3-[3-(Phenalkylamino)cyclohexyl]phenols: Synthesis, biological activity, and in silico investigation of a naltrexone-derived novel class of MOR-antagonists.

The development of novel µ-opioid receptor (MOR) antagonists is one of the main objectives of drug discovery and development. Based on a simplified version of the morphinan scaffold, 3-[3-(phenalkylamino)cyclohexyl]phenol analogs were designed, synthesized, and evaluated for their MOR antagonist activity in vitro and in silico. At the highest concentrations, the compounds decreased by 52% to 75% DAMGO-induced GTPγS stimulation, suggesting that they acted as antagonists. Moreover, Extra-Precision Glide and Generalized-Born Surface Area experiments provided useful information on the nature of the ligand-receptor interactions, indicating a peculiar combination of C-1 stereochemistry and N-substitutions as feasibly essential for MOR-ligand complex stability. Interestingly, compound 9 showed the best experimental binding affinity, the highest antagonist activity, and the finest MOR-ligand complex stability. In silico experiments also revealed that the most promising stereoisomer (1R, 3R, 5S) 9 retained 1,3-cis configuration with phenol ring equatorial oriented. Further studies are needed to better characterize the pharmacodynamics and pharmacokinetic properties of these compounds.

Human Neuronal Cell Lines as An In Vitro Toxicological Tool for the Evaluation of Novel Psychoactive Substances.

Novel psychoactive substances (NPS) are synthetic substances belonging to diverse groups, designed to mimic the effects of scheduled drugs, resulting in altered toxicity and potency. Up to now, information available on the pharmacology and toxicology of these new substances is very limited, posing a considerable challenge for prevention and treatment. The present in vitro study investigated the possible mechanisms of toxicity of two emerging NPS (i) 4'-methyl-alpha-pyrrolidinoexanophenone (3,4-MDPHP), a synthetic cathinone, and (ii) 2-chloro-4,5-methylenedioxymethamphetamine (2-Cl-4,5-MDMA), a phenethylamine. In addition, to apply our model to the class of synthetic opioids, we evaluated the toxicity of fentanyl, as a reference compound for this group of frequently abused substances. To this aim, the in vitro toxic effects of these three compounds were evaluated in dopaminergic-differentiated SH-SY5Y cells. Following 24 h of exposure, all compounds induced a loss of viability, and oxidative stress in a concentration-dependent manner. 2-Cl-4,5-MDMA activates apoptotic processes, while 3,4-MDPHP elicits cell death by necrosis. Fentanyl triggers cell death through both mechanisms. Increased expression levels of pro-apoptotic Bax and caspase 3 activity were observed following 2-Cl-4,5-MDMA and fentanyl, but not 3,4-MDPHP exposure, confirming the different modes of cell death.

Impaired brain endocannabinoid tone in the activity-based model of anorexia nervosa.

OBJECTIVE: Despite the growing knowledge on the functional relationship between an altered endocannabinoid (eCB) system and development of anorexia nervosa (AN), to date no studies have investigated the central eCB tone in the activity-based anorexia (ABA) model that reproduces key aspects of human AN. METHOD: We measured levels of two major eCBs, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), those of two eCB-related lipids, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), and the cannabinoid type-1 receptor (CB1R) density in the brain of female ABA rats, focusing on areas involved in homeostatic and rewarding-related regulation of feeding behavior (i.e., prefrontal cortex, nucleus accumbens, caudato putamen, amygdala, hippocampus and hypothalamus). Analysis was carried out also at the end of recovery from the ABA condition. RESULTS: At the end of the ABA induction phase, 2-AG was significantly decreased in ABA rats in different brain areas but not in the caudato putamen. No changes were detected in AEA levels in any region, whereas the levels of OEA and PEA were decreased exclusively in the hippocampus and hypothalamus. Furthermore, CB1R density was decreased in the dentate gyrus of hippocampus and in the lateral hypothalamus. After recovery, both 2-AG levels and CB1R density were partially normalized in some areas. In contrast, AEA levels became markedly reduced in all the analyzed areas. DISCUSSION: These data demonstrate an altered brain eCB tone in ABA rats, further supporting the involvement of an impaired eCB system in AN pathophysiology that may contribute to the maintenance of some symptomatic aspects of the disease.

PPARα regulates cholinergic-driven activity of midbrain dopamine neurons via a novel mechanism involving α7 nicotinic acetylcholine receptors.

Ventral tegmental area dopamine neurons control reward-driven learning, and their dysregulation can lead to psychiatric disorders. Tonic and phasic activity of these dopaminergic neurons depends on cholinergic tone and activation of nicotinic acetylcholine receptors (nAChRs), particularly those containing the ß2 subunit (ß2*-nAChRs). Nuclear peroxisome proliferator-activated receptors type-α (PPARα) tonically regulate ß2*-nAChRs and thereby control dopamine neuron firing activity. However, it is unknown how and when PPARα endogenous ligands are synthesized by dopamine cells. Using ex vivo and in vivo electrophysiological techniques combined with biochemical and behavioral analysis, we show that activation of α7-nAChRs increases in the rat VTA both the tyrosine phosphorylation of the ß2 subunit of nAChRs and the levels of two PPARα endogenous ligands in a Ca(2+)-dependent manner. Accordingly, in vivo production of endogenous PPARα ligands, triggered by α7-nAChR activation, blocks in rats nicotine-induced increased firing activity of dopamine neurons and displays antidepressant-like properties. These data demonstrate that endogenous PPARα ligands are effectors of α7-nAChRs and that their neuromodulatory properties depend on phosphorylation of ß2*-nAChRs on VTA dopamine cells. This reveals an autoinhibitory mechanism aimed at reducing dopamine cell overexcitation engaged during hypercholinergic drive. Our results unveil important physiological functions of nAChR/PPARα signaling in dopamine neurons and how behavioral output can change after modifications of this signaling pathway. Overall, the present study suggests PPARα as new therapeutic targets for disorders associated with unbalanced dopamine-acetylcholine systems.

Correlations between Gustatory, Olfactory, Cognitive Function, and Age in Healthy Women.

Aging is a progressive physiological degeneration associated with a decline in chemosensory processes and cognitive abilities and a reduction in synaptic plasticity. The biological bases of ageing are still not completely understood, and many theories have been proposed. This study aimed to evaluate the occurrence of age-related changes affecting the chemosensory function (gustatory and olfactory) and general cognitive abilities and their potential associations in women. To this aim, 319 women (the age ranging from 18 to 92 years) were recruited and divided into four different age groups: 18-34 years, 35-49 years, 50-64 years, and ≥65 years. Our results confirmed that in women, gustatory, olfactory, and cognitive functions decline, though in a different manner during aging. Olfactory and cognitive function showed a slight decline along the first three age classes, with a dramatic decrease after age 65 years, while gustatory function decreased more gradually. Olfactory and gustatory deficits may have a high degree of predictivity for general cognitive function as well as for specific cognitive subdomains such as visuospatial/executive abilities, language, memory, and attention. Our study highlighted the importance of using chemosensory assessments for the early diagnosis of cognitive decline and for the development of appropriate personalized risk prevention strategies.

NMDARs mediate the role of monoamine oxidase A in pathological aggression.

Converging evidence shows that monoamine oxidase A (MAO A), the key enzyme catalyzing serotonin (5-hydroxytryptamine; 5-HT) and norepinephrine (NE) degradation, is a primary factor in the pathophysiology of antisocial and aggressive behavior. Accordingly, male MAO A-deficient humans and mice exhibit an extreme predisposition to aggressive outbursts in response to stress. As NMDARs regulate the emotional reactivity to social and environmental stimuli, we hypothesized their involvement in the modulation of aggression mediated by MAO A. In comparison with WT male mice, MAO A KO counterparts exhibited increases in 5-HT and NE levels across all brain regions, but no difference in glutamate concentrations and NMDAR binding. Notably, the prefrontal cortex (PFC) of MAO A KO mice exhibited higher expression of NR2A and NR2B, as well as lower levels of glycosylated NR1 subunits. In line with these changes, the current amplitude and decay time of NMDARs in PFC was significantly reduced. Furthermore, the currents of these receptors were hypersensitive to the action of the antagonists of the NMDAR complex (dizocilpine), as well as NR2A (PEAQX) and NR2B (Ro 25-6981) subunits. Notably, systemic administration of these agents selectively countered the enhanced aggression in MAO A KO mice, at doses that did not inherently affect motor activity. Our findings suggest that the role of MAO A in pathological aggression may be mediated by changes in NMDAR subunit composition in the PFC, and point to a critical function of this receptor in the molecular bases of antisocial personality.

The PPARα agonist fenofibrate reduces the cytokine imbalance in a maternal immune activation model of schizophrenia.

Maternal infections during pregnancy may increase the risk of psychiatric disorders in offspring. We recently demonstrated that activation of peroxisome proliferator-activate receptor-α (PPARα), with the clinically available agonist fenofibrate (FEN), attenuates the neurodevelopmental disturbances induced by maternal immune activation (MIA) in rat offspring. We hypothesized that fenofibrate might reduce MIA-induced cytokine imbalance using a MIA model based on the viral mimetic polyriboinosinic-polyribocytidilic acid [poly (I:C)]. By using the Bio-Plex Multiplex-Immunoassay-System, we measured cytokine/chemokine/growth factor levels in maternal serum and in the fetal brain of rats treated with fenofibrate, at 6 and 24 h after poly (I:C). We found that MIA induced time-dependent changes in the levels of several cytokines/chemokines/colony-stimulating factors (CSFs). Specifically, the maternal serum of the poly (I:C)/control (CTRL) group showed increased levels of (i) proinflammatory chemokine macrophage inflammatory protein 1-alpha (MIP-1α), (ii) tumor necrosis factor-alpha (TNF-α), the monocyte chemoattractant protein-1 (MCP-1), the macrophage (M-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Conversely, in the fetal brain of the poly (I:C)/CTRL group, interleukin 12p70 and MIP-1α levels were lower than in vehicle (veh)/CTRL group. Notably, MIP-1α, TNF-α, keratinocyte derived chemokine (GRO/KC), GM-CSF, and M-CSF levels were lower in the poly (I:C)/FEN than in poly (I:C)/CTRL rats, suggesting the protective role of the PPARα agonist. PPARα might represent a therapeutic target to attenuate MIA-induced inflammation.

Food restriction and hyperactivity induce changes in corticolimbic brain dopamine and serotonin levels in female rats.

Compelling data support altered dopamine (DA) and serotonin (5-HT) signaling in anorexia nervosa (AN). However, their exact role in the etiopathogenesis of AN has yet to be elucidated. Here, we evaluated the corticolimbic brain levels of DA and 5-HT in the induction and recovery phases of the activity-based anorexia (ABA) model of AN. We exposed female rats to the ABA paradigm and measured the levels of DA, 5-HT, the metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA), and the dopaminergic type 2 (D2) receptors density in feeding- and reward-implicated brain regions (i.e., cerebral cortex, Cx; prefrontal cortex, PFC; caudate putamen, CPu; nucleus accumbens, NAcc; amygdala, Amy; hypothalamus, Hyp; hippocampus, Hipp). DA levels were significantly increased in the Cx, PFC and NAcc, while 5-HT was significantly enhanced in the NAcc and Hipp of ABA rats. Following recovery, DA was still elevated in the NAcc, while 5-HT was increased in the Hyp of recovered ABA rats. DA and 5-HT turnover were impaired at both ABA induction and recovery. D2 receptors density was increased in the NAcc shell. These results provide further proof of the impairment of the dopaminergic and serotoninergic systems in the brain of ABA rats and support the knowledge of the involvement of these two important neurotransmitter systems in the development and progression of AN. Thus, providing new insights on the corticolimbic regions involved in the monoamine dysregulations in the ABA model of AN.

Characterization of COR627 and COR628, two novel positive allosteric modulators of the GABA(B) receptor.

The potential efficacy of GABA(B) receptor agonists in the treatment of pain, drug addiction, epilepsy, cognitive dysfunctions, and anxiety disorders is supported by extensive preclinical and clinical evidence. However, the numerous side effects produced by the GABA(B) receptor agonist baclofen considerably limit the therapeutic use of this compound. The identification of positive allosteric modulators (PAMs) of the GABA(B) receptor may constitute a novel approach in the pharmacological manipulation of the GABA(B) receptor, leading to fewer side effects. The present study reports the identification of two novel compounds, methyl 2-(1-adamantanecarboxamido)-4-ethyl-5-methylthiophene-3-carboxylate (COR627) and methyl 2-(cyclohexanecarboxamido)-4-ethyl-5-methylthiophene-3-carboxylate (COR628), which act as GABA(B) PAMs in 1) rat cortical membranes and 2) in vivo assay. Both compounds potentiated GABA- and baclofen-stimulated guanosine 5'-O-(3-[(35)S]thio)-triphosphate binding to native GABA(B) receptors, while producing no effect when given alone. GABA concentration-response curves in the presence of fixed concentrations of COR627 and COR628 revealed an increase of potency of GABA rather than its maximal efficacy. In radioligand binding experiments [displacement of the GABA(B) receptor antagonist, 3-N-[1-((S)-3,4dichlorophenyl)-ethylaminol]-2-(S)hydroxypropyl cyclo-hexylmethyl phosphinic acid ([(3)H]CGP54626)], both COR627 and COR628 increased the affinity of high- and low-affinity binding sites for GABA, producing no effect when administered alone up to a concentration of 1 mM. In vivo experiments indicated that pretreatment with per se ineffective doses of COR627 and COR628 potentiated the sedative/hypnotic effect of baclofen. In conclusion, COR627 and COR628 may represent two additional tools for use in investigating the roles and functions of positive allosteric modulatory binding sites of the GABA(B) receptor.

Pharmacological characterization of novel synthetic opioids: Isotonitazene, metonitazene, and piperidylthiambutene as potent µ-opioid receptor agonists.

Recent trends of opioid abuse and related fatalities have highlighted the critical role of Novel Synthetic Opioids (NSOs). We studied the µ-opioid-like properties of isotonitazene (ITZ), metonitazene (MTZ), and piperidylthiambutene (PTB) using different approaches. In vitro studies showed that ITZ and MTZ displayed a higher potency in both rat membrane homogenates (EC50:0.99 and 19.1 nM, respectively) and CHO-MOR (EC50:0.71 and 10.0 nM, respectively) than [D-Ala2, NMe-Phe4, Gly-ol5]-enkephalin (DAMGO), with no difference in maximal efficacy (Emax) between DAMGO and NSOs. ITZ also has higher affinity (Ki:0.06 and 0.05 nM) at the MOR than DAMGO in both systems, whilst MTZ has higher affinity in CHO-MOR (Ki=0.23 nM) and similar affinity in rat cerebral cortex (Ki = 0.22 nM). PTB showed lower affinity and potency than DAMGO. In vivo, ITZ displayed higher analgesic potency than fentanyl and morphine (ED50:0.00156, 0.00578, 2.35 mg/kg iv, respectively); ITZ (0.01 mg/kg iv) and MTZ (0.03 mg/kg iv) reduced behavioral activity and increased dialysate dopamine (DA) in the NAc shell (max. about 200% and 170% over basal value, respectively. Notably, ITZ elicited an increase in DA comparable to that of higher dose of morphine (1 mg/kg iv), but higher than the same dose of fentanyl (0.01 mg/kg iv). In silico, induced fit docking (IFD) and metadynamic simulations (MTD) showed that binding modes and structural changes at the receptor, ligand stability, and the overall energy score of NSOs were consistent with the results of the biological assays.

COR758, a negative allosteric modulator of GABAB receptors.

Allosteric modulators of G protein coupled receptors (GPCRs), including GABABRs (GABABRs), are promising therapeutic candidates. While several positive allosteric modulators (PAM) of GABABRs have been characterized, only recently the first negative allosteric modulator (NAM) has been described. In the present study, we report the characterization of COR758, which acts as GABABR NAM in rat cortical membranes and CHO cells stably expressing GABABRs (CHO-GABAB). COR758 failed to displace the antagonist [3H]CGP54626 from the orthosteric binding site of GABABRs showing that it acts through an allosteric binding site. Docking studies revealed a possible new allosteric binding site for COR758 in the intrahelical pocket of the GABAB1 monomer. COR758 inhibited basal and GABABR-stimulated O-(3-[35Sthio)-triphosphate ([35S]GTPγS) binding in brain membranes and blocked the enhancement of GABABR-stimulated [35S]GTPγS binding by the PAM GS39783. Bioluminescent resonance energy transfer (BRET) measurements in CHO-GABAB cells showed that COR758 inhibited G protein activation by GABA and altered GABABR subunit rearrangements. Additionally, the compound altered GABABR-mediated signaling such as baclofen-induced inhibition of cAMP production in transfected HEK293 cells, agonist-induced Ca2+ mobilization as well as baclofen and the ago-PAM CGP7930 induced phosphorylation of extracellular signal-regulated kinases (ERK1/2) in CHO-GABAB cells. COR758 also prevented baclofen-induced outward currents recorded from rat dopamine neurons, substantiating its property as a NAM for GABABRs. Altogether, these data indicate that COR758 inhibits G protein signaling by GABABRs, likely by interacting with an allosteric binding-site. Therefore, COR758 might serve as a scaffold to develop additional NAMs for therapeutic intervention.

The GABAB receptor positive allosteric modulator COR659: In vitro metabolism, in vivo pharmacokinetics in rats, synthesis and pharmacological characterization of metabolically protected derivatives.

We report an in vitro phase I metabolism study on COR659 (1), a 2-acylaminothiophene derivative able to suppress alcohol and chocolate self-administration in rats, likely via positive allosteric modulation of the GABAB receptor and antagonism/inverse agonism at the cannabinoid CB1 receptor. Given the identification of the methyl ester group at C-3 of the thiophene ring as a metabolic soft spot, we also report the chemical optimization project aimed to balance metabolic stability with in vitro and in vivo potency on a set of 3-substituted COR659 analogues. High performance liquid chromatography coupled to tandem and high resolution mass spectrometry was employed for the characterization of in vitro metabolism and in vivo pharmacokinetics of COR659 in rats. In vitro [35S]GTPγS binding assays on stimulated GABAB and CB1 receptors, in combination with alcohol and chocolate self-administration experiments in rats, were employed to assess the pharmacological profile of this novel set of analogues, using COR659 as reference compound. Eight metabolites of COR659 were discovered in liver microsomal incubates; two of them (M1, M2) were identified by comparison with synthetic reference standards. M2, oxidation product of methyl group at C-5 of the thiophene ring, was a major metabolite in vitro, but showed a low systemic exposure in vivo. M1, cleavage product of the methyl ester group at C-3, revealed in vitro an unusual mechanism of metabolism by a NADPH-dependent route and, in vivo, it maintained high and persistent levels in plasma, which could represent a potential pharmacokinetic and toxicological issue. In the novel set of COR659 analogues, those bearing branched alkyl substituents on the ester group, showed an improved in vitro metabolic stability (2-4), had an in vitro GABAB PAM (2-4) and/or CB1 partial agonist/antagonist profile (2-3) and maintained the ability to reduce alcohol (2-4) and/or chocolate (4) self-administration in rats. Both PK and PD data ruled out any involvement of metabolite M1 in the in vivo potency of COR659 and 4. The present results, therefore, highlight the importance to design and synthesize novel compounds endowed with the dual activity profile and devoid of metabolic liabilities.

Predisposition to Alcohol Drinking and Alcohol Consumption Alter Expression of Calcitonin Gene-Related Peptide, Neuropeptide Y, and Microglia in Bed Nucleus of Stria Terminalis in a Subnucleus-Specific Manner.

Excessive alcohol consumption is often linked to anxiety states and has a major relay center in the anterior part of bed nucleus of stria terminalis (BNST). We analyzed the impact of (i) genetic predisposition to high alcohol preference and consumption, and (ii) alcohol intake on anterior BNST, namely anterolateral (AL), anteromedial (AM), and anteroventral (lateral + medial subdivisions: AVl, AVm) subnuclei. We used two rat lines selectively bred for low- and high-alcohol preference and consumption, named Sardinian alcohol-non preferring (sNP) and -preferring (sP), respectively, the latter showing also inherent anxiety-related behaviors. We analyzed the modulation of calcitonin gene-related peptide (CGRP; exerting anxiogenic effects in BNST), neuropeptide Y (NPY; exerting mainly anxiolytic effects), and microglia activation (neuroinflammation marker, thought to increase anxiety). Calcitonin gene-related peptide-immunofluorescent fibers/terminals did not differ between alcohol-naive sP and sNP rats. Fiber/terminal NPY-immunofluorescent intensity was lower in BNST-AM and BNST-AVm of alcohol-naive sP rats. Activation of microglia (revealed by morphological analysis) was decreased in BNST-AM and increased in BNST-AVm of alcohol-naive sP rats. Prolonged (30 consecutive days), voluntary alcohol intake under the homecage 2-bottle "alcohol vs. water" regimen strongly increased CGRP intensity in BNST of sP rats in a subnucleus-specific manner: in BNST-AL, BNST-AVm, and BNST-AM. CGRP area sum, however, decreased in BNST-AM, without changes in other subnuclei. Alcohol consumption increased NPY expression, in a subnucleus-specific manner, in BNST-AL, BNST-AVl, and BNST-AVm. Alcohol consumption increased many size/shapes parameters in microglial cells, indicative of microglia de-activation. Finally, microglia density was increased in ventral anterior BNST (BNST-AVl, BNST-AVm) by alcohol consumption. In conclusion, genetic predisposition of sP rats to high alcohol intake could be in part mediated by anterior BNST subnuclei showing lower NPY expression and differential microglia activation. Alcohol intake in sP rats produced complex subnucleus-specific changes in BNST, affecting CGRP/NPY expression and microglia and leading to hypothesize that these changes might contribute to the anxiolytic effects of voluntarily consumed alcohol repeatedly observed in sP rats.

6-Hydroxydopamine lesion in the ventral tegmental area fails to reduce extracellular dopamine in the cerebral cortex.

Dopamine and noradrenaline are both involved in modulation of superior cognitive functions that are mainly dependent on frontal cortex activity. Experimental evidence points to parallel variations in extracellular concentrations of catecholamines in the cerebral cortex, which leads us to hypothesize their corelease from noradrenergic neurons. This study aimed to verify this hypothesis, by means of cerebral microdialysis following destruction of dopaminergic innervation in rats. The unilateral injury of dopaminergic neurons, by 6-hydroxydopamine injection in the ventral tegmental area, dramatically reduced the immunoreactivity for dopamine transporter in the cerebral hemisphere ipsilateral to the lesion. Tissue dopamine content in the ipsilateral nucleus accumbens and medial prefrontal and parietal cortex was also profoundly decreased, whereas noradrenaline was only slightly affected. Despite the lower tissue content in the denervated side, the extracellular dopamine level was not changed in the cortex, although it was markedly decreased in the nucleus accumbens ipsilateral to the lesion. The effect of drugs selective for D(2)-dopaminergic (haloperidol) or alpha(2)-noradrenergic (RS 79948) receptors was verified. Haloperidol failed to modify extracellular dopamine in either cortex but increased it in the nucleus accumbens, such an increase being greatly reduced in the denervated side. On the other hand, RS 79948 increased extracellular dopamine and DOPAC in all areas tested, the increases being of the same degree in both intact and lesioned sides. The results strongly support the hypothesis that the majority of extracellular dopamine in the cortex, unlike that in the nucleus accumbens, originates from noradrenergic terminals.

Multi-faceted aspects of gamma-hydroxybutyric acid: a neurotransmitter, therapeutic agent and drug of abuse.

Gamma hydroxybutyric acid (GHB), an endogenous constituent of the mammalian brain, acts as i) a neurotransmitter or neuromodulator, ii) a medicine used for the treatment of narcolepsy and alcoholism, and iii) a drug illicitly used for its psychotropic effects. GHB is thought to act as a specific GHB receptor agonist as well as a weak gamma-aminobutyric acid type B (GABA(B)) receptor agonist. Here, I review the in vivo and in vitro pharmacological properties of GHB and its interaction with GHB and GABA(B) receptors. When exogenously administered, GHB is rapidly absorbed, crosses the blood-brain barrier, penetrates into the brain and exerts a number of pharmacological effects including anxiolysis, sedation/hypnosis and anesthesia. Due to its effects on the central nervous system, GHB has been used for the treatment of narcolepsy and as an anesthetic adjuvant. More recently, a role for GHB in the pharmacotherapy of alcohol dependence has been described. In this review, I also focus on the abuse liability and reinforcing properties of GHB in humans and laboratory animals.

Rimonabant, a potent CB1 cannabinoid receptor antagonist, is a Gαi/o protein inhibitor.

Rimonabant is a potent and selective cannabinoid CB1 receptor antagonist widely used in animal and clinical studies. Besides its antagonistic properties, numerous studies have shown that, at micromolar concentrations rimonabant behaves as an inverse agonist at CB1 receptors. The mechanism underpinning this activity is unclear. Here we show that micromolar concentrations of rimonabant inhibited Gαi/o-type G proteins, resulting in a receptor-independent block of G protein signaling. Accordingly, rimonabant decreased basal and agonist stimulated [35S]GTPγS binding to cortical membranes of CB1- and GABAB-receptor KO mice and Chinese Hamster Ovary (CHO) cell membranes stably transfected with GABAB or D2 dopamine receptors. The structural analog of rimonabant, AM251, decreased basal and baclofen-stimulated GTPγS binding to rat cortical and CHO cell membranes expressing GABAB receptors. Rimonabant prevented G protein-mediated GABAB and D2 dopamine receptor signaling to adenylyl cyclase in Human Embryonic Kidney 293 cells and to G protein-coupled inwardly rectifying K+ channels (GIRK) in midbrain dopamine neurons of CB1 KO mice. Rimonabant suppressed GIRK gating induced by GTPγS in CHO cells transfected with GIRK, consistent with a receptor-independent action. Bioluminescent resonance energy transfer (BRET) measurements in living CHO cells showed that, in presence or absence of co-expressed GABAB receptors, rimonabant stabilized the heterotrimeric Gαi/o-protein complex and prevented conformational rearrangements induced by GABAB receptor activation. Rimonabant failed to inhibit Gαs-mediated signaling, supporting its specificity for Gαi/o-type G proteins. The inhibition of Gαi/o protein provides a new site of rimonabant action that may help to understand its pharmacological and toxicological effects occurring at high concentrations.

Limited Access to a High Fat Diet Alters Endocannabinoid Tone in Female Rats.

Emerging evidence suggest an impaired endocannabinoid activity in the pathophysiology of binge eating disorder (BED). Herein, we investigated whether endocannabinoid tone could be modified as a consequence of dietary-induced binge eating in female rats. For this purpose, brain levels of the endocannabinoids anandamide (AEA) and 2-arachidonoyl glycerol (2-AG), as well as two endocannabinoid-like lipids, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), were assessed in different brain areas involved in the hedonic feeding (i.e., prefrontal cortex, nucleus accumbens, amygdala, hippocampus, and hypothalamus). The brain density of cannabinoid type-1 receptors (CB1) was also evaluated. Furthermore, we determined plasma levels of leptin, ghrelin, and corticosterone hormones, which are well-known to control the levels of endocannabioids and/or CB1 receptors in the brain. To induce binge eating behavior, rats were subject to an intermittent and limited access to a high fat diet (HFD) (margarine). Three experimental groups were used, all with ad libitum access to chow: control (CTRL), with no access to margarine; low restriction (LR), with 2 h margarine access 7 days/week; high restriction (HR), with 2 h margarine access 3 days/week. Bingeing was established when margarine intake in the HR group exceeded that of the LR group. Our results show that, compared to CTRL, AEA significantly decreased in the caudate putamen, amygdala, and hippocampus of HR group. In contrast, 2-AG significantly increased in the hippocampus while OEA decreased in the hypothalamus. Similar to the HR group, AEA and OEA decreased respectively in the amygdala and hypothalamus and 2-AG increased in the hippocampus of LR group. Moreover, LR group also had AEA decreased in the prefrontal cortex and increased in the nucleus accumbens. In both groups we found the same reduction of CB1 receptor density in the prefrontal cortex compared to CTRL. Also, LR and HR groups showed alterations in both ghrelin and corticosterone levels, while leptin remained unaltered. In conclusion, our findings show a modified endocannabinoid tone due to margarine exposure, in several brain areas that are known to influence the hedonic aspect of food. Even if not uniquely specific to binge eating, margarine-induced changes in endocannabinoid tone could contributes to the development and maintenance of this behavior.

In Vitro Functional Characterization of GET73 as Possible Negative Allosteric Modulator of Metabotropic Glutamate Receptor 5.

The present study was aimed to further characterize the pharmacological profile of N-[4-(trifluoromethyl) benzyl]-4-methoxybutyramide (GET73), a putative negative allosteric modulator (NAM) of metabotropic glutamate subtype 5 receptor (mGluR5) under development as a novel medication for the treatment of alcohol dependence. This aim has been accomplished by means of a series of in vitro functional assays. These assays include the measure of several down-stream signaling [intracellular Ca++ levels, inositol phosphate (IP) formation and CREB phosphorylation (pCREB)] which are generally affected by mGluR5 ligands. In particular, GET73 (0.1 nM-10 µM) was explored for its ability to displace the concentration-response curve of some mGluR5 agonists/probes (glutamate, L-quisqualate, CHPG) in different native preparations. GET73 produced a rightward shift of concentration-response curves of glutamate- and CHPG-induced intracellular Ca++ levels in primary cultures of rat cortical astrocytes. The compound also induced a rightward shift of concentration response curve of glutamate- and L-quisqualate-induced increase in IP turnover in rat hippocampus slices, along with a reduction of CHPG (10 mM)-induced increase in IP formation. Moreover, GET73 produced a rightward shift of concentration-response curve of glutamate-, CHPG- and L-quisqualate-induced pCREB levels in rat cerebral cortex neurons. Although the engagement of other targets cannot be definitively ruled out, these data support the view that GET73 acts as an mGluR5 NAM and support the significance of further investigating the possible mechanism of action of the compound.

Activation of GABA(B) receptors reverses spontaneous gating deficits in juvenile DBA/2J mice.

RATIONALE: Gamma-amino-butyric acid (GABA)(B) receptors play a key role in the pathophysiology of psychotic disorders. We previously reported that baclofen, the prototypical GABA(B) agonist, elicits antipsychotic-like effects in the rat paradigm of prepulse inhibition (PPI) of the startle, a highly validated animal model of schizophrenia. OBJECTIVES: We studied the role of GABA(B) receptors in the spontaneous PPI deficits displayed by DBA/2J mice. MATERIALS AND METHODS: We tested the effects of baclofen (1.25-5 mg/kg, intraperitoneal [i.p.]) in DBA/2J and C57BL/6J mice, in comparison to the antipsychotic drugs haloperidol (1 mg/kg, i.p.) and clozapine (5 mg/kg, i.p.). Furthermore, we investigated the expression of GABA(B) receptors in the brain of DBA/2J and C57BL/6J mice by quantitative autoradiography. RESULTS: Baclofen dose-dependently restored PPI deficit in DBA/2J mice, in a fashion similar to the antipsychotic clozapine (5 mg/kg, i.p.). This effect was reversed by pretreatment with the GABA(B) antagonist SCH50211 (50 mg/kg, i.p.). In contrast, baclofen did not affect PPI in C57BL/6J mice. Finally, quantitative autoradiographic analyses assessed a lower GABA(B) receptor expression in DBA/2J mice in comparison to C57BL/6J controls in the prefrontal cortex and hippocampus but not in other brain regions. CONCLUSIONS: Our data highlight GABA(B) receptors as an important substrate for sensorimotor gating control in DBA/2J mice and encourage further investigations on the role of GABA(B) receptors in sensorimotor gating, as well as in the pathophysiology of psychotic disturbances.

Dysregulation of the endogenous cannabinoid system in adult rats prenatally treated with the cannabinoid agonist WIN 55,212-2.

Cannabis is widely abused by women at reproductive age and during pregnancy. Animal studies showed a particular vulnerability of the developing brain to prenatal chronic cannabinoid administration. We determined whether prenatal exposure to WIN 55,212-2, a potent cannabinoid receptor agonist, affected (1) density, affinity and/or function of cannabinoid CB(1) receptors, (2) endogenous levels of the endocannabinoid anandamide, (3) activities of the major anandamide synthesising and hydrolysing enzymes, N-acyl-phosphatidylethanolamine-specific phospholipase D (NAPE-PLD) and fatty acid amide hydrolase (FAAH), respectively, in brain areas of adult male offspring rats. Furthermore, the effect of prenatal WIN 55,212-2 on spontaneous motility was analyzed. Pregnant rats were treated daily with WIN 55,212-2 (0.5 mg/kg, gestation day 5-20) or vehicle. [(3)H]CP 55,940 and WIN 55,212-2-stimulated [(35)S] GTPgammaS binding were carried out in cerebellum, cerebral cortex, hippocampus, striatum and limbic areas of male adult offspring. Levels of anandamide, FAAH and NAPE-PLD activity were also determined. EC(50) values for WIN 55,212-2-stimulated [(35)S]GTPgammaS binding were significantly different in hippocampus (-26%) and striatum (+27%) in WIN 55,212-2-treated rats. Cannabinoid CB(1) receptor density and affinity were not affected in any analyzed region. In the striatum, increased anandamide levels were associated with reduced FAAH and enhanced NAPE-PLD activities. Opposite changes in anandamide levels and enzymatic activities were detected in limbic areas of WIN 55,212-2-treated rats. Ambulatory activity between WIN 55,212-2- and vehicle-treated adult offspring did not vary. Our results show that prenatal exposure to cannabinoid agonist induces a long-term alteration of endocannabinoid system in brain areas involved in learning-memory, motor activity and emotional behavior.