RESUMO
Decreased activity and expression of the G-protein coupled receptor GPR88 is linked to many behavior-linked neurological disorders. Published preclinical GPR88 allosteric agonists all have in vivo pharmacokinetic properties that preclude their progression to the clinic, including high lipophilicity and poor brain penetration. Here, we describe our attempts to improve GPR88 agonists' drug-like properties and our analysis of the trade-offs required to successfully target GPR88's allosteric pocket. We discovered two new GPR88 agonists: One that reduced morphine-induced locomotor activity in a murine proof-of-concept study, and the atropoisomeric BI-9508, which is a brain penetrant and has improved pharmacokinetic properties and dosing that recommend it for future in vivo studies in rodents. BI-9508 still suffers from high lipophilicity, and research on this series was halted. Because of its utility as a tool compound, we now offer researchers access to BI-9508 and a negative control free of charge via Boehringer Ingelheim's open innovation portal opnMe.com.
Assuntos
Encéfalo , Receptores Acoplados a Proteínas G , Animais , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Camundongos , Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , Humanos , Descoberta de Drogas , Masculino , Relação Estrutura-Atividade , Camundongos Endogâmicos C57BL , Morfina/farmacologia , Morfina/farmacocinéticaRESUMO
Opioid use disorder (OUD) is a chronic relapsing disorder that is a major burden for the lives of affected individuals, and society as a whole. Opioid withdrawal is characterized by strong physical symptoms, along with signs of negative affect. Negative affect due to opioid withdrawal is a major obstacle to recovery and relapse prevention. The mechanisms behind negative affect due to either spontaneous or antagonist-precipitated opioid withdrawal are not well known, and more animal models need be developed. Here, we present behavioral models of negative affect upon naloxone-precipitated morphine withdrawal in adult male mice. Social, anxiety, and despair-like deficits were investigated following naloxone administration in mice receiving morphine under three dosing regimens; acute, chronic constant dose and chronic escalating doses. Social behaviour in the three-chamber social preference test was decreased following withdrawal from chronic and escalating but not acute morphine. Anxiety-like behaviour in the open field was increased for all three treatments. Despair-like behaviour was increased following withdrawal from chronic and escalating but not acute morphine. Altogether, these animal models will contribute to study behavioural and neuronal circuitries involved in the several negative affective signs characterizing OUD.
Assuntos
Modelos Animais de Doenças , Morfina , Naloxona , Síndrome de Abstinência a Substâncias , Animais , Masculino , Morfina/efeitos adversos , Morfina/administração & dosagem , Camundongos , Naloxona/administração & dosagem , Naloxona/farmacologia , Ansiedade , Comportamento Animal/efeitos dos fármacos , Antagonistas de Entorpecentes/administração & dosagem , Antagonistas de Entorpecentes/farmacologia , Analgésicos Opioides/administração & dosagem , Analgésicos Opioides/efeitos adversos , Comportamento Social , Dependência de Morfina/psicologia , Transtornos Relacionados ao Uso de OpioidesRESUMO
BACKGROUND: The transcription factor ΔFOSB, acting in the nucleus accumbens, has been shown to control transcriptional and behavioral responses to opioids and other drugs of abuse. However, circuit-level consequences of ΔFOSB induction on the rest of the brain, which are required for its regulation of complex behavior, remain unknown. METHODS: We used an epigenetic approach in mice to suppress or activate the endogenous Fosb gene and thereby decrease or increase, respectively, levels of ΔFOSB selectively in D1-type medium spiny neurons of the nucleus accumbens and tested whether these modifications affect the organization of functional connectivity (FC) in the brain. We acquired functional magnetic resonance imaging data at rest and in response to a morphine challenge and analyzed both stationary and dynamic FC patterns. RESULTS: The 2 manipulations modified brainwide communication markedly and differently. ΔFOSB down- and upregulation had overlapping effects on prefrontal- and retrosplenial cortex-centered networks, but also generated specific FC signatures for epithalamus (habenula) and dopaminergic/serotonergic centers, respectively. Analysis of dynamic FC patterns showed that increasing ΔFOSB essentially altered responsivity to morphine and uncovered striking modifications of the roles of the epithalamus and amygdala in brain communication, particularly upon ΔFOSB downregulation. CONCLUSIONS: These novel findings illustrate how it is possible to link activity of a transcription factor within a single cell type of an identified brain region to consequent changes in circuit function brainwide by use of functional magnetic resonance imaging, and they pave the way for fundamental advances in bridging the gap between transcriptional and brain connectivity mechanisms underlying opioid addiction.
Assuntos
Neurônios Espinhosos Médios , Núcleo Accumbens , Animais , Camundongos , Encéfalo/metabolismo , Morfina/farmacologia , Núcleo Accumbens/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Opioid use disorder is a chronic relapsing disorder. The brain adapts to opioids that are taken for pain treatment or recreational use so that abstinence becomes a true challenge for individuals with opioid use disorder. Studying brain dysfunction at this stage is difficult, and human neuroimaging has provided highly heterogeneous information. METHODS: Here, we took advantage of an established mouse model of morphine abstinence together with functional magnetic resonance imaging to investigate whole-brain functional connectivity (FC) first at rest and then in response to an acute morphine challenge during image acquisition. RESULTS: Hierarchical clustering of seed pair correlation coefficients showed modified FC in abstinent animals, brainwide and regardless of the condition. Seed-to-voxel analysis and random forest classification, performed on data at rest, indicated that the retrosplenial cortex (a core component of the default mode network) and the amygdala (a major aversion center) are the best markers of abstinence, thus validating the translatability of the study. Seed pair network clustering confirmed disruption of a retrosplenial cortex-centered network, reflecting major reorganization of brain FC. The latter analysis also identified a persistent but unreported morphine signature in abstinent mice at rest, which involves cortical and midbrain components and characterizes the enduring morphine footprint. Finally, dynamic FC analysis revealed that the intrascanner acute morphine challenge modified FC faster and more broadly in abstinent animals, demonstrating brainwide adaptations of FC reactivity to an acute opioid challenge. CONCLUSIONS: This study used a unique experimental design to demonstrate that a prior history of chronic opioid exposure leaves a durable pharmacological signature on brain communication, with implications for pain management and recovery from opioid use disorder.
RESUMO
BACKGROUND: Chronic opioid exposure leads to hedonic deficits and enhanced vulnerability to addiction, which are observed and even strengthen after a period of abstinence, but the underlying circuit mechanisms are poorly understood. In this study, using both molecular and behavioral approaches, we tested the hypothesis that neurons expressing mu opioid receptors (MORs) in the dorsal raphe nucleus (DRN) are involved in addiction vulnerability associated with morphine abstinence. METHODS: MOR-Cre mice were exposed to chronic morphine and then went through spontaneous withdrawal for 4 weeks, a well-established mouse model of morphine abstinence. We studied DRN-MOR neurons of abstinent mice using 1) viral translating ribosome affinity for transcriptome profiling, 2) fiber photometry to measure neuronal activity, and 3) an opto-intracranial self-stimulation paradigm applied to DRN-MOR neurons to assess responses related to addiction vulnerability including persistence to respond, motivation to obtain the stimulation, self-stimulation despite punishment, and cue-induced reinstatement. RESULTS: DRN-MOR neurons of abstinent animals showed a downregulation of genes involved in ion conductance and MOR-mediated signaling, as well as altered responding to acute morphine. Opto-intracranial self-stimulation data showed that abstinent animals executed more impulsive-like and persistent responses during acquisition and scored higher on addiction-like criteria. CONCLUSIONS: Our data suggest that protracted abstinence to chronic morphine leads to reduced MOR function in DRN-MOR neurons and abnormal self-stimulation of these neurons. We propose that DRN-MOR neurons have partially lost their reward-facilitating properties, which in turn may lead to increased propensity to perform addiction-related behaviors.
Assuntos
Núcleo Dorsal da Rafe , Morfina , Camundongos , Animais , Morfina/farmacologia , Receptores Opioides mu , Analgésicos Opioides , Neurônios/metabolismoRESUMO
Introduction: Tianeptine is approved in some countries to treat depression and anxiety. In addition to its activity on serotonin and glutamate neurotransmission, tianeptine has been proven to be a mu-opioid receptor (MOR) agonist, but only a few preclinical studies have characterized the opioid-like behavioral effects of tianeptine. Methods: In this study, we tested tianeptine activity on G protein activation using the [S35] GTPγS binding assay in brain tissue from MOR+/+ and MOR-/- mice. Then, to determine whether tianeptine behavioral responses are MOR-dependent, we characterized the analgesic, locomotor, and rewarding responses of tianeptine in MOR+/+ and MOR-/- mice using tail immersion, hot plate, locomotor, and conditioned place preference tests. Results: Using the [S35] GTPγS binding assay, we found that tianeptine signaling is mediated by MOR in the brain with properties similar to those of DAMGO (a classic MOR agonist). Furthermore, we found that the MOR is necessary for tianeptine's analgesic (tail immersion and hot plate), locomotor, and rewarding (conditioned place preference) effects. Indeed, these behavioral effects could only be measured in MOR+/+ mice but not in MOR-/- mice. Additionally, chronic administration of tianeptine induced tolerance to its analgesic and hyperlocomotor effects. Discussion: These findings suggest that tianeptine's opioid-like effects require MOR and that chronic use could lead to tolerance.
RESUMO
BACKGROUND: Mu opioid receptors (MORs) are key for reward processing, mostly studied in dopaminergic pathways. MORs are also expressed in the dorsal raphe nucleus (DRN), which is central for the modulation of reward and mood, but MOR function in the DRN remains underexplored. Here, we investigated whether MOR-expressing neurons of the DRN (DRN-MOR neurons) participate in reward and emotional responses. METHODS: We characterized DRN-MOR neurons anatomically using immunohistochemistry and functionally using fiber photometry in responses to morphine and rewarding/aversive stimuli. We tested the effect of opioid uncaging on the DRN on place conditioning. We examined the effect of DRN-MOR neuron optostimulation on positive reinforcement and mood-related behaviors. We mapped their projections and selected DRN-MOR neurons projecting to the lateral hypothalamus for a similar optogenetic experimentation. RESULTS: DRN-MOR neurons form a heterogeneous neuronal population essentially composed of GABAergic (gamma-aminobutyric acidergic) and glutamatergic neurons. Calcium activity of DRN-MOR neurons was inhibited by rewarding stimuli and morphine. Local photo-uncaging of oxymorphone in the DRN produced conditioned place preference. DRN-MOR neuron optostimulation triggered real-time place preference and was self-administered, promoted social preference, and reduced anxiety and passive coping. Finally, specific optostimulation of DRN-MOR neurons projecting to the lateral hypothalamus recapitulated the reinforcing effects of total DRN-MOR neuron stimulation. CONCLUSIONS: Our data show that DRN-MOR neurons respond to rewarding stimuli and that their optoactivation has reinforcing effects and promotes positive emotional responses, an activity which is partially mediated by their projections to the lateral hypothalamus. Our study also suggests a complex regulation of DRN activity by MOR opioids, involving mixed inhibition/activation mechanisms that fine-tune DRN function.
Assuntos
Núcleo Dorsal da Rafe , Receptores Opioides mu , Neurônios/fisiologia , Morfina/farmacologia , Analgésicos Opioides , RecompensaRESUMO
Opioid use disorder (OUD) is a chronic brain disease which originates from long-term neuroadaptations that develop after repeated opioid consumption and withdrawal episodes. These neuroadaptations lead among other things to the development of a negative affect, which includes loss of motivation for natural rewards, higher anxiety, social deficits, heightened stress reactivity, an inability to identify and describe emotions, physical and/or emotional pain, malaise, dysphoria, sleep disorders and chronic irritability. The urge for relief from this negative affect is one of major causes of relapse, and thus represents a critical challenge for treatment and relapse prevention. Animal models of negative affect induced by opioid withdrawal have recapitulated the development of a negative emotional state with signs such as anhedonia, increased anxiety responses, increased despair-like behaviour and deficits in social interaction. This research has been critical to determine neurocircuitry adaptations during chronic opioid administration or upon withdrawal. In this review, we summarize the recent literature of rodent models of (i) acute withdrawal, (ii) protracted abstinence from passive administration of opioids, (iii) withdrawal or protracted abstinence from opioid self-administration. Finally, we describe neurocircuitry involved in acute withdrawal and protracted abstinence. This article is part of the Special Issue on "Opioid-induced changes in addiction and pain circuits".
Assuntos
Analgésicos Opioides , Síndrome de Abstinência a Substâncias , Animais , Analgésicos Opioides/efeitos adversos , Síndrome de Abstinência a Substâncias/tratamento farmacológico , Modelos Animais , Entorpecentes , Afeto , DorRESUMO
Withdrawal from chronic opioid use often causes hypodopaminergic states and negative affect, which may drive relapse. Direct-pathway medium spiny neurons (dMSNs) in the striatal patch compartment contain µ-opioid receptors (MORs). It remains unclear how chronic opioid exposure and withdrawal impact these MOR-expressing dMSNs and their outputs. Here, we report that MOR activation acutely suppressed GABAergic striatopallidal transmission in habenula-projecting globus pallidus neurons. Notably, withdrawal from repeated morphine or fentanyl administration potentiated this GABAergic transmission. Furthermore, intravenous fentanyl self-administration enhanced GABAergic striatonigral transmission and reduced midbrain dopaminergic activity. Fentanyl-activated striatal neurons mediated contextual memory retrieval required for conditioned place preference tests. Importantly, chemogenetic inhibition of striatal MOR+ neurons rescued fentanyl withdrawal-induced physical symptoms and anxiety-like behaviors. These data suggest that chronic opioid use triggers GABAergic striatopallidal and striatonigral plasticity to induce a hypodopaminergic state, which may promote negative emotions and relapse.
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Analgésicos Opioides , Corpo Estriado , Corpo Estriado/metabolismo , Fentanila , Receptores Opioides , Afeto , Receptores Opioides mu/metabolismoRESUMO
GPR88 is an orphan G protein-coupled receptor mainly expressed in the brain, whose endogenous ligand has not yet been identified. To elucidate GPR88 functions, our group has developed RTI-13951-33 (1b) as the first in vivo active GPR88 agonist, but its poor metabolic stability and moderate brain permeability remain to be further optimized. Here, we report the design, synthesis, and pharmacological characterization of a new series of RTI-13951-33 analogues with the aim of improving pharmacokinetic properties. As a result, we identified a highly potent GPR88 agonist RTI-122 (30a) (cAMP EC50 = 11 nM) with good metabolic stability (half-life of 5.8 h) and brain permeability (brain/plasma ratio of >1) in mice. Notably, RTI-122 was more effective than RTI-13951-33 in attenuating the binge-like alcohol drinking behavior in the drinking-in-the-dark paradigm. Collectively, our findings suggest that RTI-122 is a promising lead compound for drug discovery research of GPR88 agonists.
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Desenho de Fármacos , Receptores Acoplados a Proteínas G , Animais , Camundongos , Encéfalo/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Estabilidade de Medicamentos , Consumo de Bebidas Alcoólicas/tratamento farmacológicoRESUMO
GPR88 is an orphan G protein-coupled receptor which has been implicated in a number of striatal-associated disorders. Herein we describe the synthesis and pharmacological characterization of the first GPR88 radioligand, [3H]RTI-33, derived from a synthetic agonist RTI-13951-33. [3H]RTI-33 has a specific activity of 83.4 Ci/mmol and showed one-site, saturable binding (KD of 85 nM) in membranes prepared from stable PPLS-HA-hGPR88-CHO cells. A competition binding assay was developed to determine binding affinities of several known GPR88 agonists. This radioligand represents a powerful tool for future mechanistic and cell-based ligand-receptor interaction studies of GPR88.
Assuntos
Proteínas de Transporte , Receptores Acoplados a Proteínas G , Cricetinae , Animais , Cricetulus , Receptores Acoplados a Proteínas G/agonistas , Ensaio RadioliganteRESUMO
RATIONALE: The Netrin-1/DCC guidance cue pathway is critically involved in the adolescent organization of the mesocorticolimbic dopamine circuitry. Adult mice heterozygous for Dcc show reduced dopamine release in the nucleus accumbens in response to amphetamine and, in turn, blunted sensitivity to the rewarding effects of this drug. OBJECTIVE: Here, we tested whether the protective effects of Dcc haploinsufficiency are specific to stimulant drugs of abuse or instead extrapolate to opioids and ethanol. METHODS: We used the place preference paradigm to measure the rewarding effects of cocaine (20 mg/kg), morphine (5 or 10 mg/Kg), or ethanol (20%) in adult (PND 75) male Dcc haploinsufficient mice or their wild-type litter mates. In a second experiment, we compared in these two genotypes, in vivo dopamine release in the nucleus accumbens after a single i.p. injection of morphine (10 mg/kg). RESULTS: We found reduced morphine-induced dopamine release in the nucleus accumbens of Dcc haploinsufficient male mice, but, contrary to the effects of stimulant drugs, there is no effect of genotype on morphine-induced conditioned preference. CONCLUSION: These findings show that reduced drug-induced mesolimbic dopamine in Dcc haploinsufficient male mice protects specifically against the rewarding effects of stimulant drugs, but not against the rewarding properties of morphine and ethanol. These results suggest that these drugs exert their rewarding effect via different brain circuits.
Assuntos
Cocaína , Camundongos , Masculino , Animais , Cocaína/farmacologia , Cocaína/metabolismo , Dopamina/metabolismo , Receptor DCC/genética , Receptor DCC/metabolismo , Morfina/farmacologia , Morfina/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/farmacologia , Haploinsuficiência , Etanol/farmacologia , Receptores de Superfície Celular/genética , Núcleo AccumbensRESUMO
BACKGROUND: The mu opioid receptor (MOR) is central to hedonic balance and produces euphoria by engaging reward circuits. MOR signaling may also influence aversion centers, notably the habenula (Hb), where the receptor is highly dense. Our previous data suggest that the inhibitory activity of MOR in the Hb may limit aversive states. To investigate this hypothesis, we tested whether neurons expressing MOR in the Hb (Hb-MOR neurons) promote negative affect. METHODS: Using Oprm1-Cre knockin mice, we combined tracing and optogenetics with behavioral testing to investigate consequences of Hb-MOR neuron stimulation for approach/avoidance (real-time place preference), anxiety-related responses (open field, elevated plus maze, and marble burying), and despair-like behavior (tail suspension). RESULTS: Optostimulation of Hb-MOR neurons elicited avoidance behavior, demonstrating that these neurons promote aversive states. Anterograde tracing showed that, in addition to the interpeduncular nucleus, Hb-MOR neurons project to the dorsal raphe nucleus. Optostimulation of Hb-MOR/interpeduncular nucleus terminals triggered avoidance and despair-like responses with no anxiety-related effect, whereas light-activation of Hb-MOR/dorsal raphe nucleus terminals increased levels of anxiety with no effect on other behaviors, revealing 2 dissociable pathways controlling negative affect. CONCLUSIONS: Together, the data demonstrate that Hb neurons expressing MOR facilitate aversive states via 2 distinct Hb circuits, contributing to despair-like behavior (Hb-MOR/interpeduncular nucleus) and anxiety (Hb-MOR/dorsal raphe nucleus). The findings support the notion that inhibition of these neurons by either endogenous or exogenous opioids may relieve negative affect, a mechanism that would have implications for hedonic homeostasis and addiction.
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Habenula , Receptores Opioides mu , Camundongos , Animais , Receptores Opioides mu/genética , Habenula/metabolismo , Neurônios/metabolismo , Núcleo Dorsal da Rafe , AfetoRESUMO
The mu opioid receptor (MOR) and the orphan GPR151 receptor are inhibitory G protein coupled receptors that are enriched in the habenula, a small brain region involved in aversion processing, addiction and mood disorders. While MOR expression in the brain is widespread, GPR151 expression is restricted to the habenula. In a previous report, we created conditional ChrnB4-Cre × Oprm1fl/fl (so-called B4MOR) mice, where MORs are deleted specifically in Chrnb4-positive neurons restricted to the habenula, and shown a role for these receptors in naloxone aversion. Here we characterized the implication of habenular MORs in social behaviors. B4MOR-/- mice and B4MOR+/+ mice were compared in several social behavior measures, including the chronic social stress defeat (CSDS) paradigm, the social preference (SP) test and social conditioned place preference (sCPP). In the CSDS, B4MOR-/- mice showed lower preference for the social target (unfamiliar mouse of a different strain) at baseline, providing a first indication of deficient social interactions in mice lacking habenular MORs. In the SP test, B4MOR-/- mice further showed reduced sociability for an unfamiliar conspecific mouse. In the sCPP, B4MOR-/- mice also showed impaired place preference for their previous familiar littermates after social isolation. We next created and tested Gpr151-/- mice in the SP test, and also found reduced social preference compared to Gpr151+/+ mice. Altogether our results support the underexplored notion that the habenula regulates social behaviors. Also, our data suggest that the inhibitory habenular MOR and GPR151 receptors normally promote social reward, possibly by dampening the aversive habenula activity.
Assuntos
Habenula , Receptores Acoplados a Proteínas G , Receptores Opioides mu , Animais , Camundongos , Habenula/metabolismo , Naloxona/metabolismo , Neurônios/metabolismo , Receptores Opioides mu/metabolismo , Recompensa , Receptores Acoplados a Proteínas G/metabolismoRESUMO
GPR88 is an orphan G-protein-coupled receptor that is considered a potential target to treat neuropsychiatric disorders, including addiction. Most knowledge about GPR88 function stems from knockout mouse studies, and in vivo pharmacology is still scarce. Here we examine the effects of the novel brain-penetrant agonist RTI-13951-33 on several alcohol-related behaviours in the mouse. In the intermittent-access-two-bottle-choice paradigm, the compound reduced excessive voluntary alcohol drinking, while water drinking was intact. This was observed for C57BL/6 mice, as well as for control but not Gpr88 knockout mice, demonstrating efficacy and specificity of the drug in vivo. In the drinking-in-the-dark paradigm, RTI-13951-33 also reduced binge-like drinking behaviour for control but not Gpr88 knockout mice, confirming the alcohol consumption-reducing effect and in vivo specificity of the drug. When C57BL/6 mice were trained for alcohol self-administration, RTI-13951-33 decreased the number of nose-pokes over a 4-h session and reduced the number of licks and bursts of licks, suggesting reduced motivation to obtain alcohol. Finally, RTI-13951-33 did not induce any place preference or aversion but reduced the expression of conditioned place preference to alcohol, indicative of a reduction of alcohol-reward seeking. Altogether, data show that RTI-13951-33 limits alcohol intake under distinct conditions that require consummatory behaviour, operant response or association with contextual cues. RTI-13951-33 therefore is a promising lead compound to evaluate GPR88 as a therapeutic target for alcohol use disorders. More broadly, RTI-13951-33 represents a unique tool to better understand GPR88 function, disentangle receptor roles in development from those in the adult and perhaps address other neuropsychiatric disorders.
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Alcoolismo , Animais , Camundongos , Alcoolismo/tratamento farmacológico , Camundongos Endogâmicos C57BL , Consumo de Bebidas Alcoólicas/psicologia , Etanol/farmacologia , Camundongos Knockout , Receptores Acoplados a Proteínas GRESUMO
The neural orphan G protein coupled receptor GPR88 is predominant in the striatum and cortex of both rodents and humans, and considered a potential target for brain disorders. Previous studies have shown multiple behavioral phenotypes in Gpr88 knockout mice, and human genetic studies have reported association with psychosis. Here we tested the possibility that GPR88 contributes to Attention Deficit Hyperactivity Disorder (ADHD). In the mouse, we tested Gpr88 knockout mice in three behavioral paradigms, best translatable between rodents and humans, and found higher motor impulsivity and reduced attention together with the reported hyperactivity. Atomoxetine, a typical ADHD drug, reduced impulsivity in mutant mice. Conditional Gpr88 knockout mice in either D1R-type or D2R-type medium spiny neurons revealed distinct implications of the two receptor populations in waiting and stopping impulsivity. Thus, animal data demonstrate that deficient GPR88 activity causally promotes ADHD-like behaviors, and identify circuit mechanisms underlying GPR88-regulated impulsivity. In humans, we performed a family-based genetic study including 567 nuclear families with DSM-IV diagnosis of ADHD. There was a minor association for SNP rs2036212 with diagnosis, treatment response and cognition. A stronger association was found for SNP rs2809817 upon patient stratification, suggesting that the T allele is a risk factor when prenatal stress is involved. Human data therefore identify GPR88 variants associated with the disease, and highlight a potential role of life trajectories to modulate GPR88 function. Overall, animal and human data concur to suggest that GPR88 signaling should be considered a key factor for diagnostic and treatment of ADHD.
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Transtorno do Deficit de Atenção com Hiperatividade , Animais , Humanos , Camundongos , Transtorno do Deficit de Atenção com Hiperatividade/genética , Transtorno do Deficit de Atenção com Hiperatividade/metabolismo , Corpo Estriado/metabolismo , Camundongos Knockout , Comportamento Impulsivo , Proteínas de Transporte/metabolismo , Fatores de Risco , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Melatonin, through its G protein-coupled receptor (GPCR) (MTNR1B gene) MT2 , is implicated in analgesia, but the relationship between MT2 receptors and the opioid system remains elusive. In a model of rodent neuropathic pain (spared nerve injured [SNI]), the selective melatonin MT2 agonist UCM924 reversed the allodynia (a pain response to a non-noxious stimulus), and this effect was nullified by the pharmacological blockade or genetic inactivation of the mu opioid receptor (MOR), but not the delta opioid receptor (DOR). Indeed, SNI MOR, but not DOR knockout mice, did not respond to the antiallodynic effects of the UCM924. Similarly, the nonselective opioid antagonist naloxone and the selective MOR antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) blocked the effects of UCM924 in SNI rats, but not the DOR antagonist naltrindole (NTI). Electrophysiological recordings in the rostral-ventromedial medulla (RVM) revealed that the typical reduction of the firing activity of pronociceptive ON-cells, and the enhancement of the firing of the antinociceptive OFF-cells, induced by the microinjection of the MT2 agonist UCM924 into the ventrolateral periaqueductal gray (vlPAG) were blocked by MOR, but not DOR, antagonism. Immunohistochemistry studies showed that MT2 receptors are expressed in both excitatory (CaMKIIα+ ) and inhibitory (GAD65+ ) neuronal cell bodies in the vlPAG (~2.16% total), but not RVM. Only 0.20% of vlPAG neurons coexpressed MOR and MT2 receptors. Finally, UCM924 treatment induced an increase in the enkephalin precursor gene (PENK) in the PAG of SNI mice. Collectively, the melatonin MT2 receptor agonism requires MORs to exert its antiallodynic effects, mostly through an interneuronal circuit involving MOR and MT2 receptors.
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Melatonina , Neuralgia , Camundongos , Animais , Ratos , Receptores Opioides mu/genética , Receptores Opioides mu/agonistas , Melatonina/farmacologia , Melatonina/uso terapêutico , Antagonistas de Entorpecentes/farmacologia , Antagonistas de Entorpecentes/uso terapêutico , Receptores Opioides delta , Analgésicos Opioides/uso terapêutico , Encefalinas/farmacologia , Encefalinas/uso terapêutico , Naloxona/farmacologia , Naloxona/uso terapêutico , Neuralgia/tratamento farmacológicoRESUMO
The atypical chemokine receptor 3, ACKR3, is a G protein-coupled receptor, which does not couple to G proteins but recruits ßarrestins. At present, ACKR3 is considered a target for cancer and cardiovascular disorders, but less is known about the potential of ACKR3 as a target for brain disease. Further, mouse lines have been created to identify cells expressing the receptor, but there is no tool to visualize and study the receptor itself under physiological conditions. Here, we engineered a knock-in (KI) mouse expressing a functional ACKR3-Venus fusion protein to directly detect the receptor, particularly in the adult brain. In HEK-293 cells, native and fused receptors showed similar membrane expression, ligand induced trafficking and signaling profiles, indicating that the Venus fusion does not alter receptor signaling. We also found that ACKR3-Venus enables direct real-time monitoring of receptor trafficking using resonance energy transfer. In ACKR3-Venus knock-in mice, we found normal ACKR3 mRNA levels in the brain, suggesting intact gene transcription. We fully mapped receptor expression across 14 peripheral organs and 112 brain areas and found that ACKR3 is primarily localized to the vasculature in these tissues. In the periphery, receptor distribution aligns with previous reports. In the brain there is notable ACKR3 expression in endothelial vascular cells, hippocampal GABAergic interneurons and neuroblast neighboring cells. In conclusion, we have generated Ackr3-Venus knock-in mice with a traceable ACKR3 receptor, which will be a useful tool to the research community for interrogations about ACKR3 biology and related diseases.
Assuntos
Proteínas de Bactérias/genética , Encéfalo/irrigação sanguínea , Técnicas de Introdução de Genes , Genes Reporter , Proteínas Luminescentes/genética , Receptores CXCR/genética , Animais , Proteínas de Bactérias/análise , Proteínas de Bactérias/farmacocinética , Biomarcadores , Sistemas Computacionais , Células Endoteliais/química , Células Endoteliais/citologia , Neurônios GABAérgicos/química , Neurônios GABAérgicos/citologia , Células HEK293 , Humanos , Interneurônios/química , Interneurônios/citologia , Ligantes , Proteínas Luminescentes/análise , Proteínas Luminescentes/farmacocinética , Camundongos , Especificidade de Órgãos , Receptores CXCR/análise , Proteínas Recombinantes/análise , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacocinética , Distribuição Tecidual , beta-Arrestina 1/metabolismoRESUMO
The orphan receptor GPR88 has been implicated in a number of striatal-associated disorders, yet its endogenous ligand has not been discovered. We have previously reported that the amine functionality in the 2-AMPP-derived GPR88 agonists can be replaced with an amide (e.g., 4) without losing activity. Later, we have found that the amide can be replaced with a bioisosteric 1,3,4-oxadiazole with improved potency. Here, we report a further study of amide bioisosteric replacement with a variety of azoles containing three heteroatoms, followed by a focused structure-activity relationship study, leading to the discovery of a series of novel 1,4-disubstituted 1H-1,2,3-triazoles as GPR88 agonists. Collectively, our medicinal chemistry efforts have resulted in a potent, efficacious, and brain-penetrant GPR88 agonist 53 (cAMP EC50 = 14 nM), which is a suitable probe to study GPR88 functions in the brain.