The CRTC1-SIK1 pathway regulates entrainment of the circadian clock.

Retinal photoreceptors entrain the circadian system to the solar day. This photic resetting involves cAMP response element binding protein (CREB)-mediated upregulation of Per genes within individual cells of the suprachiasmatic nuclei (SCN). Our detailed understanding of this pathway is poor, and it remains unclear why entrainment to a new time zone takes several days. By analyzing the light-regulated transcriptome of the SCN, we have identified a key role for salt inducible kinase 1 (SIK1) and CREB-regulated transcription coactivator 1 (CRTC1) in clock re-setting. An entrainment stimulus causes CRTC1 to coactivate CREB, inducing the expression of Per1 and Sik1. SIK1 then inhibits further shifts of the clock by phosphorylation and deactivation of CRTC1. Knockdown of Sik1 within the SCN results in increased behavioral phase shifts and rapid re-entrainment following experimental jet lag. Thus SIK1 provides negative feedback, acting to suppress the effects of light on the clock. This pathway provides a potential target for the regulation of circadian rhythms.

"Domain gauges": A reference system for multivariate profiling of brain fMRI activation patterns induced by psychoactive drugs in rats.

Pharmacological magnetic resonance imaging (phMRI) of the brain has become a widely used tool in both preclinical and clinical drug research. One of its challenges is to condense the observed complex drug-induced brain-activation patterns into semantically meaningful metrics that can then serve as a basis for informed decision making. To aid interpretation of spatially distributed activation patterns, we propose here a set of multivariate metrics termed "domain gauges", which have been calibrated based on different classes of marketed or validated reference drugs. Each class represents a particular "domain" of interest, i.e., a specific therapeutic indication or mode of action. The drug class is empirically characterized by the unique activation pattern it evokes in the brain-the "domain profile". A domain gauge provides, for any tested intervention, a "classifier" as a measure of response strength with respect to the domain in question, and a "differentiator" as a measure of deviation from the domain profile, both along with error ranges. Capitalizing on our in-house database with an unprecedented wealth of standardized perfusion-based phMRI data obtained from rats subjected to various validated treatments, we exemplarily focused on 3 domains based on therapeutic indications: an antipsychotic, an antidepressant and an anxiolytic domain. The domain profiles identified as part of the gauge definition process, as well as the outputs of the gauges when applied to both reference and validation data, were evaluated for their reconcilability with prior biological knowledge and for their performance in drug characterization. The domain profiles provided quantitative activation patterns with high biological plausibility. The antipsychotic profile, for instance, comprised key areas (e.g., cingulate cortex, nucleus accumbens, ventral tegmental area, substantia nigra) which are believed to be strongly involved in mediating an antipsychotic effect, and which are in line with network-level dysfunctions observed in schizophrenia. The domain gauges plausibly positioned the vast majority of the pharmacological and even non-pharmacological treatments. The results also suggest the segregation of sub-domains based on, e.g., the mode of action. Upon judicious selection of domains and careful calibration of the gauges, our approach represents a valuable analytical tool for biological interpretation and decision making in drug discovery.

Pharmacology of basimglurant (RO4917523, RG7090), a unique metabotropic glutamate receptor 5 negative allosteric modulator in clinical development for depression.

Major depressive disorder (MDD) is a serious public health burden and a leading cause of disability. Its pharmacotherapy is currently limited to modulators of monoamine neurotransmitters and second-generation antipsychotics. Recently, glutamatergic approaches for the treatment of MDD have increasingly received attention, and preclinical research suggests that metabotropic glutamate receptor 5 (mGlu5) inhibitors have antidepressant-like properties. Basimglurant (2-chloro-4-[1-(4-fluoro-phenyl)-2,5-dimethyl-1H-imidazol-4-ylethynyl]-pyridine) is a novel mGlu5 negative allosteric modulator currently in phase 2 clinical development for MDD and fragile X syndrome. Here, the comprehensive preclinical pharmacological profile of basimglurant is presented with a focus on its therapeutic potential for MDD and drug-like properties. Basimglurant is a potent, selective, and safe mGlu5 inhibitor with good oral bioavailability and long half-life supportive of once-daily administration, good brain penetration, and high in vivo potency. It has antidepressant properties that are corroborated by its functional magnetic imaging profile as well as anxiolytic-like and antinociceptive features. In electroencephalography recordings, basimglurant shows wake-promoting effects followed by increased delta power during subsequent non-rapid eye movement sleep. In microdialysis studies, basimglurant had no effect on monoamine transmitter levels in the frontal cortex or nucleus accumbens except for a moderate increase of accumbal dopamine, which is in line with its lack of pharmacological activity on monoamine reuptake transporters. These data taken together, basimglurant has favorable drug-like properties, a differentiated molecular mechanism of action, and antidepressant-like features that suggest the possibility of also addressing important comorbidities of MDD including anxiety and pain as well as daytime sleepiness and apathy or lethargy.

Sleep and circadian rhythm disruption in psychiatric and neurodegenerative disease.

Sleep and circadian rhythm disruption are frequently observed in patients with psychiatric disorders and neurodegenerative disease. The abnormal sleep that is experienced by these patients is largely assumed to be the product of medication or some other influence that is not well defined. However, normal brain function and the generation of sleep are linked by common neurotransmitter systems and regulatory pathways. Disruption of sleep alters sleep-wake timing, destabilizes physiology and promotes a range of pathologies (from cognitive to metabolic defects) that are rarely considered to be associated with abnormal sleep. We propose that brain disorders and abnormal sleep have a common mechanistic origin and that many co-morbid pathologies that are found in brain disease arise from a destabilization of sleep mechanisms. The stabilization of sleep may be a means by which to reduce the symptoms of--and permit early intervention of--psychiatric and neurodegenerative disease.

Closing thoughts for cognitive enhancement.

The wide-ranging field of cognition enhancing research along with its ethics as it stands today is summarized. In the forefront are potentially novel drugs and non-pharmacological treatments for cognitive impairment across many different psychiatric and neurologic indications. Today's research will bring new drugs to patients tomorrow, and tomorrow's research will bring new molecular targets to clinical development that should be cognitive domain-specific. There is the likelihood that special populations may be better treated and that personalized medicine for cognitive impairment could become a reality. It is conceivable that with the current research effort, cognition enhancing drugs will become available to wide-ranging populations of people with neuropsychiatric illness and to those that are healthy. In some cultures, there is a push in society to be more intelligent or have more cognitive prowess. Thus, the ethical use of cognitive enhancing drugs should be an area of debate and communication. Neuroethics is a growing field and it intends to bring together key contributors such as physicians, disease experts, regulatory officials, and policy makers to discuss how such medicines can or should be made available. Together with this, one has to consider the possibility that no single medicine or technology will have a great impact on cognition and, therefore, combination therapy of drugs plus other approaches like exercise or transcranial direct-current stimulation may be the path forward. This is another area of scientific inquiry and debate, and the results should be fruitful and helpful to patients. The science of cognition is advancing at a rapid rate, and communication of its progress along with the development of rational and ethical policies for use of cognitive enhancers will be beneficial.

Reducing GABAA α5 receptor-mediated inhibition rescues functional and neuromorphological deficits in a mouse model of down syndrome.

Down syndrome (DS) is associated with neurological complications, including cognitive deficits that lead to impairment in intellectual functioning. Increased GABA-mediated inhibition has been proposed as a mechanism underlying deficient cognition in the Ts65Dn (TS) mouse model of DS. We show that chronic treatment of these mice with RO4938581 (3-bromo-10-(difluoromethyl)-9H-benzo[f]imidazo[1,5-a][1,2,4]triazolo[1,5-d][1,4]diazepine), a selective GABA(A) α5 negative allosteric modulator (NAM), rescued their deficits in spatial learning and memory, hippocampal synaptic plasticity, and adult neurogenesis. We also show that RO4938581 normalized the high density of GABAergic synapse markers in the molecular layer of the hippocampus of TS mice. In addition, RO4938581 treatment suppressed the hyperactivity observed in TS mice without inducing anxiety or altering their motor abilities. These data demonstrate that reducing GABAergic inhibition with RO4938581 can reverse functional and neuromorphological deficits of TS mice by facilitating brain plasticity and support the potential therapeutic use of selective GABA(A) α5 NAMs to treat cognitive dysfunction in DS.

TAAR1 activation modulates monoaminergic neurotransmission, preventing hyperdopaminergic and hypoglutamatergic activity.

The trace amine-associated receptor 1 (TAAR1), activated by endogenous metabolites of amino acids like the trace amines p-tyramine and ß-phenylethylamine, has proven to be an important modulator of the dopaminergic system and is considered a promising target for the treatment of neuropsychiatric disorders. To decipher the brain functions of TAAR1, a selective TAAR1 agonist, RO5166017, was engineered. RO5166017 showed high affinity and potent functional activity at mouse, rat, cynomolgus monkey, and human TAAR1 stably expressed in HEK293 cells as well as high selectivity vs. other targets. In mouse brain slices, RO5166017 inhibited the firing frequency of dopaminergic and serotonergic neurons in regions where Taar1 is expressed (i.e., the ventral tegmental area and dorsal raphe nucleus, respectively). In contrast, RO5166017 did not change the firing frequency of noradrenergic neurons in the locus coeruleus, an area devoid of Taar1 expression. Furthermore, modulation of TAAR1 activity altered the desensitization rate and agonist potency at 5-HT(1A) receptors in the dorsal raphe, suggesting that TAAR1 modulates not only dopaminergic but also serotonergic neurotransmission. In WT but not Taar1(-/-) mice, RO5166017 prevented stress-induced hyperthermia and blocked dopamine-dependent hyperlocomotion in cocaine-treated and dopamine transporter knockout mice as well as hyperactivity induced by an NMDA antagonist. These results tie TAAR1 to the control of monoamine-driven behaviors and suggest anxiolytic- and antipsychotic-like properties for agonists such as RO5166017, opening treatment opportunities for psychiatric disorders.

The selective antagonist EPPTB reveals TAAR1-mediated regulatory mechanisms in dopaminergic neurons of the mesolimbic system.

Trace amine-associated receptor 1 (TAAR1) is a G protein-coupled receptor (GPCR) that is nonselectively activated by endogenous metabolites of amino acids. TAAR1 is considered a promising drug target for the treatment of psychiatric and neurodegenerative disorders. However, no selective ligand to identify TAAR1-specific signaling mechanisms is available yet. Here we report a selective TAAR1 antagonist, EPPTB, and characterize its physiological effects at dopamine (DA) neurons of the ventral tegmental area (VTA). We show that EPPTB prevents the reduction of the firing frequency of DA neurons induced by p-tyramine (p-tyr), a nonselective TAAR1 agonist. When applied alone, EPPTB increases the firing frequency of DA neurons, suggesting that TAAR1 either exhibits constitutive activity or is tonically activated by ambient levels of endogenous agonist(s). We further show that EPPTB blocks the TAAR1-mediated activation of an inwardly rectifying K(+) current. When applied alone, EPPTB induces an apparent inward current, suggesting the closure of tonically activated K(+) channels. Importantly, these EPPTB effects were absent in Taar1 knockout mice, ruling out off-target effects. We additionally found that both the acute application of EPPTB and the constitutive genetic lack of TAAR1 increase the potency of DA at D2 receptors in DA neurons. In summary, our data support that TAAR1 tonically activates inwardly rectifying K(+) channels, which reduces the basal firing frequency of DA neurons in the VTA. We hypothesize that the EPPTB-induced increase in the potency of DA at D2 receptors is part of a homeostatic feedback mechanism compensating for the lack of inhibitory TAAR1 tone.

CTEP: a novel, potent, long-acting, and orally bioavailable metabotropic glutamate receptor 5 inhibitor.

The metabotropic glutamate receptor 5 (mGlu5) is a glutamate-activated class C G protein-coupled receptor widely expressed in the central nervous system and clinically investigated as a drug target for a range of indications, including depression, Parkinson's disease, and fragile X syndrome. Here, we present the novel potent, selective, and orally bioavailable mGlu5 negative allosteric modulator with inverse agonist properties 2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine (CTEP). CTEP binds mGlu5 with low nanomolar affinity and shows >1000-fold selectivity when tested against 103 targets, including all known mGlu receptors. CTEP penetrates the brain with a brain/plasma ratio of 2.6 and displaces the tracer [(3)H]3-(6-methyl-pyridin-2-ylethynyl)-cyclohex-2-enone-O-methyl-oxime (ABP688) in vivo in mice from brain regions expressing mGlu5 with an average ED(50) equivalent to a drug concentration of 77.5 ng/g in brain tissue. This novel mGlu5 inhibitor is active in the stress-induced hyperthermia procedure in mice and the Vogel conflict drinking test in rats with minimal effective doses of 0.1 and 0.3 mg/kg, respectively, reflecting a 30- to 100-fold higher in vivo potency compared with 2-methyl-6-(phenylethynyl)pyridine (MPEP) and fenobam. CTEP is the first reported mGlu5 inhibitor with both long half-life of approximately 18 h and high oral bioavailability allowing chronic treatment with continuous receptor blockade with one dose every 48 h in adult and newborn animals. By enabling long-term treatment through a wide age range, CTEP allows the exploration of the full therapeutic potential of mGlu5 inhibitors for indications requiring chronic receptor inhibition.

Mapping the binding pocket of dual antagonist almorexant to human orexin 1 and orexin 2 receptors: comparison with the selective OX1 antagonist SB-674042 and the selective OX2 antagonist N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl-acetamide (EMPA).

The orexins and their receptors are involved in the regulation of arousal and sleep-wake cycle. Clinical investigation with almorexant has indicated that this dual OX antagonist is efficacious in inducing and maintaining sleep. Using site-directed mutagenesis, beta(2)-adrenergic-based OX(1) and OX(2) modeling, we have determined important molecular determinants of the ligand-binding pocket of OX(1) and OX(2). The conserved residues Asp(45.51), Trp(45.54), Tyr(5.38), Phe(5.42), Tyr(5.47), Tyr(6.48), and His(7.39) were found to be contributing to both orexin-A-binding sites at OX(1) and OX(2). Among these critical residues, five (positions 45.51, 45.54, 5.38, 5.42, and 7.39) were located on the C-terminal strand of the second extracellular loop (ECL2b) and in the top of TM domains at the interface to the main binding crevice, thereby suggesting superficial OX receptor interactions of orexin-A. We found that the mutations W214A(45.54), Y223A(5.38), F227A(5.42), Y317A(6.48), and H350A(7.39) resulted in the complete loss of both [(3)H]almorexant and [(3)H]N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl-acetamide (EMPA) binding affinities and also blocked their inhibition of orexin-A-evoked [Ca(2+)](i) response at OX(2). The crucial residues Gln126(3.32), Ala127(3.33), Trp206(45.54), Tyr215(5.38), Phe219(5.42), and His344(7.39) are shared between almorexant and 1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone (SB-674042) binding sites in OX(1). The nonconserved residue at position 3.33 of orexin receptors was identified as occupying a critical position that must be involved in subtype selectivity and also in differentiating two different antagonists for the same receptor. In summary, despite high similarities in the ligand-binding pockets of OX(1) and OX(2) and numerous aromatic/hydrophobic interactions, the local conformation of helix positions 3.32, 3.33, and 3.36 in transmembrane domain 3 and 45.51 in ECL2b provide the structural basis for pharmacologic selectivity between OX(1) and OX(2).

Biochemical and electrophysiological characterization of almorexant, a dual orexin 1 receptor (OX1)/orexin 2 receptor (OX2) antagonist: comparison with selective OX1 and OX2 antagonists.

Recent preclinical and clinical research has shown that almorexant promotes sleep in animals and humans without disrupting the sleep architecture. Here, the pharmacology and kinetics of [(3)H]almorexant binding to human orexin 1 receptor (OX(1))- and human orexin 2 receptor (OX(2))-human embryonic kidney 293 membranes were characterized and compared with those of selective OX(1) and OX(2) antagonists, including 1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone (SB-674042), 1-(6,8-difluoro-2-methyl-quinolin-4-yl)-3-(4-dimethylamino-phenyl)-urea (SB-408124), and N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl-acetamide (EMPA). The effect of these antagonists was also examined in vitro on the spontaneous activity of rat ventral tegmental area (VTA) dopaminergic neurons. [(3)H]Almorexant bound to a single saturable site on hOX(1) and hOX(2) with high affinity (K(d) of 1.3 and 0.17 nM, respectively). In Schild analyses using the [(3)H]inositol phosphates assay, almorexant acted as a competitive antagonist at hOX(1) and as a noncompetitive-like antagonist at hOX(2). In binding kinetic analyses, [(3)H]almorexant had fast association and dissociation rates at hOX(1), whereas it had a fast association rate and a remarkably slow dissociation rate at hOX(2). In the VTA, orexin-A potentiated the basal firing frequency to 175 +/- 17% of control in approximately half of the neurons tested. In the presence of 1 microM SB-674042 or SB-408124, the effect of orexin-A was only partially antagonized. However, in the presence of 1 microM EMPA or 1 microM almorexant, the effect of orexin-A was completely antagonized. In conclusion, almorexant exhibited a noncompetitive and long-lasting pseudo-irreversible mode of antagonism as a result of its very slow rate of dissociation from OX(2). The electrophysiology data suggest that OX(2) might be more important than OX(1) in mediating the effect of orexin-A on slow-firing of VTA dopaminergic neurons.

Me-talnetant and osanetant interact within overlapping but not identical binding pockets in the human tachykinin neurokinin 3 receptor transmembrane domains.

Recent clinical trials have indicated that neurokinin 3 receptor antagonists (S)-(+)-N-{{3-[1-benzoyl-3-(3,4-dichlorophenyl)-piperidin-3-yl]prop-1-yl}-4-phenylpiperidin-4-yl}-N-methylacetamine (SR142801; osanetant) and (S)-(-)-N-(alpha-ethylbenzyl)-3-hydroxy-2-phenylquinoline-4-carboxamide (SB223412; talnetant) may treat symptoms of schizophrenia. Using site-directed mutagenesis, rhodopsin-based modeling, [(3)H](S)-(-)-N-(alpha-ethylbenzyl)-3-methoxy-2-phenylquinoline-4-carboxamide (Me-talnetant) and [(3)H]osanetant binding, and functional Schild analyses, we have demonstrated the important molecular determinants of neurokinin B (NKB), Me-talnetant, and osanetant binding pockets. The residues Asn138(2.57), Asn142(2.61), Leu232(45.49), Tyr315(6.51), Phe342(7.39), and Met346(7.43) were found to be crucial for the NKB binding site. We observed that the M134(2.53)A, V169(3.36)M, F342(7.39)M, and S341(7.38)I/F342(7.39)M mutations resulted in the complete loss of [(3)H]Metalnetant and [(3)H]osanetant binding affinities and also abolished their functional potencies in an NKB-evoked accumulation of [(3)H]inositol phosphates assay, whereas the mutations V95(1.42)A, N142(2.61)A, Y315(6.51)F, and M346(7.43)A behaved differently between the interacting modes of two antagonists. V95(1.42)A and M346(7.43)A significantly decreased the affinity and potency of Me-talnetant. Y315(6.51)F, although not affecting Me-talnetant, led to a significant decrease in affinity and potency of osanetant. The mutation N142(2.61)A, which abolished the potency and affinity of osanetant, led to a significant increase in the affinity and potency of Me-talnetant. The proposed docking mode was further validated using (S)-2-(3,5-bis-trifluoromethyl-phenyl)-N-[4-(4-fluoro-2-methyl-phenyl)-6-((S)-4-methanesulfonyl-3-methyl-piperazin-1-yl)-pyridin-3-yl]-N-methyl-isobutyramide (RO49085940), from another chemical class. It is noteworthy that the mutation F342(7.39)A caused an 80-fold gain of RO4908594 binding affinity, but the same mutation resulted in the complete loss of the affinity of Me-talnetant and partial loss of the affinity of osanetant. These observations show that the binding pocket of Me-talnetant and osanetant are overlapping, but not identical. Taken together, our data are consistent with the proposed docking modes where Me-talnetant reaches deeply into the pocket formed by transmembrane (TM)1, -2, and -7, whereas osanetant fills the pocket TM3, -5, and -6 with its phenyl-piperidine moiety.

Trace amine-associated receptor 1 modulates dopaminergic activity.

The recent identification of the trace amine-associated receptor (TAAR)1 provides an opportunity to dissociate the effects of trace amines on the dopamine transporter from receptor-mediated effects. To separate both effects on a physiological level, a Taar1 knockout mouse line was generated. Taar1 knockout mice display increased sensitivity to amphetamine as revealed by enhanced amphetamine-triggered increases in locomotor activity and augmented striatal release of dopamine compared with wild-type animals. Under baseline conditions, locomotion and extracellular striatal dopamine levels were similar between Taar1 knockout and wild-type mice. Electrophysiological recordings revealed an elevated spontaneous firing rate of dopaminergic neurons in the ventral tegmental area of Taar1 knock-out mice. The endogenous TAAR1 agonist p-tyramine specifically decreased the spike frequency of these neurons in wild-type but not in Taar1 knockout mice, consistent with the prominent expression of Taar1 in the ventral tegmental area. Taken together, the data reveal TAAR1 as regulator of dopaminergic neurotransmission.

The acoustic startle reflex in Sprague-Dawley rats is altered by permanent middle cerebral artery occlusion.

The startle reflex is an unconditioned, quantifiable behavior used to study sensory modalities. We examined whether the acoustic startle reflex (ASR) was sensitive to lesions induced by focal cerebral ischemia. Sprague-Dawley rats were pre-screened for startle reflex responses 3-6 days prior to surgery and there were no differences in mean startle amplitude across groups. Animals were subjected to permanent middle cerebral artery occlusion (pMCAo) or a sham surgical procedure. Twenty-four hours later rats were evaluated for ASR prior to sacrifice. Infarct volumes were subsequently determined by quantitative image analysis of 2,3,5-triphenyltetrazolium chloride-stained brain sections. Infarct volumes of rats undergoing pMCAO ranged from 0 to 48%. Data were divided into three groups based upon percent infarction: mild (0-20%), moderate (21-35%), and severe (>35%). A within-subject analysis revealed a significant decrease in mean startle amplitude of only severely infarcted rats relative to their pre-surgery startle responses. Furthermore, the lesioned brain areas observed in these animals provide an anatomical basis for these results. Our findings demonstrate that ASR is affected in a model of stroke. Further work is needed to characterize this behavioral test and to determine whether it may have application as a surrogate endpoint for clinical stroke studies.

Metabotropic glutamate receptor 5 as drug target for Fragile X syndrome.

Fragile X syndrome (FXS) is the most common monogenic form of inherited mental retardation caused by a trinucleotid repeat expansion and transcriptional shutdown of the FMR1 gene. FXS patients present a complex and often severe neuropsychiatric phenotype yet have mild somatic symptoms, normal life expectancies, and no indications of neurodegeneration. The therapeutic potential of mGlu5 inhibitors was proposed in the 'mGluR theory of FXS' based on early insights into the molecular pathophysiology of FXS. Studies in Fragile X mental retardation 1 (Fmr1) knock-out mice, a widely used disease model, demonstrated that mGlu5 inhibitors can correct a broad range of disease-related phenotypes. Recent clinical trials, however, with two different mGlu5 inhibitors (basimglurant and mavoglurant) showed no therapeutic benefit in FXS patients for reasons as yet unclear.

Trace amine-associated receptor 1 activation silences GSK3ß signaling of TAAR1 and D2R heteromers.

Trace amine-associated receptor 1 (TAAR1) activation by selective endogenous agonists modulates dopaminergic neurotransmission. This results in antipsychotic-like behavior in vivo which might be initiated by an interaction of TAAR1 and dopamine D2L receptor (D2R). Here we analyzed the functional link between TAAR1 and D2R using highly potent and selective TAAR1 agonists, and newly generated tools such as TAAR1 knock-out and TAAR1 overexpressing rats as well as specific anti-rat TAAR1 antibodies. We provide data from co-immunoprecipitation experiments supporting a functional interaction of the two receptors in heterologous cells and in brain tissue. Interaction of TAAR1 with D2R altered the subcellular localization of TAAR1 and increased D2R agonist binding affinity. Using specific ß-arrestin 2 (ßArr2) complementation assays we show that the interaction of TAAR1 with D2R reduced ßArr2 recruitment to D2R. In addition, we report that besides Gαs-protein signaling TAAR1 also signals via ßArr2. In the presence of D2R, cAMP signaling of TAAR1 was reduced while its ßArr2 signaling was enhanced, resulting in reduced GSK3ß activation. These results demonstrate that ßArr2 signaling may be an important pathway for TAAR1 function and that the activation of the TAAR1-D2R complex negatively modulates GSK3ß signaling. Given that patients with schizophrenia or bipolar disorder show increased GSK3ß signaling, such a reduction of GSK3ß signaling triggered by the interaction of D2R with activated TAAR1 further supports TAAR1 as a target for the treatment of psychiatric disorders.

Homer1/mGluR5 activity moderates vulnerability to chronic social stress.

Stress-induced psychiatric disorders, such as depression, have recently been linked to changes in glutamate transmission in the central nervous system. Glutamate signaling is mediated by a range of receptors, including metabotropic glutamate receptors (mGluRs). In particular, mGluR subtype 5 (mGluR5) is highly implicated in stress-induced psychopathology. The major scaffold protein Homer1 critically interacts with mGluR5 and has also been linked to several psychopathologies. Yet, the specific role of Homer1 in this context remains poorly understood. We used chronic social defeat stress as an established animal model of depression and investigated changes in transcription of Homer1a and Homer1b/c isoforms and functional coupling of Homer1 to mGluR5. Next, we investigated the consequences of Homer1 deletion, overexpression of Homer1a, and chronic administration of the mGluR5 inverse agonist CTEP (2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine) on the effects of chronic stress. In mice exposed to chronic stress, Homer1b/c, but not Homer1a, mRNA was upregulated and, accordingly, Homer1/mGluR5 coupling was disrupted. We found a marked hyperactivity behavior as well as a dysregulated hypothalamic-pituitary-adrenal axis activity in chronically stressed Homer1 knockout (KO) mice. Chronic administration of the selective and orally bioavailable mGluR5 inverse agonist, CTEP, was able to recover behavioral alterations induced by chronic stress, whereas overexpression of Homer1a in the hippocampus led to an increased vulnerability to chronic stress, reflected in an increased physiological response to stress as well as enhanced depression-like behavior. Overall, our results implicate the glutamatergic system in the emergence of stress-induced psychiatric disorders, and support the Homer1/mGluR5 complex as a target for the development of novel antidepressant agents.

Metabotropic glutamate receptor 5 negative allosteric modulators: discovery of 2-chloro-4-[1-(4-fluorophenyl)-2,5-dimethyl-1H-imidazol-4-ylethynyl]pyridine (basimglurant, RO4917523), a promising novel medicine for psychiatric diseases.

Negative allosteric modulators (NAMs) of metabotropic glutamate receptor 5 (mGlu5) have potential for the treatment of psychiatric diseases including depression, fragile X syndrome (FXS), anxiety, obsessive-compulsive disorders, and levodopa induced dyskinesia in Parkinson's disease. Herein we report the optimization of a weakly active screening hit 1 to the potent and selective compounds chloro-4-[1-(4-fluorophenyl)-2,5-dimethyl-1H-imidazol-4-ylethynyl]pyridine (basimglurant, 2) and 2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine (CTEP, 3). Compound 2 is active in a broad range of anxiety tests reaching the same efficacy but at a 10- to 100-fold lower dose compared to diazepam and is characterized by favorable DMPK properties in rat and monkey as well as an excellent preclinical safety profile and is currently in phase II clinical studies for the treatment of depression and fragile X syndrome. Analogue 3 is the first reported mGlu5 NAM with a long half-life in rodents and is therefore an ideal tool compound for chronic studies in mice and rats.

Comparative analysis of acute and chronic administration of haloperidol and clozapine using [3H] 2-deoxyglucose metabolic mapping.

In an effort to compare and contrast the mechanisms of action of typical and atypical antipsychotic drugs, [3H] 2-deoxyglucose metabolic mapping was employed following acute and chronic administration of haloperidol (1 mg/kg i.p. acute and 0.5 mg/kg i.p. chronic) and clozapine (20 mg/kg i.p., both acute and chronic). Optical density ratios (ODR) were measured in 62 brain structures. An overall decrease in ODR was observed in many of the regions analyzed. Acute haloperidol elicited significant decreases, particularly in the thalamus and hippocampus. Acute clozapine decreased glucose uptake in the caudate putamen, hippocampus, central gray, locus coreleus, and the thalamus. In both chronically treated haloperidol and clozapine animals, significant decreases in ODR were seen in the thalamus and hippocampal areas most dramatically, with other changes in the superior colliculus, retrospenial cortex, and the cerebellum. Clozapine caused significant effects in 32 nuclei acutely and only 19 nuclei chronically. Haloperidol caused significant effects in 23 nuclei acutely and 15 nuclei chronically. The pattern of change induced by haloperidol and clozapine were remarkably similar when considering their pharmacology is somewhat different. Both antipsychotics elicited fewer significant changes upon chronic administration.