Structural basis of species-selective antagonist binding to the succinate receptor.

The tricarboxylic acid cycle intermediate succinate is involved in metabolic processes and plays a crucial role in the homeostasis of mitochondrial reactive oxygen species1. The receptor responsible for succinate signalling, SUCNR1 (also known as GPR91), is a member of the G-protein-coupled-receptor family2 and links succinate signalling to renin-induced hypertension, retinal angiogenesis and inflammation3-5. Because SUCNR1 senses succinate as an immunological danger signal6-which has relevance for diseases including ulcerative colitis, liver fibrosis7, diabetes and rheumatoid arthritis3,8-it is of interest as a therapeutic target. Here we report the high-resolution crystal structure of rat SUCNR1 in complex with an intracellular binding nanobody in the inactive conformation. Structure-based mutagenesis and radioligand-binding studies, in conjunction with molecular modelling, identified key residues for species-selective antagonist binding and enabled the determination of the high-resolution crystal structure of a humanized rat SUCNR1 in complex with a high-affinity, human-selective antagonist denoted NF-56-EJ40. We anticipate that these structural insights into the architecture of the succinate receptor and its antagonist selectivity will enable structure-based drug discovery and will further help to elucidate the function of SUCNR1 in vitro and in vivo.

Chemical genetic approach identifies microtubule affinity-regulating kinase 1 as a leucine-rich repeat kinase 2 substrate.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of autosomal-dominant forms of Parkinson's disease. LRRK2 is a modular, multidomain protein containing 2 enzymatic domains, including a kinase domain, as well as several protein-protein interaction domains, pointing to a role in cellular signaling. Although enormous efforts have been made, the exact pathophysiologic mechanisms of LRRK2 are still not completely known. In this study, we used a chemical genetics approach to identify LRRK2 substrates from mouse brain. This approach allows the identification of substrates of 1 particular kinase in a complex cellular environment. Several of the identified peptides are involved in the regulation of microtubule (MT) dynamics, including microtubule-associating protein (MAP)/microtubule affinity-regulating kinase 1 (MARK1). MARK1 is a serine/threonine kinase known to phosphorylate MT-binding proteins such as Tau, MAP2, and MAP4 at KXGS motifs leading to MT destabilization. In vitro kinase assays and metabolic-labeling experiments in living cells confirmed MARK1 as an LRRK2 substrate. Moreover, we also showed that LRRK2 and MARK1 are interacting in eukaryotic cells. Our findings contribute to the identification of physiologic LRRK2 substrates and point to a potential mechanism explaining the reported effects of LRRK2 on neurite morphology.

Blocking metabotropic glutamate receptor subtype 7 (mGlu7) via the Venus flytrap domain (VFTD) inhibits amygdala plasticity, stress, and anxiety-related behavior.

The metabotropic glutamate receptor subtype 7 (mGlu7) is an important presynaptic regulator of neurotransmission in the mammalian CNS. mGlu7 function has been linked to autism, drug abuse, anxiety, and depression. Despite this, it has been difficult to develop specific blockers of native mGlu7 signaling in relevant brain areas such as amygdala and limbic cortex. Here, we present the mGlu7-selective antagonist 7-hydroxy-3-(4-iodophenoxy)-4H-chromen-4-one (XAP044), which inhibits lateral amygdala long term potentiation (LTP) in brain slices from wild type mice with a half-maximal blockade at 88 nm. There was no effect of XAP044 on LTP of mGlu7-deficient mice, indicating that this pharmacological effect is mGlu7-dependent. Unexpectedly and in contrast to all previous mGlu7-selective drugs, XAP044 does not act via the seven-transmembrane region but rather via a binding pocket localized in mGlu7's extracellular Venus flytrap domain, a region generally known for orthosteric agonist binding. This was shown by chimeric receptor studies in recombinant cell line assays. XAP044 demonstrates good brain exposure and wide spectrum anti-stress and antidepressant- and anxiolytic-like efficacy in rodent behavioral paradigms. XAP044 reduces freezing during acquisition of Pavlovian fear and reduces innate anxiety, which is consistent with the phenotypes of mGlu7-deficient mice, the results of mGlu7 siRNA knockdown studies, and the inhibition of amygdala LTP by XAP044. Thus, we present an mGlu7 antagonist with a novel molecular mode of pharmacological action, providing significant application potential in psychiatry. Modeling the selective interaction between XAP044 and mGlu7's Venus flytrap domain, whose three-dimensional structure is already known, will facilitate future drug development supported by computer-assisted drug design.

A screen for enhancers of clearance identifies huntingtin as a heat shock protein 90 (Hsp90) client protein.

Mechanisms to reduce the cellular levels of mutant huntingtin (mHtt) provide promising strategies for treating Huntington disease (HD). To identify compounds enhancing the degradation of mHtt, we performed a high throughput screen using a hippocampal HN10 cell line expressing a 573-amino acid mHtt fragment. Several hit structures were identified as heat shock protein 90 (Hsp90) inhibitors. Cell treatment with these compounds reduced levels of mHtt without overt toxic effects as measured by time-resolved Förster resonance energy transfer assays and Western blots. To characterize the mechanism of mHtt degradation, we used the potent and selective Hsp90 inhibitor NVP-AUY922. In HdhQ150 embryonic stem (ES) cells and in ES cell-derived neurons, NVP-AUY922 treatment substantially reduced soluble full-length mHtt levels. In HN10 cells, Hsp90 inhibition by NVP-AUY922 enhanced mHtt clearance in the absence of any detectable Hsp70 induction. Furthermore, inhibition of protein synthesis with cycloheximide or overexpression of dominant negative heat shock factor 1 (Hsf1) in HdhQ150 ES cells attenuated Hsp70 induction but did not affect NVP-AUY922-mediated mHtt clearance. Together, these data provided evidence that direct inhibition of Hsp90 chaperone function was crucial for mHtt degradation rather than heat shock response induction and Hsp70 up-regulation. Co-immunoprecipitation experiments revealed a physical interaction of mutant and wild-type Htt with the Hsp90 chaperone. Hsp90 inhibition disrupted the interaction and induced clearance of Htt through the ubiquitin-proteasome system. Our data suggest that Htt is an Hsp90 client protein and that Hsp90 inhibition may provide a means to reduce mHtt in HD.

LRRK2 protein levels are determined by kinase function and are crucial for kidney and lung homeostasis in mice.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset Parkinson's disease (PD), but the underlying pathophysiological mechanisms and the normal function of this large multidomain protein remain speculative. To address the role of this protein in vivo, we generated three different LRRK2 mutant mouse lines. Mice completely lacking the LRRK2 protein (knock-out, KO) showed an early-onset (age 6 weeks) marked increase in number and size of secondary lysosomes in kidney proximal tubule cells and lamellar bodies in lung type II cells. Mice expressing a LRRK2 kinase-dead (KD) mutant from the endogenous locus displayed similar early-onset pathophysiological changes in kidney but not lung. KD mutants had dramatically reduced full-length LRRK2 protein levels in the kidney and this genetic effect was mimicked pharmacologically in wild-type mice treated with a LRRK2-selective kinase inhibitor. Knock-in (KI) mice expressing the G2019S PD-associated mutation that increases LRRK2 kinase activity showed none of the LRRK2 protein level and histopathological changes observed in KD and KO mice. The autophagy marker LC3 remained unchanged but kidney mTOR and TCS2 protein levels decreased in KD and increased in KO and KI mice. Unexpectedly, KO and KI mice suffered from diastolic hypertension opposed to normal blood pressure in KD mice. Our findings demonstrate a role for LRRK2 in kidney and lung physiology and further show that LRRK2 kinase function affects LRRK2 protein steady-state levels thereby altering putative scaffold/GTPase activity. These novel aspects of peripheral LRRK2 biology critically impact ongoing attempts to develop LRRK2 selective kinase inhibitors as therapeutics for PD.

Adriforant is a functional antagonist of histamine receptor 4 and attenuates itch and skin inflammation in mice.

BACKGROUND: Histamine has been postulated to play a role in atopic dermatitis via histamine receptor 4, mediating pruritic and inflammatory effects. The H4R antagonist adriforant (PF-3893787 or ZPL389) indicated clinical efficacy in a Ph2a study in atopic dermatitis. Preclinical investigations of adriforant had been scarce as experiments in transfectants with H4R from several species suggested partial agonism, not seen in human cells. OBJECTIVE: During the Ph2b trial in AD, we performed experiments to understand the pharmacology of adriforant in primary murine cells and in vivo models. We assessed its effects on ERK phosphorylation and transcriptional changes in bone marrow-derived mast cells, histamine-dependent Ca2+ flux in neurons and histamine-induced itch response. In addition, its impact on MC903-induced skin inflammation was evaluated. RESULTS: We show that, contrary to transfectants, adriforant is a competitive antagonist of the murine histamine receptor 4, antagonizes histamine-induced ERK phosphorylation, normalizes histamine-induced transcriptional changes in mast cells and reduces histamine-dependent Ca2+ flux in neurons. Administration to mice reduces acute histamine-induced itch response. In addition, adriforant ameliorates inflammation in the mouse MC903 model. CONCLUSIONS: Our results suggest that functional inhibition of histamine receptor 4 by adriforant reduces itch and inflammation in vivo. The effects observed in mice, however, did not translate to clinical efficacy in patients as the Ph2b clinical trial with adriforant did not meet pre-specified efficacy endpoints. Given the complex pathogenesis of AD, antagonism of histamine receptor 4 alone appears insufficient to reduce disease severity in AD patients, despite the effects seen in mouse models.

Exploring subtype selectivity and metabolic stability of a novel series of ligands for the benzodiazepine binding site of the GABAA receptor.

A novel series of agonists at the benzodiazepine binding site of the GABA(A) receptor was prepared by functionalizing a known template. Adding substituents to the pyrazolone-oxygen of CGS-9896 led to a number of compounds with selectivities for either α2- or α1-containing GABA(A) receptor subtypes offering an entry into indications such as anxiety and insomnia. In this communication, structure-activity relationship and efforts to increase in vitro stabilities are discussed.

Differential compartmentalization and distinct functions of GABAB receptor variants.

GABAB receptors are the G protein-coupled receptors for the main inhibitory neurotransmitter in the brain, gamma-aminobutyric acid (GABA). Molecular diversity in the GABAB system arises from the GABAB1a and GABAB1b subunit isoforms that solely differ in their ectodomains by a pair of sushi repeats that is unique to GABAB1a. Using a combined genetic, physiological, and morphological approach, we now demonstrate that GABAB1 isoforms localize to distinct synaptic sites and convey separate functions in vivo. At hippocampal CA3-to-CA1 synapses, GABAB1a assembles heteroreceptors inhibiting glutamate release, while predominantly GABAB1b mediates postsynaptic inhibition. Electron microscopy reveals a synaptic distribution of GABAB1 isoforms that agrees with the observed functional differences. Transfected CA3 neurons selectively express GABAB1a in distal axons, suggesting that the sushi repeats, a conserved protein interaction motif, specify heteroreceptor localization. The constitutive absence of GABAB1a but not GABAB1b results in impaired synaptic plasticity and hippocampus-dependent memory, emphasizing molecular differences in synaptic GABAB functions.

Generalization of amygdala LTP and conditioned fear in the absence of presynaptic inhibition.

Pavlovian fear conditioning, a simple form of associative learning, is thought to involve the induction of associative, NMDA receptor-dependent long-term potentiation (LTP) in the lateral amygdala. Using a combined genetic and electrophysiological approach, we show here that lack of a specific GABA(B) receptor subtype, GABA(B(1a,2)), unmasks a nonassociative, NMDA receptor-independent form of presynaptic LTP at cortico-amygdala afferents. Moreover, the level of presynaptic GABA(B(1a,2)) receptor activation, and hence the balance between associative and nonassociative forms of LTP, can be dynamically modulated by local inhibitory activity. At the behavioral level, genetic loss of GABA(B(1a)) results in a generalization of conditioned fear to nonconditioned stimuli. Our findings indicate that presynaptic inhibition through GABA(B(1a,2)) receptors serves as an activity-dependent constraint on the induction of homosynaptic plasticity, which may be important to prevent the generalization of conditioned fear.

Discovery and Optimization of Novel SUCNR1 Inhibitors: Design of Zwitterionic Derivatives with a Salt Bridge for the Improvement of Oral Exposure.

G-protein-coupled receptor SUCNR1 (succinate receptor 1 or GPR91) senses the citric cycle intermediate succinate and is implicated in various pathological conditions such as rheumatoid arthritis, liver fibrosis, or obesity. Here, we describe a novel SUCNR1 antagonist scaffold discovered by high-throughput screening. The poor permeation and absorption properties of the most potent compounds, which were zwitterionic in nature, could be improved by the formation of an internal salt bridge, which helped in shielding the two opposite charges and thus also the high polarity of zwitterions with separated charges. The designed compounds containing such a salt bridge reached high oral bioavailability and oral exposure. We believe that this principle could find a broad interest in the medicinal chemistry field as it can be useful not only for the modulation of properties in zwitterionic compounds but also in acidic or basic compounds with poor permeation.

Reduction of alcohol's reinforcing and motivational properties by the positive allosteric modulator of the GABA(B) receptor, BHF177, in alcohol-preferring rats.

BACKGROUND: The positive allosteric modulators of the GABA(B) receptor, CGP7930 and GS39783, have been found to reduce alcohol self-administration in alcohol-preferring rats. The present study was designed to assess the effect of the newly synthesized positive allosteric modulator of the GABA(B) receptor, BHF177, on alcohol's reinforcing and motivational properties in selectively bred Sardinian alcohol-preferring (sP) rats. METHODS: sP rats were initially trained to respond on a lever [on a fixed ratio 4 (FR4) schedule of reinforcement] to orally self-administer alcohol (15%, v/v) or sucrose (1 to 3%, w/v) in daily 30-minute sessions. Once responding reached stable levels, rats were allocated to 2 different experiments: in the first experiment, rats were exposed to sessions with the FR4 schedule of reinforcement; in the second experiment, rats were exposed to sessions with a conventional progressive ratio (PR) schedule of reinforcement. In both experiments, the effect of BHF177 (0, 12.5, 25, and 50 mg/kg; i.g.) on responding for alcohol and sucrose (FR experiment: 1%, w/v; PR experiment: 3%, w/v) was determined. RESULTS: In the FR experiment, pretreatment with 25 and 50 mg/kg BHF177 produced a 30 and 45% reduction, respectively, in responding for alcohol. In the PR experiment, pretreatment with 50 mg/kg BHF177 resulted in a 35% reduction in breakpoint for alcohol (defined as the lowest response requirement not achieved by each rat and used as index of the motivational strength of alcohol). In both experiments, the effect of BHF177 on alcohol self-administration was specific, since responding for sucrose was unaltered by BHF177 pretreatment. CONCLUSIONS: The present results extend to BHF177 the capacity of the 2 previously tested positive allosteric modulators of the GABA(B) receptor, CGP7930 and GS39783, to specifically suppress alcohol's reinforcing and motivational properties in alcohol-preferring rats.

Antagonizing Retinoic Acid-Related-Orphan Receptor Gamma Activity Blocks the T Helper 17/Interleukin-17 Pathway Leading to Attenuated Pro-inflammatory Human Keratinocyte and Skin Responses.

The nuclear hormone receptor retinoic acid receptor-related-orphan-receptor-gamma t (RORγt) is the key transcription factor required for Th17 cell differentiation and for production of IL-17 family cytokines by innate and adaptive immune cells. Dysregulated Th17 immune responses have been associated with the pathogenesis of several inflammatory and autoimmune diseases such as psoriasis, psoriatic arthritis, and ankylosing spondylitis. In this article, we describe the in vitro pharmacology of a potent and selective low molecular weight RORγt inhibitor identified after a structure-based hit-to-lead optimization effort. The compound interfered with co-activator binding to the RORγt ligand binding domain and impaired the transcriptional activity of RORγt as evidenced by blocked IL-17A secretion and RORE-mediated transactivation of a luciferase reporter gene. The inhibitor effectively reduced IL-17A production by human naive and memory T-cells and attenuated transcription of pro-inflammatory Th17 signature genes, such as IL17F, IL22, IL26, IL23R, and CCR6. The compound selectively suppressed the Th17/IL-17 pathway and did not interfere with polarization of other T helper cell lineages. Furthermore, the inhibitor was selective for RORγt and did not modify the transcriptional activity of the closely related family members RORα and RORß. Using human keratinocytes cultured with supernatants from compound treated Th17 cells we showed that pharmacological inhibition of RORγt translated to suppressed IL-17-regulated gene expression in keratinocyte cell cultures. Furthermore, in ex vivo immersion skin cultures our RORγt inhibitor suppressed IL-17A production by Th17-skewed skin resident cells which correlated with reduced human ß defensin 2 expression in the skin. Our data suggests that inhibiting RORγt transcriptional activity by a low molecular weight inhibitor may hold utility for the treatment of Th17/IL-17-mediated skin pathologies.

Structure-Based and Property-Driven Optimization of N-Aryl Imidazoles toward Potent and Selective Oral RORγt Inhibitors.

Retinoic acid receptor-related orphan receptor gamma-t (RORγt) is considered to be the master transcription factor for the development of Th17 cells that produce proinflammatory cytokines such as IL-17A. Overproportionate Th17 cell abundance is associated with the pathogenesis of many inflammatory conditions including psoriasis. In a high-throughput fluorescence resonance energy transfer (FRET) screen, we identified compound 1 as a hit with promising lipophilic efficiency (LipE). Using structure-based drug design based on a number of X-ray cocrystal structures, we morphed this hit class into potent imidazoles, exemplified by compound 3. To improve the poor absorption, distribution, metabolism, and excretion (ADME) properties of neutral imidazoles, we extended our ligands with carboxylic acid substituents toward a polar, water-rich area of the protein. This highly lipophilicity-efficient modification ultimately led to the discovery of compound 14, a potent and selective inhibitor of RORγt with good ADME properties and excellent in vivo pharmacokinetics. This compound showed good efficacy in an in vivo delayed-type hypersensitivity pharmacology model in rats.

Metabotropic glutamate 2/3 receptors in the ventral tegmental area and the nucleus accumbens shell are involved in behaviors relating to nicotine dependence.

The motivation to maintain nicotine self-administration and dependence may involve alterations in glutamatergic neurotransmission. Metabotropic glutamate (mGlu) 2/3 receptors 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 motivational processes. We found that acute systemic, as well as intra-VTA or intra-NAc, administration of the mGlu2/3 receptor agonist LY379268 [(-)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate] decreased nicotine, but not food, self-administration in rats. 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 [35S]GTPgammaS binding assay. Furthermore, repeated treatment with LY379268 reduced nicotine self-administration at the beginning of a 14 d treatment period; however, the number of nicotine infusions earned gradually returned to baseline levels, indicating tolerance to the effects of repeated LY379268 treatment. Finally, LY379268 administration decreased both cue-induced reinstatement of nicotine- and food-seeking behavior. Together, these findings indicate an important role for mGlu2/3 receptors in the posterior VTA and the NAc shell in the mediation of the rewarding effects of nicotine and potentially in cue-induced nicotine-seeking behavior.

Positive modulation of GABA(B) receptors decreased nicotine self-administration and counteracted nicotine-induced enhancement of brain reward function in rats.

Acute administration of gamma-aminobutyric acid (GABA)-B receptor agonists decreases nicotine, cocaine, ethanol, and heroin self-administration and also decreases food-maintained responding and suppresses locomotor activity at high doses. GABA(B) receptor-positive modulators may represent potentially improved therapeutic compounds because of their fewer side effects than receptor agonists. The present study investigated the effects of administration of the GABA(B) receptor-positive modulators 2,6-di-tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol (CGP7930) and N-[(1R,2R,4S)-bicyclo[2.2.1]hept-2-yl]-2-methyl-5-[4-(trifluoromethyl)phenyl]-4-pyrimidinamine (BHF177) and coadministration of the GABA(B) receptor-positive modulator N,N'-dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine (GS39783) with the GABA(B) receptor agonist (3-amino-2[S]-hydroxypropyl)-methylphosphinic acid (CGP44532) on nicotine- and food-maintained responding under fixed ratio (FR) 5 and progressive ratio schedules of reinforcement. Furthermore, the effects of BHF177 and CGP44532 on nicotine-induced enhancement of brain reward function were evaluated. The results indicated that administration of CGP7930 decreased nicotine self-administration under an FR5 schedule. Administration of either GS39783 or CGP44532 selectively decreased nicotine self-administration, whereas coadministration of these compounds had additive effects. BHF177 administration selectively decreased nicotine- but not food-maintained responding under FR5 and progressive ratio schedules. The nicotine-induced enhancement of brain reward function was blocked by BHF177 or CGP44532, although the highest doses of both compounds, particularly CGP44532, decreased brain reward function when administered alone, suggesting an additive, rather than interactive, effect. Overall, the present results indicate that GABA(B) receptor-positive modulators, similarly to GABA(B) receptor agonists, attenuated the reinforcing and reward-enhancing effects of nicotine, perhaps with higher selectivity than GABA(B) receptor agonists. Thus, GABA(B) receptor-positive modulators may be useful antismoking medications.

PET Imaging of T Cells: Target Identification and Feasibility Assessment.

Imaging T cells using positron emission tomography (PET) would be highly useful for diagnosis and monitoring in immunology and oncology patients. There are, however, no obvious targets that can be used to develop imaging agents for this purpose. We evaluated several potential target proteins with selective expression in T cells, and for which lead molecules were available: protein kinase C isozyme θ (PKC θ), lymphocyte-specific protein tyrosine kinase (Lck), zeta-chain-associated protein kinase 70 (ZAP70), and interleukin-2-inducible T-cell kinase (Itk). Ultimately, we focused on Itk and identified a tool molecule with properties suitable for in vivo imaging of T cells: (5aR)-5,5-difluoro-5a-methyl-N-(1-((S)-3-(methylsulfonyl)phenyl)(tetrahydro-2H-pyran-4-yl)methyl)-1H-pyrazol-4-yl)-1,4,4a,5,5a,6-hexahydrocyclopropa[f]indazole-3-carboxamide (23). Although it does not have the optimal profile for clinical use, this molecule indicates that it might be possible to develop Itk-selective PET ligands for imaging the distribution of T cells in patients.

Optimizing a Weakly Binding Fragment into a Potent RORγt Inverse Agonist with Efficacy in an in Vivo Inflammation Model.

The transcription factor RORγt is an attractive drug-target due to its role in the differentiation of IL-17 producing Th17 cells that play a critical role in the etiopathology of several autoimmune diseases. Identification of starting points for RORγt inverse agonists with good properties has been a challenge. We report the identification of a fragment hit and its conversion into a potent inverse agonist through fragment optimization, growing and merging efforts. Further analysis of the binding mode revealed that inverse agonism was achieved by an unusual mechanism. In contrast to other reported inverse agonists, there is no direct interaction or displacement of helix 12 observed in the crystal structure. Nevertheless, compound 9 proved to be efficacious in a delayed-type hypersensitivity (DTH) inflammation model in rats.

GABA(B(1)) receptor isoforms differentially mediate the acquisition and extinction of aversive taste memories.

Conditioned taste aversion (CTA) is a form of aversive memory in which an association is made between a consumed substance and a subsequent malaise. CTA is a critical mechanism for the successful survival, and hence evolution, of most animal species. The role of excitatory neurotransmitters in the neurochemical mechanisms of CTA is well recognized; however, less is known about the involvement of inhibitory receptor systems. In particular, the potential functions of metabotropic GABA(B) receptors in CTA have not yet been fully explored. GABA(B) receptors are metabotropic GABA receptors that are comprised of two subunits, GABA(B(1)) and GABA(B(2)), which form heterodimers. The Gabbr1 gene is transcribed into two predominant isoforms, GABA(B(1a)) and GABA(B(1b)), which differ in sequence primarily by the inclusion of a pair of sushi domains (also known as short consensus repeats) in the GABA(B(1a)) N terminus. The behavioral function of mammalian GABA(B(1)) receptor isoforms is currently unknown. Here, using a point mutation strategy in mice, we demonstrate that these two GABA(B(1)) receptor isoforms are differentially involved in critical components of CTA. In contrast to GABA(B(1b))-/- and wild-type mice, GABA(B(1a))-/- mice failed to acquire CTA. In contrast, GABA(B(1b))-/- mice robustly acquired CTA but failed to show any extinction of this aversion. The data demonstrate that GABA(B) receptors are involved in both the acquisition and extinction of CTA; however, receptors containing the GABA(B(1a)) or the GABA(B(1b)) isoform differentially contribute to the mechanisms used to learn and remember the salience of aversive stimuli.

GABA(B) receptor-positive modulation decreases selective molecular and behavioral effects of cocaine.

Exposure to cocaine induces selective behavioral and molecular adaptations. In rodents, acute cocaine induces increased locomotor activity, whereas prolonged drug exposure results in behavioral locomotor sensitization, which is thought to be a consequence of drug-induced neuroadaptive changes. Recent attention has been given to compounds activating GABA(B) receptors as potential antiaddictive therapies. In particular, the principle of allosteric positive GABA(B) receptor modulators is very promising in this respect, as positive modulators lack the sedative and muscle relaxant properties of full GABA(B) receptor agonists such as baclofen. Here, we investigated the effects of systemic application of the GABA(B) receptor-positive modulator GS39783 (N,N'-dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4, 6-diamine) in animals treated with acute and chronic cocaine administration. Both GS39783 and baclofen dose dependently attenuated acute cocaine-induced hyperlocomotion. Furthermore, both compounds also efficiently blocked cocaine-induced Fos induction in the striatal complex. In chronic studies, GS39783 induced a modest attenuation of cocaine-induced locomotor sensitization. Chronic cocaine induces the accumulation of the transcription factor deltaFosB and upregulates cAMP-response-element-binding protein (CREB) and dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32). GS39783 blocked the induction/activation of DARPP-32 and CREB in the nucleus accumbens and dorsal striatum and partially inhibited deltaFosB accumulation in the dorsal striatum. In summary, our data provide evidence that GS39783 attenuates the acute behavioral effects of cocaine exposure in rodents and in addition prevents the induction of selective long-term adaptive changes in dopaminergic signaling pathways. Further investigation of GABA(B) receptor-positive modulation as a novel therapeutic strategy for the treatment of cocaine dependence and possibly other drugs of abuse is therefore warranted.

Behavioral evaluation of mice deficient in GABA(B(1)) receptor isoforms in tests of unconditioned anxiety.

RATIONALE: Emerging data support a role for GABA(B) receptors in anxiety. GABA(B) receptors are comprised of a heterodimeric complex of GABA(B1) and GABA(B2) receptor subunits. The predominant neuronal GABA(B1) receptor isoforms are GABA(B(1a)) and GABA(B(1b)). Recent findings indicate specific roles for these isoforms in conditioned fear responses, although their influence on behavior in tests of unconditioned anxiety is unknown. OBJECTIVE: The aim of this study was to examine the role of the GABA(B(1)) isoforms in unconditioned anxiety. MATERIALS AND METHODS: Mice deficient in the GABA(B(1a)) or GABA(B(1b)) receptor isoforms were examined in a battery of anxiety tests. RESULTS: In most tests, genotype did not significantly affect anxious behavior, including the elevated plus maze, marble burying, and stress-induced hypothermia tests. Corticosterone and adrenocorticotropic hormone levels were similarly unaffected by genotype. Female, but not male, GABA(-/-)B(1a) and GABA(-/-)B(1b) mice showed increased anxiety relative to wild-type controls in the elevated zero maze. In the staircase test, male GABA(-/-)B(1b) mice defecated more than male GABA(-/-)B(1a) mice, although no other test parameter was influenced by genotype. In the light-dark box, female GABA(-/-)B(1a) mice spent less time in the light compartment compared to the GABA(-/-)B(1b) females, whereas male GABA(-/-)B(1b) mice made fewer light-dark transitions than GABA(-/-)B(1a) males. CONCLUSIONS: Specific roles for either GABA(B(1)) isoform in unconditioned anxiety were not explicit. This differs from their contribution in conditioned anxiety and from the anxious phenotype of GABA(B1) and GABA(B2) subunit knockout mice. The findings suggest that the GABA(B(1)) isoforms have specific relevance for anxiety with a cognitive component, rather than for innate anxiety per se.