245 words Tables: 6 Figures: 14 Abbreviations: ACh: acetylcholine; AMPA: -Amino-3-hydroxy-5-methyl-4isoxazolepropionic acid; DHPG: 3,4-Dihydroxyphenylglycine; DOPAC: 3,4Dihydroxyphenylacetic acid; GABA: gamma aminobutyric acid; 5-HT: 5-hydroxytryptamine or serotonin; 5-HIAA: 5-hydroxyindolacetic acid; LY341495: (2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid; mGlu: metabotropic glutamate; LY354740.H2O: (1S,2S,5R,6S) 2Aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid monohydrate; LY379268: (1R,4R,5S,6R)-4-Amino-2-oxabicyclo[3.1.0]hexane-4,6-dicarboxylic acid; LY3020371: (1S,2R,3S,4S,5R,6R)-2-amino-3-[(3,4-difluorophenyl)sulfanylmethyl]-4-hydroxybicyclo[3.1.0]hexane-2,6-dicarboxylic acid; LY3027788: bis(((isopropoxycarbonyl)oxy)methyl) (1S,2R,3S,4S,5R,6R)-2-amino-3-(((3,4-difluorophenyl)thio)methyl)-4-hydroxybicyclo[3.1.0]hexane-2,6-dicarboxylate; MAP2: Microtubule-associated protein; 2NA: noradrenaline or norepinephrine; NPPA: natriuretic peptide A; NBQX: 1,2,3,4tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide; TMH: tele-methylhistamine or 1-methyl-histamine. This article has not been copyedited and formatted. The final version may differ from this version. JPET Fast Forward. Published on January 30, 2017 as DOI: 10.1124/jpet.116.238121 at A PE T Jornals on A uust 0, 2023 jpet.asjournals.org D ow nladed from

The ability of the N-methyl-D-aspartate receptor antagonist ketamine to alleviate symptoms in patients suffering from treatment-resistant depression (TRD) is well documented. In this paper, we directly compare in vivo biologic responses in rodents elicited by a recently discovered metabotropic glutamate (mGlu) 2/3 receptor antagonist 2-amino-3-[(3,4-difluorophenyl) sulfanylmethyl]-4-hydroxy-bicyclo[3.1.0] hexane-2,6-dicarboxylic acid (LY3020371) with those produced by ketamine. Both LY3020371 and ketamine increased the number of spontaneously active dopamine cells in the ventral tegmental area of anesthetized rats, increased O2 in the anterior cingulate cortex, promoted wakefulness, enhanced the efflux of biogenic amines in the prefrontal cortex, and produced antidepressant-related behavioral effects in rodent models. The ability of LY3020371 to produce antidepressant-like effects in the forced-swim assay in rats was associated with cerebrospinal fluid (CSF) drug levels that matched concentrations required for functional antagonist activity in native rat brain tissue preparations. Metabolomic pathway analyses from analytes recovered from rat CSF and hippocampus demonstrated that both LY3020371 and ketamine activated common pathways involving GRIA2 and ADORA1. A diester analog of LY3020371 [bis(((isopropoxycarbonyl) oxy)-methyl) (1S, 2R, 3S, 4S, 5R, 6R)-2-amino-3-(((3,4-difluorophenyl) thio) methyl)-4-hydroxy-bicyclo[3.1.0] hexane-2,6-dicarboxylate (LY3027788)] was an effective oral prodrug; when given orally, it recapitulated effects of intravenous doses of LY3020371 in the forced-swim and wake-promotion assays, and augmented the antidepressant-like effects of fluoxetine or citalopram without altering plasma or brain levels of these compounds. The broad overlap of biologic responses produced by LY3020371 and ketamine supports the hypothesis that mGlu2/3 receptor blockade might be a novel therapeutic approach for the treatment of TRD patients. LY3020371 and LY3027788 represent molecules that are ready for clinical tests of this hypothesis.

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

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system, activating ionotropic (iGlu) and metabotropic (mGlu) glutamate receptors. Targeting iGlu receptors has proven difficult, as these receptors play such a critical role in fast synaptic transmission. In recent years, there has been a switch in the focus of the pharmaceutical industry to target mGlu receptors, as these receptors have a more modulatory role in the brain. Eight subtypes of the G protein-coupled mGlu receptors have been cloned and classified into three groups according to their second messenger association, sequence homology and agonist selectivity. Group I (mGlu(1) and mGlu(5)) receptors are positively coupled to phosphatidylinositol (PO hydrolysis, while group II (mGlu(2) and mGlu(3)) and group III (mGlu(4), mGlu(6), mGlu(7) and mGlu(8)) receptors are negatively coupled to adenylate cyclase and thought to act as presynaptic autoreceptors, regulating glutamate transmission. There has been rapid progress in understanding the biology of mGlu receptors and their function in the central nervous system (CNS), and the field now has highly potent and selective ligands for many of the mGlu receptor subtypes. Pharmacological tools include both orthosteric and allosteric agonists and antagonists. These molecules have been explored across a range of CNS conditions, the findings of which will be discussed briefly in this review. Recently, some of the most promising molecules have progressed into clinical trials (mGlu(2/3) receptor agonists for the treatment of anxiety and schizophrenia and mGlu(5) receptor antagonists for acute migraine and L-DOPA-induced dyskinesia) and achieved proof of concept. These encouraging results suggest mGlu receptors as novel drug targets with application in a range of psychiatric and neurological conditions.

Background and purpose:Increased glutamatergic innervation of the substantia nigra pars reticulata (SNpr) and pars compacta (SNpc) may contribute to the motor deficits and neurodegeneration, respectively, in Parkinson's disease (PD). This study aimed to establish whether activation of pre-synaptic group III metabotropic glutamate (mGlu) receptors reduced glutamate release in the SN, and provided symptomatic or neuroprotective relief in animal models of PD.Experimental approach:Broad-spectrum group III mGlu receptor agonists, O-phospho-l-serine (l-SOP) and l-2-amino-4-phosphonobutyrate (l-AP4), were assessed for their ability to inhibit KCl-evoked [3H]-d-aspartate release in rat nigral prisms or inhibit KCl-evoked endogenous glutamate release in the SNpr in vivo using microdialysis. Reversal of akinesia in reserpine-treated rats was assessed following intranigral injection of l-SOP and l-AP4. Finally, the neuroprotective effect of 7 days' supra-nigral treatment with l-AP4 was examined in 6-hydroxydopamine (6-OHDA)-lesioned rats.Key results:l-SOP and l-AP4 inhibited [3H]-d-aspartate release by 33 and 44% respectively. These effects were blocked by the selective group III mGlu antagonist (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG). l-SOP also reduced glutamate release in the SNpr in vivo by 48%. Injection of l-SOP and l-AP4 into the SNpr reversed reserpine-induced akinesia. Following administration above the SNpc, l-AP4 provided neurochemical, histological and functional protection against 6-OHDA lesion of the nigrostriatal tract. Pretreatment with CPPG inhibited these effects.Conclusions and implications:These findings highlight group III mGlu receptors in the SN as potential targets for providing both symptomatic and neuroprotective relief in PD, and indicate that inhibition of glutamate release in the SN may underlie these effects.

Current antipsychotics provide symptomatic relief for patients suffering from schizophrenia and related psychoses; however, their effectiveness is variable and many patients discontinue treatment due to side effects. Although the etiology of schizophrenia is still unclear, a leading hypothesis implicates an imbalanced dopaminergic system. Muscarinic acetylcholine (ACh) receptors regulate dopamine levels in key areas of the brain involved in psychosis, with the M(4) subtype emerging as a key regulator of dopaminergic hyperactivity. Unfortunately, no selective small molecule tools exist to provide pharmacological validation of this hypothesis. Here, we describe the discovery of a small molecule modulator, LY2033298, that is highly selective for human M(4) receptors by virtue of targeting an allosteric site on this receptor. Pharmacological assays confirmed the selectivity of LY2033298 for the M(4) receptor and revealed the highest degree of positive allosteric enhancement of ACh potency thus far identified. Radioligand binding assays also show this compound to directly potentiate agonist binding while having minimal effects on antagonist binding. Mutational analysis identified a key amino acid (D(432)) in the third extracellular loop of the human M(4) receptor to be critical for selectivity and agonist potentiation by LY2033298. Importantly, LY2033298 was active in animal models predictive of clinical antipsychotic drug efficacy indicating its potential use as a first-in-class, selective, allosteric muscarinic antipsychotic agent.

Recent evidence that 5-HT2 receptors exert a negative influence on central 5-hydroxytryptamine (5-HT) neurones suggests that 5-HT2 receptor antagonists may augment the effects of serotonin selective reuptake inhibitors (SSRIs). The present study investigated whether pre-treatment with 5-HT2 receptor antagonists enhances the effect of SSRI administration on hippocampal extracellular 5-HT of freely moving rats. Administration of the SSRI citalopram at a low (2mg kg−1) and higher (4mg kg−1) dose, increased dialysate 5-HT by 5- and 8-fold, respectively. Pre-treatment with the 5-HT2 receptor antagonist ketanserin (4mg kg−1) augmented the effect of 4mg kg−1 but not 2mg kg−1 citalopram. The effect of 4mg kg−1 citalopram was also augmented by pre-treatment with either the 5-HT2C receptor antagonist SB 242084 (0.5mg kg−1) or the 5-HT2A receptor antagonist MDL 100907 (0.5mg kg−1). As with citalopram, fluoxetine elevated dialysate 5-HT at both a low (5mg kg−1) and higher (20mg kg−1) dose. However, neither dose of fluoxetine was augmented by ketanserin (4mg kg−1). These results confirm recent findings that 5-HT2 receptor antagonists augment the effect of citalopram on extracellular 5-HT, and indicate the involvement of 5-HT2C and possibly 5-HT2A receptors. The lack of augmentation of fluoxetine might reflect the intrinsic 5-HT2 receptor antagonist properties of this drug.

Increased psychophysiological resistance to chronic stress has been related to increased 5-HT release in the dorsal hippocampus. This study investigated the changes in 5-HT release and turnover in the hippocampus evoked by acute and repeated exposure to an inescapable stressor, an elevated open platform, and compared them to the changes evoked in the frontal cortex. Repeated exposure to this stressor results in habituation of the plasma corticosterone response to the test, with full habituation being observed after 20 trials. Repeated exposure to the stressor for 5 or 10 occasions increased 5-HT turnover in the hippocampus. By contrast, 5-HT turnover in frontal cortex was increased by acute exposure to the stressor. Microdialysis studies showed that acute stress increased 5-HT overflow in prefrontal cortex but not dorsal hippocampus whereas repeated daily (10 days) exposure to the stressor increased basal extracellular 5-HT in the dorsal hippocampus, but not the prefrontal cortex. Prior exposure to the stressor on up to 10 occasions enhanced the plasma corticosterone response to a challenge in an elevated plus-maze performed 24 h later whereas repeated, but not acute, exposure to the stressor, elicited anxiolytic-like behavioural responses in this test. It is concluded that acute exposure to this form of inescapable stress selectively stimulates the 5-HT projections to the frontal cortex; repeated stress elicits a sustained increase in 5-HT release and turnover in the hippocampus. The data are consistent with the hypothesis that increased 5-HT release in the hippocampus may be implicated in the mechanisms underlying habituation to inescapable stress.

A series of N-alkyl-N-arylmethylpiperidin-4-amines have been prepared and are demonstrated to be inhibitors of both serotonin and norepinephrine reuptake.

Whilst local intrastriatal infusion of nicotine consistently elicits striatal dopamine release, systemic administration often fails to do so. Since chronic nicotine administration is known to result in desensitisation-induced upregulation of nicotinic acetylcholine receptors (nAChRs), the present study investigated whether chronic pre-treatment could enhance the response to systemic nicotine and, if so, whether increases in specific nAChR subunit mRNA levels in the substantia nigra pars compacta (SNc) may underlie this effect. In vivo microdialysis studies in male Sprague-Dawley rats revealed that following 4 days pre-treatment with nicotine (0.8 mg kg(-1)s.c.), local intrastriatal nicotine infusion (3 mM) elicited significantly higher dopamine efflux compared to vehicle pre-treated controls (peak release: 1273 +/- 199% basal versus 731 +/- 113% basal), whereas systemic nicotine challenge (0.8 mg kg(-1)s.c.) elicited no response. In contrast, following 8 days pre-treatment with nicotine (0.8 mg kg(-1)s.c.), systemic nicotine challenge (0.8 mg kg(-1)s.c.) now produced significantly higher dopamine efflux than that of vehicle pre-treated controls (147 +/- 30% basal versus 91 +/- 5% basal). Eight days pre-treatment with nicotine also significantly elevated the levels of alpha6 (approximately 55%) and beta3 (approximately 43%) nAChR subunit mRNA in the SNc, suggesting that up-regulation of these nAChR subunit genes in the nigrostriatal tract may contribute to the enhanced nicotine-evoked striatal dopamine release.

A series of naphthalenyloxy-arylpropylamines have been prepared and are demonstrated to be inhibitors of both serotonin and norepinephrine reuptake. One member of this series, duloxetine (Cymbalta™) has proven to be effective in clinical trials for the treatment of depression.

The effect of the group II metabotropic receptor agonist (-)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY379268), on basal and phencyclidine-induced dopamine efflux were measured in the shell and core subdivisions of the nucleus accumbens--regions which are associated with limbic and motor functions, respectively. Extracellular levels of dopamine were measured using microdialysis in conscious animals, and LY379268 was delivered locally by inclusion in the artificial cerebrospinal fluid (aCSF) flowing through the microdialysis probe. Local administration of LY379268 in the concentration range 10 nM-10 microM reduced basal levels of dopamine in the nucleus accumbens shell, whilst having no effect in the nucleus accumbens core. In the nucleus accumbens shell, basal levels were reduced to approximately 60% compared to the pre-injection control, with a maximal reduction occurring at concentrations of LY379268 > or =100 nM. The response to LY379268 (100 nM) was reversible, with levels returning to baseline following its removal from the aCSF. In a separate experiment, local perfusion of the nucleus accumbens shell with LY379268 (at both 1 and 10 microM) reduced the magnitude of the response to a subsequent systemic administration of phencyclidine (5 mg/kg i.p.). The reduction in the peak dopamine response was only evident with doses of LY379268 that also reduced basal dopamine efflux--LY379268 being ineffective against PCP at 10 nM. However, in animals pre-treated with LY379268 at 1 or 10 microM, PCP still evoked a dopamine response, and in these animals the relative extent of the response was not significantly different between the respective treatment groups. In contrast, in the nucleus accumbens core the magnitude of the dopamine response to PCP was unaffected by local application of LY379268 (at 1 or 10 microM). Our data suggest that within the nucleus accumbens, there exists a distinct regional difference in the control of dopamine release by group II mGluRs, with the nucleus accumbens shell being preferentially affected. Moreover, the selective action of LY379268 on dopamine levels in the nucleus accumbens shell may have implications for the potential antipsychotic activity of group II mGluR agonists.