Douglas A. Craig

GABA B receptors function as a heteromeric assembly of the subunits GABA B R1 and GABA B R2

The principal inhibitory neurotransmitter GABA (γ-aminobutyric acid) exerts its effects through two ligand-gated channels, GABAA and GABAC receptors, and a third receptor, GABAB (ref. 1), which acts through G proteins to regulate potassium and calcium channels. Cells heterologously expressing the cloned DNA encoding the GABABR1 protein exhibit high-affinity antagonist-binding sites, but they produce little of the functional activity expected from studies of endogenous GABAB receptors in the brain. Here we describe a new member of the GABAB polypeptide family, GABABR2, that shows sequence homology to GABABR1. Neither GABABR1 nor GABABR2, when expressed individually, activates GIRK-type potassium channels; however, the combination of GABABR1 and GABABR2 confers robust stimulation of channel activity. Both genes are co-expressed in individual neurons, and both proteins co-localize in transfected cells. Moreover, immunoprecipitation experiments indicate that the two polypeptides associate with each other, probably as heterodimers. Several G-protein-coupled receptors (GPCRs) exist as high-molecular-weight species, consistent with the formation of dimers by these receptors, but the relevance of these species for the functioning of GPCRs has not been established. We have now shown that co-expression of two GPCR structures, GABABR1 and GABABR2, belonging to the samesubfamily is essential for signal transduction by GABAB receptors.

Neurogenesis-Dependent and -Independent Effects of Fluoxetine in an Animal Model of Anxiety/Depression

Understanding the physiopathology of affective disorders and their treatment relies on the availability of experimental models that accurately mimic aspects of the disease. Here we describe a mouse model of an anxiety/depressive-like state induced by chronic corticosterone treatment. Furthermore, chronic antidepressant treatment reversed the behavioral dysfunctions and the inhibition of hippocampal neurogenesis induced by corticosterone treatment. In corticosterone-treated mice where hippocampal neurogenesis is abolished by X-irradiation, the efficacy of fluoxetine is blocked in some, but not all, behavioral paradigms, suggesting both neurogenesis-dependent and -independent mechanisms of antidepressant action. Finally, we identified a number of candidate genes, the expression of which is decreased by chronic corticosterone and normalized by chronic fluoxetine treatment selectively in the hypothalamus. Importantly, mice deficient in one of these genes, beta-arrestin 2, displayed a reduced response to fluoxetine in multiple tasks, suggesting that beta-arrestin signaling is necessary for the antidepressant effects of fluoxetine.

Efficacy of the MCHR1 antagonist N-[3-(1-{[4-(3,4-difluorophenoxy)phenyl]methyl}(4-piperidyl))-4-methylphenyl]-2-methylpropanamide (SNAP 94847) in mouse models of anxiety and depression following acute and chronic administration is independent of hippocampal neurogenesis.

Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide that plays a role in the modulation of food intake and mood. In rodents, the actions of MCH are mediated via the MCHR1 receptor. The goal of this study was to investigate the effects of acute (1 h) and chronic (28 days) p.o. dosing of a novel MCHR1 antagonist, N-[3-(1-{[4-(3,4-difluorophenoxy)-phenyl]methyl}(4-piperidyl))-4-methylphenyl]-2-methylpropanamide (SNAP 94847), in three mouse models predictive of antidepressant/anxiolytic-like activity: novelty suppressed feeding (NSF) in 129S6/SvEvTac mice and light/dark paradigm (L/D) and forced swim test (FST) in BALB/cJ mice. A significant increase in the time spent in the light compartment of the L/D box was observed in response to acute and chronic treatment with SNAP 94847. An anxiolytic/antidepressant-like effect was found in the NSF test after acute and chronic treatment, whereas no effect was observed in the FST. Because neurogenesis in the dentate gyrus has been shown to be a requirement for the effects of antidepressants in the NSF test, we investigated whether neurogenesis was required for the effect of SNAP 94847. We showed that chronic treatment with SNAP 94847 stimulated proliferation of progenitors in the dentate gyrus. The efficacy of SNAP 94847 in the NSF test, however, was unaltered in mice in which neurogenesis was suppressed by X-irradiation. These results indicate that SNAP 94847 has a unique anxiolytic-like profile after both acute and chronic administration and that its mechanism of action is distinct from that of selective serotonin reuptake inhibitors and tricyclic antidepressants.

The Novel Neuropeptide Y Y5 Receptor Antagonist Lu AA33810 [N-[[trans-4-[(4,5-Dihydro[1]benzothiepino[5,4-d]thiazol-2-yl)amino]cyclohexyl]methyl]-methanesulfonamide] Exerts Anxiolytic- and Antidepressant-Like Effects in Rat Models of Stress Sensitivity

Neuropeptide Y (NPY) regulates physiological processes via receptor subtypes (Y1, Y2, Y4, Y5, and y6). The Y5 receptor is well known for its role in appetite. Based on expression in the limbic system, we hypothesized that the Y5 receptor might also modulate stress sensitivity. We identified a novel Y5 receptor-selective antagonist, Lu AA33810 [N-[[trans-4-[(4,5-dihydro[1]-benzothiepino[5,4-d]thiazol-2-yl)amino]cyclohexyl]methyl]-methanesulfonamide], that bound to cloned rat Y5 receptors (Ki = 1.5 nM) and antagonized NPY-evoked cAMP and calcium mobilization in vitro. Lu AA33810 (3-30 mg/kg p.o.) blocked feeding elicited by intracerebroventricular injection of the Y5 receptor-selective agonist [cPP1-7,NPY19-23,Ala31,Aib32,Gln34]-hPancreatic Polypeptide in Sprague-Dawley rats. In vivo effects of Lu AA33810 were correlated with brain exposure ≥ 50 ng/g and ex vivo Y5 receptor occupancy of 22 to 95%. Lu AA33810 was subsequently evaluated in models of stress sensitivity. In Fischer 344 rats, Lu AA33810 (30 mg/kg p.o.) attenuated increases in plasma ACTH and corticosterone elicited by intracerebroventricular injection of [cPP1-7,NPY19-23,Ala31,Aib32,Gln34]-hPancreatic Polypeptide. In Sprague-Dawley rats subjected to the social interaction test, Lu AA33810 (3-30 mg/kg p.o.) produced anxiolytic-like effects after acute or chronic treatment. In Flinders sensitive line rats, chronic dosing of Lu AA33810 (10 mg/kg/day i.p.) produced anxiolytic-like effects in the social interaction test, plus antidepressant-like effects in the forced swim test. In Wistar rats exposed to chronic mild stress, chronic dosing of Lu AA33810 (3 and 10 mg/kg/day i.p.) produced antidepressant-like activity, i.e., normalization of stress-induced decrease in sucrose consumption. We propose that Y5 receptors may function as part of an endogenous stress-sensing system to mediate social anxiety and reward or motivational deficits in selected rodent models.

Signal Transduction by GABAB Receptor Heterodimers

GABAB receptors are G-protein-coupled receptors that mediate inhibition throughout the central and peripheral nervous systems. A single cloned receptor, GABABR1, which has at least three alternatively spliced forms, appears to account for the vast majority of binding sites in the brain for high-affinity antagonists. In heterologous expression systems GABABR1 is poorly expressed on the plasma membrane and largely fails to couple to ion channels. A second gene, GABABR2, which exhibits moderately low homology to GABABR1, permits surface expression of GABABR1 and the appearance of baclofen-sensitive K+ and Ca+1 currents. We review the data that supports a model of the native GABAB receptor as a heterodimer composed of GABABR1 and GABABR2 proteins. New data from mutagenesis experiments are presented that point to amino acid residues on GABABR1 critical for ligand activation of the heterodimer. The possible role of GABABR2 in signal transduction is also discussed. The interdependent nature of the two subunits for receptor function makes the GABAB receptor a useful model to explore the larger significance of GPCR dimerization for G-protein activation.

The effects of stressful stimuli and hypothalamic–pituitary–adrenal axis activation are reversed by the melanin-concentrating hormone 1 receptor antagonist SNAP 94847 in rodents

Melanin-concentrating hormone (MCH) is an orexigenic and dipsogenic neuropeptide that has been reported to mediate acute behavioral and neuroendocrine stress-related responses via MCH(1) receptor activation in rodents. The purpose of the present investigation was to use the MCH(1) receptor antagonist SNAP 94847 (N-(3-{1-[4-(3,4-difluoro-phenoxy)-benzyl]-piperidin-4-yl}-4-methyl-phenyl)-isobutyramide) to determine the effects of MCH(1) receptor blockade on MCH-evoked adrenocorticotropic hormone (ACTH) release, chronic mild stress-induced anhedonia, stress-induced hyperthermia and forced swim stress-induced immobility. The appropriate dose range for testing SNAP 94847 was determined by measuring MCH-evoked water drinking. The corresponding occupancy of MCH(1) receptors in rat striatum was also measured across a broad dose range. Orally administered (p.o.) SNAP 94847 (1-10 mg/kg) corresponds to 30-60% occupancy at MCH(1) receptors and significantly blocks water drinking induced by the intracerebroventricular (i.c.v.) injection of MCH. MCH (i.c.v.) significantly elevates plasma levels of ACTH in rats, and SNAP 94847 (2.5 mg/kg, p.o.) blocks MCH-evoked ACTH release. Using the chronic mild stress paradigm, we show that repeated daily exposure to environmental stressors for 5 weeks significantly suppresses sucrose intake in rats, and that SNAP 94847 (1 mg/kg, BID) for 1-5 weeks restores baseline sucrose intake. Moreover, a single administration of SNAP 94847 attenuates stress-induced hyperthermia and the behavioral effects of forced swim stress with minimal effective doses of 2.5 and 30 mg/kg (p.o.), respectively. The regulation of ACTH release and reversal of the effects of chronic and acute stress by SNAP 94847 are suggestive of a role for MCH(1) receptor blockade in the treatment of disorders characterized by high allostatic load.

The MCH1 receptor antagonist SNAP 94847 induces sensitivity to dopamine D2/D3 receptor agonists in rats and mice.

Antidepressant treatment of two or more weeks in rats has been shown to enhance the locomotor-stimulating effects of dopamine D(2)/D(3) receptor agonists. This action has been attributed to an increased sensitivity of postsynaptic dopamine receptors in the nucleus accumbens, thought to represent an essential mechanism by which antidepressants act therapeutically to enhance reward and motivation. We tested whether the melanin-concentrating hormone receptor(1) (MCH(1)) antagonist SNAP 94847, reported to have antidepressant-like activity in several preclinical behavioral models, mimics this key feature of established antidepressants. Locomotor responses to the dopamine D(2)/D(3) agonist quinpirole following acute or chronic administration of fluoxetine (18 mg/kg/day) or SNAP 94847 (20 mg/kg/day) were assessed in habituated Sprague-Dawley rats, as well as BALB/c and CD-1 mice. Rats showed a significant increase in quinpirole-induced locomotor activity following chronic (2 weeks), but not acute (1 h) fluoxetine or SNAP 94847 administration. BALB/c mice treated for 21 days with fluoxetine or SNAP 94847 showed marked increases in quinpirole-induced locomotor activity, with the onset of hyper-locomotion appearing earlier in the time course after SNAP 94847 compared to fluoxetine. Administration of either compound for 7 days was also sufficient to augment the quinpirole response in BALB/c mice. Fluoxetine and SNAP 94847 (21 days) failed to modify quinpirole responses in CD-1 mice, and the compounds were ineffective after acute administration in both mouse strains. This report demonstrates in two rodent species that chronic treatment with an MCH(1) receptor antagonist, as with clinically proven antidepressants, produces sensitization to the locomotor effects of dopamine D(2)/D(3) agonists.

The Role of Melanin-Concentrating Hormone-1 Receptors in the Voiding Reflex in Rats

We have used the selective melanin-concentrating hormone-1 (MCH1) receptor antagonist SNAP 7941 [((+)-methyl (4S)-3-{[(3-{4-[3-(acetylamino)phenyl]-1-piperidinyl}propyl) amino]carbonyl}-4-(3,4-difluorophenyl)-6-(methoxymethyl)-2-oxo-1,2,3,4-tetrahydro-5-pyrimidinecarboxylate hydrochloride)] to investigate the role of the hypothalamic neuropeptide MCH in the control of voiding in rats. Intravenous administration of SNAP 7941 (3 and 10 mg/kg i.v.) produced dose-related inhibition of rhythmic, distension-induced voiding contractions in anesthetized rats. In conscious rats in which repeated voiding cycles were evoked by continuous slow transvesicular infusion of saline, intragastric SNAP 7941 [0.03–1 mg/kg intragastrically (i.g.)] produced sustained increases in infusion capacity (maximum = 220% basal), comparable with the effects of the 5-hydroxytryptamine1A antagonist WAY 100635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl-cyclohexanecarboxamide maleate salt), and the muscarinic antagonist, oxybutynin (4-diethylaminobut-2-ynyl 2-cyclohexyl-2-hydroxy-2-phenylacetate hydrochloride). SNAP 7941 produced similar results when administered at a low dose (0.01 nmol) into the lateral ventricle (intracerebroventricular). The opposite effect was produced when MCH (20 nmol) was delivered intracerebroventricularly, resulting in a 34% decrease in apparent bladder capacity with increased urinary frequency. The effect of MCH was blocked by the prior intragastric administration of SNAP 7941 (0.1 mg/kg), but oxybutynin (1 mg/kg) was ineffective. Finally, in conscious spontaneously hypertensive rats, SNAP 7941 (0.1 mg/kg i.g.) produced a 31% reduction in micturition frequency, accompanied by a 36% increase in bladder capacity, with no effect on total volume voided over 6 h. The data indicate that MCH acts via MCH1 receptors within the CNS to modulate the voiding reflex in rats. The striking effects of the MCH1 antagonist SNAP 7941 to increase bladder capacity and reduce voiding frequency indicate that MCH1 antagonists may offer a potential novel approach for treating overactive bladder syndrome.