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1.
Nature ; 630(8015): 141-148, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38778097

RESUMO

Fentanyl is a powerful painkiller that elicits euphoria and positive reinforcement1. Fentanyl also leads to dependence, defined by the aversive withdrawal syndrome, which fuels negative reinforcement2,3 (that is, individuals retake the drug to avoid withdrawal). Positive and negative reinforcement maintain opioid consumption, which leads to addiction in one-fourth of users, the largest fraction for all addictive drugs4. Among the opioid receptors, µ-opioid receptors have a key role5, yet the induction loci of circuit adaptations that eventually lead to addiction remain unknown. Here we injected mice with fentanyl to acutely inhibit γ-aminobutyric acid-expressing neurons in the ventral tegmental area (VTA), causing disinhibition of dopamine neurons, which eventually increased dopamine in the nucleus accumbens. Knockdown of µ-opioid receptors in VTA abolished dopamine transients and positive reinforcement, but withdrawal remained unchanged. We identified neurons expressing µ-opioid receptors in the central amygdala (CeA) whose activity was enhanced during withdrawal. Knockdown of µ-opioid receptors in CeA eliminated aversive symptoms, suggesting that they mediate negative reinforcement. Thus, optogenetic stimulation caused place aversion, and mice readily learned to press a lever to pause optogenetic stimulation of CeA neurons that express µ-opioid receptors. Our study parses the neuronal populations that trigger positive and negative reinforcement in VTA and CeA, respectively. We lay out the circuit organization to develop interventions for reducing fentanyl addiction and facilitating rehabilitation.


Assuntos
Fentanila , Receptores Opioides mu , Reforço Psicológico , Animais , Feminino , Masculino , Camundongos , Analgésicos Opioides/farmacologia , Analgésicos Opioides/administração & dosagem , Núcleo Central da Amígdala/citologia , Núcleo Central da Amígdala/efeitos dos fármacos , Núcleo Central da Amígdala/metabolismo , Dopamina/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Fentanila/farmacologia , Camundongos Endogâmicos C57BL , Núcleo Accumbens/citologia , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Transtornos Relacionados ao Uso de Opioides/metabolismo , Transtornos Relacionados ao Uso de Opioides/patologia , Optogenética , Receptores Opioides mu/metabolismo , Síndrome de Abstinência a Substâncias/metabolismo , Síndrome de Abstinência a Substâncias/patologia , Área Tegmentar Ventral/citologia , Área Tegmentar Ventral/efeitos dos fármacos , Área Tegmentar Ventral/metabolismo
2.
J Neurosci ; 43(10): 1692-1713, 2023 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-36717230

RESUMO

The brain µ-opioid receptor (MOR) is critical for the analgesic, rewarding, and addictive effects of opioid drugs. However, in rat models of opioid-related behaviors, the circuit mechanisms of MOR-expressing cells are less known because of a lack of genetic tools to selectively manipulate them. We introduce a CRISPR-based Oprm1-Cre knock-in transgenic rat that provides cell type-specific genetic access to MOR-expressing cells. After performing anatomic and behavioral validation experiments, we used the Oprm1-Cre knock-in rats to study the involvement of NAc MOR-expressing cells in heroin self-administration in male and female rats. Using RNAscope, autoradiography, and FISH chain reaction (HCR-FISH), we found no differences in Oprm1 expression in NAc, dorsal striatum, and dorsal hippocampus, or MOR receptor density (except dorsal striatum) or function between Oprm1-Cre knock-in rats and wildtype littermates. HCR-FISH assay showed that iCre is highly coexpressed with Oprm1 (95%-98%). There were no genotype differences in pain responses, morphine analgesia and tolerance, heroin self-administration, and relapse-related behaviors. We used the Cre-dependent vector AAV1-EF1a-Flex-taCasp3-TEVP to lesion NAc MOR-expressing cells. We found that the lesions decreased acquisition of heroin self-administration in male Oprm1-Cre rats and had a stronger inhibitory effect on the effort to self-administer heroin in female Oprm1-Cre rats. The validation of an Oprm1-Cre knock-in rat enables new strategies for understanding the role of MOR-expressing cells in rat models of opioid addiction, pain-related behaviors, and other opioid-mediated functions. Our initial mechanistic study indicates that lesioning NAc MOR-expressing cells had different effects on heroin self-administration in male and female rats.SIGNIFICANCE STATEMENT The brain µ-opioid receptor (MOR) is critical for the analgesic, rewarding, and addictive effects of opioid drugs. However, in rat models of opioid-related behaviors, the circuit mechanisms of MOR-expressing cells are less known because of a lack of genetic tools to selectively manipulate them. We introduce a CRISPR-based Oprm1-Cre knock-in transgenic rat that provides cell type-specific genetic access to brain MOR-expressing cells. After performing anatomical and behavioral validation experiments, we used the Oprm1-Cre knock-in rats to show that lesioning NAc MOR-expressing cells had different effects on heroin self-administration in males and females. The new Oprm1-Cre rats can be used to study the role of brain MOR-expressing cells in animal models of opioid addiction, pain-related behaviors, and other opioid-mediated functions.


Assuntos
Dependência de Heroína , Heroína , Ratos , Masculino , Feminino , Animais , Heroína/farmacologia , Analgésicos Opioides/farmacologia , Núcleo Accumbens , Receptores Opioides/metabolismo , Ratos Transgênicos , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Dor/metabolismo
3.
Cell ; 137(6): 1148-59, 2009 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-19524516

RESUMO

Delta and mu opioid receptors (DORs and MORs) are inhibitory G protein-coupled receptors that reportedly cooperatively regulate the transmission of pain messages by substance P and TRPV1-expressing pain fibers. Using a DOReGFP reporter mouse we now show that the DOR and MOR are, in fact, expressed by different subsets of primary afferents. The MOR is expressed in peptidergic pain fibers, the DOR in myelinated and nonpeptidergic afferents. Contrary to the prevailing view, we demonstrate that the DOR is trafficked to the cell surface under resting conditions, independently of substance P, and internalized following activation by DOR agonists. Finally, we show that the segregated DOR and MOR distribution is paralleled by a remarkably selective functional contribution of the two receptors to the control of mechanical and heat pain, respectively. These results demonstrate that behaviorally relevant pain modalities can be selectively regulated through the targeting of distinct subsets of primary afferent pain fibers.


Assuntos
Dor/fisiopatologia , Receptores Opioides delta/fisiologia , Receptores Opioides mu/fisiologia , Analgesia , Analgésicos Opioides/farmacologia , Animais , Técnicas de Introdução de Genes , Temperatura Alta , Masculino , Mecanorreceptores/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Morfina/farmacologia , Nociceptores/fisiologia , Dor/induzido quimicamente , Receptores Opioides delta/agonistas , Receptores Opioides mu/agonistas , Medula Espinal/patologia , Medula Espinal/fisiologia , Substância P/metabolismo , Canais de Cátion TRPV/metabolismo
4.
Mol Psychiatry ; 27(11): 4662-4672, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36075963

RESUMO

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.


Assuntos
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/metabolismo
5.
Bioorg Med Chem Lett ; 80: 129120, 2023 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-36587872

RESUMO

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 Radioligante
6.
Cereb Cortex ; 32(3): 479-489, 2022 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-34247243

RESUMO

GPR88 is an orphan G-protein-coupled receptor (GPCR) highly expressed in striatal medium spiny neurons (MSN), also found in cortical neurons at low level. In MSN, GPR88 has a canonical GPCR plasma membrane/cytoplasmic expression, whereas in cortical neurons, we previously reported an atypical intranuclear localization. Molecular size analysis suggests that GPR88, expressed in plasma membrane of MSN or in nuclear compartment of cortical neurons, corresponds to the full-length protein. By transfection of cortical neurons, we showed that GPR88 fluorescent chimeras exhibit a nuclear localization. This localization is contingent on the third intracytoplasmic loop and C-terminus domains, even though these domains do not contain any known nuclear localization signals (NLS). Using yeast two-hybrid screening with these domains, we identified the nuclear proteins ATRX, TOP2B, and BAZ2B, all involved in chromatin remodeling, as potential protein partners of GPR88. We also validated the interaction of GPR88 with these nuclear proteins by proximity ligation assay on cortical neurons in culture and coimmunoprecipitation experiments on cortical extracts from GPR88 wild-type (WT) and knockout (KO) mice. The identification of GPR88 subcellular partners may provide novel functional insights for nonclassical modes of GPCR action that could be relevant in the maturating process of neocortical neurons.


Assuntos
Proteínas Nucleares , Receptores Acoplados a Proteínas G , Animais , Córtex Cerebral/metabolismo , Corpo Estriado/metabolismo , Camundongos , Camundongos Knockout , Proteínas Nucleares/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo
7.
Addict Biol ; 27(6): e13227, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36301207

RESUMO

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.


Assuntos
Alcoolismo , Animais , Camundongos , Alcoolismo/tratamento farmacológico , Camundongos Endogâmicos C57BL , Consumo de Bebidas Alcoólicas/psicologia , Etanol/farmacologia , Camundongos Knockout , Receptores Acoplados a Proteínas G
8.
J Physiol ; 598(1): 189-205, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31589332

RESUMO

KEY POINTS: The main cause of death from opioid overdose is respiratory depression due to the activation of µ-opioid receptors (MORs). We conditionally deleted MORs from neurons in two key areas of the brainstem respiratory circuitry (the Kölliker-Fuse nucleus (KF) and pre-Bötzinger complex (preBötC)) to determine their role in opioid-induced respiratory disturbances in adult, awake mice. Deletion of MORs from KF neurons attenuated respiratory rate depression at all doses of morphine. Deletion of MORs from preBötC neurons attenuated rate depression at the low dose, but had no effect on rate following high doses of morphine. Instead, high doses of morphine increased the occurrence of apnoeas. The results indicate that opioids affect distributed key areas of the respiratory network in a dose-dependent manner and countering the respiratory effects of high dose opioids via the KF may be an effective approach to combat overdose. ABSTRACT: The primary cause of death from opioid overdose is respiratory failure. High doses of opioids cause severe rate depression and increased risk of fatal apnoea, which correlate with increasing irregularities in breathing pattern. µ-Opioid receptors (MORs) are widely distributed throughout the brainstem respiratory network, but the mechanisms underlying respiratory depression are poorly understood. The medullary pre-Bötzinger complex (preBötC) and the pontine Kölliker-Fuse nucleus (KF) are considered critical for inducing opioid-related respiratory disturbances. We used a conditional knockout approach to investigate the roles and relative contribution of MORs in KF and preBötC neurons in opioid-induced respiratory depression in awake adult mice. The results revealed dose-dependent and region-specific opioid effects on the control of both respiratory rate and pattern. Respiratory depression induced by an anti-nociceptive dose of morphine was significantly attenuated following deletion of MORs from either the KF or the preBötC, suggesting cumulative network effects on respiratory rate control at low opioid doses. Deletion of MORs from KF neurons also relieved rate depression at near-maximal respiratory depressant doses of morphine. Meanwhile, deletion of MORs from the preBötC had no effect on rate following administration of high doses of morphine. Instead, a severe ataxic breathing pattern emerged with many apnoeas. We conclude that opioids affect distributed areas of the respiratory network and opioid-induced respiratory depression cannot be attributed to only one area in isolation. However, countering the effects of near maximal respiratory depressant doses of opioids in the KF may be a powerful approach to combat opioid overdose.


Assuntos
Analgésicos Opioides/efeitos adversos , Morfina/efeitos adversos , Receptores Opioides mu/metabolismo , Centro Respiratório/efeitos dos fármacos , Insuficiência Respiratória/induzido quimicamente , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores Opioides mu/genética , Vigília
9.
Neurobiol Dis ; 134: 104681, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31759136

RESUMO

Hyperactivity of glutamatergic corticostrial pathways is recognized as a key pathophysiological mechanism contributing to development of PD symptoms and dopaminergic neurotoxicity. Subset of corticostriatal projection neurons uses Zn2+ as a co-transmitter alongside glutamate, but the role of synaptically released Zn2+ in PD remains unexplored. We used genetically modified mice and pharmacological tools in combination with 6-hydroxydopamine (6-OHDA) lesion models of PD to investigate the contribution of synaptic zinc to disease associated behavioral deficits and neurodegeneration. Vesicular zinc transporter-3 (ZnT3) knockout mice lacking releasable Zn2+ were more resistant to locomotor deficit and memory impairment of nigrostriatal dopamine (DA) denervation compared to wildtype littermates. The loss of striatal dopaminergic fibers was comparable between genotypes, indicating that synaptically released Zn2+ contributes to behavioral deficits but not neurotoxic effects of 6-OHDA. To gain further insight into the mechanisms of Zn2+ actions, we used the extracellular Zn2+ chelator CaEDTA and knock-in mice lacking the high affinity Zn2+ inhibition of GluN2A-containing NMDA receptors (GluN2A-NMDARs). Acute chelation of extracellular Zn2+ in the striatum restored locomotor deficit of 6-OHDA lesion, confirming that synaptic Zn2+ suppresses locomotor behavior. Disruption of the Zn2+-GluN2A interaction had, on the other hand, no impact on locomotor deficit or neurotoxic effect of 6-OHDA. Collectively, these findings provide clear evidence for the implication of striatal synaptic Zn2+ in the pathophysiology of PD. They unveil that synaptic Zn2+ plays predominantly a detrimental role by promoting motor and cognitive deficits caused by nigrostriatal DA denervation, pointing towards new therapeutic interventions.


Assuntos
Cognição , Locomoção , Doença de Parkinson/metabolismo , Doença de Parkinson/psicologia , Zinco/metabolismo , Animais , Comportamento Animal , Proteínas de Transporte de Cátions/genética , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Masculino , Memória/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxidopamina/administração & dosagem , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/metabolismo , Transtornos Parkinsonianos/psicologia , Vesículas Sinápticas/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo
11.
J Neurosci ; 36(12): 3541-51, 2016 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-27013682

RESUMO

Ligand-specific recruitment of arrestins facilitates functional selectivity of G-protein-coupled receptor signaling. Here, we describe agonist-selective recruitment of different arrestin isoforms to the delta opioid receptor in mice. A high-internalizing delta opioid receptor agonist (SNC80) preferentially recruited arrestin 2 and, in arrestin 2 knock-outs (KOs), we observed a significant increase in the potency of SNC80 to inhibit mechanical hyperalgesia and decreased acute tolerance. In contrast, the low-internalizing delta agonists (ARM390, JNJ20788560) preferentially recruited arrestin 3 with unaltered behavioral effects in arrestin 2 KOs. Surprisingly, arrestin 3 KO revealed an acute tolerance to these low-internalizing agonists, an effect never observed in wild-type animals. Furthermore, we examined delta opioid receptor-Ca(2+)channel coupling in dorsal root ganglia desensitized by ARM390 and the rate of resensitization was correspondingly decreased in arrestin 3 KOs. Live-cell imaging in HEK293 cells revealed that delta opioid receptors are in pre-engaged complexes with arrestin 3 at the cell membrane and that ARM390 strengthens this membrane interaction. The disruption of these complexes in arrestin 3 KOs likely accounts for the altered responses to low-internalizing agonists. Together, our results show agonist-selective recruitment of arrestin isoforms and reveal a novel endogenous role of arrestin 3 as a facilitator of resensitization and an inhibitor of tolerance mechanisms. SIGNIFICANCE STATEMENT: Agonists that bind to the same receptor can produce highly distinct signaling events and arrestins are a major mediator of this ligand bias. Here, we demonstrate that delta opioid receptor agonists differentially recruit arrestin isoforms. We found that the high-internalizing agonist SNC80 preferentially recruits arrestin 2 and knock-out (KO) of this protein results in increased efficacy of SNC80. In contrast, low-internalizing agonists (ARM390 and JNJ20788560) preferentially recruit arrestin 3 and, surprisingly, KO of arrestin 3 produces acute tolerance and impaired receptor resensitization to these agonists. Arrestin 3 is in pre-engaged complexes with the delta opioid receptor at the cell membrane and low-internalizing agonists promote this interaction. This study reveals a novel role for arrestin 3 as a facilitator of receptor resensitization.


Assuntos
Arrestinas/metabolismo , Benzamidas/administração & dosagem , Hiperalgesia/fisiopatologia , Percepção da Dor , Piperazinas/administração & dosagem , Receptores Opioides delta/antagonistas & inibidores , Receptores Opioides delta/metabolismo , Animais , Tolerância a Medicamentos , Feminino , Masculino , Camundongos , Camundongos Knockout , Isoformas de Proteínas
12.
N Engl J Med ; 381(21): 2067-2069, 2019 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-31747732
13.
14.
Addict Biol ; 22(5): 1205-1217, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27126842

RESUMO

Unified theories of addiction are challenged by differing drug-seeking behaviors and neurobiological adaptations across drug classes, particularly for narcotics and psychostimulants. We previously showed that protracted abstinence to opiates leads to despair behavior and social withdrawal in mice, and we identified a transcriptional signature in the extended amygdala that was also present in animals abstinent from nicotine, Δ9-tetrahydrocannabinol (THC) and alcohol. Here we examined whether protracted abstinence to these four drugs would also share common behavioral features, and eventually differ from abstinence to the prototypic psychostimulant cocaine. We found similar reduced social recognition, increased motor stereotypies and increased anxiety with relevant c-fos response alterations in morphine, nicotine, THC and alcohol abstinent mice. Protracted abstinence to cocaine, however, led to strikingly distinct, mostly opposing adaptations at all levels, including behavioral responses, neuronal activation and gene expression. Together, these data further document the existence of common hallmarks for protracted abstinence to opiates, nicotine, THC and alcohol that develop within motivation/emotion brain circuits. In our model, however, these do not apply to cocaine, supporting the notion of unique mechanisms in psychostimulant abuse.


Assuntos
Tonsila do Cerebelo/metabolismo , Analgésicos Opioides/efeitos adversos , Comportamento Animal , Agonistas de Receptores de Canabinoides/efeitos adversos , Depressores do Sistema Nervoso Central/efeitos adversos , Inibidores da Captação de Dopamina/efeitos adversos , Agonistas Nicotínicos/efeitos adversos , Comportamento Social , Síndrome de Abstinência a Substâncias/fisiopatologia , Abstinência de Álcool , Animais , Ansiedade/psicologia , Encéfalo/metabolismo , Cocaína/efeitos adversos , Dronabinol/efeitos adversos , Comportamento de Procura de Droga , Emoções , Etanol/efeitos adversos , Masculino , Camundongos , Morfina/efeitos adversos , Motivação , Nicotina/efeitos adversos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Comportamento Estereotipado/fisiologia , Síndrome de Abstinência a Substâncias/metabolismo , Síndrome de Abstinência a Substâncias/psicologia
15.
J Neurosci ; 35(37): 12917-31, 2015 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-26377476

RESUMO

The endogenous dynorphin-κ opioid receptor (KOR) system encodes the dysphoric component of the stress response and controls the risk of depression-like and addiction behaviors; however, the molecular and neural circuit mechanisms are not understood. In this study, we report that KOR activation of p38α MAPK in ventral tegmental (VTA) dopaminergic neurons was required for conditioned place aversion (CPA) in mice. Conditional genetic deletion of floxed KOR or floxed p38α MAPK by Cre recombinase expression in dopaminergic neurons blocked place aversion to the KOR agonist U50,488. Selective viral rescue by wild-type KOR expression in dopaminergic neurons of KOR(-/-) mice restored U50,488-CPA, whereas expression of a mutated form of KOR that could not initiate p38α MAPK activation did not. Surprisingly, while p38α MAPK inactivation blocked U50,488-CPA, p38α MAPK was not required for KOR inhibition of evoked dopamine release measured by fast scan cyclic voltammetry in the nucleus accumbens. In contrast, KOR activation acutely inhibited VTA dopaminergic neuron firing, and repeated exposure attenuated the opioid response. This adaptation to repeated exposure was blocked by conditional deletion of p38α MAPK, which also blocked KOR-induced tyrosine phosphorylation of the inwardly rectifying potassium channel (GIRK) subunit Kir3.1 in VTA dopaminergic neurons. Consistent with the reduced response, GIRK phosphorylation at this amino terminal tyrosine residue (Y12) enhances channel deactivation. Thus, contrary to prevailing expectations, these results suggest that κ opioid-induced aversion requires regulation of VTA dopaminergic neuron somatic excitability through a p38α MAPK effect on GIRK deactivation kinetics rather than by presynaptically inhibiting dopamine release. SIGNIFICANCE STATEMENT: Kappa opioid receptor (KOR) agonists have the potential to be effective, nonaddictive analgesics, but their therapeutic utility is greatly limited by adverse effects on mood. Understanding how KOR activation produces dysphoria is key to the development of better analgesics and to defining how the endogenous dynorphin opioids produce their depression-like effects. Results in this study show that the aversive effects of κ receptor activation required arrestin-dependent p38α MAPK activation in dopamine neurons but did not require inhibition of dopamine release in the nucleus accumbens. Thus, contrary to the prevailing view, inhibition of mesolimbic dopamine release does not mediate the aversive effects of KOR activation and functionally selective κ opioids that do not activate arrestin signaling may be effective analgesics lacking dysphoric effects.


Assuntos
Aprendizagem da Esquiva/fisiologia , Dopamina/fisiologia , Neurônios Dopaminérgicos/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Receptores Opioides kappa/fisiologia , Área Tegmentar Ventral/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , (trans)-Isômero de 3,4-dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclo-hexil)-benzenoacetamida/farmacologia , Potenciais de Ação/efeitos dos fármacos , Analgésicos não Narcóticos/farmacologia , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Condicionamento Clássico/efeitos dos fármacos , Condicionamento Clássico/fisiologia , Dopamina/metabolismo , Ativação Enzimática , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Técnicas de Silenciamento de Genes , Ativação do Canal Iônico/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Núcleo Accumbens/metabolismo , Fosforilação/efeitos dos fármacos , Potássio/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Receptores Opioides kappa/deficiência , Receptores Opioides kappa/genética , Proteínas Recombinantes de Fusão/farmacologia , Teste de Desempenho do Rota-Rod , Neurônios Serotoninérgicos/fisiologia , Área Tegmentar Ventral/citologia , Proteínas Quinases p38 Ativadas por Mitógeno/deficiência , Proteínas Quinases p38 Ativadas por Mitógeno/genética
16.
J Neurosci ; 35(33): 11682-93, 2015 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-26290245

RESUMO

The nociceptin/orphanin FQ (NOP) receptor, the fourth member of the opioid receptor family, is involved in many processes common to the opioid receptors including pain and drug abuse. To better characterize receptor location and trafficking, knock-in mice were created by inserting the gene encoding enhanced green fluorescent protein (eGFP) into the NOP receptor gene (Oprl1) and producing mice expressing a functional NOP-eGFP C-terminal fusion in place of the native NOP receptor. The NOP-eGFP receptor was present in brain of homozygous knock-in animals in concentrations somewhat higher than in wild-type mice and was functional when tested for stimulation of [(35)S]GTPγS binding in vitro and in patch-clamp electrophysiology in dorsal root ganglia (DRG) neurons and hippocampal slices. Inhibition of morphine analgesia was equivalent when tested in knock-in and wild-type mice. Imaging revealed detailed neuroanatomy in brain, spinal cord, and DRG and was generally consistent with in vitro autoradiographic imaging of receptor location. Multicolor immunohistochemistry identified cells coexpressing various spinal cord and DRG cellular markers, as well as coexpression with µ-opioid receptors in DRG and brain regions. Both in tissue slices and primary cultures, the NOP-eGFP receptors appear throughout the cell body and in processes. These knock-in mice have NOP receptors that function both in vitro and in vivo and appear to be an exceptional tool to study receptor neuroanatomy and correlate with NOP receptor function. SIGNIFICANCE STATEMENT: The NOP receptor, the fourth member of the opioid receptor family, is involved in pain, drug abuse, and a number of other CNS processes. The regional and cellular distribution has been difficult to determine due to lack of validated antibodies for immunohistochemical analysis. To provide a new tool for the investigation of receptor localization, we have produced knock-in mice with a fluorescent-tagged NOP receptor in place of the native NOP receptor. These knock-in mice have NOP receptors that function both in vitro and in vivo and have provided a detailed characterization of NOP receptors in brain, spinal cord, and DRG neurons. They appear to be an exceptional tool to study receptor neuroanatomy and correlate with NOP receptor function.


Assuntos
Proteínas de Fluorescência Verde/metabolismo , Microscopia de Fluorescência/métodos , Neurônios/citologia , Neurônios/metabolismo , Receptores Opioides/metabolismo , Frações Subcelulares/metabolismo , Animais , Células Cultivadas , Técnicas de Introdução de Genes , Proteínas de Fluorescência Verde/genética , Masculino , Camundongos , Camundongos Transgênicos , Imagem Molecular/métodos , Receptores Opioides/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Frações Subcelulares/ultraestrutura , Distribuição Tecidual , Receptor de Nociceptina
17.
Mol Pain ; 122016.
Artigo em Inglês | MEDLINE | ID: mdl-27030709

RESUMO

BACKGROUND: Opioids are the gold standard for the treatment of acute pain despite serious side effects in the central and enteric nervous system. µ-opioid receptors (MOPs) are expressed and functional at the terminals of sensory axons, when activated by exogenous or endogenous ligands. However, the presence and function of MOP along nociceptive axons remains controversial particularly in naïve animals. Here, we characterized axonal MOPs by immunofluorescence, ultrastructural, and functional analyses. Furthermore, we evaluated hypertonic saline as a possible enhancer of opioid receptor function. RESULTS: Comparative immunolabeling showed that, among several tested antibodies, which all provided specific MOP detection in the rat central nervous system (CNS), only one monoclonal MOP-antibody yielded specificity and reproducibility for MOP detection in the rat peripheral nervous system including the sciatic nerve. Double immunolabeling documented that MOP immunoreactivity was confined to calcitonin gene-related peptide (CGRP) positive fibers and fiber bundles. Almost identical labeling and double labeling patterns were found using mcherry-immunolabeling on sciatic nerves of mice producing a MOP-mcherry fusion protein (MOP-mcherry knock-in mice). Preembedding immunogold electron microscopy on MOP-mcherry knock-in sciatic nerves indicated presence of MOP in cytoplasm and at membranes of unmyelinated axons. Application of [D-Ala(2), N-MePhe(4), Gly-ol]-enkephalin (DAMGO) or fentanyl dose-dependently inhibited depolarization-induced CGRP release from rat sciatic nerve axons ex vivo, which was blocked by naloxone. When the lipophilic opioid fentanyl was applied perisciatically in naïve Wistar rats, mechanical nociceptive thresholds increased. Subthreshold doses of fentanyl or the hydrophilic opioid DAMGO were only effective if injected together with hypertonic saline. In vitro, using ß-arrestin-2/MOP double-transfected human embryonic kidney cells, DAMGO as well as fentanyl lead to a recruitment of ß-arrestin-2 to the membrane followed by a ß-arrestin-2 reappearance in the cytosol and MOP internalization. Pretreatment with hypertonic saline prevented MOP internalization. CONCLUSION: MOPs are present and functional in the axonal membrane from naïve animals. Hypertonic saline acutely decreases ligand-induced internalization of MOP and thereby might improve MOP function. Further studies should explore potential clinical applications of opioids together with enhancers for regional analgesia.


Assuntos
Analgesia , Axônios/metabolismo , Receptores Opioides mu/química , Receptores Opioides mu/metabolismo , Tonsila do Cerebelo/efeitos dos fármacos , Tonsila do Cerebelo/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Axônios/efeitos dos fármacos , Axônios/ultraestrutura , Comportamento Animal/efeitos dos fármacos , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Endocitose/efeitos dos fármacos , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Feminino , Fentanila/farmacologia , Técnicas de Introdução de Genes , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Nociceptividade/efeitos dos fármacos , Potássio/farmacologia , Ratos Wistar , Reprodutibilidade dos Testes , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/metabolismo , beta-Arrestinas/metabolismo
18.
Artigo em Inglês | MEDLINE | ID: mdl-26861145

RESUMO

BACKGROUND: A difficult problem in treating opioid addicts is the maintenance of a drug-free state because of the negative emotional symptoms associated with withdrawal, which may trigger relapse. Several lines of evidence suggest a role for the metabotropic glutamate receptor 5 in opioid addiction; however, its involvement during opioid withdrawal is not clear. METHODS: Mice were treated with a 7-day escalating-dose morphine administration paradigm. Following withdrawal, the development of affective behaviors was assessed using the 3-chambered box, open-field, elevated plus-maze and forced-swim tests. Metabotropic glutamate receptor 5 autoradiographic binding was performed in mouse brains undergoing chronic morphine treatment and 7 days withdrawal. Moreover, since there is evidence showing direct effects of opioid drugs on the metabotropic glutamate receptor 5 system, the presence of an metabotropic glutamate receptor 5/µ-opioid receptor interaction was assessed by performing metabotropic glutamate receptor 5 autoradiographic binding in brains of mice lacking the µ-opioid receptor gene. RESULTS: Withdrawal from chronic morphine administration induced anxiety-like, depressive-like, and impaired sociability behaviors concomitant with a marked upregulation of metabotropic glutamate receptor 5 binding. Administration of the metabotropic glutamate receptor 5 antagonist, 3-((2-Methyl-4-thiazolyl)ethynyl)pyridine, reversed morphine abstinence-induced depressive-like behaviors. A brain region-specific increase in metabotropic glutamate receptor 5 binding was observed in the nucleus accumbens shell, thalamus, hypothalamus, and amygdala of µ-opioid receptor knockout mice compared with controls. CONCLUSIONS: These results suggest an association between metabotropic glutamate receptor 5 alterations and the emergence of opioid withdrawal-related affective behaviors. This study supports metabotropic glutamate receptor 5 system as a target for the development of pharmacotherapies for the treatment of opioid addiction. Moreover, our data show direct effects of µ-opioid receptor system manipulation on metabotropic glutamate receptor 5 binding in the brain.


Assuntos
Emoções/efeitos dos fármacos , Morfina/efeitos adversos , Receptor de Glutamato Metabotrópico 5/metabolismo , Receptores Opioides mu/genética , Síndrome de Abstinência a Substâncias/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Encéfalo/metabolismo , Masculino , Camundongos , Camundongos Knockout , Morfina/farmacologia , Regulação para Cima/efeitos dos fármacos
19.
Brain Behav Immun ; 57: 227-242, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27139929

RESUMO

Opioids are the most powerful analgesics. As pain is driven by sensory transmission and opioid receptors couple to inhibitory G proteins, according to the classical concept, opioids alleviate pain by activating receptors on neurons and blocking the release of excitatory mediators (e.g., substance P). Here we show that analgesia can be mediated by opioid receptors in immune cells. We propose that activation of leukocyte opioid receptors leads to the secretion of opioid peptides Met-enkephalin, ß-endorphin and dynorphin A (1-17), which subsequently act at local neuronal receptors, to relieve pain. In a mouse model of neuropathic pain induced by a chronic constriction injury of the sciatic nerve, exogenous agonists of δ-, µ- and κ-opioid receptors injected at the damaged nerve infiltrated by opioid peptide- and receptor-expressing leukocytes, produced analgesia, as assessed with von Frey filaments. The analgesia was attenuated by pharmacological or genetic inactivation of opioid peptides, and by leukocyte depletion. This decrease in analgesia was restored by the transfer of wild-type, but not opioid receptor-lacking leukocytes. Ex vivo, exogenous opioids triggered secretion of opioid peptides from wild-type immune cells isolated from damaged nerves, which was diminished by blockade of Gαi/o or Gßγ (but not Gαs) proteins, by chelator of intracellular (but not extracellular) Ca(2+), by blockers of phospholipase C (PLC) and inositol 1,4,5-trisphosphate (IP3) receptors, and was partially attenuated by protein kinase C inhibitor. Similarly, the leukocyte depletion-induced decrease in exogenous opioid analgesia was re-established by transfer of immune cells ex vivo pretreated with extracellular Ca(2+) chelator, but was unaltered by leukocytes pretreated with intracellular Ca(2+) chelator or blockers of Gαi/o and Gßγ proteins. Thus, both ex vivo opioid peptide release and in vivo analgesia were mediated by leukocyte opioid receptors coupled to the Gαi/o-Gßγ protein-PLC-IP3 receptors-intracellular Ca(2+) pathway. Our findings suggest that opioid receptors in immune cells are important targets for the control of pathological pain.


Assuntos
Analgesia , Cálcio/metabolismo , Leucócitos/metabolismo , Neuralgia/metabolismo , Peptídeos Opioides/metabolismo , Receptores Opioides/metabolismo , Transdução de Sinais , Animais , Modelos Animais de Doenças , Camundongos , Neuralgia/tratamento farmacológico , Receptores Opioides/agonistas , Método Simples-Cego
20.
J Pharmacol Exp Ther ; 355(2): 206-11, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26350161

RESUMO

The corticotropin-releasing factor (CRF) and kappa-opioid receptor (KOR) systems are both implicated in stress-related behaviors and drug dependence. Although previous studies suggest that antagonism of each system blocks aspects of experimental models of drug dependence, the possible interaction between these systems at the neuronal level has not been completely examined. We used an in vitro brain slice preparation to investigate the interaction of these two peptide systems on inhibitory neurotransmission in the central nucleus of the amygdala (CeA). Application of exogenous CRF increased the mean frequency of GABAergic miniature inhibitory postsynaptic currents (mIPSC) by 20.2%, suggesting an increase in presynaptic GABA release. Although the pharmacological blockade of KORs by norBNI alone did not significantly affect mIPSC frequency, it significantly enhanced the effect of CRF (by 43.9%, P = 0.02). Similarly, the CRF effects in slices from KOR knockout (KO) mice (84.0% increase) were significantly greater than in wild-type (WT) mice (24.6%, P = 0.01), although there was no significant difference in baseline mIPSC frequency between slices from KOR KO and WT mice. The increase in CRF action in the presence of norBNI was abolished by a CRF-1 receptor antagonist but was unaffected by a CRF-2 receptor antagonist. We hypothesize that CRF facilitates the release of an endogenous ligand for KORs and that subsequent activation of KOR receptors modulates presynaptic effects of CRF in CeA. These results suggest that potential pharmacotherapies aimed at neurobehavioral and addictive disorders may need to involve both the KOR/dynorphin and the CRF systems in CeA.


Assuntos
Núcleo Central da Amígdala/metabolismo , Hormônio Liberador da Corticotropina/metabolismo , Receptores Opioides kappa/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Potenciais Pós-Sinápticos Inibidores , Camundongos Endogâmicos C57BL , Camundongos Knockout , Potenciais Pós-Sinápticos em Miniatura , Naltrexona/análogos & derivados , Naltrexona/farmacologia , Receptores de Hormônio Liberador da Corticotropina/antagonistas & inibidores , Receptores Opioides kappa/antagonistas & inibidores , Receptores Opioides kappa/genética , Transmissão Sináptica
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