C. Michalski3, G. Battaglia4, L. Lionetto7, M. Simmaco7, A. Pittaluga5, M. Cannella4, M. Motolese4, F. Nicoletti4,6, S. Morley-Fletcher1 and S. Maccari1 1

Neural Plasticity Team - UMR CNRS/USTL n° 8576 Structural and Functional Glycobiology Unit, North University of Lille FR; 2 Dept. of Biomedical Sciences, Medical School, University of Foggia, IT; 3Cell Signaling Team-UMR CNRS/USTL n° 8576 FR; 4 I.N.M. Neuromed, Pozzilli, IT; 5Dept. of Experimental Medicine, University of Genova, IT; 6Dept. of Pharmacology and 7DESMO, Sapienza University of Rome IT6 We used adult rats subjected to prenatal restraint stress (PRS rats) to examine the impact of early life stress on the function of the basal ganglia motor circuit in the adult life. Male adult PRS rats (57 months of age) were highly resistant to haloperidol-induced catalepsy and showed an increase in oral stereotypies induced by apomorphine. At least at 30 min after drug injection (2 mg/kg, i.p.)


haloperidol levels in the plasma and striatum did not differ between control and PRS rats, suggesting that the reduction in haloperidolinduced catalepsy in PRS rats depended on abnormalities of the basal ganglia motor circuit. PRS rats showed no changes in the transcripts of mGlu4 receptors, mGlu5 receptors, CB1 receptors, and preproenkephalin A, and no changes in the mGlu5 receptor levels in the striatum. However, PRS rats showed a significant reduction in striatal A2A adenosine receptors. In addition, these rats showed a reduction in depolarization-induced dopamine release in striatal superfused striatal synaptosomes (with no changes in glutamate and aspartate release), associated with increases in tyrosine hydroxylase expression and dopamine/DOPAC/HVA levels in the striatum. There were no changes in the expression of the dopamine transporter, DAT. We conclude that early life stress causes neuroadaptive changes in the A2A-mediated control of the indirect pathway of the basal ganglia motor circuit that make these animals resistant to haloperidol-induced catalepsy. It will be interesting to examine whether early life stress influences the prevalence of extrapyramidal side effects in patients treated with classical antipsychotic drugs.

112.mGluR1 Positive Melanoma-Initiating Cells Show Drug Resistance Ability E. Mastrantoni1, D. Melchiorri1 and S. Chen2 1

Dept. of Human Physiology and Pharmacology, University of Rome “Sapienza”, Rome, Italy; 2Dept. of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ, USA Several studies have demonstrated the central role of glutamate signaling in cancer. Results from previous studies suggest the ectopic expression of the GRM1 gene is sufficient to transform melanocytes in vitro and cause malignant melanoma in vivo (Pollock et al. 2003). In addition, it has been documented that the GRM1 gene is aberrantly expressed in about 60% human melanomas cell lines and biopsies (Pollock et al. 2003, Ortiz P. 2007). We were able to isolate a drug-resistant slow cycling cell population from the mGluR1-positive metastatic human melanoma cell line C8161, using the spheres formation assay. We isolated melanoma cells capable of surviving and proliferating as nonadherent spheres, similar to the neurospheres and mammospheres generated from brain and breast cancer tumors, respectively. Expression analysis for stemness markers (Nestin, CD133) did not show any difference between the C8161-derived spheres and the original monolayer culture. The spheres showed higher tumorigenicity in vivo, when implanted s.c. in nude mice at 104 cells/site, a cell number that normally does not give rise to tumor growth. Cytofluorimetric analysis showed that, compared to monolayer cultures, a larger percentage of the C8161-derived melanoma sphere cells (65%) are in G1 phase of the cell cycle, a typical feature of stem cells. The slow-cycling status of melanoma sphere cells was further confirmed by Western blot analysis showing higher expression levels of p27 and lower expression levels of MITF, the Microphthalmia-associated transcription factor that is known to induce a p27Kip1-dependent G1 arrest. C8161derived melanoma spheres showed a drug resistant phenotype as opposed to the adherent monolayer cells. We tested commonly used chemotherapeutic drugs (etoposide, temozolomide) and riluzole (a glutamate release inhibitor currently in phase II clinical trial in advanced melanoma patients). In the presence of these drugs, C8161-derived melanoma spheres consistently displayed an increased resistance with higher survival rates than monolayer cultures treated under the same conditions. Our data demonstrate that these spheroid aggregates are composed of cells which features that are associated with the typical properties of cancer stem cells, such as expression of stem cell markers, tumor-initiating potential in vivo, and drug resistance. Given the central role of mGluR1 and


glutamate in melanoma cell biology, we assessed mGluR1 expression in this subset of C8161-derived cells. Western blot analysis at different in vitro cell passages showed persistent mGluR1 expression. However, this cell population did not show enhanced sensitivity when challenged with the mGluR1 receptor antagonist BAY36-7620, as opposed to the increased cell death observed in the original monolayer cultured cells. This specific subset of C8161-derived cells represents an interesting model for the study of the interactions between mGluR1 receptor and chemoresistance in putative melanoma stem cells.

113.Group-II mGlu Receptors and Schizophrenia-Related Genes

Epigenetics of

Francesco Matriscianoa,b, Patricia Tuetinga, Stefania Maccarid, Ferdinando Nicolettib,c, and Alessandro Guidottia a

The Psychiatric Institute, Department of Psychiatry, College of Medicine, The University of Illinois at Chicago, Chicago, IL 60612, USA; bDepartment of Physiology and Pharmacology, University of Rome “ Sapienza”, Piazzale Aldo Moro, 5, 00185 Rome, Italy; c I.N.M. Neuromed, Pozzilli, Italy; dNeuroplasticity Team – CNRS UMR 8576/UGSF, North University of Lille1, France Altered epigenetic mechanisms contribute to the pathogenesis of schizophrenia and currently used medications exert chromatin remodeling effects. We have shown recently that pharmacological activation of mGlu2/3 receptors increases the expression of Gadd45-, a molecular player of DNA demethylation, in the mouse frontal cortex and hippocampus, as well as the amount of Gadd45- bound to specific promoter regions of reelin, brain-derived neurotrophic factor (BDNF), and glutamate decarboxylase-67 (GAD67). In addition, the mGlu2/3 receptor agonist, LY379268, mimics the action of clozapine and valproate in reducing cytosine methylation in the promoter of the three genes [1] Here, we extended the study to a mouse model of early life stress. Repeated episodes of restraint stress delivered to the mothers during pregnancy cause neuroadaptive mechanisms in the offspring, which appear to be the consequence of a pathological epigenetic programming. We found that the offspring of pregnant mice exposed to restraint stress (named “PRS mice”) developed a schizophrenia-like phenotype, characterized by a decreased expression of brain derived neurotrophic factor (BDNF) and glutamic acid decarboxylase (GAD)-67, an increased expression of type-1 DNA methyl transferase (DNMT1) in the frontal cortex, and a deficit in social interaction, locomotor activity, and prepulse inhibition. PRS mice also showed a decreased expression of mGlu2 and mGlu3 receptors in the frontal cortex, which was manifested at birth and persisted in adult life. This decrease was associated with an increased binding of DNMT1 to CpG-rich regions of mGlu2 and mGlu3 receptor promoters and an increased binding of MeCP2 to the mGlu2 receptor promoter. Systemic treatment with LY379268 (0.5 mg/kg, i.p., twice daily for five days), corrected all the biochemical and behavioral abnormalities shown in PRS mice. Our data suggest that epigenetic changes in mGlu2 and mGlu3 receptors lie at the core of the pathological programming induced by early life stress, and reinforce the view that mGlu2/3 receptor agonists may act as “epigenetic drugs” in the treatment of schizophrenia


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H. Rudykb, B. Clarkb, C. Beadleb, D. Tupperb, S. Bakerb, J. Haoa, M. Reinharda, S. Henrya, S. Masseya, L. Taboadac, C. Lamasc, C. Dominguezc, C. Monteroc, A. Marcosc, J. Blancoc, S. Atwelld, D. Thompsond, S. Emtaged, F. Lud, S. Swansona, D. Bedwella, A. Paka and L. Prietoc Lilly Research Laboratories, Eli Lilly and Company, aIndianapolis, IN, USA, bWindlesham, UK, cAlcobendas, Spain, dSan Diego, CA, USA LY2812223 is a functionally selective orthosteric mGlu2 receptor agonist (see Monn et al.; Prieto et al.; Heinz et al.; Felder et al.; this meeting, for in vitro characterization). LY2812223 was tested in rodent assays predictive of clinical antipsychotic efficacy, such as reversal of phencyclidine- and amphetamine-induced locomotor activity, inhibition of conditioned avoidance responding (CAR), and stimulation of monoamine efflux in rat prefrontal cortex. LY2812223 was also assessed for anxiolytic- and antidepressantlike activity in stress-induced hyperthermia and forced swim test assays. Finally, LY2812223 was profiled in the rat formalininduced pain assay. LY2812223 attenuated phencyclidine (PCP)induced activity potently in both rats and mice with ED50 values of 1.2 mg/kg and 4.3 mg/kg, respectively. Similarly, amphetamineinduced activity was dose-dependently reversed by LY2812223 in rats (2.6 mg/kg) and mice (12.4 mg/kg). LY2812223 exhibited an atypical antipsychotic-like profile in a mouse CAR paradigm, significantly reducing avoidance responses following a 10 mg/kg dose without the presence of escape failures at 30 mg/kg which inhibited avoidance responses by almost 80%. Consistent with literature mGlu2/3 agonists, LY2812223 significantly attenuated formalin-evoked pain at 3 mg/kg and stress-induced hyperthermia in rats from 30-82% within a dose range of 1-10 mg/kg. However, unlike previous reports with balanced mGlu2/3 agonists, LY2812223 significantly reduced immobility in the forced swim test at 10 mg/kg, indicating an antidepressant-like profile; activity of LY2812223 was lost in mGlu2 knockout mice. To increase bioavailability, a prodrug (LY2979165.HCl) was made to improve absorption across the intestine. Oral LY2979165.HCl (dosed as active equivalents of LY2812223) produced effects consistent with systemic LY2812223 administration: ED50 of 2.9 mg/kg in blocking PCP activity; significantly reducing stress-induced hyperthermia (10 mg/kg) and immobility (3 mg/kg) in the rat forced swim test; and significantly increasing monoamine release in rat prefrontal cortex. The in vivo pharmacological profile of LY2812223 and its prodrug LY2979165.HCl confirms the broad efficacy reported with literature mGlu2/3 agonists in efficacy models of psychosis, pain, and anxiety. However, the antidepressant-like efficacy profile of LY2812223/LY2979165.HCl differentiates it from other orthosteric agonist compounds of this class.

115.Behavioral and Neurochemical Pharmacology of a Novel Group II Metabotropic Glutamate Receptor Agonist and its Prodrug D. McKinziea, B. Johnsona, K. Knitowskia, D. Shawa, L. Rorick-Kehna, J. Katnera, K. Perrya, M. Johnsona, L. Prietob and J. Monna Lilly Research Laboratories, Eli Lilly & Company, aIndianapolis, IN, USA, bAlcobendas, Spain

[1] Matrisciano, et al. Mol. Pharmacol., 2011.

114.In Vivo Pharmacological Characterization of a Novel Group II Metabotropic Glutamate Receptor Agonist and its Oral Prodrug D. McKinziea, J. Monna, B. Johnsona, J.M. Witkina, K. Knitowskia, D. Shawa, L. Rorick-Kehna, J. Katnera, K. Perrya, X. Lia, C. Overshinera, L. Waltonb, T. Manb,

Balanced metabotropic glutamate receptor-2/3 (mGlu2/3) orthosteric agonists (e.g., LY354740, LY379268, LY404039, MGS0028) have been studied extensively in preclinical paradigms and exhibit a broad range of activity in animal models predictive of therapeutic benefit for psychiatric and neurological disorders. For instance, mGlu2/3 agonists are consistently reported to have an antipsychotic-like, anxiolytic-like, analgesic, and anti-addictive properties. This poster provides in vivo characterization of the


selective orthosteric mGlu2 agonist LY2812223, and its prodrug LY2979165, in pharmacology models relevant to psychosis and pain. For the behavioral pharmacology of LY2812223 in models of anxiety and depression, see Witkin et al (poster this meeting). See Heinz et al and Felder et al (this meeting) for the in vitro characterization of LY2812223. Systemic dosing of LY2812223 potently attenuated increased activity induced by phencyclidine (1.2 mg/kg ED50 in rats; 4.3 mg/kg ED50 in mice) and amphetamine (2.6 mg/kg ED50 in rats; 12.4 mg/kg ED50 in mice). The ability of LY2812223 to reverse psychostimulant activity was blocked by the mGlu2/3 antagonist LY341495. In the mouse Conditioned Avoidance Response (CAR) assay, LY2812223 significantly and selectively (i.e., no escape failures observed) reduced avoidance behavior following a 10 mg/kg dose, with almost an 80% reduction in avoidance responses at 30 mg/kg. In the rat formalin model of nociception, LY2812223 significantly reduced pain-associated behaviors by around 75% at 3 and 10 mg/kg doses. Oral administration of the LY2979165.HCl prodrug was effectively absorbed and hydrolysized into the active compound (see Prieto et al., this meeting). LY2979165.HCl (dosed as active equivalents of LY2812223) produced comparable efficacy in blocking phencyclidine-induced hyperactivity in rats (2.9 mg/kg ED50). Consistent with clinically-active atypical antipsychotic agents, LY2979165.HCl significantly induced prefrontal cortical monoamine efflux following 10 mg/kg (dopamine 150% of baseline; norepinephrine 130% of baseline) and 30 mg/kg (dopamine 250% of baseline; norepinephrine 140% of baseline; serotonin 175% of baseline). Literature reports characterizing the effects of balanced orthosteric mGlu2/3 agonists in mice lacking mGlu2 or mGlu3 receptors indicate that the mGlu2 receptor is critical for maintaining efficacy in preclinical models of psychosis and pain. These data show that a functionally selective mGlu2 agonist exhibits a pharmacological profile that is comparable to that of balanced mGlu2/3 agonists.

116.Use of Pharmacodynamic Measures to Demonstrate Functional Central Activity of a Novel Group II Metabotropic Glutamate Receptor Agonist Prodrug D. McKinziea, B. Johnsona, L. Thompsona, W. Seidelb, D.M. Edgarb, K. Waffordb, B. Gitterd, J. Hornd, J. Petersd, J. Meyerd, P. Territoe, L. Prietoc and J. Monna a

Lilly Research Laboratories, Eli Lilly & Company, Indianapolis, IN, USA; bWindlesham, UK; cAlcobendas, Spain; dNeuroscience & Nuclear Medicine, Discovery & Translational Services, Covance Laboratories, Greenfield, IN, USA; eDepartment of Radiology, Indiana University School of Medicine, Indianapolis, IN, USA To date, central target engagement determination of orthosteric metabotropic glutamate (mGlu) Group II receptors has proven challenging due to a lack of selective radiotracers for use in PET methodologies. The present studies used pharmacodynamic preclinical models of functional central activity to assess effects of the selective mGlu2 agonist LY2812223 or its prodrug LY2979165.HCl. Compound was evaluated in three rat assays believed to be mediated by central mechanisms: 1) induction of monoamine turnover in cerebrospinal fluid (CSF), 2) changes in sleep electroencephalogram (sEEG) patterns, and 3) effects upon basal and ketamine-evoked metabolic activity using 2deoxyglucose (2-DG) autoradiography. Both the active compound LY2812223 (ip. route; 1, 3, and 10 mg/kg doses; 60 min pretreatment) and the prodrug LY2979165.HCl (oral route; 3, 10, and 30 mg/kg active equivalent doses; 120 min pretreatment) were administered and CSF withdrawn from the cisterna magna of rats and dopamine (DOPAC & HVA), serotonin (5-HIAA), and norepinephrine (MHPG) metabolites analyzed. LY2812223 produced dose-related increases in dopamine turnover: DOPAC


levels significantly increased by 165% and 312% (from vehicle control levels) at 3 and 10 mg/kg; HVA levels increased by 134% and 234% at 3 and 10 mg/kg. 5-HIAA levels did not significantly change and MHPG was increased by 136% at the 10 mg/kg dose. The prodrug (LY2979165.HCl) produced similar increases in DOPAC (124% and 183% at 10 and 30 mg/kg, respectively) and HVA (147% and 193% at 10 and 30 mg/kg, respectively). MHPG levels were not altered by LY2979165.HCl and 5-HIAA increased by 136% only at the 30 mg/kg dose. DOPAC and HVA changes in CSF correlated (r2 values > 0.60) with plasma active compound levels.Oral LY2979165.HCl (1, 3, and 10 mg/kg) impacted various sEEG parameters, 5 hours into the light phase of a 12:12 light:dark cycle. The 10 mg/kg LY2979165.HCl dose significantly inhibited REM sleep over the remaining 7-hour light phase without impacting NREM significantly. EEG delta power during NREM sleep showed a nonsignificant tendency to increase following 10 mg/kg. Oral LY2979165.HCl (3, 10, and 30 mg/kg; 120 min pretreatment) attenuated increased neural metabolism induced by 25 mg/kg ketamine (i.v.) in brain regions thought to be dysregulated in schizophrenia. For instance, LY2979165.HCl reduced ketamine-evoked local cerebral glucose utilization (LCGU) by 15% (10 mg/kg) and 35% (30 mg/kg) in the prefrontal cortex; by 18% (10 mg/kg) and 38% (30 mg/kg) in the cingulate cortex; and, 8% (10 mg/kg) and 27% (30 mg/kg) in the N. Accumbens. These results indicate that the oral prodrug LY2979165.HCl produces consistent pharmacodynamic effects in preclinical assays thought to be mediated by central mechanisms.

117.Role of mGluR8 in Hippocampal Synaptic Transmission and Plasticity M. Mercier, Z. Bortolotto, G. Collingridge and D. Lodge School of Physiology and Pharmacology, University of Bristol, Medical Sciences Building, University Walk, Bristol, BS8 1TD, UK Metabotropic glutamate receptors (mGluRs) are classified into three groups (I-III) based on sequence homology, transduction mechanisms and pharmacological profiles. Group III mGluRs (mGluR4/6/7/8) have received far less attention than either groups I (mGluR1/5) or II (mGluR2/3), in part due to the paucity of selective pharmacological tools available for these receptors. However, important advances have recently been made into the development of group III mGluR subtype-selective compounds, enabling more targeted research into the functional roles and therapeutic potential of these receptors, which may be involved in a variety of neurological conditions including anxiety and epilepsy. One such compound is (S)-3,4-dicarboxyphenylglycine ((S)-3,4DCPG), identified as an mGluR8-selective agonist, along with its -methyl analogue (RS)--methyl-3,4-dicarboxyphenylglycine (3,4MDCPG), a suggested mGluR8 antagonist [1]. The effects of (S)3,4-DCPG were originally characterised in the rat spinal cord, and found to induce a dose-dependent depression of excitatory transmission, consistent with the known role of group III mGluRs as presynaptic autoreceptors. Here, we sought to further characterise the actions of both (S)-3,4-DCPG and 3,4-MDCPG in the lateral perforant path (LPP) input to the dentate gyrus, a hippocampal pathway known to express high levels of mGluR8 [2], in order to gain a better understanding of the role of this receptor in hippocampal synaptic transmission. Using field recordings in rat hippocampal slices, we show a dose-dependent (S)-3,4-DCPGinduced depression of field excitatory postsynaptic potentials (fEPSPs) in the lateral but not the medial component of the perforant path. The (S)-3,4-DCPG concentration-response curve in the LPP is biphasic, consistent with previous findings [1], with EC50 values of 0.2±2.2 M and 9.7±3.3 M for the high and low affinity components, respectively. Only the high affinity component was antagonised by the mGluR8-selective antagonist 3,4-MDCPG. Consistent with this, hippocampal slices from mGluR8 knock-out (-/-)


mice revealed that only the high affinity component of the curve is selective for mGluR8, with the lower affinity component remaining in mGluR8-/- animals. Using concentrations of (S)-3,4DCPG and 3,4-MDCPG identified as mGluR8-selective in these studies, we explored the role of this receptor in hippocampal synaptic plasticity, in particular long-term depression (LTD), and thus aim to further our understanding of the functional role of this group III mGluR. [1] Thomas, N.K, et al. Neuropharmacology, 2001, 40(3), 311-318. [2] Shigemoto, R, et al. J. Neurosci., 1997, 17(19), 7503–7522.

118.Progress Towards a Structural Understanding of Selective Activation of mGlu3 over mGlu2 Receptors E.J. Miller1, J. DiRaddo2, S. Pshenichkin2, B. Wroblewska2, E. Grajkowska2, J.P. Snyder1, D.C. Liotta1 and J.T. Wroblewski2 1

Department of Chemistry, Emory University, Atlanta, GA; Department of Pharmacology, Georgetown University, Washington, D.C 20057, USA 2

Within the past decade, metabotropic glutamate receptors have been demonstrated as prospective drug targets for the treatment of Parkinson’s Disease. In particular, stimulation of group II mGluRs in rodent models of PD has resulted in the inhibition of dopaminergic neurodegeneration. The relative therapeutic value of targeting mGlu2 vs. mGlu3 receptors, however, remains to be determined. Discovery of group II mGluR subtype-selective ligands will potentially facilitate data generation necessary to answer this question. Here we report N-acetylaspartylglutamate (NAAG) as an agonist selective for mGlu3 over mGlu2 receptors, and 2hydroxymethylglutamate (HMG) as an mGlu3 agonist and mGlu2 antagonist. In attempts to understand the structural interactions that dictate differential receptor activation, computational modeling has been employed. Docking HMG and NAAG into the crystal structure of the mGlu3 receptor, followed by amino acid residue energy minimization resulted in the determination of particularly important ligand-residue interactions. Comparison of these models to that of the glutamate-bound receptor illustrates divergent binding site interactions, and further allows preliminary hypothesis formulation concerning differential ligand binding at mGlu3 and mGlu2 receptors. Incorporation of sequence analysis and directed mutagenesis will provide additional information towards the elucidation of these intricacies. Greater understanding of the structural basis for selective stimulation of mGlu3 over mGlu2 receptors might allow the determination of a preferred metabotropic drug target, and moreover could potentially fuel the development of novel ligands that demonstrate greater potency and selectivity, perhaps leading to the discovery of new small molecule therapeutics for treatment of Parkinson’s Disease. Supported partially by NIH grant NS37436.

119.Binding Mode of a Novel MPEP-Derived Metabotropic Glutamate Receptor Subtype 5 (mGluR5) NAM, 1,3-Bis(pyridin-2-ylethynyl)Benzene C. Mølck1,2,*, K. Harpsøe1, D.E. Gloriam1, B.H. Kaae1, H.N. Jimenez3, M.A. Uberti3, S. Topiol3, R.P. Clausen1, U. Madsen1, S.M. Nielsen2, J.M. Mathiesen1,2 and H. Bräuner-Osborne1 1 Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Fruebjergvej 3 and Universitetsparken 2, 2100 Copenhagen, Denmark 2 Department of Molecular and Cellular Pharmacology (884), H. Lundbeck A/S, Ottiliavej 9, 2500 Valby, Denmark 3 Lundbeck Research USA, Inc., 215 College Road, Paramus, New Jersey 07652- 1431, USA*[email protected]


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Metabotropic glutamate receptor (mGluR) subtype 5 is a potential drug target in diseases such as depression, anxiety, schizophrenia and neurodegenerative disease. Lack of subtype-selective orthosteric ligands for mGluRs has lead to an increased interest for allosteric ligands as drug candidates. In this study the binding sites of 3 novel MPEP-derived negative allosteric modulators (NAMs) have been characterized. The NAMs are structurally very similar all being 1,3-bis(pyridinylethynyl) benzenes. Competitive binding studies confirmed that the binding sites of all 3 NAMs overlapped with the MPEP binding site in the 7TM domain of mGluR5. The position of the N-atoms in the pyridyl rings significantly affected potency (app. 24-fold), 1,3-bis(pyridin-2-ylethynyl)benzene being the most potent NAM with an IC50 value in the nanomolar range. Non-rhodopsin multi-template homology modelling, site-directed mutagenesis and a functional Ca2+ mobilization assay were used to determine the binding mode of 1,3-bis(pyridin-2-ylethynyl) benzene. The residues I651 (TM3), Y659 (TM3), N747 (TM5) and S809 (TM7) were found to have central roles in defining the ligand site. Mutational analysis suggested that the higher potency of 1,3bis(pyridin-2-ylethynyl)benzene was due to hydrogen bond interactions with S809 as the mutation S809A made 1,3bis(pyridin-2- ylethynyl)benzene equipotent to the 2 other NAMs (IC50: 1 – 2.5 μM). MPEP was also included in the mutational analysis for comparison with binding modes of the novel MPEPderived NAMs. The binding mode of MPEP resembled 1,3bis(pyridin- 2-ylethynyl)benzene with S809 being an important hydrogen bond donor for MPEP. There were however also mutations with differential effects on MPEP and 1,3- bis(pyridin-2ylethynyl)benzene indicating differences in binding modes, one difference being an additional hydrogen bond interaction of 1,3bis(pyridin-2-ylethynyl)benzene with N747.

120. Enhanced mGlu5-Receptor dependent LTD in the Hippocampus of a Mouse model of Angelman Syndrome G. Molinaro1, S. Piccinin2,3, M. Pignatelli2,3, I. Panaccione2,3, L. Di Menna1, B. Riozzi1, M. Bernabucci1, L. Di Nuzzo1, M. Cannella1, V. Bruno1,2, F. Nicoletti1,2 and R. Nisticò3,4 1 INM Neuromed, Pozzilli , Italy; 2Department of Physiology and Pharmacology, University “Sapienza”, Rome, Italy; 3IRCSS S Lucia Foundation, Rome , Italy; 4Department of Pharmacobiology, University of Calabria, Rende, Italy

Angelman syndrome (AS) is a genetic disorder caused by loss of function mutations or deletions of the Ube3A gene, that is maternally expressed in almost all neurons. Patients with AS are characterized by motor dysfunction, ataxia, mental retardation, seizures, and often exhibit socialization and communication deficits that meet the diagnostic criteria for autism. Data suggest that Ube3A may impact synaptic connectivity, function, and plasticity through the ubiquitination and degradation of a number of synaptic protein including Ephexin-5 and Arc, which results in deficits in excitatory synapse formation and leads to excessive endocytosis of GluA1-containing AMPARs. We examined group-I mGlu receptordependent LTD at the Schaffer collateral/CA1 pyramidal cell synapses of mice lacking maternally expressed Ube3A. LTD induced by the mGlu1/5 receptor agonist, DHPG, was significantly enhanced in Ube3A-lacking mice, whereas LTD induced by low frequency stimulation (i.e. NMDA-dependent LTD) was unchanged. In both control and mutant mice, DHPG-induced LTD was abolished by MPEP, as well as by the MAP kinase inhibitor, UO126, or the mTOR inhibitor, rapamycin. So far, we were unable to find changes in DHPG-stimulated polyphosoinositide hydrolysis, ERK1/2 phosphorylation, and CAMK-II phosphorylation, or in Arc levels in hippocampal slices prepared from PND30 Ube3A-lacking mice under the same conditions used for electrophysiological experiments. Thus, the molecular events leading to the amplified


mGlu5-receptor dependent LTD in Ube3A-lacking mice are still unknown. These data support the hypothesis that changes in mGlu5-receptor dependent synaptic plasticity are not exclusive to Fragile-X syndrome, but may extend to other forms of monogenic autism.

121.Discovery and in vitro Characterization of a Novel Group II Metabotropic Glutamate Receptor Agonist and its Oral Prodrug J. Monn1, D. McKinzie1, C. Felder1, B. Heinz1, X. Wang1, R. Vivier2, H. Sanger2, L. Walton2, T. Man2, H. Rudyk2, B. Clark2, C. Beadle2, D. Tupper2, S. Baker2, J. Hao1, M. Reinhard1, S. Henry1, S. Massey1, L. Taboada3, C. Lamas3, C. Dominguez3, C. Montero3, A. Marcos3, J. Blanco3, S. Atwell4, D. Thompson4, S. Emtage4, F. Lu4, S. Swanson1, D. Bedwell1, A. Pak1 and L. Prieto3 1

Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, USA, 2Windlesham, UK, 3Alcobendas, Spain, 4San Diego, CA, USA

As part of our ongoing efforts to identify novel orthosteric mGlu2/3 receptor agonists we have discovered compound LY2812223, a molecule which displays an in vitro pharmacologic profile distinct from previously described compounds in this class (see Heinz et al; Felder et al, this meeting). LY2812223 displaced high affinity [3H] 459477 binding in RGT cells expressing recombinant human mGlu2 (Ki = 151 nM) or mGlu3 (Ki = 92.7 nM) receptors. In whole cell functional assays, LY2812223 elicited potent hmGlu2 receptor-mediated agonist responses in two assay formats, namely the inhibition of forskolin-stimulated cAMP formation in RGT cells (EC50 = 5.57 nM) and the stimulation of Ca2+ fluorescence in RGT cells co-expressing Gq15 (EC50 = 23 nM). LY2812223 produced a potent inhibition of forskolin-stimulated cAMP in RGT cells expressing hmGlu3 receptors (EC50 = 133 nM), but with unanticipated submaximal efficacy (Emax = 57%). Furthermore, in RGT cells co-expressing hmGlu3 and G q15, LY2812223 produced no discernable agonist response at concentrations up to 250 μM. Cultured mouse cortical neurons can develop spontaneous Ca2+ oscillations that are NMDA antagonist and mGlu2/3 agonistsensitive. In neurons grown from mGlu3 (-/-) mice, LY2812223 elicited a potent (EC50 = 20.6 nM) and near maximal agonist response, whereas it produced only a slight inhibition of Ca2+ oscillations in cultured neurons derived from mGlu2 (-/-) mice. Interestingly, in cultures derived either from WT mice or rat, LY2812223 produced a potent (EC50 ~ 30 nM), yet only partial (Emax = 50 - 60%), blockade of Ca2+ oscillations, suggesting that this molecule may function as a partial agonist in cortical neurons. While orthosteric agonists for mGlu2/3 receptors have many favorable pharmacokinetic attributes, absorption following oral administration can be problematic. We have utilized a prodrug approach to overcome this potential liability (see Prieto et al, this meeting), resulting in the discovery of compound LY2979165. This molecule inhibits the intestinal proton-dependent dipeptide transporter PepT1-mediated transport of [14C]-GlySar uptake into HeLa cells and is effectively converted to LY2812223 upon incubation with human intestinal tissue in vitro. Collectively, the in vitro characteristics of LY2812223 appear to be unique compared to other mGlu2/3 receptor agonists described to date as this molecule appears to display considerable functional selectivity for mGlu2 receptors and to produce submaximal (~50%) agonist responses in cultured cortical neurons. These findings have encouraged us to assess the consequences of these pharmacological actions on biological effects in vivo. Results from these studies have revealed a unique in vivo profile for this molecule. In addition to producing potent antipsychotic and anxiolytic effects in rodents, LY2812223 also demonstrated an antidepressant-like behavioral


response that is not seen with previously disclosed mGlu2/3 agonists (see McKinzie et al; Witkin et al, this meeting).

122.Epigenetic Programming Induced by Prenatal Stress Leads to a Reduced Expression of Synaptic-Vesicle Associated Proteins and a Selective Impairment of Glutamate Release in the Adult Hippocampus S. Morley-Fletcher1, J. Marrocco1, J. Mairesse1, A. Pittaluga2, M. Summa2, R. Ngomba3, F. Nicoletti3,4 and S. Maccari1 1

Neural Plasticity Team UMRCNRS 8576 North Univ. of Lille, Villeneuve d'Ascq, FR; 2Dept Exptl. Medecine, Univ. of Genova, IT; 3 I.N.M. Neuromed, Pozzilli, IT; 4Dept. of Pharmacology, Sapienza University of Rome IT Both human and animal studies suggest that exposure to stressful events during critical periods of brain development triggers an epigenetic programming leading to low resilience to stress in the adult life. Abnormalities of synaptic transmission and plasticity in the hippocampus represent an integral part of this epigenetic program. Rats exposed to prenatal restraint stress (PRS) develop long-lasting biochemical and behavioral changes that comprise a dysfunction of the hypothalamo-pituitary-adrenal (HPA) axis, a generalized disorganization of circadian rhythms and the sleepwake cycle, an age-dependent impairment in spatial learning, and a lifelong reduction of hippocampal neurogenesis. Some of these changes are reversed by chronic antidepressant treatment. We report here that PRS rats show a reduction in hippocampal levels of synapsins, synaptophisin, and synaptobrevin, three proteins that regulate intracellular trafficking and membrane fusion of synaptic vesicles. Interestingly, these changes were associated with a substantial reduction in depolarization-evoked glutamate release from hippocampal synaptosomes, with no changes in GABA release. These observations indicate that PRS leads to enduring changes in glutamate release in the hippocampus and suggest that an impairment of glutamatergic transmission is a key component of the epigenetic program induced by early life stress.

123.The Selective mGluR2/3 Antagonist, LY341495, Enhances Chemosensitivity of Colon Cancer Stem Cells to Standard Chemotherapy P. Mosillo1, P. Spinsanti1, F. Serone1, F. Azzollini1, B. Turriziani1, L. Ricci-Vitiani2, F. Nicoletti1,3 and D. Melchiorri1,4 1

Dept. Physiology & Pharmacology, The University of Rome, “Sapienza”, 2I.S.S. Rome, Italy, 3I.R.C.C.S. Neuromed, Pozzilli, 4 I.R.C.C.S. San Raffaele Pisana, Rome The recent isolation of cancer stem cells from human bioptical specimens of colorectal cancer (CRC) has led to the hypothesis that CSCs are responsible of both CRC spreading and chemoresistance. CD133+-CSCs, derived from CRC patients, were cultured in vitro under non differentiating conditions and exposed to either 5fluorouracil (5-FU), the mainstay treatment for CRC, or irinotecan (IR), a drug with significant activity in advanced CRC. In addition, cisplatin (CS) a chemotherapic drug with reduced activity towards CRC, was tested as a negative control. Exposure of CSCs to different concentrations of 5-FU (0.05, 0.5 and 5 g/ml) or IR (0.5 or 5 g/ml) for 48h induced a dose-related cell death as assessed by MTT assay. No cell death was detected following CS treatment (0.5 or 5 g/ml). In the attempt to increase chemosensitivity of CSCs, we focused on metabotropic glutamate receptors (mGluRs), which were recently shown to be expressed by different tumors. Western blot and RT-PCR analysis showed that mGluR2/3 was the only mGluR subtype consistently expressed by cultured CD133+-CSCs.


We exposed CSCs to a combination of the selective mGluR2/3 agonist, LY379268 (1 M), the selective mGluR2/3 antagonist, LY341495 (1 M) and the three cytotoxic drugs. LY341495, which was ineffective by itself, significantly increased cell death if added in combination with either 5-FU and IR, but did not induce cell death combined with CS. No amplification of cell death was detected when LY379268 was combined with any of the three chemotherapic drugs. However, administration of LY379268 to CSCs treated with LY341495 and 5-FU or IR, reverted LY341495induced amplification of cell death, indicating the involvement of mGluR2/3 in the increased chemosensitivity elicited by LY341495. Type 2/3-mGluRs are coupled to activation of the phosphatidylinositide-3 kinase (PI-3K) pathway. Co-administration of the selective inhibitor of PI-3K activity, LY290042 (100 nM), with 5-FU led to an increase in cell death similar to what observed following the co-administration of LY341495 and 5-FU. No effect on cell death was observed when LY290042 was administred by itself. These results suggest that mGluR2/3 blockade enhances the toxic action of 5-FU through the inhibition of the PI-3K pathway. Increased expression of dihydrouracil dehydrogenase (DPD) is known to contribute to 5-FU resistance. Exposure of cells to LY341495 + 5-FU greatly reduced DPD expression as compared to treatment with 5-FU, suggesting that amplification of 5-FU-induced cell death by LY341495 is due, at least in part, to a decreased metabolism of 5-FU. To explore the molecular mechanisms underlying the reduced expression of DPD in cells treated with 5FU and LY341495, cultured CSCs were exposed to LY294002 in the presence or absence of 5-FU for 48h. LY294002, when combined with 5-FU decresead DPD levels similarly to what observed following co-administration of 5-FU and LY341495, suggesting that the decrease of DPD expression induced by the combination of 5-FU and LY341495 is mediated by the inhibition of the PI-3K pathway. These results were confirmed in cells transfected with AKT, the target of PI-3K. These data show that mGluRs2/3 are potential targets for innovative antineoplastic therapies aiming at overcoming resistance to chemotoxic drugs.

124.Scaffold Remodeling During Long Term Potentiation Enables Synaptic Glutamate Receptor Cross-Talk E. Moutin1,2,3, F. Raynaud1,2,3, J. Roger1,2,3, E. Pellegrino1,2,3, V. Homburger1,2,3, V. Ollendorff4, J. Bockaert1,2,3, L. Fagni1,2,3 and J. Perroy1,2,3 1 CNRS, UMR-5203, Institut de Génomique Fonctionnelle, F34000 Montpellier, France; 2INSERM, U661, F-34000 Montpellier, France; 3Universités de Montpellier 1 & 2, UMR-5203, F-34000 Montpellier, France; 4Institut National de la Recherche Agronomique, UMR866; Différenciation cellulaire et croissance, Montpellier, France

At brain synapses, scaffolding proteins function not only as anchors, but also as signaling proteins for neurotransmitter receptors. As synapses are dynamic structures, it is a fundamental issue to study the dynamics of such synaptic scaffolds and their role in synaptic receptor function. In this study we combined BRET imaging and electrophysiological recordings to understand the role of scaffold remodeling in hippocampal glutamatergic synaptic transmission. We characterized the spatio-temporal dynamics of protein-protein interactions within Homer-containing scaffold complexes. We showed that assembly/disassembly of this scaffold complex governs physical and functional crosstalk between ionotropic (NMDA) and metabotropic (mGlu5a) glutamate receptors in dendritic spines. This synaptic remodeling occurs during Long Term Potentiation (LTP) and enables inhibition of NMDA current during LTP expression. The present findings highlight a new role of post-synaptic scaffolding proteins on glutamate receptor crosstalk in synaptic function. During LTP, disengagement of mGlu5a receptor from Homer-containing


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scaffold enables functional association of the receptor with NMDA receptor. This remodeling represents a form of homeostatic control of synaptic excitability.

125.Acetyl-l-Carnitine Causes Antidepressant-like effect Mediated by Group-II mGlu Receptors in Spontaneously Depressed FSL Rats C. Nasca1, D. Xenos1, Y. Barone2, A. Caruso1, S. Scaccianoce1, G. Battaglia3, S.Rosati1, M. Ciampoli1, F. Matrisciano1,4, A. Mathé5, G. De Lorenzo2, C. Niolu2, A. Pittaluga6, S. Di Prisco6, A. Siracusano2, A. Koverech7 and F. Nicoletti1,3 1

Dept. Physiology and Pharmacology, Univ. of Rome Sapienza, Italy; 2Dept. of Psychiatry, University of Rome Tor Vergata, Italy; 3 INM Neuromed, Pozzilli, Italy; 4Karolinska Institutet, Clinical Neuroscience, Psychiatry, Karolinska University Hospital Huddinge, Stockholm, Sweden; 5Dept. of Psychiatry, University of Illinois at Chicago, Chicago, IL; 6Department of Experimental Medicine, Pharmacology and Toxicology Section, Center of Excellence for Biomedical Research, University of Genova, Italy, 7 Sigma-Tau, Pomezia, Italy L-acetylcarnitine (LAC), a drug used in the treatment of neuropathic pain, causes analgesia by acetylating p65 (a member of the NFB family of trascription factors), thereby inducing the expression of mGlu2 receptors in the spinal cord (Chiechio et al., Trends Pharmacol. Sci. 31, 2010). Here, we examined the effect of LAC on depression-like behaviour in spontaneously depressed Flinders Sensitive Line (FSL) rats. LAC injected i.p. at the dose of 100 mg/kg, once a day for 21 days, significantly reduced the immobility time at the forced swim test. The antidepressant effect was visible after 3 days, and became more robust after two and three weeks of treatment. This temporal profile differed from that exhibited by the classical antidepressant, chlorimipramine, which showed antidepressant-like effects only after 3 weeks of treatment. In FSL rats, a 21-day treatment with LAC increased the number of proliferating neuroprogenitors in the hippocampus, as shown by BrdU immunostaining. In addition, LAC treatment reversed the deficit in the evoked release of glutamate seen in hippocampal synaptoneurosomes of FSL rats. The antidepressant-like activity of LAC was confirmed using mice subjected to four weeks of unpredictable stress (two episodes of stress per day). Remarkably, the antidepressant activity of LAC in FSL rats was attenuated by a single injection of the preferential mGlu2/3 receptor antagonist, LY341495 (1 mg/kg, i.p.). LAC treatment increased the levels of mGlu2 mRNA and mGlu2/3 receptor protein in the frontal cortex and hippocampus of FLS rats, which constitutively showed a defective expression of mGlu2/3 receptors. LAC was inactive in non-depressed Flinders Resistant Line rats. These data suggest that LAC behaves as a fast-acting antidepressant drug, and its effect involves an up-regulation of mGlu2 receptors.

126.mGluR5 and CB1 Interactions in the Medial Prefrontal Cortex in Pain V. Neugebauer1, H. Sun1, W. Ren1, S.R. Stauffer2 and P.J. Conn2 1

Dept. of Neuroscience & Cell Biology, University of Texas Medical Branch, Galveston, TX, USA; 2Vanderbilt Center for Neuroscience Drug Discovery, Dept. of Pharmacology, Vanderbilt University Medical Center, Nashville, TN, USA The medial prefrontal cortex (mPFC) serves executive cognitive functions such as emotion-based decision making and control of emotional processes. mPFC dysfunction and impaired cognitive control of emotions play an important role in a number of neuropsychiatric disorders. Pain, a multidimensional experience


with a strong emotional component, has also been associated with impaired mPFC function and decision-making. A recent study from our laboratory showed that hyperactivity in the amygdala, an emotional brain center closely interconnected with the mPFC, leads to the deactivation of the mPFC [1] through a mechanism that involves feedforward inhibition of mPFC pyramidal cells [2]. We propose that restoring normal mPFC function can restore cortical control of pain and its emotional component. Group I metabotropic glutamate receptors (mGluRs) can modulate excitatory and inhibitory transmission in the cortex and have emerged as important targets for the treatment of neuropsychiatric disorders associated with cognitive deficits. We hypothesize that mPFC pyramidal cell output can be increased by increasing mGluR5 function while controlling abnormal synaptic inhibition through CB1 activation. To study mPFC function, whole-cell voltage- and current-clamp recordings were made from visually identified pyramidal cells in layer V of the mPFC in brain slices from normal rats (controls) and from rats with arthritis (5-6 h postinduction). In normal control slices, an orthosteric agonist for mGluR5 (CHPG) increased excitability (frequency-current [F-I] relationship) and synaptically evoked spiking (E-S coupling) but had little effect on excitatory synaptic transmission (excitatory postsynaptic currents, EPSCs). Positive allosteric modulators (PAMs) for mGluR5 such as VU’172 increased evoked EPSCs and E-S coupling and amplitude, but not frequency, of miniature EPSCs, suggesting a postsynaptic action. PAMs also enhanced depolarization-induced suppression of inhibition (DSI), which is mediated by type 1 cannabinoid receptors (CB1) because it was blocked by a CB1 antagonist (AM251). In slices from arthritic rats, CHPG or VU’172 alone had little effect on pyramidal cells and DSI was lost, which is consistent with abnormal synaptic inhibition of mPFC pyramidal output in pain. Coactivation of CB1 (with ACEA) and mGluR5 (with CHPG or VU’172) increased EPSCs, E-S coupling and F-I relationship, and restored DSI. The results show that mGluR5 acting in concert with CB1 can overcome pain-related abnormal inhibition of mPFC pyramidal cells and increase pyramidal cell output to engage cortical control of pain processing, which may be a novel strategy for pain relief. [1] Ji, G, et al. J. Neurosci., 2010, 30: 5451-5464. [2] Sun, H, Neugebauer, V. J. Neurophysiol., 2011 (in press).

127.Modulation of Spike and Wave Discharges by Individual mGlu Receptor Subtypes in the WAG/Rij Rat Model of Absence Epilepsy R.T. Ngomba1 and G. van Luijtelaar2 1

I.N.M. Neuromed, Pozzilli, Italy; 2Donders Centre for Cognition, Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen, Nijmegen, The Netherlands Metabotropic glutamate (mGlu) receptors modulate synaptic responses in the thalamocorticcal circuitry and are therefore strategically positioned to influence the occurrence of spike-andwave discharges (SWDs) associated with absence epilepsy. The modulatory role of individual mGlu receptor subtypes on excitatory and inhibitory synaptic transmission in the thalamocortical circuitry makes subtype-selective mGlu receptor ligands potential candidates as novel anti-absence drugs. We examined the role of mGlu receptors in absence epilepsy using WAG/Rij rat model, which develop spontaneous SWDs after 3 months of age to put into evidence links of mGlu receptors to the pathophysiology of pathological SWDs moving from the anatomical localization and function of distinct mGlu receptor subtypes in the thalamocortical network. Selective enhancers of mGlu1 or mGlu5 receptors reduced SWDs in WAG/Rij rats, and their action was prevented by mGlu1 and mGlu5 receptor NAMs, respectively. However, while mGlu1 receptor NAMs amplified spontaneous seizures when injected alone, the mGlu5 receptor NAM, MTEP was inactive. This


suggests that mGlu1 receptors exert a tonic control on absence seizures, whereas mGlu5 receptors can influence seizure activity only if pharmacologically activated. mGlu4 receptors are directly involved in the generation of absence seizures, and their absence prevents the occurrence of SWDs in animal models of absence epilepsy. Pharmacological activation of mGlu4 receptors enhanced the frequency of SWDs in WAG/Rij rats. Activation of mGlu2/3 receptors with the agonist, LY379268, also enhanced the frequency of absence seizures, whereas receptor blockade with LY341495 produced opposite effects. A role for mGlu7 receptors in absence epilepsy is suggested by the elegant experiments carried out by Joel Bockaert and his collegues showing that receptor uncoupling with PICK-1 causes absence seizures. It is unknown how precisely mGlu7 receptor ligands affect absence epilepsy, since our unpublished data from WAG/Rij rats shows that acute receptor blockade with the preferential mGlu7 receptor NAM, MMPIP increased together the incidence and duration of SWDs. Likewise, similar modulation of SWDs was observed after receptor activation with the agonist, AMN082. The role of mGlu8 receptors in absence seizures remains to be investigated.

128.Determining the Role of Metabotropic Glutamate Receptors in Glioma H.H. Nickols1,2, J.M. Rook2, N. Byun2 and P.J. Conn1,2 1

Department of Pathology, Microbiology and Immunology, Division of Neuropathology, Vanderbilt University Medical Center, Medical Center North (MCN) Room C-2318C, 1161 21st Avenue South, Nashville, TN 37232-2561; 2Department of Pharmacology and Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, Light Hall (MRB-IV) Room 1215, 2215 B Garland Avenue, Nashville, TN 37232

Pharmacological therapy for the treatment of human gliomas, specifically glioblastoma, is severely limited. Antagonists of the Group II metabotropic glutamate receptors (mGlu2/3) have been shown to inhibit glioma cell growth both in vivo and in vitro, demonstrating their potential as therapeutic agents for malignant gliomas [1,2]. Expression of both Group II mGlu and mGlu5 in human gliomas provides therapeutic opportunities for glioma treatment [3]. The mGlu3 metabotropic glutamate receptor is expressed in primary human glioblastoma cultures, human glioma cell lines, including glioma initiating cells, and is the predominant mGlu subtype expressed in human astrocytes. Both mGlu2 and mGlu5 are variably expressed in glioma cell lines. Glutamate transporter expression is differentially regulated by mGlu3 and mGlu5 in vitro, with mGlu5 agonists resulting in the downregulation of glutamate transporter expression [4]. This downregulation of glutamate transporter expression provides a growth advantage for gliomas since gliomas with reduced cell surface glutamate transporter expression exhibit more rapid growth. We are investigating the expression of metabotropic glutamate receptors in human glioma cell lines and primary human gliomas. We have found expression of both mGlu2 and 3 in a range of primary human gliomas by RT-PCR. In addition, we have seen expression of mGlu5 by Western blot analysis in a primary human glioblastoma cell line. We are further exploring mGlu expression and signaling pathways in gliomas. The expression of mGlus provide an opportunity for glioma imaging. An mGlu5 PET ligand may provide a tool for glioma imaging as well as potential in vivo determination of mGlu5 expression within gliomas relative to normal tissue. Using the rat C6 glioma cell line, known to express mGlu5, with orthotopic implantation into the frontal cortex of Wistar rats, studies are ongoing to investigate the potential of mGlu5 agents in the in vivo imaging of gliomas using PET and MRI. The expression of mGlus in human glioma and the availability of allosteric agents provide exciting therapeutic opportunities for glioma treatment development.

Abstracts of the 7th International Meeting on Metabotropic Glutamate Receptors [1] Arcella, A, et al. J. Neurochem., 2003, 84, 1288. [2] Ciceroni, C, et al. Neuropharmacology, 2008, 55, 568. [3] Condorelli, D.F, et al. Neurochem. Res., 1997, 22: 1127. [4] Aronica, E, et al. Eur. J. Neurosci., 2003, 17, 2106. Support: NIH/NIDA 5RO1DA023926 and NIH 5RO1MH074953 to P.J.C.; Department of Pathology, Microbiology and Immunology startup funds.

129.LY2491503, A Novel mGluR1 Antagonist Persistent Pain: Preclinical and Clinical Studies

for

E. S. Nisenbaum2, S. Iyengar2, R. M. Simmons2, D. Li2, M. J. Fisher2, S. Kuklish2, V. N. Barth2, M. G. Chambers2, B. A. Heinz2, S. A. Monk2, L. Arendt-Nielsen1 and T. W.Stephens2 1

C4Pain Hobrovej 42D, 3 9000 Aalborg Denmark, 2Eli Lilly and Company, Indianapolis, IN USA

Persistent pain is postulated to depend in part on long-term increases in synaptic efficacy of glutamatergic inputs to somatosensory neurons of the spinal cord following intense peripheral noxious stimuli, tissue injury or nerve damage. This enhanced synaptic transmission termed central sensitization leads to a reduction in pain thresholds (i.e., allodynia), an intensification of pain responses (i.e., hyperalgesia) and an expansion of pain sensitivity to non-injured areas. Considerable research has focused on the role of mGluR1 in these processes because they are expressed on peripheral sensory nerve fibers and sensory regions of the spinal cord and activation of mGluR1 is critical for the induction and maintenance of central sensitization. In addition, mGluR1 agonists induce painful responses in animals, whereas mGluR1 antisense, antibodies and antagonists suppress nocifensive behaviors in multiple animal models of pain. Collectively, these data indicate that antagonism of mGluR1 may provide a novel therapeutic approach for persistent pain conditions. The present studies investigated the pharmacokinetic (PK) properties and pharmacodynamic activity of a novel mGluR1 antagonist, LY2491503 in preclinical models of chronic pain and in human experimental pain (HEP) models in healthy volunteers (HVs). Initial in vitro studies demonstrated that LY2491503 is a potent antagonist at both human (IC50=8.9 nM) and rat (IC50=1.5 nM) mGluR1 with >1000-fold selectivity as an antagonist vs. other mGluRs (except mGluR5, IC50=1250 nM). PK studies showed that the compound is orally bioavailable in rats (92% F) with a moderate elimination half-life (3.1 hr). LY2491503 displayed dose- and timedependent occupancy of CNS mGluR1 (ED50= 7.9 mg/kg, p.o.) (but not mGluR5) that persisted for at least three hours and was positively correlated with efficacy in the formalin model of persistent pain (ED50=8.6 mg/kg, p.o.). LY2491503 suppressed mechanical allodynia behavior in the L5/L6 spinal nerve ligation model of neuropathic pain comparable to efficacy achieved with gabapentin. The compound also reduced asymmetrical hindpaw weight-bearing pain in the monoiodoacetate model of osteoarthritis, but did not affect thermal hyperalgesia in the carrageenan model of inflammatory pain. No motor impairment was observed at doses up to 7-fold higher than the ED50 in the formalin model. Electrophysiological studies demonstrated that LY2491503 (1 mg/kg, i.v.) reduced the wind-up in spike discharge of spinal dorsal horn neurons in response repetitive stimulation of peripheral sensory nerve fibers. PK assessment in HVs showed that plasma exposure was greater than predicted by allometric scaling and increased proportionately with dose. The primary adverse events were dizziness and fatigue that increased in incidence with higher doses. Possible adverse effects on cognitive function also were detected. LY2491503 was evaluated in two HEP models using a partial cross-over design with HVs receiving either 7 days of dosing with LY2491503 (10, 30, 60, 120 mg) or placebo, followed by a 3-day washout period and then 1 day of dosing with gabapentin (120 mg) or placebo. In the temporal summation HEP model, higher doses


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(60, 120 mg/kg) of LY2491503 unexpectedly reduced pain thresholds to repeated electrical stimulation of the sural nerve; whereas gabapentin (120 mg) had the opposite effect. In addition, LY2491503 had no consistent effect the area of secondary mechanical hyperalgesia following intradermal administration of capsaicin to the forearm. The present results show that LY2491503 displays robust efficacy in multiple preclinical models of persistent pain that is correlated with mGluR1CNS occupancy. Although mGluR1 occupancy was not measured in HVs, plasma exposures at the time of pain assessment were comparable to exposures in rats associated with > 70% CNS mGluR1 occupancy. These data highlight a potential discrepancy between mGluR1-mediated efficacy observed in quantitative sensory testing paradigms evaluated in preclinical rodent models and in HVs. The extent to which this molecule/mechanism would be beneficial in human chronic pain conditions remains an open question.

130.Association between a Genetic Marker in HTR2A and Response to the mGlu2/3 Agonist LY2140023 Monohydrate in the Treatment of Schizophrenia L.K. Nisenbaum, F. Zhao, A.C.M. Downing, L.M. Munsie, P. Chen, B.A. Fijal, M.R. Smith, B.A. Millen, D.H. Adams and B.J. Kinon Lilly Corporate Center, Eli Lilly and Company, Indianapolis, IN 46285, USA Introduction: LY2140023 monohydrate (hereafter referred to as LY2140023) is the oral prodrug of LY404039, a selective agonist for metabotropic glutamate 2/3 (mGlu) receptors. LY2140023 is currently under development for the treatment of schizophrenia and has the potential to introduce a novel mechanism of action for antipsychotics. In an effort to identify genetic markers associated with LY2140023 response, two pharmacogenetic studies were conducted – a retrospective analysis based on a 4-week phase-2 proof-of-concept trial H8Y-BD-HBBD [1], and a prospective analysis based on a 24-week phase-2 safety trial H8Y-MC-HBBR. Methods: In the HBBD genetic study, genetic variants in eight candidate genes were investigated in 193 DNA samples collected from clinical trials HBBD and F1D-US-HGLF. The primary analysis examined the association between genetic variants and change in the Positive and Negative Syndrome Scale (PANSS) total score in Caucasians treated with placebo, LY2140023 or olanzapine using an ANCOVA model under the additive allelic assumption. In the HBBR genetic study of 253 DNA samples, the top findings from the HBBD genetic study were examined in 53 Non-Hispanic Caucasians treated with LY2140023 and 63 Non-Hispanic Caucasians treated with standard of care (SOC) atypical antipsychotics. The genetic association was tested using an MMRM model under the additive allelic assumption at a 0.05 one-sided alpha level. Results: The HBBD analysis identified 23 single nucleotide polymorphisms (SNPs) that were associated with a change in PANSS total score in response to LY2140023 at Week 4, with the serotonin 2A receptor gene (HTR2A) SNP rs7330461 being the most significant association (unadjusted p> glutamate aspartate DHPG ACPD acidic homocysteine derivatives (L-homocysteic acid, L-cysteic acid and L-cysteine sulfinic acid). However, only glutamate, aspartate and acidic homocysteine derivatives but not quisqualate, DHPG and ACPD were able to protect neurons from K5 toxicity.These results strongly suggest that the protective action of mGlu1 receptors is mediated by a signal transduction mechanism distinct from G protein-mediated PI hydrolysis. Our recent data suggest that the -arrestin-mediated activation of MAPK and ERK 1/2 phosphorylation may be the primary pathway involved in mGlu1 receptor-induced neuroprotection. In addition, differential ability of agonists to stimulate PI hydrolysis and neuroprotection indicates the existence of a ligand bias in the activation of different signal transduction pathways of mGlu1 receptors. Supported by NIH grant NS37436.

152.Fenobam Binds to sites not Recognized by ABP688 and other mGluR5 NAMs, but by AZ11696415 in the Primate brain P. Raboisson1, A. Johnson1, A. Juréus1, S. Pierrou2, D. Wensbo1, P. Ström1, J. Malmquist1, J. Sandell1, J.D. Andersson3, K. Varnäs3 and C. Halldin3 and L. Farde1 1

AstraZeneca R&D, Södertälje, Sweden 2AstraZeneca R&D, Mölndal, Sweden 3Karolinska Institutet, Department of Clinical Neuroscience, Stockholm, Sweden

Over the years, several chemical classes of selective noncompetitive metabotropic receptor subtype 5 (mGluR5) antagonists also called negative allosteric modulators (NAM) have been identified through focused drug discovery efforts by industry and academic centers. As reported by other companies [1], the high throughput screening of the AstraZeneca compound collection revealed that fenobam, a non-benzodiazepine anxiolytic developed in the late 70’s, was a mGluR5 NAM as well [2]. Some mGluR5 NAMs, such as M-MTEP and ABP688 have been developed as useful [3H] or [11C] radioligands for imaging studies. These ligands compete with MPEP on its allosteric binding site at mGluR5.

151.Ligand Bias of mGlu1 Receptors Agonists in Stimulating PI Hydrolysis and Inducing Neuroprotection in Rat Cerebellar Neurons S. Pshenichkin, A.C. Emery, H. Hathaway, G.R. Takoudjou, J. DiRaddo, E.Grajkowska and J.T.Wroblewski Department of Pharmacology, Georgetown University, Washington, DC 20057, USA Previously, we have reported that glutamate, acting at mGlu1 receptors, protects primary cultures of rat cerebellar granule cells from toxicity induced by trophic deprivation. Here, we report differences in the ability of various mGlu1 receptor agonists to activate G protein-mediated PI hydrolysis and to protect cerebellar granule neurons from toxicity induced by low K+. Cerebellar granule neurons survive in vitro in Neurobasal medium supplemented with B27 and 25 mM K+ (K25 conditions). In mature cultures (7DIV) lowering K+ to a physiological concentration (5 mM) along with withdrawal of B27 (K5 conditions) results in apoptotic cell death, which is accompanied by an increased expression of mGlu1 receptors. Under these conditions, addition of glutamate protects cells from apoptosis and this effect is blocked by several selective noncompetitive antagonists of mGlu1 receptors (CPCCOEt, YM298198 and JNJ16259685), but is not affected by

AZ11696415 is a novel mGluR5 NAM developed by AstraZeneca. Using in vitro functional assays, AZ11696415 has been identified as a potent (IC50 7 and 3 nM at human and rat mGluR5, respectively) and selective NAM. In its [3H] version AZ11696415 has been proven to be a useful ligand for in vitro autoradiography in rodent. AZ11696415 has also been labelled with carbon-11 for PET studies in primates. We assessed the ability of various mGluR5 NAMs, including MTEP, ABP688, AZ11696415 and fenobam to compete with [3H]ABP688, [3H]M-MTEP and [3H]AZ11696415 in rat and cynomolgus monkey brain slices using in vitro autoradiography, or in cynomolgus and human brain membrane preparation using a filtration assay. In the rat striatum all mGluR5 antagonists tested could displace [3H]AZ11696415, [3H]ABP688 or [3H]M-MTEP specific binding. In contrast, in primate brain tissues, only fenobam could completely displace [3H]AZ11696415, while [3H]ABP688 or [3H]M-MTEP could be completely displaced by the other mGluR5 NAMs. Concurring results were obtained in vivo in cynomolgus monkey using PET. These results suggest that, in the primate brain AZ11696415 and fenobam bind to sites not


recognized by e.g. ABP688 or M-MTEP. The nature of these sites remains to be elucidated. [1] Porter, et al. J. Pharmacol. Exp. Ther., 2005. [2] Wållberg, et al. Bioorg. Med. Chem. Lett., 2006.

153.Altered mGluR5-Homer Interactions Contribute to mGluR5 Dysfunction in Fragile X Syndrome Jennifer A. Ronesi1, Katie A. Collins1, Seth A. Hays1, Nien-Pei Tsai1, Weirui Guo1, Shari G. Birnbaum2, Jia- Hua Hu3, Paul F. Worley3, Jay R. Gibson1, and Kimberly M. Huber1 1 Departments of Neuroscience, 2Department of Psychiatry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA; 3The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA

Enhanced mGluR5 dependent plasticity occurs in the mouse model of Fragile X Syndrome, Fmr1 knockout mice. Remarkably, mGluR5 antagonism has been demonstrated to be effective in reversing symptoms of Fragile X in both animal models and patients. However, there are also deficits in other mGluR5 functions in Fmr1 KO mice, such as activation of protein translation. Therefore, mGluR5 dysfunction in Fragile X is complex and little is known about the mechanisms that underlie mGluR5 dysfunction in the disease. A clue comes from work by Giuffrida et al. [1] who reported decreased association of mGluR5 with the postsynaptic scaffolding protein Homer and increased association with the short Homer1a isoform in Fmr1 KO mice. We find that the altered balance of mGluR5-Homer interactions mediates several phenotypes in Fmr1 KO mice that have been attributed to altered mGluR5 function. In contrast, other aspects of mGluR5 dysfunction in Fmr1 KO mice, such as altered mGluR-LTD cannot be accounted for by abnormal mGluR5-Homer scaffolds and therefore implicate an essential role for Fmr1. [1] Giuffrida, R, et al. J. Neurosci., 2005, 25(39), 8908-16.

154.Compensatory Molecular and Functional Mechanisms in Neurons of the Grm1crv4 Mouse, a Murine Model for Ataxia Lacking the mGlu1 Receptor P. I. A. Rossi1,2, I. Musante1,2, M. Summa3, A. Pittaluga3, M. Ikehata4,5, M. P. Rastaldi4,5, R. Ravazzolo1,2 and A. Puliti1,2 1

Molecular Genetics and Cytogenetics Unit, Gaslini Institute, Genoa, Italy; 2Department of Pediatric Sciences, University of Genoa, Genoa, Italy; 3Department of Experimental Medicine, Section of Pharmacology and Toxicology, University of Genoa, Genoa, Italy; 4Renal Research Laboratory, Fondazione IRCCS Ospedale Maggiore Policlinico, Milan, Italy; 5Fondazione D’Amico per la Ricerca sulle Malattie Renali, Milan, Italy The metabotropic glutamate type 1 (mGlu1) and 5 (mGlu5) receptors are the only members of group I mGlu receptors, a subfamily of G protein-coupled receptors implicated in synaptic plasticity, and in mechanisms of feed-back control of glutamate release. mGlu1 and mGlu5 receptors exhibit a largely complementary distribution throughout the central nervous system, more evident in the cerebellum, where mGlu1 receptor is most intensely expressed while the mGlu5 receptor is not. Despite the different distribution, they show a similar subcellular localization relative to transmitter release sites, and use common transducing pathways. We recently described the Grm1crv4 mouse mutant carrying a spontaneous inactivating mutation of the mGlu1 receptor [1].


Homozygous Grm1crv4/crv4 mice exhibit a complex phenotype with ataxia. To better define the pathophysiological mechanisms of ataxia in these mice, we evaluated expression and function of the other member of group I mGlus, the mGlu5 receptor, in Grm1crv4 mouse cerebellar and brain cortices. Western blot and immunofluorescence analyses show mGlu5 receptor overexpression in the homozygous Grm1crv4/crv4 mice. Real-time quantitative RT-PCR results indicate that up-regulation is already evident at RNA level, suggesting a cellular compensatory mechanism due to the absence of active mGlu1 receptor molecules. To note, these results are reminescent of those observed in a neuroimmunological disease, the multiple sclerosis (MS). Indeed, in MS patients and in the EAE mice, a model of MS, the presence of mGlu1 receptors is reduced but mGlu5 receptors expression enhanced [2]. Mechanisms that affect glutamate bioavailability and glutamatergic transmission at the synaptic level may contribute to the development and/or to the rescue of the affected phenotype. The effects of mGlu1 and mGlu5 receptor activation on the depolarization-evoked release of [3H]Daspartate from Grm1crv4 mouse cortical cerebellar and brain synaptosomes were investigated. The results confirm in the mouse mutant an enhanced glutamate release from cortical cerebellar and brain synaptosomes as respect to wild type, that is abolished by mGlu5 receptor specific inhibitors. Finally, in the Grm1crv4 mouse, the constitutive expression of mGlu5 receptor in the cerebellum and cerebral cortex in the absence of the mGlu1 receptors, seems to compensate, at least in part, the control of the partner receptor on the glutamate release at pre-synaptic level. Supported by RFPS-4631972 grant, Italian Ministry of Health. [1] Conti, et al. Int. J. Mol. Med., 2006, 18 (4), 593-600. [2] Fazio, et al. Neuropharmacology, 2008, 55(4), 491-9.

155.Unveiling Physiological Roles for mGlu Receptors in the Thalamus Using Positive Allosteric Modulators T.E. Salt and C.S. Copeland Visual Neuroscience, UCL Institute of Ophthalmology, University College London, UK Previous work from this laboratory and others has indicated that mGlu receptors belonging to all three groups can modulate various facets of sensory processing in the thalamus, and mGlu receptors can be activated synaptically under some conditions. For example, we have shown, using selective antagonists, that mGlu1 receptors contribute to cortico-thalamic responses of thalamic relay neurones, and that Group II (mGlu2/mGlu3) receptors modulate inhibitory processes in the ventrobasal thalamus [1]. The more recent availability of positive allosteric modulators (PAMs) for these receptors now offers the possibility of more detailed pharmacological analysis of responses and elucidation of novel synaptic mechanisms. We have explored some of these possibilities in the ventrobasal thalamus in vivo with the selective mGlu1 PAM Ro674853 [2] and the selective mGlu2 PAM LY487379 [3]. Extracellular single neurone recordings were made in vivo in the ventrobasal thalamus (VB) of urethane-anaesthetized adult Wistar rats with multi-barrel iontophoretic electrodes. Iontophoretic applications of glutamate receptor agonists, antagonists and PAMs were made into the vicinity of recorded neurones through the outer barrels of the electrode assemblies. Procedures were Home Office (UK) approved and accorded with the Animals (Scientific Procedures) Act 1986. Iontophoretic application of the mGlu1 PAM Ro67-4853 was found to selectively enhance postsynaptic excitatory responses to the Group I agonist DHPG, an action that is consistent with the known effects of this agonist via mGlu1 receptors in rat VB. Similarly, the mGlu2 PAM LY487379 potentiated the inhibitory effects of the Group II agonist LY354740 on GABAergic inhibition in VB, indicating that the agonist effects are mediated at least in part via


mGlu2 receptors. These findings demonstrate that it is possible to enhance mGlu receptor mediated responses with PAMs in vivo in the thalamus, and thus we investigated whether it would be possible to modulate synaptic responses with these agents. Synaptic responses were evoked by either short duration (10-30ms) or long duration (1000ms) stimulation of either a single vibrissa or by paired stimulation to two adjacent vibrissae in order to reveal excitatory responses and GABAergic inhibitory interactions between responses. Application of Ro67-4853 was found to enhance excitatory responses to long duration stimulation (to 131% of control) with little effect on short-duration responses, and these effects were reversed by the mGlu1 antagonist LY367385. Application of LY487379 enhanced responses to both types of stimuli (130% and 132% of control, respectively) in a manner consistent with a reduction in GABAergic inhibition, and this could be antagonised by LY341495. These findings demonstrate that mGlu PAMs can exert effects on synaptic responses in vivo, and further that mGlu1 and mGlu2 receptors can be specifically activated in a stimulus-specific manner in the thalamus. [1] Salt, T. E. Phil. Trans. Royal Soc. London Series B, 2002, 357, 1759-1766. [2] Knoflach, F. et al. Proc. Natl. Acad. Sci., (USA), 2001, 98, 13402-13407. [3] Johnson, M. P. et al. J. Med. Chem., 2003, 46, 3189-3192.

156.Pharmacological Activation of Metabotropic Glutamate Receptor Subtype 5 Reduces Spike and Wave Discharges in the WAG/Rij rat Model of Absence Epilepsy I. Santolini1, V. D’Amore1, C.M. van Rijn2, A. Simonyi3, A. Prete1,4, P.J. Conn5,6, C.W. Lindsley5,6, S. Zhou5,6, P.N. Vinson5,6, A.L. Rodriguez5,6, C.K. Jones5,6, S.R. Stauffer5,6, R. Gradini1, F. Nicoletti1,7, G. van Luijtelaar2 and R.T. Ngomba1 1

I.N.M. Neuromed, Pozzilli, 86077, Italy; 2Donders Centre for Cognition, Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen, Nijmegen, The Netherlands; 3 Department of Biochemistry. University of Missouri, Columbia, MO; 5Department of pharmacology, Vanderbilt University and 6 Vanderbilt Program in Drug Discovery, Nashville, TN 37232 USA; 4Child Neurology and Sleep Pedediatric Centre, Chair of Pediatrics, Ospedale S. Andrea, and 7Departments of Physiology and Pharmacology University of Rome, Sapienza, Italy Absence epilepsy is generated by the cortico-thalamo-cortical network under the form of spike and wave discharges (SWDs) as captured on the cortical EEG. This loop is under the control of a reverberant excitatory glutamatergic projections that are shaped by inhibitory GABAergic interneurons both in the thalamus and cortex. The control of firing properties of this network has been demonstrated to be also under the influence of the group I metabotropic glutamate receptors. Furthermore, the use of animal models to identify molecular and cellular mechanism has sometimes been controversial due to lack of supplementary specific group I mGlu receptor subtype ligands and also how genes are being expressed in different animal models. We have used the WAG/Rij rat model and recently synthesized group I mGlu receptor ligands to study the in vivo influence of mGlu5 receptor on SWDs. Symptomatic WAG/Rij rats showed lower levels of mGlu5 receptor protein in ventrobasal thalamic nuclei as compared to age-matched controls, the reduced expression of mGlu5 receptors in the ventrobasal thalamus of WAG/Rij rats was confirmed at mRNA level by in situ hybridization. Systemic injection of the selective mGlu5 receptor NAM, MTEP (30 or 10 mg/kg, i.p.), did not influence the number of SWDs; in contrast, pharmacological activation of mGlu5 receptors with a selective and systemically


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active receptor PAM, VU0360172-6 (10 or 3 mg/kg, i.p.), drastically reduced the frequency of SWDs during the first 2 hours after administration. No reduction was seen when the PAM was combined with MPEP. These data suggest that mGlu5 receptors exert a phasic control over the generation of SWDs, and that mGlu5 receptor PAMs are potential candidates to the treatment of absence epilepsy.

157.Neuroprotection Following Postconditioning Via Group I mGlu Receptor Activation is Coupled to the PI3K/Akt/GSK3 Signalling Pathway in a rat Organotypic Hippocampal slice Model of Cerebral Ischemia T. Scartabelli, E. Gerace, E. Landucci, F. Moroni and D.E. Pellegrini-Giampietro Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy Ischemic postconditioning is defined as a repetitive series of brief interruptions of reperfusion applied immediately after ischemia, [1]. We previously reported that it is possible to evoke a neuroprotective postconditioning response in organotypic hippocampal slices exposed to a lethal period (30 min) of OGD by applying, 5 min later, a low dose (10 M) of DHPG and that this protective mechanism implicate the activation of the mGlu1/mGlu5-PI3K-Akt signalling pathway [2]. Here, we extend these data by exploring the production of reactive oxygen species (ROS) and the glycogen synthase kinase 3 (GSK3) signalling pathway. To assess whether DHPG postconditioning exerted neuroprotective actions by counteracting an increase in ROS levels, we tested its effects on ROS accumulation. Exposure of organotypic hippocampal slices to 30 min OGD induced a massive increase in the formation of ROS, which was particularly intense 24 h later in the CA1 region. Neuroprotection induced by postconditioning was associated with a reduction in the formation of ROS, that started 6 h after the postconditioning treatment and was maximal 24 h later. The PI3K inhibitor LY294002 (10 M) was able to prevent this reduction, indicating that there is a link between the PI3K/Akt pathway and the formation of ROS in the protective mechanisms of postconditioning. Subsequently, we performed Western blot experiments to analyze the inactivation of GSK3. Our data demonstrate that hippocampal slices exposed to our postconditioning protocol or to 10 μM DHPG for 30 min resulted in a transient increased phosphorylation of GSK3. In conclusion, we show that the neuroprotection afforded by DHPG postconditioning is associated with a decrease in ROS production and with an activation of the PI3K/Akt signalling pathway, which results in the phosphorylation (and thus inactivation) of GSK3, and possibly in the accumulation of -catenin in the nucleus.

158.LY2607540 (THIIC): A Novel, Brain Penetrant, Positive Allosteric Modulator of the Metabotropic Glutamate Receptor 2 J. Schkeryantz, K. Svensson, L. Rorick Kehn, M. Fell, J.M. Witkin, C. Felder, D. Mayhugh, A. Khilevich, S. Swanson, J. Catlow, B. Heinz and Xushan Wang Eli Lilly and Company, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Indiana, 46033, U.S.A Activation of the group II mGluRs (mGlu2/3) by orthosteric agonists has led to notable in vivo effects in a number of animal models of schizophrenia and anxiety. Consistent with these preclinical results LY2140023, an orally active amide pro-drug of LY404039, has shown evidence of improvement in both positive


and negative symptoms of schizophrenia in a double-blind placebo controlled clinical study in schizophrenic patients. In addition, LY544344, an amide pro-drug of LY354740 was efficacious in a human trial on general anxiety disorder compared with placebo treated patients. Positive allosteric activation of the mGlu2 receptor is a potential alternative to orthosteric mGluR2/3 activation in the clinic. A positive allosteric modulator (PAM) of the mGlu2 receptor is postulated to bind to a topologically distinct site on the glutamate receptor to potentiate the agonist response but possesses minimal or no activity on its own. Lilly was one of the first to disclose a PAM of the mGlu2 receptor with in vitro and in vivo data reported on the meta-pyridylsulphonamide series. The carboxylic acid containing hydroxyacetophenone class of molecules is a second series of mGluR2 PAMs we have identified. This series of molecules are not only potent mGluR2 potentiators but possess significant CysLT1 antagonist activity and are effective in a dural plasma protein extravasation (PPE) model of migraine (see Johnson et al, this meeting). Expanding the SAR on the hydroxyacetophenone series to increase both the brain penetration and selectivity for mGlu2 receptor (vs CysLT1) led to the identification of molecules with in vivo activity in rodent models of mood disorders and schizophrenia. The SAR leading from the acid containing acetophenones to the identification of LY2607540 will be presented highlighting the challenges associated with crossing the blood brain barrier as well overcoming an unusual in-vivo Baeyer-Villiger like metabolic transformation. In addition, the favorable PK characteristics and in vivo results in animal models of schizophrenia and mood discorders will be presented. LY2607540, N-(4-((3-hydroxy-4-isobutyryl-2-(trifluoromethyl)phenoxy)methyl) benzyl)-1-methyl-1H-imidazole-4-carboxamide (THIIC) is a potent positive allosteric modulator of the mGlu2 receptor (EC50 = 22.5 nM in an intracellular calcium based functional assay) possessing no activity as a positive or negative allosteric modulator of the mGlu1/3/4/5/8 or GABAB receptors at concentrations of 12.5 M. Moreover LY2607540 was devoid of agonist activity on its own when tested at any of the above mentioned receptors.

159.A new Approach to Analyze Cell Surface mGluRs Reveals Allosteric Switches During Activation and Possible Heterodimers P. Scholler1,2, E. Doumazane1, L. Fabre1, G. Donsimoni2, J. M. Zwier2, E. Trinquet2, P. Rondard1 and J.-P. Pin1 1 Institut de Génomique Fonctionnelle, Department of Molecular Pharmacology, CNRS UMR5203 - Inserm U661 - Universités de Montpellier 1 & 2, Montpellier, France, 2Cisbio Bioassays, Codolet, France

The activation mechanism of metabotropic glutamate receptors (mGluRs) and whether they are able to form heteromers are still poorly understood. Here we report a new method to analyze the mGluRs at the surface of living cells: we label the extracellular part of mGluR subunits with fluorophores compatible with timeresolved FRET (trFRET) thanks to suicide enzymes, SNAP- and CLIP-tags, fused at the N-termini. This new approach allows, at the cell surface, the studying of: 1) the unexpected ability of mGluRs to form functional heterodimers, and 2) the conformational changes of the extracellular domain upon binding of ligands. Using this new multilabeling approach, we show that mGlu subunits can heteromerize, and our trFRET data are consistent with mGluR subunits forming strict homodimeric receptors on single expression, and a combination of strict heterodimeric and strict homodimeric receptors on co-expression. The existence of such mGlu heterodimers is further confirmed by biochemical and functional complementation studies. A comprehensive analysis of all possible pairs of two mGluR subunits shows that at the cell surface, group II and group III mGlu (2, 3, 4, 7, 8) subunits can form strict


heterodimeric receptors, whereas they cannot assemble with group I mGlu (1, 5) subunits. Thanks to this approach and by monitoring the efficiency of inter-subunit FRET, we can also analyze the conformational changes of mGlu2 during activation. We demonstrate that in living cells, in accordance to previous crystallographic studies, the relative movement between the two extracellular domains is directly associated with the activation of the receptor. The efficacy of different ligands acting on mGlu2 (agonists, antagonists, positive or negative allosteric modulators) is highly correlated with the trFRET signal changes measured with our sensor of activation. This was further demonstrated using receptor mutants that either prevent the relative movement of the subunits, or mutants in which the two subunits are locked in the active orientation. Finally, we show that positive allosteric modulators acting in the membrane domain, as well as mutations, facilitate trFRET change induced by orthosteric agonists, revealing allosteric communication within the receptor. Altogether, these data provide important new information on the functioning of these receptors, offering new possibilities to develop new types of drugs modulating their activity. This work was supported by a grant from the ‘Agence National pour la Recherche’ (09-BIOT-018).

160.Strain-Dependency of the Analgetic Effect of Parecoxib is Related to Differences in the Cannabinoid System H. Schröder, V. Höllt and A. Becker Dept. Pharmacology & Toxicology, Medical Faculty, O.-v.Guericke University, D-39120 Magdeburg, Germany Loose ligation of the sciatic nerve (CCI) is an accepted model in the study of neuropathic pain. Using CCI we investigated nociceptive behaviour and the effects of the COX2 inhibitor Parecoxib (Par) on Wistar (Wist) and Sprague Dawley rats. In CCI rats from both strains, cold allodynia and a reduced thermal pain threshold were detected, whereas no effect was found in the hot plate test. The thermal pain threshold was used to determine the antinociceptive effects of morphine, gabapentin, and Par 5 days after surgery. Morphine and Gabapentine were effective in both strains, whereas an antinociceptive effect of Par was found in CCI Wist rats only. The antinociceptive activity caused by Par was attenuated by the CB1 antagonist Rimonabant. Receptor binding experiments showed increased CB1 binding in CCI Wist rats treated with Par. Surprisingly, Par exerted a specific affinity to the cannabinoid receptor measured in CB1 transfected HEK 293 cells and brain tissue. It has to be mentioned, that in comparison with WIN 55.2122 (a specific cannabinoid receptor ligand) the COX 2 inhibitor is about 100 times less potent. This suggests that differences in the analgesic effects of the COX 2 inhibitor Par are at least attributed to the strain-dependent function of the cannabinoid system.

161.Successful Use of Biotech Discovery Approaches for the Identification of Potent Sweet Taste Enhancers G. Servant Senomyx, Inc. 4767 Nexus Centre Drive, San Diego CA 92121 USA Sweet taste is mediated by an obligatory heterodimeric receptor composed of two subunits called T1R2 and T1R3. These 2 subunits correspond to class C GPCRs characterized by a venus fly trap domain (VFD) at the N-terminus linked to the c-terminal heptahelical transmembrane domain (TMD) via a cystein rich domain (CRD). We used a cell based assay for the human sweet taste receptor, high throughput screening, assay-guided chemical optimization and taste testing with trained panelists to identify positive allosteric modulators (PAMs) that could allow a reduction of the amount of sweeteners used in consumer products. We present here a unique class of PAMs that considerably potentiate sucralose


or sucrose effects in the assay but that have no or little agonist activity on their own. In taste tests these PAMs do not have intrinsic sweetness but significantly increase the sweetness of alow amount of sucralose or sucrose. Mutagenesis and molecular modeling suggest that these PAMs act as molecular glue near the opening of the VFT, stabilizing the closed and active conformation via hydrophobic interactions. In consumer product prototypes, these PAMs aresignificantly more effective than other classical taste enhancers at reducing calories in consumer products without compromising on the true taste of sugar.

162.Expression and Distribution of Group-II Metabotropic Glutamate Receptors in the Bladder D. Sferra1, C. Zappulla2, F. Biagioni2, P. Scarselli2, S. Notartomaso2, G. Mallel3, P. Menè3, V. Russo1, F. Fazio2, F. Nicoletti2,4, F. Fornai2,5 and R.Di Marco1,2 1

Department of Health Sciences, University of Molise, Campobasso, Italy; 2I.N.M.Neuromed, Pozzilli, Italy; 3Division of Nephrology, IInd School of Medicine, University of Rome “Sapienza”, S. Andrea Hospital, Rome, Italy; 4Department of Human Physiology and Pharmacology, University of Rome 'La Sapienza', Rome, Italy; 5 Department of Human Morphology and Applied Biology, University of Pisa, Pisa, Italy Metabotropic glutamate (mGlu) receptors have been identified in peripheral organs including the liver, gut, pancreas, heart, and lymphoid organs [1]. Here, we examined the distribution and function of mGlu receptor subtypes in the bladder. Real-time PCR analysis showed the treanscripts of mGlu3 and mGlu7 receptors in the bladder of normal C57bl/6 mice, but not the transcripts of mGlu1 and mGlu5 receptors. The presence of mGlu3 receptors in the mouse bloadder was confirmed by immunoblot analysis using a polyclonal mGlu2/3 antibody. Immunohistochemical analysis showed the presence of mGlu2/3 receptor immunoreactivity in clavated and basal cells, whereas umbrella cells of the urinary bladder were only weakly stained. Immunostaining was not visible in mGlu3 receptor KO mice, whereas it persisted in the bladder of mice lacking mGlu2 receptors. Intrabladder injection of LY379268 (80 pmol/40 ml) reduced cAMP levels in the presence of the phosphodiesterase inhibitor, IBMX. These data suggest the presence of functional mGlu3 receptors in the mouse bladder. [1] Julio-Pieper, et al. Pharmacol. Rev., 2011.

163.Defining the Translational Pharmacology of Negative Allosteric Modulators of the Metabotropic Glutamate Receptor Subtype 5 in Rats and Nonhuman Primates C. Shaffer, D.L. Smith, J. Cianfrogna, E. Miller, S. Grimwood, K. Zasadny, G. Currier, D. McGinnis, P. Seymour, P. Trapa, D.A. Smith and M. Zaleska Neuroscience Research Unit, Pfizer Global Research & Development, Groton Laboratories, Eastern Point Road, Groton, CT, 06339 USA Metabotropic glutamate receptor subtype 5 (mGluR5) is implicated in a variety of central nervous system (CNS) diseases whose underlying pathophysiology is hypothesized to be due to glutamatergic dysregulation [1]. Antagonism of mGluR5, most notably by negative allosteric modulation (NAM) [2], is a proposed therapeutic approach for CNS disorders such as Parkinson’s Disease L-DOPA-induced dyskinesia (PD LID) [3], Fragile X syndrome, Treatment-resistant Depression and Autism Spectrum Disorder. Recently reported clinical efficacy with the mGluR5 NAM AFQ056 in both PD LID [4] and Fragile X syndrome [5] lends initial validation to these neuropharmacological concepts. However, mGluR5 NAMs have also shown sedation and cognitive


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impairment preclinically [6]. Collectively, these data suggest the importance of carefully understanding the exposure-response relationships of mGluR5 NAMs preclinically to aid optimal clinical dosing. Defining these associations is tremendously enhanced by the availability of well-characterized mGluR5 NAM radioligands, which afford exposure-receptor occupancy (RO) correlations. Here we report the exposure-RO-response continuum for AFQ056 and a novel proprietary compound (1) across both rats and nonhuman primates (nhp), with cross-species exposures normalized to unbound brain compound concentrations (Cb,u) to allow direct comparison to in vitro-derived binding and functional values. Neuropharmacokinetics, in vivo RO, rotarod performance and effects on spatial working memory via a radial arm maze were evaluated in rats, while pharmacokinetics, PET RO and effects in a PD LID model [7] were determined for nhp. Holistically, these datasets provide the exposure-RO-response continuum for both AFQ056 and 1 across rats and nhp, and provide considerable insight into the RO to target clinically for evaluating mGluR5 NAM-mediated PD LID efficacy and potential adverse events (sedation and/or cognitive disruption). [1] Marino, M.J. and Conn, J.P. Curr. Opin. Pharmacol., 2006, 6, 98–102. [2] Conn, J.P, et al. Nat. Rev. Drug Discov., 2009, 8, 41–54. [3] Calabresi, P, et al. Lancet Neurol., 2010, 9, 1106–1117. [4] Berg, D, et al. Mov. Disord., 2010 (in press). [5] Jacquemont, S, et al. Sci. Transl. Med. Neurol., 2011 (in press). [6] Simonyi, A, et al. Eur. J. Pharmacol., 2010, 639, 17–25. [7] Rylander, D, et al. Neurobiol. Dis., 2010, 39, 352–361.

164.Investigation of the Group II Metabotropic Glutamate Receptor Subtype Responsible for Increased Cyclic AMP Production and Reduced Excitatory activity caused by co-Activation of -Adrenergic and mGlu2/3 Receptors D.J. Sheffler, A.G. Walker and P.J. Conn Department of Pharmacology and Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, Light Hall (MRB-IV) Room 1215, 2215 B Garland Avenue, Nashville, TN 37232 We have previously demonstrated that co-activation of -adrenergic (AR) and group II metabotropic glutamate receptors (mGlu2/3) leads to a dramatic increase in cyclic AMP (cAMP) accumulation in rat hippocampus. Furthermore, co-application of the AR agonist isoproterenol (ISO) and mGlu2/3 preferring agonist DCG-IV was shown to decrease excitatory transmission at the Schaffer collateral (SC)-CA1 synapse in electrophysiological recordings. These effects were shown to be mediated through a form of glial-neuronal communication whereby co-activation of these receptors leads to increased cAMP production within glia, release or cAMP metabolites into the extracellular space, activation of presynaptic adenosine A1 receptors, and subsequent suppression of glutamate release at the SC-CA1 synapse. Receptor expression and cellular localization studies suggest that mGlu3 is the primary group II mGlu expressed on glia in hippocampus and could be the mGlu subtype mediating these effects. We have recently begun exploring this hypothesis using newer generation group II pharmacological agents. Biochemical studies measuring cAMP accumulation with the earlier generation group II agonist DCG-IV have been replicated. Additionally, the newer generation group II agonist LY379268 was found to dose-dependently increase cAMP responses elicited by the AR agonist ISO in rat cortex, rat hippocampus, and mouse cortex without having a response alone. These LY379268 potentiated cAMP responses are not potentiated by BINA, the mGlu2 selective positive allosteric modulator, indicating that mGlu2 may not contribute to this response. Experiments utilizing a fixed concentration of LY541850 (30M), a mixed mGlu2 orthosteric agonist and mGlu3 orthosteric


antagonist, demonstrate a right-shift in the LY379268 doseresponse for potentiation of ISO-induced cAMP production, consistent with antagonist activity at mGlu3. Whole-cell recordings were obtained from CA1 pyramidal neurons and evoked excitatory postsynaptic currents (eEPSCs) were measured in response to SC stimulation. As previously reported ISO (1M) alone slightly increased eEPSC amplitude relative to baseline. Preliminary data also indicate that DCG-IV (1M) or LY379268 (1M or 100nM) alone had no effect on eEPSC amplitude. Co-application of ISO and DCG-IV or ISO and LY379268 suppressed eEPSCs amplitude. Further experiments are being performed utilizing subtype selective allosteric modulators, and mGlu2 and mGlu3 knockout mice to fully evaluate the specific mGlu subtype responsible for these glial-neuronal effects. Vanderbilt is a Specialized Chemistry Center within the Molecular Libraries Probe Centers Network. Support: NIH/NINDS grant R01 NS031373 to P.J.C.

165.Neuroprotective Effects of Metabotropic Glutamate Receptor 5 (mGluR5) Antagonist Against Dopaminergic and Noradrenergic Neuronal Degeneration in the MPTPtreated Monkey Model of Parkinson’s Disease Y. Smith1,2, G. Masilamoni1, J. Bogenpohl1, D. Alagille3 and G. Tamagnan3 1

Yerkes National Primate Research Center and 2Department of Neurology, Emory University, Atlanta, GA 30329, USA; 3Institute for Neurodegenerative Disorders, New Haven, CT, USA The degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc) and noradrenergic cells in the locus coeruleus (LC) are two main pathological features of Parkinson's disease (PD). Despite significant advances in the development of symptomatic antiparkinsonian therapies, there is an important need for therapeutic agents that slow down and alter the course of the neurodegenerative process in PD. In light of promising rodent data suggesting that mGluR5 antagonist has significant neuroprotective effects against midbrain dopaminergic cell loss in MPTP (1-methyl4-phenyl-1,2,3,6-tetrahydropyridine)-treated mice [1], the goal of the present study was to test whether the highly specific mGluR5 antagonist, 3-[(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine (MTEP), reduces dopaminergic and noradrenergic neuronal loss in monkeys rendered parkinsonian by chronic treatment with low doses of MPTP. Weekly intramuscular MPTP injections (0.2-0.5 mg/kg body wt), in combination with daily administration of MTEP or vehicle, were performed until the appearance of parkinsonian motor symptoms in either of the two experimental groups (MPTP/MTEP vs MPTP/vehicle). After 21 weeks of MPTP treatment, all MPTP/vehicle-treated animals displayed parkinsonian motor symptoms (i.e. rigidity, bradykinesia, postural instability, etc…), whereas none of the MPTP/MTEP-treated monkeys were significantly affected. These behavioral observations were consistent with in vivo positron emission tomography (PET) dopamine transporter (DAT) imaging data, and with postmortem stereological counts of midbrain dopaminergic neurons, as well as striatal intensity measurements of DAT and tyrosine hydroxylase (TH) immunoreactivity, which were all significantly higher in MPTP/MTEP-treated animals than in MPTP/vehicle-treated monkeys. The MTEP treatment also had a robust effect on the MPTP-induced loss of norepinephrine (NE) neurons in the LC and adjoining A5 and A7 cell groups. In MPTP/vehicle-treated animals, almost 40% loss of TH-positive NE neurons was found in LC/A5/A7 cell groups, whereas the extent of neuronal loss was lower than 15% of control values in MPTP/MTEP-treated monkeys.

166.Estrogen and mGlu1 Receptors are Interdependent in Protecting Cortical Neurons Against -Amyloid


S.F. Spampinato1, G. Molinaro3, S. Merlo1, L. Iacovelli4, F. Caraci1,2, G. Battaglia3, F. Nicoletti3,4, V. Bruno4, A. Copani2 and M.A. Sortino1 1

Departments of 1Clinical and Molecular Biomedicine, and Pharmaceutical Sciences, University of Catania; 3Istituto Neurologico Mediterraneo Neuromed Pozzilli; 4Department of Physiology and Pharmacology, University of Rome Sapienza, Italy 2

The neuroprotective activity of estrogen is known to be exerted through activation of classical estrogen receptors (ER and ER), intracellular receptors controlling gene transcription, but also through membrane localized ERs, able to elicit rapid signalling events. Thus, mitogen activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K/Akt) and cAMP-response element binding protein (CREB) have been suggested to play a role. ERs have long been known to trans-activate surface receptor proteins and more recently the ability of ERs to interact with G-protein coupled receptors (GPCR), specifically metabotropic glutamate (mGlu) receptors, has been shown. The specific aim of the present study has been to establish whether ERs interact with mGlu1 receptors in neurons and whether this interaction is involved in the protective effect of estrogen against amyloid- (Aß)-induced neurodegeneration. ER and mGlu1 receptors are both expressed in cultured cortical neurons, whereas only ER are present in astrocytes, as from western blot and double immunocytochemistry. The two receptors co-immunoprecipitate in extracts from pure cultures of cortical neurons, an effect that is increased by exposure to receptor agonists, either 17estradiol (17E2) or 3,5dihydroxyphenilglycine (DHPG). In mixed cortical cultures treatment with 100 nM Aß1-42 or 25 M A25-35 for 24 h increased by 2-3 fold neuronal death, assessed by counting trypan blue-labeled cells. Pretreatment for 30 min with 10 nM 17E2, its not cell permeable analog, 17E2-BSA, the ER selective agonist PPT (100 nM) or 100 M DHPG caused about a 30% reduction of neuronal death. When the 17E2 and DHPG were added together, neuroprotection observed was comparable to that produced by each treatment alone. The neuroprotective effect was prevented by respective receptor antagonists, but also 1 M ICI182,780 (ER antagonist) prevented DHPG-induced effect whereas the mGlu1 receptor antagonist, JNJ16259685 (100 nM) reduced 17E2 effect. To investigate on the signaling pathway involved, attention has been focused on the PI3K/Akt, Known to be linked to mechanisms of neuroprotection. Treatment with the Akt inhibitor, 10-DEBC hydrochloride (10 M), abolished the neuroprotective effect of 17E2 and DHPG (applied alone or in combination). Treatment of pure cortical neurons with either 17E2 (10 nM) or DHPG (100 M) increased Akt phosphorylation and co-addition of 17E2 and DHPG again produced a less than additive effect. ICI182,780 and JNJ16259685 abolished activation of the PI3K/Akt pathway produced by their respective agonists, but also the effect observed after stimulation of the other receptor. The same study was carried out in HEK-293 cells transiently transfected with both ER and mGlu1 receptors. Both 17E2 and quisqualate (200 M), increased pAkt, but, in this case, co-treatment with quisqualate and 17E2 produced an effect greater than that seen with either drug applied alone. Interestingly, the effect of 17E2 and quisqualate was abrogated by pre-treatment with ICI182,780 and/or JNJ16259685, whereas each antagonist was only slightly effective in reducing pAkt induced by stimulation of the other receptor.

167.mGlu7 Receptor Activation Produces Potential Anxiolytic and Anti-Obsessive-Compulsive Effects in Mice K. Stachowicz, JM. Wieroska, A. Sawiska and A. Pilc


Institute of Pharmacology, Polish Academy of Sciences, Smtna 12, 31-343 Krakow, Poland and Jagiellonian Univ., Coll. Med., Fac. of Health Sci., Krakow, Polan Our earlier studies demonstrated that selective mGlu7 receptor agonist, N,N’-dibenzyhydryl-ethane-1,2-diamine dihydrochloride (AMN082) showed anxiolytic-like effects in the modified stressinduced hyperthermia (SIH) and in the four-plate (FPT) tests in mice [1]. The data also indicated that activation of the mGluR7 produces anxiolytic-like effects via the modulation of the aminobutyric acid system (AMN082-mediated anxiolytic-like effects were blocked by the benzodiazepine receptor antagonist, flumazenil). In the present study we have continued research over the AMN082 mechanism of action. The anxiolytic-like effects of AMN082 (6 mg/kg i.p.) in the SIH paradigm [2] was blocked by WAY100635 a 5-HT1A receptor antagonist N--N-(2-pyridynyl) cyclohexane-carboxamide (0.1 mg/kg s.c.) and 5-HT2A/2C receptor antagonist ritanserin (0.5 mg/kg i.p.). In the SIH test in mice we found that AMN082 (6 mg/kg i.p.) did not induced an anxiolytic-like effect after depletion of serotonin (5-HT) with parachlorophenylalanine (p-CPA).11111. Moreover, we examined AMN082 in marble-burying test (MBT) [3], used for detecting the potential anti-obsessive-compulsive effects. AMN082 showed dose-dependent potency (3 and 6 mg/kg i.p.) comparable to effects mediated by a fluvoxetine (10 mg/kg i.p.). The results of our studies indicate that activation of the mGlu7 receptor produces anxiolyticlike effects via the modulation of the 5-HT system and not only through the GABA-ergic system. Simultaneously, activation of these receptors may exert anti-obsesive-compulsive disorder effects, however the possibility that AMN082 acts via its active metabolite has to be taken into account [4]. [1] Stachowicz, K, et al. Behav. Pharmacol., 2008, 19, 597-603. [2] Van der Heyden, J.A.M, et al. Phys. Behav., 1997, 62, 463-70. [3] Millan, M.J, et al. Pharmacol. Exp. Ther. 2000, 295, 1206-4. [4] Sukoff, Rizzo, S.J, et al. J.Pharmacol. Exp. Ther., 2011, 338, 345-52. Supported by Grant POIG 01.01.02-12-004/09

168.Identification and Optimization of mGlu5 PAMs which Activate Receptor at Allosteric Sites Outside the MPEP-Site S.R. Stauffer1,2,3, S. Zhou1,2, J. Manka1,2, A. Rodriguez1,2, M. J. Noetzel,2 K. J. Gregory2, P.N. Vinson1,2 , C. M. Niswender1,2, E. Dawson1,2, C. K. Jones1,2, R. Lambert, 1,2 A. G. Thompson1,2, J. S. Daniels1,2, T. Bridges1,2, H. Lavreysen4, J. M. Bartolomé4, C. Mackie4, T. Steckler4, G. MacDonald4, C.W. Lindsley1,2,3 and P.J. Conn1,2 1

Vanderbilt Center for Neuroscience Drug Discovery, 2Vanderbilt University Medical Center Department of Pharmacology, 3Vanderbilt University Department of Chemistry, Nashville, TN 37212. 4 Neuroscience Drug Discovery, Janssen RND, Beerse, Belgium Positive allosteric modulators (PAMs) of the group I metabotropic glutamate receptor mGlu5 provide a compelling therapeutic approach with the potential to treat the three symptom domains of schizophrenia- including positive, negative and cognitive impairments. Nearly all mGlu5 PAMs reported to have preclinical utility in vivo thus far, including DFB, CDPPB, ADX47273, and CPPZ, have been shown to competitively interact with the well characterized MPEP allosteric binding site which is localized within the 7TM domain [1]. Two of these scaffolds, DFB and ADX47273, have been shown through chemical modification to display a propensity to induce subtle ‘molecular switches’ in their mode of pharmacology in certain contexts (e.g. PAM to NAM),


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raising important questions and potential challenges in lead optimization and clinical candidate selection. In an effort to identify additional unique mGlu5 chemotypes which potentiate mGlu5 responses via interactions outside the MPEP site, we have focused on novel HTS hits as well as known non-MPEP allosteric ligands, including CPPHA [2] and a recent series of alkyl ether benzamides [3], as starting points for further optimization and/or potential scaffold hopping. Progress to date in the pharmacological, physicochemical, and in vivo characterization of key compounds from within these efforts will be presented. [1] Stauffer, ACS Chem. Neurosci., 2011, ASAP accepted. [2] O'Brien, J. A, et al. J. Pharmacol. Exp. Ther., 2004, 309, 568. [3] Hammond, A. S, et al. ACS Chem. Neurosci., 2010, 702. sources include NIH and Johnson and Johnson Pharmaceuticals. Vanderbilt is a Specialized Chemistry Center within the Molecular Libraries Probe Centers Network.

169.mGluR5 Positive Allosteric Modulators: An Industrial Perspective T. Steckler1, C.K. Jones2, S.R. Stauffer2, J.M. Bartolomé1, C.M. Niswender2, H. Lavreysen1, G. Macdonald1, C. Mackie3, P. Vinson2, J.S. Daniels2, C.W. Lindsley2 and P.J. Conn2 1

Neuroscience Drug Discovery, Janssen RND, Beerse, Belgium; Department of Pharmacology and Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, Nashville, TN; 3Discovery ADME/Tox, Janssen RND, Beerse, Belgium

2

Accumulating evidence indicates that selective activation of the metabotropic glutamate receptor subtype 5 (mGluR5) may provide a novel therapeutic strategy for the treatment of the psychotic symptoms and cognitive impairments observed in individuals with neuropsychiatric disorders, including schizophrenia. mGluR5 is expressed in limbic brain regions, including the parahippocampal region, hippocampus, amygdala, striatum and thalamus. It is primarily postsynaptically localized, modulating neuronal excitability, at least in part by potentiating NMDA receptor function. Both mGluR5 agonists and positive allosteric modulators (PAMs) have been described. PAMs can be hypothesized to be of greater utility than agonists because a PAM should only be active in the presence of the endogenous ligand glutamate, i.e., it will boost the activity of the endogenous ligand rather than having a direct stimulatory effect at the orthosteric binding site. Because of this, it is expected that the effect of a PAM is physiologically more relevant as it provides a more phasic rather than a tonic stimulation of the receptor. Consequently, there should be less of a risk for overstimulation due to the need for the endogenous ligand to be present and less risk of receptor desensitization. However, while efficacy has been documented with several mGluR5 PAMs, limited information has been provided on possible side effects. Moreover, PAMs were hypothesized to be advantageous over orthosteric mGluR5 agonists because of better selectivity for the mGluR5 receptor subtype, and to possibly overcome some other agonistrelated issues, such as poor bioavailability and brain penetration. Solubility, bioavailability and brain penetration have also been issues with mGluR5 PAMs, although more recently described ligands show improved properties. While in general the selectivity of PAMs for the mGluR5 is high, there are also compounds that have additional properties at other mGluRs, as well as compounds that do not just act as pure PAMs but also have some mGluR5 agonist activity on their own. Because different PAMs may bind to different pockets within the seven-transmembrane domain of the mGluR5, this may pose additional challenges for translational strategies, especially the development of PET ligands needed to study target occupancy. Besides these challenges, substantial progress has been made and the target remains to be of high


interest, although direct clinical evidence of the mGluR5 PAM approach is still outstanding.

170.Presynaptic mGlu7 Receptor Inhibits the KClInduced Release of GABA in Mouse Hippocampus M. Summa1, S. Di Prisco1, C. Usai3 and A. Pittaluga1,2 1 Department of Experimental Medicine, Section of Pharmacology and Toxicology, University of Genoa, Genoa, Italy, 2Center of Excellence for Biomedical Research, University of Genoa, Genoa, Italy,3Institute of Biophysics, National Research of Council, Genoa, Italy

The existence of presynaptic mGlu7 heteroreceptors controlling GABA release in the hippocampus has been inferred on the basis of histochemical and functional observations [1-3], but a direct demonstration is still lacking. We have tested this hypothesis by investigating the impact of selective ligand on the release of GABA (measured as preloaded [3H]GABA) elicited by a mild depolarizing stimulus from adult mouse hippocampal synaptosomes. Transient exposure (90 sec) of synaptosomes to a solution containing 12 mM KCl elicits a Ca2+-dependent, exocytotic-like release of GABA. The exocytotic nature of the release is supported by the finding that this event is inhibited by the GABAB agonist (±) baclofen. When AMN082 was added contemporary to the depolarizing stimulus, a significant inhibition of GABA outflow could be observed. AMN082 concentration-dependently (1 nM-1μM) inhibited the 12 mM K+-evoked [3H]GABA overflow, the maximum inhibitory effect (-31.26 ± 1.97, result expressed as % inhibition) being observed when the agonist was added at 0.1 μM. Inhibition by 0.1 μM AMN082 was concentration-dependently (0.1 nM- 1 μM) recovered by the mGlu7 allosteric modulator MMPIP; concentration as low as 10 nM of MMPIP completely recovered the inhibitory effect exerted by the agonist. Based on data in literature suggesting that AMN082-mediated effect also could involve NMDA, 5HT1A or 5HT2A/2C receptors, we investigated whether these receptors could participate in the effect observed. Selective antagonists for the three receptors, however, failed to affect the AMN082-induced inhibition of the K+-evoked [3H]GABA outflow from mouse hippocampal synaptosomes. Western blotting analysis of the presynaptic component of mouse hippocampal synaptosomes confirmed the presence of mGlu7 receptor protein at this level. Confocal analysis showed significant co-localization of mGlu7 immunoreactivity with syntaxin-1A and V-GAT immunoreactivity in adult mouse hippocampal synaptosomes, indicating that presynaptic mGlu7 receptors exist in GABAergic terminals. All together these results allow to conclude that presynaptic mGlu7 heteroreceptors controlling GABA exocytosis exist in GABA nerve endings isolated from the hippocampus of adult mice. [1] Shigemoto, et al. J. Neurosci., 1997, 17(19), 7503–7522. [2] Gereau, R.W, and Conn, P.J. J. Neurosci., 1995, (10), 6879-89. [3] Cossart, et al. Neuron, 2001, 29, 497-508.

171.mGlu1 and Cell Cycle Interactions in the Developmental Apoptosis of Rat Cerebellar Granule Neurons G.R. Takoudjou, J. DiRaddo, S. Pshenichkin, H. Hathaway, E. Grajkowska and J.T. Wroblewski. Department of Pharmacology, Georgetown University, Washington, DC 20057, USA Previously, we have shown that mGlu1 receptors play a dual role in neuronal apoptosis: mGlu1 receptor overexpression led to massive cell death while several mGlu1 receptor agonists protected neurons from this receptor-induced apoptosis. In this study, we used primary cultures of cerebellar granule neurons as a model of neuronal


apoptosis and survival. These neurons survive and differentiate well in a medium supplemented with high potassium concentrations (25 mM, K25 conditions) while physiological K+ concentrations (5 mM, K5 conditions) lead to an increasing in mGlu1 receptor expression and robust cell death. Under this conditions glutamate strongly protected neurons from K5 toxicity and this protective effect of glutamate was blocked by the mGlu1 receptor selective antagonist CPCCOEt. This suggests a role for mGlu1 receptors in glutamate-induced protection during neuronal development and low K5 conditions may be regarded as an in vitro model of developmental apoptosis. Our previous observations have also indicated that under these conditions enhanced mGlu1 expression is influenced by the E2F1 transcription factor, suggesting the involvement of proteins associated with the cell cycle. In fact, transferring granule cells from K25 to K5 conditions caused a differential, time-dependent, regulation of several such proteins. This included an increase in the phosphorylation of the retinoblastoma protein, as well as increase of free E2F1. Also the expression of cyclin E was increased after 6 hours while cyclinA was downregulated after 4 hours. Under these conditions, we also observed enhanced DNA synthesis, as measured by BrdU incorporation. Quantitative PCR studies also showed increases in cdk2 expression, which were reduced by glutamate acting at mGlu1 receptors.Moreover, a selective cdk2 inhibitor reversed K5 toxicity, while selective cdk1 and cdk4/6 inhibitors failed to provide this protective effect. These results indicate that mGlu1 receptors are involved in a complex manner in the apoptosis of cerebellar neurons which may result from an aberrant reentry into the cell cycle which leads to apoptotic death of post-mitotic cells. The interactions between mGlu1 receptors and cell cycle proteins may involve both negative, agonist-independent, and positive, glutamate-mediated, signaling of mGlu1 receptors. Supported by NIH grant NS37436.

172.Group II Metabotropic Glutamate Receptors Signal via Src kinase to Modulate NMDAR-Currents in CA1 Neurons C.H. Trepanier1,2, M.F. Jackson2, P.J. Conn3, T. Nakazawa4, T. Yamamoto4 and J.F. MacDonald2 1

Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON, Canada 2 Robarts Research Institute, Molecular Brain Research Group, University of Western Ontario, London, ON, Canada 3Vanderbilt University Medical Center, Department of Pharmacology and Vanderbilt Center for Neuroscience Drug Discovery, Nashville, TN 4 Department of Medical Chemistry, Kansai Medical University, Moriguchi, Osaka, Japan. Group II metabotropic glutamate receptors (mGluRs) have emerged as important targets for the treatment of schizophrenia [1-3]. Although predominantly located presynaptically, there is some evidence for postsynaptic expression of these receptors where they couple to Gi/o protein and inhibition of adenylyl cyclase [4]. Since hypofunction of postsynaptic NMDAR function has also been implicated in the etiology of schizophrenia, we examined whether postsynaptic group II mGluRs (mGluR2/3) regulate N-methyl-Daspartate receptors (NMDARs) function in identified CA1 hippocampal neurons. Surprisingly, application of the selective group II mGluR agonist, LY 379 268 (10 nM) for 5 min, significantly enhanced the peak of NMDA-evoked currents. To establish which G-protein mediates the observed enhancement, we applied the Gi inhibitor pertussis toxin (5 ug/ml) inside the patch pipette and showed that it blocked the enhancement by LY 379 268. Similarly, application of recombinant RGS4 (1 ug/ml) also prevented the enhancement by group II mGluRs. Intracellular application of the Src inhibitory peptide Src(40-58) blocked the mGluR2/3 effect on NMDAR currents. Furthermore, the mGluR2/


3-mediated potentiation did not discriminate between subtypes of NMDARs as the regulation was blocked by both the GluN2Aantagonist Zn2+ (0.5 uM) and the GluN2B-antagonist Ro 25-6981 (0.5 uM). Consistent with the subtype-specific antagonists, the mGluR2/3-mediated enhancement of NMDAR currents was abolished in neurons isolated from mice expressing a knock-in mutation of the NR2B Tyr1472 site to phenylalanine compared to wild-type controls. Overall these results demonstrate that activation of mGluR2/3 recruits Src kinase to potentiate GluN2A- and GluN2B-containing NMDA currents in CA1 neurons. This may represent a novel mechanism to correct the hypoglutamatergic state found in schizophrenia. [1] Vinson, P.N., Conn, J.P. Neuropharmacology, 2011, xxx, 1-12 . [2] Patil, S.T, et al. Nat. Med., 2007, 13, 1102-7. [3] Chavez-Noriega, L.E, et al. Curr. Drug Targets CNS Neurol. Disord., 2002, 1, 261-81. [4] Niswender, C.M., Conn, J.P. Annu. Rev. Pharmacol. Toxicol., 2010, 50, 295-322.

173.mGlu4 Receptor Potentiation for the Treatment of Parkinson’s Disease M. A. Uberti1, M. D. Bacolod1, H. N. Jimenez1, M. Cajina1, D. Doller1, P. Gubellini2 and K. E.Bennouar2 1

Lundbeck Research USA, Paramus, NJ, USA and 2Institut de Biologie du Développement de Marseille-Luminy (IBDML) UMR6216 Case 907, Parc Scientifique de Luminy13009 Marseille – France Parkinson’s disease (PD) is the second most common neurodegenerative disorder. It is characterized by dysfunction of motor and non-motor symptoms. Dopamine replacement therapy, including L-DOPA and dopamine agonists, is the most common treatment, however, over time these treatments lose efficacy and induce debilitating side-effects. Thus, there is an increasing interest to identify novel non-dopaminergic therapeutic agents for the treatment of motor symptoms in PD. Previous efforts using rodent behavioral models generated support for the concept that activation of metabotropic glutamate receptor 4 (mGlu4) by orthosteric agonists has the potential to alleviate PD symptoms when injected in the striatum or the globus pallidus of rodents [1]. Our current studies have shown that a brain penetrant mGlu4 receptor positive allosteric modulator (PAM), Lu AF21934, can dose-dependently reduce synaptic transmission in cortico-striatal slice electrophysiology and potentiate the inhibitory effect of the orthosteric mGlu4 receptor agonist LSP1-2111. In behavioral studies, Lu AF21934 dose-dependently inhibited motor deficits induced by haloperidol. Lu AF21934 was also tested in unilaterally 6-OHDA lesioned rats using the cylinder test. This study was designed to explore the hypothesis that a combination of a mGlu4 PAM and LDOPA would potentiate the akinetic effects of a sub-threshold dose of L-DOPA alone. The results from this study demonstrated the pro-kinetic effects of a combination PAM + L-DOPA treatment. This synergistic effect suggests that mGlu4 PAMs could be a potential therapeutic treatment for PD. The implications of these studies will be discussed. [1] Dario, C, et al. J. Neurochem., 2009, 109, 1096-1105.

174.Acetyl-L-Carnitine Promotes Adult Hippocampal Neurogenesis in vitro via NF- B-Mediated Upregulation of mGlu2 Receptors M.M. Valente1,2, V. Bortolotto1,2, P.L. Canonico2, A. Koverech3 and M. Grilli1,2 1 Laboratory of Neuroplasticity & Pain, 2DiSCAFF & DFB Center, University of Piemonte Orientale “A. Avogadro”, Novara, Italy, 3 Medical Direction, Sigma-Tau, Pomezia, Italy


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Attention has been raised on the pharmacological properties of Acetyl-L-carnitine (ALC) in several neuropsychiatric disorders, including chronic neuropathic pain and mood disturbances. Recently, the analgesic effects of ALC have been explained by epigenetic mechanisms, namely drug-induced upregulation of mGlu2 receptors expression in dorsal root ganglia, via acetylation of the NF-B p65 transcription factor [1]. Since the proposed role of NF-B proteins in adult neurogenesis, in stress-induced disregulated neurogenesis and depressive behaviour [2,3], we decided to explore the potential activity of ALC as a modulator of adult hippocampal neurogenesis. We tested ALC (10 μM-1 mM) activity in an in vitro model of adult neural progenitor cells (NPC) from mouse hippocampus. The drug increased, in a concentrationdependent manner, the number of mature and immature neurons derived from adult NPC, with a maximal effect elicited at 1 mM, and, in parallel, decreased the number of undifferentiated progenitor cells. Interestingly, under the same experimental conditions, L-carnitine (LC) had no effect on NPC neuronal differentiation. Moreover, the mGlu2/3 antagonist LY341495 (0.1-10 M) counteracted the proneurogenic activity of ALC, suggesting that it was probably mediated by group II mGlu receptors. In the same cellular model, both the mGlu2/3 agonist, LY379268 (0.1-10 M) and the mGlu2 positive allosteric modulator LY487379 (0.1-1 M) demonstrated proneurogenic activity, suggesting a specific role of the mGlu2 receptor subtype in such effect. Finally, we demonstrated that a 24h treatment with 300 μM ALC upregulated mGlu2 expression in adult hippocampal NPC and that NF-B activation lied downstream the signaling pathway mediating ALC proneurogenic effects. In conclusion, for the first time, we propose a new pharmacological activity for ALC as a positive modulator of hippocampal neurogenesis in vitro, potentially via mGlu2 receptor and NF-B signaling activation. In addition these data would support a role for mGlu2 receptor as molecular regulator of adult hippocampal neurogenesis. In the future, we would like to investigate whether this novel activity may contribute to ALC efficacy in relevant animal models of depression and cognitive impairment. [1] Chiechio, S, et al. Trends Pharmacol. Sci., 2010, 31, 153-160. [2] Denis-Donini, S, et al. J. Neurosci., 2008, 28, 3911-3919. [3] Koo, J.W, et al. PNAS, 2010, 107, 2669-2674.

175. In vitro Characterization of the Binding of the mGlu2 Receptor Positive Allosteric Modulator [3H]JNJ40068782 to Native and Recombinant mGlu2 Receptors I. Van der Lindena, L. Peetersa, I. Biesmansa, J.I. Andrésb, M. Vliegenc, P. Te Rielea, X. Langloisa, J.R. Atacka and H. Lavreysena a

Neuroscience, bC.R.E.A.Te and cIsotope Synthesis, Janssen Research and Development, a division of Janssen Pharmaceutica NV, Turnhoutseweg 30, 2340 Beerse, Belgium

mGlu2 receptors are promising targets for the treatment of psychiatric and neurological diseases associated with dysfunction of glutamatergic neuro-transmission. Although mGlu2/3 agonists represent one approach to altering glutamate neurotransmission in the treatment of anxiety and schizophrenia, the positive allosteric modulation of mGlu2 receptor function is an attractive alternative. JNJ-40068782 has been identified as a new selective mGlu2 receptor positive allosteric modulator (PAM), which potentiates glutamate-induced signaling with an EC50 of 150 nM in human mGlu2 receptor-expressing cells. In order to further characterize the allosteric interaction of JNJ-40068782 with the mGlu2 receptor, we evaluated the binding of [3H]JNJ-40068782 to cloned receptors stably expressed in cells as well as in native rodent brain tissue. Binding of [3H]JNJ-40068782 to recombinant human mGlu2 receptors expressed in CHO cells was saturable and was best-fitted to a single-site model with a KD of 13 nM. Similar KD values were


found for rat and mouse cortex homogenates. In rat brain sections, the anatomical distribution of [3H]JNJ-40068782 binding was consistent with that previously described for mGlu2 receptors. As PAMs can increase the functional potency of agonists, we evaluated the effect of glutamate on the binding of [3H]JNJ-40068782, in both human or rat mGlu2 receptor stably-transfected CHO cells and rat cortical membranes. In the presence of 100 M glutamate, a 5 fold increase in affinity of the PAM was seen for both cloned rat and human mGlu2 receptors. In contrast, glutamate had no effect on [3H]JNJ-40068782 binding affinity observed in rat cortex, indicating that allosteric interaction between agonists and PAMs may be context-dependent or masked by the presence of endogenous glutamate. We furthermore tested [3H]JNJ-40068782 binding in the presence of various other PAMs, including BINA and LY487379 and examined whether also negative allosteric modulators like Ro676221 were able to displace the radioligand. Interestingly, all compounds were able to displace [3H]JNJ40068782 binding. While these data may suggest that they all bind to common determinants within the same recognition site, the true nature of interaction, being either competitive or non-competitive, is not yet clear. The IC50 values of compounds inhibiting [3H]JNJ40068782 binding were comparable between human mGlu2 receptor CHO cells and rat cortex. Additionally, for human mGlu2 receptor-expressing cells, the IC50 values obtained in the binding assay corresponded well to IC50 values obtained with a functional [35S]GTPS binding assay.

176.Spinal mGlu4 Activation Inhibits Hyperalgesia in Animal Models of Chronic Pain by a Modulation of the Glutamatergic Neurotransmission B. Vilar1,2, J. Busserolles3,4,5, B. Ling3,4,5, E. Chapuy3,4,5, F. C. Acher6, J. P. Pin1,2, A. Eschalier3,4,5 and C. Goudet1,2 1

Univ Montpellier I& II, CNRS UMR5203, Institut de Génomique Fonctionnelle, Montpellier, France; 2INSERM, U661, Montpellier, France; 3INSERM, UMR766, Pharmacologie Fondamentale et Clinique de la Douleur, Clermont-Ferrand, France; 4Univ Clermont 1, Faculté de Médecine, Laboratoire de Pharmacologie Médicale, Clermont-Ferrand, France; 5CHU Clermont-Ferrand, Service de Pharmacologie, Clermont-Ferrand, France; 6UMR8601CNRS, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, Université Paris V, Paris, France Glutamate plays a key role in the modulation of nociceptive processing. This excitatory amino acid exerts its action through two distinct types of receptors, the ionotropic and the metabotropic glutamate receptors (mGluRs). Eight mGluRs have been identified and divided in three groups. Here, we focused on group-III mGluRs, which contains mGlu4, 6, 7 & 8. These receptors are mostly presynatic and are present in most structures implicated in pain neuraxis including the spinal cord (except mGlu6). Previous studies have shown that group-III mGluRs spinal activation has an antihyperalgesic effect in neuropathic and inflammatory pain models but no effect in acute pain models in rodents [1]. However, little is known about the precise role of the different group-III mGluRs subtypes, partly due to the poor pharmacology for these receptors. The aim of this study is to discriminate the role and the cellular and molecular mechanisms of mGlu4 in pain regulation at the spinal cord level. Interestingly, immunohistochemistry study revealed that mGlu4 is expressed in a subpopulation of sensory neurons involved in the modulation of pain hypersensitivity. Accordingly, electrophysiological recordings in spinal cord slice selective activation of mGlu4 strongly reduces the evoked excitatory postsynaptic currents amplitude (eEPSCs) in lamina II neurons through mechanisms involving voltage-gated calcium channels and this inhibition is significantly increased in slices from inflamed animals. Moreover, selective activation of mGlu4 by a specific agonist (LSP4-2022) has an antihyperalgesic effect on


chronic pain models (Carrageenan and unilateral peripheral mononeuropathy) comparable to that observed following whole group-III mGluRs activation (ACPT1). The antihyperalgesic effect of ACPT1 is strongly reduced in rats traited by mGlu4 antisenses and lost in mGlu4 KO mice. Taken together, these results underline the crucial role of mGlu4 among group III mGluRs in pain modulation. [1] Goudet, et al. Pain, 2008.

177.Optimization of mGlu5 Positive Allosteric Modulator for in vivo Studies P. N. Vinson1,2, J. M. Rook2, S. R. Stauffer1,3, J. Manka1, S. Zhou1, K. Gogi1,2, C. M. Niswender1,2, C. K. Jones1,2, J. S. Daniels1,2, T. Bridges1,2, H. Lavreysen4, J. M. Bartolomé4, C. Mackie4, T. Steckler4, G. MacDonald4, C.W. Lindsley1,3 and P. J. Conn1,2 1

Vanderbilt Center for Neuroscience Drug Discovery, 2Vanderbilt University Medical Center Department of Pharmacology, 3 Vanderbilt University Department of Chemistry, Nashville, TN. 4 Neuroscience Drug Discovery, Janssen RND, Beerse, Belgium Several positive allosteric modulators (PAMs) of the metabotropic glutamate receptor subtype 5 have been validated in preclinical models predictive of antipsychotic activity as well as models that investigate potential cognitive enhancement [1] indicating that mGlu5 PAMs show promise as a novel therapeutic approach for schizophrenia. However, the mGlu5 PAMs currently used as tool compounds suffer from low potency, low exposure, and/or low solubility which limit their use in in vivo models. An mGlu5 PAM with more attractive properties allowing easier formulation and lower doses to achieve efficacy is desired. A recently disclosed mGlu5 PAM, VU0360172, [2] has been chosen for further optimization of potency and physicochemical properties to result in a compound that may be used for more in-depth studies of the effects of positive modulation of mGlu5. From a set of over 80 compounds based on the core scaffold of VU0360172 (Fig. 1), compounds for further investigation were chosen based on potency (EC50 of potentiating an EC20 of glutamate) and cLogP. These compounds will move forward to determine which possess the most promising exposure and formulatability profiles before being tested in animal models predictive of antipsychotic activity (e.g. reversal of amphetamine-induced hyperlocomotion) and cognitive enhancement. The goal of this work is to develop a best in class mGlu5 PAM to enable both in vitro and in vivo studies to better understand the mechanism and behavioral effects of mGlu5 potentiation. [1] Vinson, P. N. and Conn. P. J. Neuropharmacology, In Press. [2] Rodriguez, A. L, et al. Mol. Pharmacol., 2010, 78(6), 1105-1123. Funding sources include NIH and Johnson and Johnson Pharmaceuticals. Vanderbilt is a Specialized Chemistry Center within the Molecular Libraries Probe Centers Network.

Fig. (1). Structure of VU0360172 with the core group shown in blue.

178.mGlu2/3 agonist and Antagonist Effects on Sleep and Wakefulness in Rodents –Dependence on Receptor Subtype K. A. Wafford, E. Shanks, W. Seidel, A. McCarthy, D. Kellett and D. M. Edgar Lilly UK, Erl Wood Manor, Windlesham, Surrey, GU20 6PH, UK


Metabotropic glutamate receptors agonists and antagonist have previously been demonstrated to influence the parameters of sleep and wake in rodents. Pharmacological manipulation of mGlu2 and mGlu3 having large effects on the extent of different vigilance states. For example the agonist LY379268 has been shown to inhibit REM sleep [1] and the antagonist LY341495 was demonstrated to increase arousal level [1]. In this study we have measured sleep vigilance state using EEG and EMG together with simultaneous measures of locomotor activity, body temperature, feeding and drinking to investigate the effects of LY379268 and LY341495 in rat. We have utilised the same measures to profile the mGlu2-/-, mGlu3-/- and mGlu2/3 -/- mice compared to wild type for any changes in sleep/wake behaviour, together with effects of pharmacological agents in these mice. The agonist LY379268 produced a marked inhibition of REM sleep immediately following dosing which was dose dependent between 1-10mg/kg and at the high dose sustained for up to 30 hrs post treatment with no rebound. Interference with NREM sleep also occurred after 2 hours and was maintained into the dark cycle. This response was accompanied by an increase in body temperature and decreased locomotor activity producing prolonged hypoactivity at 10mg/kg. The antagonist LY341495 dosed between 1and 10mg/kg produced a dosedependent increase in wakefulness with a corresponding drop in both NREM and REM sleep. The increase in wakefulness was sustained for 6hrs with no rebound hypersomnolence observed in NREM sleep. REM sleep was compensated for in the subsequent dark period. Locomotor activity was increased but no hyperactivity produced while awake. Body temperature increased slightly after dosing however remained below the circadian maximum. mGlu2-/and mGlu3-/- mice demonstrated no significant differences compared to wildtype in sleep/wake cycles with respect to NREM sleep, REM sleep, sleep and wake bout length, locomotor and body temperature cycles. mGlu2/3-/- mice exhibited a 2 hr phase delay in body temperature rhythm. The pharmacological effects elicited by LY379268 were similar in mice to those observed in rat with the exception of a reduction in body temperature rather than an increase seen in rat. Efficacy of LY379268 on all measures was substantially reduced in the mGlu2-/- and mGlu2/3-/- mice. A small reduction was also seen in the mGlu3-/- mice, suggesting that the major effects on sleep/wake parameters of the agonist are mGlu2 receptor mediated. LY341495 increased wakefulness in wildtype mice which was reduced to a small extent in both mGlu2-/- mice and mGlu3-/- mice suggesting contribution from both receptor subtypes. Some increase in wakefulness was still observed in the mGlu2/3-/- mice indicating the potential contribution from additional targets of this compound. [1] Feinberg, et al. JPET, 2005, 312, 826-833.

179.Glutamate Addiction in mGluR1 Expression Human Melanoma Cells Brian Wall, Janet Wangari-Talbot and Suzie Chen Susan Lehman Cullman Laboratory for Cancer Research, Ernest Mario School of Pharmacy, Rutgers, the State University of New Jersey, Piscataway, NJ 08854, U.S.A Despite the advancements of medical technologies, melanoma remains one of the least treatable cancers. Although surgical excision and adjuvant therapies have improved the management of this disease, patients with advanced stages of melanoma have poor long-term prognosis and high mortality rates due to the intrinsic resistance of melanoma to the apoptosis that is commonly induced by anticancer drugs. We have previously developed a transgenic mouse model that spontaneously develops cutaneous, malignant melanoma with 100% penetrance in the absence of any exogenous carcinogens. We showed that the ectopic expression of murine metabotropic glutamate receptor 1 (mGLuR1) in melanocytes is


65

sufficient to induce spontaneous melanoma development in vivo. We also demonstrated that ~60% of human melanoma cell lines and biopsies display aberrant mGluR1 expression. mGluR1 is predominantly associated with excitatory synaptic neurotransmission in the mammalian central nervous system. The signaling cascades activated by its natural ligand, glutamate, are important in regulating the growth and differentiation of a wide variety of both neuronal and non-neuronal cells. We hypothesize that one of the consequences of deregulated mGluR1 expression in melanocytes is activation of pathways leading to an increase in cell proliferation and/or decrease in apoptosis, thus resulting in cell transformation and subsequent tumor formation. Riluzole is an FDA approved drug for the treatment of amyotrophic lateral sclerosis (ALS). One of the modes of action of Riluzole is to disrupt glutamate receptor activities by reducing the availability of glutamate. We propose that the apoptotic responses observed in Riluzole-treated mGluR1positive melanoma cells could be one of the consequences of irreparable damage to the genome via elevated intracellular glutamate levels, ultimately resulting in a reduction in GSH synthesis and an altered ROS homeostasis.

180.Dysregulation of mGluR2/3 and mGluR5 is a factor in Compulsive Behavior Motivated by Drugs of Abuse and Conventional Reward: Implications for the Treatment Target Potential of mGluRs F. Weiss Molecular and Integrative Neurosciences Department, The Scripps Research Institute, La Jolla, CA, 92037, USA Metabotropic glutamate receptors (mGluRs) are abundant in brain regions mediating incentive motivation and drive, as well as circuitries regulating anxiety and behavioral responses to stress. MGluR dysfunction in these brain regions has been implicated drug and ethanol (EtOH) addiction. We focus here on mGluR2/3 that negatively modulate glutamate transmission, reducing synaptic availability of glutamate, and on MGluR5 that positively modulate glutamate transmission, increasing neural excitability. MGluR2/3 agonists and mGluR5 antagonists (NAMs) reverse several neurobehavioral effects of cocaine and EtOH, and are particularly effective in attenuating reinstatement of drug seeking induced by drug cues and stress. We sought to establish the effects of the mGluR5 negative allosteric modulator (NAM), MTEP, and the mGluR2/3 agonist LY379268 (compounds that reduce neural excitability -- although via different mechanisms) on selfadministration of cocaine, EtOH, highly palatable natural reward, and on reinstatement motivated by these reinforcers. Emphasis in these studies was on differences in the effects of mGluR manipulation on behavior in non-dependent vs. post-dependent rats in order to identify changes in function of these mGluRs induced by chronic drug exposure. Three major findings were obtained. First, both LY379268 and MTEP attenuated drug self-administration and reinstatement in nondependent rats. Strikingly, LY379268 produced substantially stronger inhibition of EtOH-motivated behaviors in rats with a history of dependence than in nondependent rats, an effect that was paired with increased mGluR2/3 functional activity. In contrast, the efficacy of MTEP to attenuate these behaviors was significantly reduced dependent compared to nondependent rats, and this effect was accompanied by decreased dimeric mGluR5 expression in the NAcore. Second, a history of escalated cocaine self-administration conveyed “compulsive” character to behavior motivated by natural reward in that, different from drug-naïve rats, conditioned natural reward seeking developed resistance to extinction, similar to behavior normally associated with drug seeking only. In addition, natural reward seeking, a behavior not normally sensitive to reversal by mGluR2/3 agonists in drug-naïve animals, became sensitive to reversal by LY379268 in rats with a



history of escalated cocaine self-administration. Lastly, in studies designed to characterize the treatment target potential of mGluR2/3, we found that tolerance develops to the inhibitory effects of LY379268 on cue-induced cocaine reinstatement with daily treatment, but that tolerance is completely abolished if LY379268 is administered intermittently at two or three day intervals. Overall, the findings further confirm that mGluR2/3 and mGluR5 represent promising treatment targets for cue- and stress-induced drug seeking. In addition, the findings reveal that chronic ethanol and cocaine intoxication neuroadaptively alters the function on mGluR2/3 and mGluR5. Moreover, chronic cocaine results in a “switch” toward compulsive-like behavior motivated not only by drugs of abuse but also natural reward. These changes may be of significance for understanding the neurobiological basis of compulsive drug seeking associated with a history of drug dependence. Supported by NIH grants: AA10531, AA018010, DA07348, and DA08467.

181.Hit to lead Activities in a Novel Series of mGlu 2/3 Receptor Negative Allosteric Modulators: PyrazoloPyrimidines J. Wichmann, T. M. Ballard, S. Gatti, M. Graf, J. Huwyler, F. Knoflach and T. J. Woltering Hoffmann-La Roche Ltd., pRED Basel, Grenzacherstrasse 124, CH-4070 Basel, Switzerland In a HTS campaign using the rat mGlu2 receptor FLIPR assay a pyrazolo-pyrimidine derivative was discovered as a singleton out of a library purchased from ChemBridge. Due to the interesting scaffold, low molecular weight and high chemical tractability the compound appeared to be a valuable starting point for hit to lead activities. The activity of the compound could be increased by incorporating substitution at the phenyl ring, and in vivo activity could be achieved by the replacement of the cyano group by pyridines. For example, one representative of this pyridine subclass, RO4654252, is a potent mGlu2 receptor negative allosteric modulator which displayed in vivo activity after oral administration in a rat LMA model. CF3

CF3 N

N N

N N

N N

F3C N

HTS hit binding cAMP

IC50 = 560 nM IC50 = 428 nM

RO4654252 binding IC50 = 24 nM cAMP IC50 = 11 nM ED50 = 25 mg/kg p.o. (rats)

The Medicinal Chemistry aspects of the hit to lead activities as well as the accompanying in vitro pharmacology profiling of the novel class of mGlu2/3 receptor negative allosteric modulators will be discussed. For advanced compounds in vivo and DMPK data will be presented.

182.The involvement of mGlu4, yet not the mGlu7 Receptor, in the Potential Antipsychotic-Like Action of mGlu Group III Receptor Ligands: Focusing on LSP12111 and AMN082 Activity J.M. Wieroska1, K. Stachowicz1, P. Braski1. A. Paucha-Poniewiera1, T. Lech1, F.C. Acher2 and A. Pilc1,3 1

Institute of Pharmacology, Polish Academy of Sciences, 31-343 Kraków, Poland; 2 Laboratoire de Chimie et BiochimiePharmacologiques et Toxicologiques, UMR 8601 CNRS, Universite Paris

Descartes, Paris, France, 3Jagiellonian University, Medical College, Faculty of Health Sciences, 31-531 Kraków, Poland Behavioral studies show that modulation of the glutamatergic system might be an efficient way to achieve antipsychotic activity in rodents. Such an activity was shown for selective, allosteric mGlu5 receptor agonists and mGlu2/3 agonists/positive modulators. Recently, also the third group of mGlu receptors has been investigated, as the new, selective and brain penetrating compounds became available. It was shown that ACPT-I, a non-selective group III agonist, exerted antipsychotic activity in mice. Here, we decided to investigate the possible role of the more selective group III mGlu receptors ligands, LSP1-2111, the orthosteric mGlu4 receptor preferential agonist and AMN082, the mGlu7 receptor positive modulator. We used MK-801- and amphetamine-induced hyperactivity tests, as well as DOI-induced head twitches in mice as a models for positive symptoms of psychosis in mice. LSP1-2111 (1, 2 and 5 mg kg-1) dose-dependently inhibited both MK-801 and amphetamine-induced hyperactivities. Moreover, the drug antagonized DOI-induced head twitches. In opposite, AMN082 (3 and 6 mg kg-1), had no effect on amphetamine-induced hyperactivity, but induced an enhancement of MK-801-induced hyperactivity and DOI-induced head twitches in mice. In C57Bl/6J mGlu7 receptor knockout animals the specificity of the effect of AMN082 was confirmed, as the drug induced no effects in those animals. Moreover, mGlu7 KO animals were less sensitive for DOI-induced effect than their wild type littermates. Altogether, we propose that among group III mGlu receptors, mGlu4 receptor is a promising target for the development of novel antipsychotic drugs. The study was supported by POIG. 01.01.02-12-004/09 “Depression-Methods-Therapy” grant, task 3.7.

183. An Unanticipated Antidepressant-Like Effect of an mGlu2 Receptor Agonist J.M. Witkin1, X. Li1, C. Overshiner1, K. Ellis1, D. Bedwell1, S. Swanson1, L. Rorick-Kehn1, J. Katner1, K.W. Perry1, L. Prieto2, D.L. McKinzie1 and J. Monn1 Lilly Research Laboratories, Eli Lilly and Company, 1Indianapolis, IN, USA, and 2Alcobendas, Spain LY2812223 binds to hmGlu2 and hmGlu3 receptors with comparable potency (~100 nM) but acts as a preferential full agonist of hmGlu2 receptors with less pronounced agonist activity at hmGlu3 receptors; although the EC50 = 133 nM in forskolinstimulated cAMP in RGT cells expressing hmGlu3 receptors, LY2812223 had a reduced efficacy (Emax = 57%) (see B. Heinz et al; C. Felder et al; J. Monn et al this meeting). Since balanced, maximal efficacy mGlu2/3 receptor agonists are uniformly devoid of activity in acute antidepressant screens in rodents, LY2812223 was evaluated for its ability to produce antidepressant-like effects in the mouse forced-swim test. LY2812223 produced antidepressantlike effects in mice with an ED60 of 6.1 + 2.5 mg/kg and maximal effect of 50.5 + 11.5%, comparable to that of imipramine. LY2812223 produced significant decreases in immobility in wildtype mice but was devoid of effect in mice without mGlu2 receptors. No additive or synergistic effects were observed in the mouse forced swim test when an inactive dose of LY2812223 (3 mg/kg) was given in conjunction with inactive doses of fluoxetine (10 mg/kg, i.p.) or imipramine (5 mg/kg, i.p.). Comparable findings were obtained with 379268, a full mGlu2/3 receptor agonist. Brain and plasma levels of LY2812223, imiprmaine, or fluoxetine, were not significantly affected by the drug combinations although there was a tendency for levels to increase when the drugs were given together. A peptide prodrug, LY2979165 (both zwitterion and HCl salt form), was generated that liberated LY2812223 upon oral dosing in rats (see L. Prieto et al; D. McKinzie et al, this meeting).


LY2979165.HCl dose-dependently decreased immobility time in rats with a maximal efficacy of 32%, comparable to that of imipramine (30%). The minimal effective dose was 4.1 mg/kg (equivalent to 3 mg/kg of the active moiety of LY2812223). In contrast to full mGlu2/3 agonists, the mglu2 preferring LY2812223 and its orally-active peptide prodrug produce antidepressant-like effects in the forced-swim test. LY2979165.HCl also engendered antidepressant-related neurochemical and EEG changes in rats (see D. McKinzie, this meeting). LY2979165.HCl increased the efflux of the biogenic amines dopamine, serotonin, and norepinephrine in rat prefrontal cortex following 10 and 30 mg/kg (active equivalents) (see McKinzie et al., this meeting). In addition to the unprecedented antidepressant-like effects of the mGlu2 full agonist, it is noteworthy that these molecules also produced anti-stress/antianxiety-like effects in stress-induced lactate efflux and stressinduced hyperthermia assays in rats. Moreover, antipsychotic-like efficacy typical of other mGlu2/3 receptor agonists was also observed (see D. McKinzie et al, this meeting); a molecule with this combination of in vivo activities might have unique and novel clinical efficacy.

184.1,3-Dihydro-benzo[b][1,4]Diazepin-2-one Derivatives as Potent Non-Competitive Metabotropic Glutamate Receptor 2/3 Antagonists T. J. Woltering, J.Wichmann, E. Goetschi, J. Huwyler, F. Knoflach, T. M. Ballard and S. Gatti F. Hoffmann-La Roche Ltd., pRED Basel, Grenzacherstrasse 124, CH-4070 Basel, Switzerland We have synthesized and characterized the properties of a series of 1,3-Dihydro-benzo[b][1,4]diazepin-2-one derivatives which are potent and selective non-competitive mGlu2/3 receptor antagonists. Attachment of an 8-(2-aryl)-ethynyl-moiety produced compounds like 2 inhibiting the binding of [3H]-LY354740 to rat mGlu2 with low nanomolar affinity and consistent functional effect at both mGlu2 and mGlu3 [1]. The selectivity of this new class of noncompetitive mGlu2/3 receptor antagonists was demonstrated versus mGlu1, mGlu4 mGlu5 and mGlu8 receptors and ionotropic glutamate receptors. H N

Me

O

F

Ph

H N

N

H N

N CN

1 IC50 = 6.4 M

F

O

2 IC50 = 34 nM

HO

H N

O

N

N 3 IC50 = 26 nM

N

N

O

NN N

F3C

H N

Me

N

O

N

4 IC50 = 16 nM ED50 = 3.3 mg/kg p.o.

Me

Me IC50 = 2 nM ED50 ~ 3 mg/kg p.o.

Replacement of a cyano group by a 5- and 6-membered heterocycle at the position R4 and further modification to improve the physicochemical properties led eventually to compounds like 3 with the ability to reverse LY354740-mediated inhibition of field excitatory postsynaptic potentials in the rat dentate gyrus [2]. Finally replacement of the (2-aryl)-ethynyl-moiety in 8-position with smaller less lipophilic substituents produced compounds like 4 where for the first time in vivo activity could be demonstrated by reversal of the LY354740-induced hypoactivity in mice after oral administration [3]. Optimization with particular emphasis on derivatizations compatible with brain penetration and in vivo activity led to compounds bearing a para-pyridine, like RO4491533, which consistently showed in vivo activity in rat behavioral tests after oral administration, e.g. blockade of the mGluR2/3 agonist LY354740-induced hypoactivity and improvement of a working memory deficit induced either by LY354740 or scopolamine in the delayed match to position (DMTP) task. Moreover, combination studies with a cholinesterase inhibitor show apparent synergistic effects on working memory impairment induced by scopolamine [4].


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[1] Woltering, T. J, et al. S. Bioorg. Med. Chem. Lett., 2007, 17(24), 68116815. [2] Woltering, T. J, et al. Bioorg. Med. Chem. Lett., 2008, 18(3), 1091-1095. [3] Woltering, T. J, et al. Bioorg. Med. Chem. Lett., 2008, 18(8), 2725-2729. [4] Woltering, T. J.; Wichmann, J.; Goetschi, E.; Knoflach, F.; Ballard, T. M.; Huwyler, J.; Gatti, S. Bioorg. Med. Chem. Lett., 2010, 20(23), 6969.

185.Development and Cross-species Evaluation of GTP 35S Autoradiography Methods to Measure Functional Activity of Selective mGlu2 Potentiators in Native Tissues R. Wright, M. Johnson, M. Muhlhauser and K. Svensson Neuroscience Discovery Biology, Eli Lilly & Company, Indianapolis IN 46285 As part of our ongoing efforts to develop and study the pharmacology of selective mGlu2 positive allosteric modulators, we needed to establish the means to demonstrate functional activity in native tissues. A method to measure GTP35S binding in slidemounted sections from frozen brain tissue was developed and characterized. In initial studies, GTP35S binding in mouse frontal cortex was stimulated by the mGlu2/3 selective agonist LY379268 (EC50 =26 nM, Bmax=82% over Basal) and was substantially increased (EC50=3.5 nM, Bmax=274% over basal) upon addition of the selective mGlu2 potentiator LY566332 (CBiPES) at 10 μM. LY566332 had some intrinsic activity on its own (EC50=1800 nM, Bmax=86% over basal) and this binding was increased in the presence of 10 mM LY379268 (EC50=220 nM, Bmax=321% over basal). The agonist-stimulated and potentiator-enhanced binding was localized in brain regions that correspond to binding by the highly-selective mGlu2/3 receptor agonist ligand [3H]-LY459477 in mGlu3 receptor knockout mice. The greatest levels of enhanced binding occurred in the prefrontal/limbic regions of the cortex, lacunosum moleculare of the hippocampus, striatum and granular layers of the cerebellum. The binding in these mGlu2-expressing brain regions was blocked by the mGlu2/3 orthosteric antagonist LY341495 (10 μM) and absent in mGlu2 receptor knockout mice. Further studies were conducted to examine the effects of mGlu2 receptor potentiators LY566332 and LY2607540 on glutamate and other mGlu receptor agonist-induced GTP35S binding in brain sections from transgenic mice and from other species. This presentation will illustrate the utility of GTP35S autoradiography for quantifying and comparing the effects of selective mGlu2 receptor potentiators on the functional activation of mGlu2 receptors in multiple species, including rodent, non-human primate and human brain tissues.

186.Selective Agonists of mGlu3 Receptors: NAcetylaspatylglutamate (NAAG) and 2-Hydroxymethylglutamate (HMG) B. Wroblewska1, S. Pshenichkin1, E.J. Miller2, J. DiRaddo1, E.Grajkowska1 and J.T. Wroblewski1 1

Department of Pharmacology, Georgetown University, Washington, DC 20057, 2Department of Chemistry, Emory University, Atlanta, GA, USA The understanding of the physiological role of group II metabotropic glutamate receptors is hampered by the lack of selective agonists able to distinguish between mGlu2 and mGlu3 receptors. We have previously reported that the endogenous dipeptide N-acetylaspatylglutamate (NAAG) activated mGlu3 receptors expressed in CHO cells, without affecting cells expressing mGlu2 receptors. Recently, two published reports attributed the effects of NAAG on mGlu3 receptors to the contamination of commercial preparations of NAAG with glutamate. To re-evaluate


the selectivity at mGlu3 receptor we have used highly purified preparations of NAAG. We also performed experiments in the presence of glutamate-pyruvate transaminase which removes all traces of glutamate. These new results confirm our previous observations that NAAG is an agonist of mGlu3 receptors and that it’s activity is not due to contamination with glutamate. We also show that a new compound, 2-hydroxymethylglutamate (HMG), is a selective agonist of mGlu3 receptors. When tested in cell lines expressing the various mGluRs, HMG showed no significant activity at group I mGluRs, while among group III mGluRs it was a very weak agonist of mGlu4, without activity at mGlu6, mGlu7 and mGlu8 receptors. At group II mGluRs, HMG showed an unusual pattern of selectivity activating mGlu3, while inhibiting mGlu2 receptors. HMG was also inactive at ionotropic glutamate receptors (NMDA, AMPA or kainate) in primary cultures of cerebellar neurons.Neuroprotective properties of HMG were tested in three in vitro models of neurotoxicity in mixed neuronal-glial mice cortical cultures. Neurotoxicity was evoked by (1) 10 min application of 75 μM NMDA, (2) 60 min glucose and oxygen deprivation, and (3) 48 h application of 25 μM -amyloid peptide. Cell death was quantified by measurement of lactate dehydrogenase. In all three models HMG showed strong neuroprotective action, although the maximal efficacy varied depending on the model used. Additional experiments showed that the efficacy of HMG depended on the presence of glial cells, suggesting that the neuroprotective mechanism may involve the activation of glial mGlu3 receptors followed by the release of trophic factors. These results are consistent with our previous observations that the neuroprotective action of NAAG is depends on the presence of glial cells. Since the neuroprotective actions of NAAG are similar to the actions of a selective mGlu3 agonist (HMG), these data provide additional evidence that NAAG is a selective endogenous agonist of mGlu3 receptors. Supported by NIH grant NS37436.

187.Unique inhibitory Synapse with Particularly Rich Endocannabinoid Signaling Machinery on Pyramidal Neurons in Basal Amygdaloid Nucleus T. Yoshida1,2, M. Uchigashima1, M. Yamasaki1, I. Katona3, M. Yamazaki4, K. Sakimura4, M. Kano5, M. Yoshioka2 and M.Watanabe1 1

Departments of Anatomy and 2Neuropharmacology, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan, 3Institute of Experimental Medicine, Hungarian Academy of Sciences, Szigony utca 43, 1083 Budapest, Hungary, 4Department of Cellular Neurobiology, Brain Research Institute, Niigata University, Niigata 951-8585, Japan, 5Department of Neurophysiology, Graduate School of Medicine, University of Tokyo, Tokyo 1130033, Japan 2-Arachidonoylglycerol (2-AG) is the endocannabinoid that mediates retrograde suppression of synaptic transmission in the brain. 2-AG is synthesized in activated postsynaptic neurons by sn1-specific diacylglycerol lipase (DGL), binds to presynaptic cannabinoid CB1 receptors, suppresses neurotransmitter release, and is degraded mainly by monoacylglycerol lipase (MGL). In the basolateral amygdala complex, CB1 is particularly enriched in axon terminals of cholecystokinin (CCK)-positive GABAergic interneurons, induces short- and long-term depression at inhibitory synapses, and is involved in extinction of fear memory. Here we clarified a unique molecular convergence of DGL, CB1, and MGL at specific inhibitory synapses in the basal nucleus (BA), but not lateral nucleus, of the basolateral amygdala. The synapses, termed invaginating synapses, consisted of conventional symmetrical contact and unique perisynaptic invagination of nerve terminals into perikarya. At invaginating synapses, DGL was preferentially


recruited to concave somatic membrane of postsynaptic pyramidal neurons, while invaginating presynaptic terminals highly expressed CB1, MGL, and CCK. No such molecular convergence was seen for flat perisomatic synapses made by parvalbumin-positive interneurons. On the other hand, DGL and CB1 were expressed weakly at axo-spinous excitatory synapses. Consistent with these morphological data, thresholds for DGL-mediated depolarizationinduced retrograde suppression were much lower for inhibitory synapses than for excitatory synapses in BA pyramidal neurons. Moreover, depolarization-induced suppression was readily saturated for inhibition, but never for excitation. These findings suggest that perisomatic inhibition by invaginating synapses is a key target of 2-AG-mediated control of the excitability of BA pyramidal neurons. In the talk, we will also introduce some other brain regions with such intensive convergence of endo cannabinoid signaling molecules.

188.Histamine: an Endogenous Modulator of Metabotropic Glutamate Receptors S. Yin1,2, R. Zamorano1,2, P. J. Conn1,2 and C. M. Niswender1,2 1 Department of Pharmacology, 2Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, Nashville, TN 37232, USA

The metabotropic glutamate receptors (mGluRs) are a group of Family C G-protein-coupled receptors that are activated by glutamate. As a member of the group III mGluRs, mGluR4 is expressed predominantly presynaptically and has been shown to rectify inappropriate GABA release at the striatopallidal synapse in Parkinson’s Disease. While synthetic small molecules have been discovered that specifically potentiate the mGluR4 activity, it is also possible that endogenous compounds also exist which can modulate receptor activity in a positive or negative fashion. We screened a series of neurotransmitters to determine if these endogenous ligands could modulate glutamate activity at mGluR4. Interestingly, the neurotransmitter histamine appeared to potentiate mGluR4 activity with similar efficacy when compared to known small molecule positive allosteric modulators of mGluR4. These data suggest that histamine could serve as an endogenous modulator that plays an important role in regulating mGluR4 function. Ongoing studies are focused on developing a detailed understanding of the mechanism by which histamine potentiates mGluR4 responses and how this impacts different aspects of mGluR4 signaling. Supported by grants from the NIH. Vanderbilt is a Specialized Chemistry Center in the MLPCN.

189.Beneficial Effect of mGlu4 Receptor Activation on Multiple Animal Models o Autoimmune Disease Zappulla C1, Notartomaso S1, Cetrangolo G2, Fazio F1, Acaro A2, Galgani M3, Scarselli P1, Busceti C1, Lepore A4, Formisano S4, Nicoletti F1,5, Gentile F 2 and Di Marco R1,2 1

I.N.M. Neuromed, Pozzilli; 2Department of Health Sciences (SpeS), University of Molise, Campobasso; 3Institute of Experimental Endocrinology and Oncology (I.E.O.S.) - CNR, Naples; 4Department of Cell and Molecular Biology and Pathology (DBPCM), University of Naples “Federico II”; 5Dept. of Physiology and Pharmacology University of Rome Sapienza, Italy


The mGlu4 receptor, is gaining increasing interest for its immonomodulatory properties. We show here the effects of the treatment with a selective mGlu4 receptor enhancer, (-)-N-phenyl-7(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC), on two models of Experimental Autoimmune Neuritis (EAN), a Tcell-mediated autoimmune disease reminiscent of the clinical, pathological and electrophysiological features of the human Guillain-Barré syndrome (GBS). Acute EAN induced in Lewis rats by active immunization leads to a monophasic disease with transient flaccid paresis of the tail and limbs due to an acute and mainly demyelinating inflammatory polyradiculoneuropathy. A new and biphasic form of EAN is described in dark agouti rats (DA rats) those develop mild episodes of disease; DA-EAN may serve as a model for relapsing inflammatory demyelinating polyneuropathies such as chronic inflammatory demyelinating polyneuropathy (CIDP). In both experimental models we treated animals with PHCCC (3 mg/kg, s.c., once a day), either under prophylactic regimen (from the day of immunization) or therapeutically (at the onset of symptoms). Both treatments, when compared to the respective control groups, were associated with significant improvements in the clinical scores, as well as in the histological hallmarks of neuroinflammation, and with reductions in the number and severity of relapses in DA-EAN models. In order to assess whether similar effects could be reproduced on different preclinical models of autoimmunity, we extended the study to experimental autoimmune thyroiditis (EAT) in CBA/J(H-2k) mice, a murine model of Hashimoto’s thyroiditis, which can be induced in mice with H-2k and H-2s haplotypes by immunization with mouse thyroglobulin (mTg) or human Tg (hTg) in complete Freund’s adjuvant (CFA). EAT is characterized by the infiltration of thyroid by mononuclear cells, the production of specific antibodies and secondary proliferative responses of lymphocytes against Tg in vitro. Once immunized with 100 μg of hTg in CFA subcutaneously, mice were treated daily with PHCCC (3mg/kg). Boosting on day 10 was accomplished with 50 μg of hTg in IFA s.c. Histological examination of thyroid lobe showed decreased mononuclear infiltration of thyroid tissue in PHCCC treated mice, compared with untreated mice. In addition, the analysis of multiple cytokine concentrations revealed marked decreases in the secretion of inflammatory cytokines, such as IL-6 and IL-17, but not IFN-, in mice treated with PHCCC, compared with untreated mice. IL-2 secretion was not affected by treatment. These data indicate a role of mGlu4 receptors in the pathophysiology of inflammatory/ autoimmune diseases and suggest a potential role of mGlu4 receptor enhancers in the treatment of experimental neuroinflammation.

190.Role of Group II Metabotropic Receptors in LongTerm Plasticity of Presynaptic Vesicular Release at Glutamatergic Synapses X.L. Zhang1, R.E. Nicholls2, C.P. Bailey3, C. Upreti1, W. Müller4, E.R. Kandel2 and P.K. Stanton1 1

New York Medical College, Valhalla, NY, USA; 2Columbia University, NY, NY, USA 3University of Bath, Bath, UK; 4University of New Mexico, Albuquerque, NM, USA Long-term plasticity of synaptic transmission can consist of multiple changes and loci, both presynaptic and postsynaptic. Different forms of long-term potentiation (LTP) and long-term depression (LTD) of glutamatergic synaptic transmission can be induced by activation of either N-methyl-D-aspartate (NMDAR) or group I metabotropic (mGluR) glutamate receptors. Utilizing two-photon laser scanning microscopic (TPLSM) imaging of presynaptic terminals labelled with fluorescent indicators, we discovered that NMDAR-dependent LTP and LTD can be expressed as a combination of alterations in both presynaptic glutamate release and postsynaptic sensitivity to glutamate. TPLSM imaging


69

of vesicular release using either FM1-43 or the pH-sensitive vesicular release marker SynaptopHluorin reveals components of LTP and LTD expressed as selective changes in vesicular release from the rapidly-recycling vesicle pool loaded by action potentials or hypertonic shock. We found that activation of either presynaptic group II mGluRs or A1 adenosine receptors, when paired with elevation of intracellular [cyclic GMP], is sufficient to induce LTD. These G protein-coupled receptors, when activated, release Gi2 and G in the presynaptic terminal. Gi2 inhibition of adenylate cyclase promotes, but does not induce, LTD. G is known to both inhibit voltage-dependent calcium channels, and to bind directly to the C-terminus of the SNARE protein SNAP-25. We discovered that, at Schaffer collateral-CA1 synapses in the hippocampus, the C terminus of SNAP-25 is necessary for the expression of LTD, but not LTP. Using type A botulinum toxin (BoNT/A) to enzymatically cleave the 14 amino acid C-terminus of SNAP-25 eliminated the ability of low-frequency synaptic stimulation to induce LTD. Presynaptic infusion by electroporation of CA3 pyramidal neurons with either the 14 amino acid C-terminus of SNAP-25, or the G scavenging peptide mSIRK, also blocked the induction of LTD, without persistently altering presynaptic calcium influx. Taken together, our data show that involvement of presynaptic group II mGluRs is a necessary, but not sufficient, condition for long-term plasticity of vesicular transmitter release. The larger view of studies of activity-dependent synaptic plasticity shows us that persistent changes in both transmitter release and postsynaptic receptor sensitivities are regulated by patterns of synaptic activation, and that they can even shift in opposing directions as a function of transsynaptic signaling molecules that produce a bidirectional dialogue across the synapse.

191.Loss of mGluR-Dependent Long-Term Depression in the ACC after Digit amputation Min Zhuo Department of Physiology, Faculty of Medicine, University of Toronto, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada ACC is a crucial cortical region involved in brain-related physiological and pathological conditions such as emotional fear and chronic pain. Both long-term depression (LTD) and long-term potentiation (LTP) have been reported in the ACC of adult animals. Using traditional single recording electrode approach, we reported previously that peripheral injury (digit amputation) caused loss of LTD induced by low-frequency stimulation. Due to the limit of the recording method, it is unknown if such loss of LTD is widespreading within the ACC. In the current study, we used a multielectrode array (MED64) system to map cortical LTD in a spatiotemporal manner. The MED64 system enabled us to observe the network properties of ACC by stimulating deep layer V and detecting field excitatory postsynaptic potentials (fEPSP) from other 63 sites within the ACC. We found that low frequency stimulation (1Hz) applied on deep layer V induced LTD in nearby layers II/III and layers V/VI regions of the ACC. Pharmacological results showed that L-type voltage-gated calcium channels, metabotropic glutamate receptors (mGluRs) especially mGluR1 are required for the induction of ACC LTD. Blocking NMDA receptors also reduced LTD. Amputation of the distal tail impaired LTD that persisted for at least two weeks. Our results provide the first study on the network properties of ACC LTD in adult mice. and demonstrate that tail amputation causes LTD impairment within the ACC circuit.

192.Role of Optineurin and Huntingtin in the Regulation of mGluR5 signalling Stephen S. G. Ferguson and J. Allyn Taylor


Centre for Cell Biology, Molecular Brain Research Group, Robarts Research Institute, 100 Perth Dr., London, Ontario, N6A 5K8 Group I metabotropic glutamate receptors (mGluR1 and mGluR5) are members of the G protein-coupled receptor (GPCR) superfamily, but other than a seven transmembrane spanning topology do not have structural motifs common to other GPCRs. Group I mGluRs have extended carboxyl-teriminal tails that have the potential to interact with multiple intracellular signalling proteins. We have utilized both proteomic and yeast two-hybrid based genetic screening approaches to identify optineurin as a mGluR1/5 interacting protein. Optineurin is a protein that was originally identified as a positive regulator of TNF-mediated apoptosis, but that has now been recognized as a causative factor for normal pressure open angled glaucoma and as a huntingtin interacting protein. Huntington’s disease is associated with the progressive neuronal cell death in both the neocortex and the striatum of patients due to a polyglutamine expansion of the huntingtin aminoterminus. We have demonstrated that optineurin contributes to the


desensitization of Group I mGluR signalling and that mutant huntingtin and not wild-type huntingtin potentiates optineurinmediated mGluR desensitization in culture. Consistent with this observation, mGluR5 signalling is severely impaired in striatal neuronal cultures from mutant human huntingtin (HdhQ111/Q111) knock-in mice, compared with wild-type human huntingtin (HdhQ20/Q20) knock-in mice. We have also used acute striatal slices derived from HdhQ20/Q20 and HdhQ111/Q111 mice to demonstrate that mGluR5-mediated G protein-signalling is selectively attenuated in mutant huntingtin knockin mice that are between 2 and 9 months of age. This attenuation of mGluR5a signalling is lost in mutant huntingtin mice that are more than 11 months of age, which is consistent with disease progression with age. The decrease in mGluR5-stimulated inositol phosphate formation is accompanied by a generalized dysregulation of Ca2+ signalling, as well as increased mGluR5 coupling to both ERK1/2 and Akt. Taken together these observations suggest that adaptations in mGluR5 signalling favour neuronal survival at early ages of life.

Abstracts of the 7th International Meeting on Metabotropic

A Abi-Saab, W. Acaro, A. Acher, F.C. Ackermann B. Adams, C.L. Adams, D.H. Adelfinger, L. Ago, Y. Ahnaou, A. Aiba, A. Alagille, D. Alvarado-Munoz, I.P. Amalric, M. Ametamey, S.M. Andersson, J.D. Anderson, S. Andree, T. H. Andrés, J.I. Anthony, N.J. Antonysamy, S. April, M. Araque, A. Arcella, A. Ardecky, R. Arendt-Nielsen, L. Atack, J.R. Atwell, S. Auberson, Y.P. Aubert, L. Auerbach, B.D. Austin, J. Ayan-Oshodi, M. Azzollini, F.

69 189 1,71,101,176,182 103 95 130 2 3 94 4 84,165 137 40,71 41 152 103 82 175 141 104 97,98 5 30 33 129 15,94,142,175 104,114,121,149 41 16 6,10 97 103 123

B Bacolod, M.D. Badger II, J. D. Bahi, A. Bailey, C.P. Baker, S.J. Baker, S. Baldwin, R. Ballard, T.M. Banères, J.L. Bannerman, D.

11,97,173 29 2 190 36 114,121,149 84 7,181,184 47,146 93

71

Barker, G.R.I. 26 Barret, O. 8,84 Barth, V.N. 46,129 Barone, Y. 125 Bartolomé, J.M. 168,169,177 Bashir, Z.I. 140 Baskys, A. 28 Bates, B.S. 55,87 Batis, J. 84 Battaglia, G. 9,23,30,52,101,111,125,137,140,145,166 Beadle, C. 114,121,149 Bear, M.F. 6,10 Beato, M. 109 Beck, J. 105,106 Becker, A. 160 Becker, J. 38 Beckmann, J.S. 68 Bedwell, D. 114,121,149,183 Belladonna, M.L. 51 Bender, C.N. 82 Benk, B. 59 Bennouar, K.E. 11,173 Berg, D. 69 Bergonzelli, G.E. 108 Bernabucci, M. 12,52,120,133 Berry-Kravis, E. 68 Bertrand, H.-O. 1,71,101 Bessiron, T. 71 Bettler, B. 13 Bhatia, V.K. 21 Biagioni F, F. 162 Bianchi, R. 51 Biechele, T.L. 14 Bielik, A. 59 Biemans, B. 16 Biesmans, I. 15,94,175 Bilbe, G. 61,68 Birnbaum, S.G. 153 Bissantz, C. 105,106 Blanco, J. 114,121,149 Bleher, B. 38 Blobaum A.L. 17,50,55,87,131 Bobok, A. 18,59 Bockaert, J. 124 Bojarski, A. 20 Boléa, C. 24 Bogenpohl, J. 165 Bolinger, J.L. 50

72

Bonanno, G. Bonelli, M. Bonifacino, T. Bonnet, B. Borro, M. Bortolotto, Z.A. Bouret, S.G. Bournique, B. Bouwknecht, J.A. Bouwalerh, H. Boyle, N.J. Brabet, I. Branchi, I. Brandon, N.J. Braski, P. Branson, J. Brasjno, G. Bräuner-Osborne H. Brewer, K.A. Bridges, T. Broad, L.M. Brodbeck, R.M. Brown, R.M. Browne, S. Brückner, S. Brun, A. Bruno, V. Bryce, D. Bubser, M. Bucci, D. Buck, A. Bugno, R. Burdi, D.F. Burger, C. Burkhardt, S. Burnat, G. Busceti, C. Busserolles, J. Bussy, G. Byers, F.W. Byun, N.

Author Index

19 30 19 45 137 26,75,76,102,117,174 108 24,57 15 108,111 97 1,71 108 82 20,138,139,182 68 73 21,119 131 168,177 76 97,98 95 24 24 68 9,23,52,101,120,166 82 131 9,23 41 20 77 41 38 20 189 176 68 55,87 128

C Cajina, M. Calaza, K.C. Camp, N.D. Campo, B. Cannella, M. Canonico, P.L.

11,98,173 74 14 22,24,57 23,111,120,133 174

Caraci, F. 166 Carniglia, L. 44 Carpenter, R.L. 87 Caruso, A. 125 Caruso, C. 44 Cassano, T. 111 Catlow, J. 158 Cavallone, L.F. 65 Célanire, S. 24 Cenci, M.A. 25 Ceolin, L. 26,102 Cesarini, S. 1 Cetrangolo, G. 189 Chambers, M.G. 129 Chandrasena, G. 98 Charvin, D. 24,45,57 Chaki, S. 3,27 Chan, P. 28 Chang, K. 29 Chapuy, E. 176 Chen, F. 68 Chen, P. 130 Chen, S. 112,179 Cheng, F. 28 Cheung, Y. 131 Chiechio, S. 133 Chien, A.J. 14 Chin, C.-L. 36 Cho, H.P. 72,132 Choi, K. Y. 29 Chorobik, P. 20 Christiansenm, B. 21 Chrucicka, B. 20 Ciampoli, M. 125 Cianfrogna, J. 163 Ciceroni, C. 30 Cid, J. 94,142 Clark, B. 114,121,149 Clausen, R.P. 119 Clawson, D. 104 Clemmensen, C. 21 Colleen, M. 17 Collingridge, G.L. 26,75,76,102,117 Collini, S. 12 Collins, K.A. 153 Colvin, E.M. 76 Conn, P.J. 17,31,33,37,50,55,72,85,87,110,126,128, 131,132,156,164,168,169,172,177,188 Conti, F. 19 Copani, A. 133,166


Copeland, C.S. Cornish, K. Corti, C. Cosford, N. Courtiol, T. Crock, L.W. Cryan, J.F. Csoti, I. Curie, A. Curini, M. Currier, G.

32,155 68 93 33,110 1,71 65 34,135 69 68 101 163

D Dahl, R. D’Amore, V. Daniel, H. Daniels, J.S. Danysz, W. Darnaudéry, M. Darren, W. Dautzenberg, F. Dawson, E.S. Day, M. Dean, R. De Blasi, A. Dekundy, A. Dhanya, R. Delange, K. Dell’Anno, M.T. Delille, H.K. De Lorenzo, G. Deltheil, T. De Maria, R. De Mello, F.G. De Novellis, V. Deschwanden, A. Des Portes, V. Detheux, M. Diener, C. Dietz, M. Dilella, A. Dillinger, A. Di Luca, M. Di Marco, R. Di Menna, L. Dimitrov, D. Dinan, T. G. Di Nuzzo, L. Di Paolo, T.

33 52,156 71 17,50,55,87,131,132,168,169,177 35 108 50 94 37,131,168 36 103 83 35 33 68 39 38 125 40,71 30 74 107 41 68 77 16 2 24 2 64 51,52,101,133,162,189 52,120 109 135 120 69

73

Di Prisco, S. 125,170 DiRaddo, J. 42,49,63,78,118,150,151,171,186 Doller, D. 11,97,98,173 Domány, G. 59 Dominguez, C. 114,121,149 Dong, E.N. 72 Donovan-Rodriguez, T. 45 Donsimoni, G. 43,159 Doumazane, E. 43,47,146,159 Downing, A.C.M. 130 Drinkenburg, W. 94 D’Souza, D.M. 110 Duncan, J. 95 Dunlop, J. 82 Durand, D. 44 Duvey, G. 45,57,94 Duvoisin, R.M. 48 Dwyer, J.M. 82 E Eckstein, J. Edgar, D.M. Eessalu, T. Eggert, K. El-Moustaine, D. Ellis, K. Emery, A.C. Emmitte, K. A. Engers, D.W. Emtage, S. Eschalier, A.

103 116,144,178 46 69 47,146 183 42,49,78,150,151 55,87 17,50,131 114,121,149 176

F Fabre, L. Fagni, L. Fallarino, F. Farde, L. Fass, D.M. Fazio, F. Felder, C. Fell, M. Felts, A.S. Ferguson, S.S.G. Ferraguti, F. Feyissa, A.M. Fijal, B.A. Finn, T. Fioretti, M.C.

43,159 124 51 152 14 51,52,101,133,162,189 53,54,79,121,158 158 55,87 192 56 41 130 57 51

74

Fischer, C. Fisher, M.J. Fisone, G. Fizia, K. Floesser, A. Flor, P.J. Fonsi, M. Forest, T. Formisano, S. Fornai, F. Fox, G.B. Friedman, L.K.

Author Index

16 129 39 2 68 2,61 24,57 141 189 162 36 58

Granier, S. Gravius, A. Gregg, L.C. Gregory, K.J. Grilli, M. Grimwood, S. Grohmann, U. Grueter, B.A. Gubellini, P. Guidotti, A. Guimarães-Souza, E. M. Guo, W. Gyertyán, I.

47,146 35 134 31,72,132,168 174 163 51 73 11,173 113 74 153 18

G H Gabriëls, L. Gagliardi, S. Gál, K. Galgani, M. Gardino, P. F. Gardoni, F. Gardner, E.L. Gasparini, F. Gass, P. Gatti, S. Gelb, T. Gentile, F. Gentile, G. Gerace, E. Gereau IV, R.W. Gertner, M.J. Gheyi, T. Gibson, C. Gibson Jay, R. Giovine, A. Girard, F. Giribaldi, F. Gitter, B. Gloriam, D.E. Godau, J. Goetschi, E. Gogi, K. Gogliotti, R.D. Gomez-Mancilla, B. González-Maeso, J. Goudet, C. Gradini, R. Graf, M. Grajkowska, E.

142 45 18,59 189 74 64 60 2,61,68,69 62,145 7,105,106,181,184 63 189 137 64,92,143,157 65 66 104 24 153 108 24,57 19 67,116 21,119 69 7,105,184 177 17,37,50,131 68,69 70 1,71,101,176 108,133,156 181 42,49,63,78,118,150,151,171,186

Hadjikhani, N. Hagerman, R.J. Haggarty, S.J. Halldin, C. Hanna, L. Hansen, K.B. Hao, J. Hardy, L.W. Hariry, S. Harpsøe1, K. Harrison, P. Hasler, G. Hathaway, H. Hays, S.A. He, Y. Healy, A. Hechenberger, M. Heinz, B. Henry, S. Henstridge, C. Herath, A. Hess, F. Hilse, T. Ho, M. Hohmann, A.G. Höllt, V. Homburger, V. Hopkins, C.R. Horn, J. Hostetler, E. Hu, J.-H. Huang, S. Huber, H.

68 68 14 152 75,76,102 21 114,121,149 77 69 119 93 41 42,49,63,78,150,151,171 153 68 87 35 53,54,79,121,129,158 114,121,149 134 33 24 68 103 134 160 124 17,37,50,131 67,116 80 153 47,146 69


Huber, K.M. Huganir, R.L. Hughes, Z.A. Huguenard, J. Hugues, A. Hunt, R. Huszar, S.L. Hutson, P.H. Huwyler, J.

153 81 82 96 141 77 141 141 7,181,184

I Iacovelli, L. Iijima, M. Ikehata, M. Iyengar, S.

52,83,166 27 154 129

J Jackson, K. Jackson, M.F. Jacobson, M.A. Jacquemont, S. Jadhav, S. Jaeschke, G. Jean, A. Jennings, D. Jimenez, H.N. Johansen, L.D. Johayem, A. Johns, D. Johnson, A. Johnson, B. Johnson, K.A. Johnson, M.P. Johnson, K.W. Johnson, M. Jones, C.K. Jones, K. Jones, M.W. Julio-Pieper, M. Jung, K.M. Juréus, A.

103 172 141 68 17,50,55 16 43 84 11,119,173 21 41 68,69 152 114,115,116 85 86 86 115,185 17,31,50,55,87,131,156,168,169,177 177 144 34 134 152

K Kaae1, B.H. Kammermeier, P.J. Kandel, E.R.

119 88 190

75

Kano, M. Karmacharya, R. Karolewicz, B. Katner, J. Katona, I. Katner, J. Kaufmann, K.W. Kearins, M. Kellett, D. Kenji, U. Keith, A. Kerkerian-Le Goff, L. Keser, G.M. Kew, J. Khilevich, A. King, R. Kinon, B.J. Kiss, B. Klein, K.U. Knitowski, K. Knoflach, F. Knöpfel, T. Koike, H. Kolber, B.J. Kouranova, E. Koverech, A. Kuczenski, R. Kuhn, R. Kuklish, S. Kulikauskas, R.M. Kurczab, R.

89,187 14 41 114,183 134,187 114,115 72 104 144,178 28 178 11 59 93 158 140 90,130 18,59 35 114,115 7,105,181,184 109 27 65 82 125,174 110 61 129 14 20

L Laloux, C. Lamas, C. Lambeng, N. Lambert, R. Landucci, E. Lane, T. Langlois, X. Lanzetta, D. Larocca, L.M. Lasaga, M. Laurenza, M. Lavreysen, H. Lawrence, A.J. Lawson, K.P. Le, U.M.

108 114,121,149 45,91 168 64,92,143,157 93 15,94,175 8 30 44 30 15,94,142,168,169,175,177 95 55,87 50

76

Lech, T. Ledent, C. Lee, C. Lee, J. Lemasson, I.A. Leonard, S.K. Lepore, A. Le Poul, E. Lew, R. Li, G. Li, D. Li, J. Li, X. Li, Y. Liechti, M. Lindemann, L. Linden, D.J. Lindsley, C.W. Lindsley, S.R. Ling, B. Lionetto, L. Liotta, D.C. Lis, E. Liu, G. Liu, J. Liverton, N. Lodge, K.E. Lodge, D. Lotarski, S. Lowe, S. Lu, F. Lu, S. Lucas, S.J. Ludányi, A. Lundström, L. Luo, B. Luongo, L. Lütjens, R. Lyon, L.

Author Index

182 95 96 63 1 82 189 24,45,57,94 77 97,98 82,129 60 28,60,114,183 28 110 16 99 17,31,37,50,55,72,87,100,131, 132, 156,168,169,177 17,50 176 101,111 118 24 28 47,146 24 141 26,75,76,102,117 82 103 104,114,121,149 82 102 134 105,106 24 107 94 93

M Macaluso, A. Maccari, S. MacDonald, G. MacDonald, J.F. Mackie, C.

12 108,111,113,122 31,94,168,169,177 172 168,169,177

Mackie, K. 134 Madsen, U. 119 Magliaro, B.C. 141 Maione, S. 107 Mairesse, J. 108,111,122 Malenka, R.C. 73 Mallel, G. 162 Malmquist, J. 152 Man, T. 114,121,149 Manahan-Vaughan, D. 148 Manka, J. 31,72,168,177 Mannaioni, G. 64,92,143 Manzoni, O. 134 Marcaggi, P. 109 Marcos, A. 114,121,149 Marek, G. 36 Marek, G.J. 36,38 Marek, K. 84 Markou, A. 33,110 Marrocco, J. 108,111,122 Martinet, D. 68 Masi, A. 64 Masilamoni, G. 165 Massey, S. 114,121,149 Mastrantoni, E. 30,112 Mastroiacovo, F. 101 Masure, S. 142 Mathé, A. 125 Mathiesen, J.M. 119 Matrisciano, F. 113,125,145 Matsuda, T. 3 Mattson, B.A. 141 Mauro, G. 52 Mayhugh, D. 158 McAllister, K. 61 McCarthy, A. 178 McGinnis, D. 163 McKinzie, D. 53,54,67,79,103,114,115,116,121,149,183 McKinzie, D.L. 183 McLean, H. 71 Megens, A. 15,94 Meiler, J. 72 Mela, F. 35 Melon, C. 11 19 Melone, M. Menon, U.N. 50,55 Melchiorri, D. 30,112,123 Menè, P. 162


77

Menzel, K. 141 Mercier, M. 117 Merlo, S. 166 Messer, J. 105 Meyer, J. 67,68,116 Mezler, M. 36,38 Michalski, J-C. 111 Milanese, M. 19 Millen, B.A. 130 Miller, E. 163 Miller, E.J. 42,118,186 Mølck, C 119 Molinaro, G. 9,23,52,101,120,137,140,145,166 Monk, S.A. 129 Monn, J. 53,54,67,76,79,104,114,115,116,121,149,183 Montana, M.C. 65 Montero, C. 114,121,149 Moog, R. 7 Moon, R.T. 14 Moore, E.C. 14 Morelli-Canelo, M. 69 Morley-Fletcher, S. 108,111,122 Moroni, F. 64,92,143,157 Morrison, R.D. 17,50,87,131 Mosillo, P. 123 Motolese, M. 23,111,133,137 Mouillac, B. 47,146 Moutin, E. 124 Muhlhauser, M. 185 Müller, W. 190 Munsie, L.M. 130 Murray, T.K. 144 Musante, I. 19,154 Muster, J. 14 Mutoh, H. 109

Nicoletti F.

N

Pak, A. Palazzo, E. Pallini, R. Pallotta, M.T. Pallottino, S. Palucha-Poniewiera, A. Panaccione, I. Parmentier-Batteur, S. Parsons, C.G. Passer, R. Paulding, C.J. Pedersen, D.S. Peeters, L.

Nakazawa, T Nasca, C. Natanegara, F. Neal, S.J. Neale, S.A. Neri, G. Neugebauer, V. Ngomba, R. Niccolini, C. Nicholls, R.E. Nickols, H.

172 125,145 103 82 32 68 126 101,122,127,156 30 190 33,128

Nielsen, S.M. Niolu, C. Nisenbaum, E. Nisenbaum, E.S. Nisenbaum, L.K. Nisticò, R. Niswender, C.M. Noetzel, M.J. Nógrádi, K. Notartomaso, S. Nowak, G. Nyilas, R.

9,12,23,30,51,52,83,101, 108,111,113,120,122,123, 125,137,140,145,156,162, 166,189 119 125 54 129 130 120,145 17,31,37,50,72,85,87,131,132, 168,169,177,188 31,72,132,168 59 51,52,101,133,162,189 138 134

O O’Brien, J.A. O’ Connor, R.M. Ofner, S. Olive, M.F. Ollendorff, V. Olsen, M. O’Neill, M.J. O’Neil, S.V. Orabona, C. Orlando, R. Orsini, M. Osterweil, E.K. Overshiner, C. Oueslati, N.

141 135 61 136 124 82 144 82 51 137 77 6,10 114,183 1,71

P 114,121,149 107 30 51 9,39 20,138,139,182 120,140 141 35 16 68 21 94,142,175

78

Author Index

Pellegrino, E. 124 Pellegrini-Giampietro, D.E. 64,92,143,157 Peng, X.-Q. 60 Perkins, A.N. 86 Perry, B. 45 Perroy, J. 124 Perry, K. 103,114,115,183 Pesenti, C. 45 Peters, J. 67,116 Phillips, K.G. 144 Piccinin, S. 120,145 Pickel, J.M. 29 Pierrou, S. 152 Pignatelli, M. 120,145 Pilc, A. 20,138,139,167,182 Pin, J.-P. 1,43,47,71,101,146,159,176 Piomelli, D. 134 Pittaluga, A. 19,111,122,125,154,170 Poli, S. 24,45,57,147 Polsky-Fisher, S. 24 Pomierny-Chamioo, L. 138 Popkirov, S.G. 148 Prete, A. 156 Prieto, L. 53,54,79,114,115,116,121,149,183 Pshenichkin, S. 42,49,63,78,118,150,151,171,186 Puccetti, P. 51 Pulicicchio, C. M. 82 Puliti, A. 19,154 Pusceddu, M. 135 Pu, X. 97,98

Reynolds, I. Rhoads, E. Ricci-Vitiani, L. Ries, V. Rigault, D. Ring, R.H. Riozzi, B. Robin, N.C. Roche, K.W. Rodriguez, A. Rodríguez, A.L. Roger, J. Rondard, P. Ronesi, J.A. Rook, J.M. Rorick Kehn, L. Rosati, O. Rosati, S. Rosenzweig-Lipson, S. Rossi, F. Rossi, P. Rossi, P.I.A. Rudenko, V. Rudyk, H. Rush, R. Russell, D. Russell, M. Russo, V.

Q

Sághy, K. Sakimura, K. Salovich, J.M. Salt, T.E. Sandell, J. Sanger, H. Santolini, I. Saraswat, L. Scaccianoce, S. Scarselli, P. Scartabelli, T. Schechter, L.E. Schkeryantz, J. Schmitt, M. Schneider, C. Schober, D. Schoemaker, H.

Quets, A. Quinton, M. S.

53,54 77

R Raber, J. Raboisson, P Ramos, F.J. Rastaldi, M. P. Ravazzolo, R. Raynaud, F. Reiff, S.D. Reinhard, M. Ren, W. Reynaert, M.L.

48 152 68 154 154 124 72 114,121,149 126 108,111

24 46 30,123 69 1,71 82 9,120,140 14 29 168 55,87,156 124 43,47,146,159 153 72,87,128,132,177 114,115,158,183 101 125 82 107 19 154 58 114,121,149 8 84 104 162

S 18 187 131 32,155 152 76,79,121 156 77 125 101,162,189 64,92,143,157 82 86,158 7 69 53,54 38


Scholler, P. Schröder, H. Seibyl, J. Seidel, W. Selvam, C. Semenova, S. Sepers, M. Serone, F. Servant, G. Seymour, P. Sferra, D. Shaffer, C. Shanks, E. Shaw, D. Sharp, T. Sheffler, D. Sheffler, D.J. Shumway, M. Sidique, S. Simmaco, M. Simmons, R.M. Simonyi, A. Siracusano, A. Siuda, E. Sawiska, A. Smajilovic, S. Smith, D.L. Smith, D.G. Smith, J.A. Smith, M.R. Smith, D.A. Smith, Y. Snyder, J.P. Sortino, M.A. Sovago, J. Spampinato, S.F. Spear, K. Spiller, K. Spinsanti, P. Spooren, W. Springer, D. Stachowicz, K. Stagnitti, M. Stamelou, M. Stanton, P.K. Staro, J. Stauffer, S.R. Steckler, T.

43,47,146,159 160 8,84 116,178 1 33,110 134 123 161 163 162 163 178 114,115 93 33 110,164 87 33 101,111,137 129 156 125 54 138,167 21 82,163 97 37 130 163 165 118 166 41 166 77 60 30,123 16 82 138,167,182 95 69 190 20 31,37,72,126,132,156,168,169,177 31,168,169,177

79

Stephens, T.W. Stockmeier, C.A. Storch, A. Storto, M. Ström, P Stump, C.A. Sukoff Rizzo, S.J. Summa, M. Sun, H. Svensson, K. Swanson, S.

129 41 69 9 152 141 82 122,154,170 126 53,103,158,185 86,114,121,149,158,183

T Taboada, L. Takeuchi, K. Takoudjou, G.R. Takuma, K. Tamagnan, G. Tang, L. Taraska, C.M. Taylor, J.A. Te Riele, P. Territo, P. Terstappen, G.C. Thompson, A.D. Thompson, D. Thompson, L. Thompson, A.G. Tober, C. Topiol, S. Torrioli, M.G. Trabanco, A.A. Trabucco, A. Traficante, A. Trapa, P. Trenkwalder, C. Trepanier, C.H. Tresadern, G. Treyer, V. Trinquet, E. Tsai, N.-P. Tu, Y. Tueting, P. Turle-Lorenzo, N. Tupper, D. Turriziani, B. Tynebor, R.M.

114,121,149 66 42,49,78,150,151,171 3 8,84,165 57 14 192 15,94,175 67,116 38 50,131 114,121,149 116 168 35 119 68 15,94 133 30 163 69 172 142 41 43,47,146,159 153 53,54 113 40 114,121,149 123 141

80

Author Index

U Uberti, M.A. Uchigashima, M. Uddin, N. Unger, L. Upadhyay, J. Upreti, C. Ursu, D. Usai, C. Uslaner, J.H.

11,97,98,119,173 187 8 38 36 190 76 170 141

V Vacca, C. Valente, M.M. Van Camp, G. Vandemeulebroecke, M. van der Elst, M. Vandergriff, A. Van der Linden, I. van Luijtelaar, G. van Rijn, C.M. Varnäs, K. Vastag, M. Vilar, B. Vinson, P.N. Vivier, R. Vliegen, M. Vollmayr, B. Volpi, C. Vranesic, I.

51 174 108,111 69 35 79 94,175 127,156 156 152 59 71,176 31,72,132,156,168,169,177 76,79,121 175 145 51 61

W Wafford, K.A Walker, A.G. Wall, B. Walton, L. Wang, T. Wang, X. Wangari-Talbot, J. Warburton, E.C. Wassvik, C.M. Watanabe, M. Watt, M. Weaver, C.D. Weiss, F. Wellendorph, P.

116,144,178 164 179 114,121,149 28 79,121,158 179 26 15 134,187 53,54 50 180 21

Wensbo, D. Wettstein, J.G. White, B.D. Whiteside, G.T. Wichmann, J. Wieroska, J.M. Williams, T.A. Williams, R. Witkin, J.M. Wolfe, B.B. Woltering, T.J. Wolz, M. Worley, P.F. Wouters, R. Wright, R. Wroblewska, B. Wroblewski, J.T.

152 105,106 14 82 7,105,106,181,184 138,139,167,182 141 31 114,158,183 49,150 7,105,106,181,184 69 153 15 53,185 118,186 42,49,63,78,118,150,151,171,186

X Xenos, D. Xi, Z.-X. Xiang, Z. Xiao, H.

125 60 85,132 53,54

Y Yamamoto, T. Yamasaki, M. Yamazaki, M. Yang, L. Yasuda, R.P. Yin, S. Yoshida, T. Yoshioka, M. Yu, S. Yuen, E.

172 187 187 33 49 188 187 187 28 103

Z Zaleska, M. Zamorano, R. Zammataro, M. Zappulla, C. Zasadny, K. Zemribo, R. Zhang, A. Zhang, M. Zhang, X.F.

163 50,131,188 133 52,133,162,189 163 35 104 36 141


Zhang, X.L. Zhao, F. Zhou, H. Zhou, S. Zhou, Y. Zhou, Y.S.

190 130 97,98 31,156,168,177 17,50 72

81

Zhuo, M. Zianni, E. Zubal, G. Zukin, R.S. Zwier, J.M.

191 64 84 66 43,47,146,159