RESUMO
The small G-protein Ras-related C3 botulinum toxin substrate 1 (Rac1) promotes the formation of filamentous actin (F-actin). Actin is a major component of dendritic spines, and we previously found that alcohol alters actin composition and dendritic spine structure in the nucleus accumbens (NAc) and the dorsomedial striatum (DMS). To examine if Rac1 contributes to these alcohol-mediated adaptations, we measured the level of GTP-bound active Rac1 in the striatum of mice following 7â weeks of intermittent access to 20% alcohol. We found that chronic alcohol intake activates Rac1 in the DMS of male mice. In contrast, Rac1 is not activated by alcohol in the NAc and DLS of male mice or in the DMS of female mice. Similarly, closely related small G-proteins are not activated by alcohol in the DMS, and Rac1 activity is not increased in the DMS by moderate alcohol or natural reward. To determine the consequences of alcohol-dependent Rac1 activation in the DMS of male mice, we inhibited endogenous Rac1 by infecting the DMS of mice with an adeno-associated virus (AAV) expressing a dominant negative form of the small G-protein (Rac1-DN). We found that overexpression of AAV-Rac1-DN in the DMS inhibits alcohol-mediated Rac1 signaling and attenuates alcohol-mediated F-actin polymerization, which corresponded with a decrease in dendritic arborization and spine maturation. Finally, we provide evidence to suggest that Rac1 in the DMS plays a role in alcohol-associated goal-directed learning. Together, our data suggest that Rac1 in the DMS plays an important role in alcohol-dependent structural plasticity and aberrant learning.
Assuntos
Corpo Estriado , Camundongos Endogâmicos C57BL , Plasticidade Neuronal , Proteínas rac1 de Ligação ao GTP , Animais , Masculino , Camundongos , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Plasticidade Neuronal/fisiologia , Plasticidade Neuronal/efeitos dos fármacos , Feminino , Corpo Estriado/metabolismo , Corpo Estriado/efeitos dos fármacos , Etanol/farmacologia , Aprendizagem/fisiologia , Aprendizagem/efeitos dos fármacos , Neuropeptídeos/metabolismo , Neuropeptídeos/genética , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/efeitos dos fármacosRESUMO
The psychedelic alkaloid ibogaine has anti-addictive properties in both humans and animals1. Unlike most medications for the treatment of substance use disorders, anecdotal reports suggest that ibogaine has the potential to treat addiction to various substances, including opiates, alcohol and psychostimulants. The effects of ibogaine-like those of other psychedelic compounds-are long-lasting2, which has been attributed to its ability to modify addiction-related neural circuitry through the activation of neurotrophic factor signalling3,4. However, several safety concerns have hindered the clinical development of ibogaine, including its toxicity, hallucinogenic potential and tendency to induce cardiac arrhythmias. Here we apply the principles of function-oriented synthesis to identify the key structural elements of the potential therapeutic pharmacophore of ibogaine, and we use this information to engineer tabernanthalog-a water-soluble, non-hallucinogenic, non-toxic analogue of ibogaine that can be prepared in a single step. In rodents, tabernanthalog was found to promote structural neural plasticity, reduce alcohol- and heroin-seeking behaviour, and produce antidepressant-like effects. This work demonstrates that, through careful chemical design, it is possible to modify a psychedelic compound to produce a safer, non-hallucinogenic variant that has therapeutic potential.
Assuntos
Comportamento Aditivo/tratamento farmacológico , Desenho de Fármacos , Ibogaína/análogos & derivados , Ibogaína/efeitos adversos , Alcoolismo/tratamento farmacológico , Animais , Antidepressivos/farmacologia , Arritmias Cardíacas/induzido quimicamente , Técnicas de Química Sintética , Depressão/tratamento farmacológico , Modelos Animais de Doenças , Feminino , Alucinógenos/efeitos adversos , Dependência de Heroína/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasticidade Neuronal/efeitos dos fármacos , Segurança do Paciente , Receptor 5-HT2A de Serotonina/metabolismo , Agonistas do Receptor 5-HT2 de Serotonina/farmacologia , Transtornos Relacionados ao Uso de Substâncias/tratamento farmacológico , Natação , Tabernaemontana/químicaRESUMO
Neurotrophic growth factors were originally characterized for their support in neuronal differentiation, outgrowth, and survival during development. However, it has been acknowledged that they also play a vital role in the adult brain. Abnormalities in growth factors have been implicated in a variety of neurological and psychiatric disorders, including alcohol use disorder (AUD). This work focuses on the interaction between alcohol and growth factors. We review literature suggesting that several growth factors play a unique role in the regulation of alcohol consumption, and that breakdown in these growth factor systems is linked to the development of AUD. Specifically, we focus on the brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF2), and insulin growth factor 1 (IGF-1). We also review the literature on the potential role of midkine (MDK) and pleiotrophin (PTN) and their receptor, anaplastic lymphoma kinase (ALK), in AUD. We show that alcohol alters the expression of these growth factors or their receptors in brain regions previously implicated in addiction, and that manipulations on these growth factors and their downstream signaling can affect alcohol-drinking behaviors in animal models. We conclude that there is a need for translational and clinical research to assess the therapeutic potential of new pharmacotherapies targeting these systems.
Assuntos
Alcoolismo/fisiopatologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , RNA Mensageiro/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Fyn is a member of the Src family of protein tyrosine kinases (PTKs) that plays an important role not only in normal synaptic functions but also in brain pathologies including alcohol use disorder. We previously reported that repeated cycles of binge drinking and withdrawal activate Fyn in the dorsomedial striatum (DMS) of rodents, and that Fyn signaling in the DMS contributes to rat alcohol intake and relapse. Here, we used AZD0530, a CNS penetrable inhibitor of Src PTKs developed for the treatment of Alzheimer disease and cancer and tested its efficacy to suppress alcohol-dependent molecular and behavioral effects. We show that systemic administration of AZD0530 prevents alcohol-induced Fyn activation and GluN2B phosphorylation in the DMS of mice. We further report that a single dose of AZD0530 reduces alcohol operant self-administration and promotes extinction of alcohol self-administration without altering basal and dopamine D1 receptor-dependent locomotion. Together, our findings suggest that AZD0530, through its inhibitory actions on Fyn kinase, dampens alcohol seeking and drinking.
Assuntos
Comportamento Animal/efeitos dos fármacos , Benzodioxóis/farmacologia , Depressores do Sistema Nervoso Central/administração & dosagem , Comportamento de Procura de Droga/efeitos dos fármacos , Etanol/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-fyn/antagonistas & inibidores , Quinazolinas/farmacologia , Animais , Condicionamento Operante/efeitos dos fármacos , Extinção Psicológica/efeitos dos fármacos , Locomoção/efeitos dos fármacos , Camundongos , Neostriado/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , AutoadministraçãoRESUMO
We previously reported that the kinase AKT is activated in the nucleus accumbens (NAc) of rodents in response to excessive consumption of alcohol. One of the important downstream targets of AKT is the mammalian Target Of Rapamycin in Complex 1 (mTORC1), which was also activated by alcohol intake. mTORC1 controls dendritic protein translation, and we showed that the mTORC1-dependent translational machinery is activated in the NAc in response to alcohol intake. Importantly, systemic or intra-NAc inhibition of the AKT/mTORC1 pathway attenuated alcohol-drinking behaviors. Here, we mapped the activation patterns of AKT and mTORC1 in corticostriatal regions of rodents consuming large amounts of alcohol. We found that the activation of AKT and mTORC1 in response to cycles of binge drinking of 20 percent alcohol was centered in the NAc shell. Both kinases were not activated in the dorsolateral striatum (DLS); however, AKT, but not mTORC1, was activated in the dorsomedial striatum (DMS) of mice but not rats. Interestingly, excessive intake of alcohol produced a selective activation of the AKT/mTORC1 pathway in the orbitofrontal cortex (OFC), which was not observed in medial prefrontal cortex (mPFC). Furthermore, this signaling pathway was not activated in the NAc shell or OFC of rats consuming moderate amounts of alcohol nor was it activated in rats consuming sucrose. Together, our results suggest that excessive alcohol intake produces a brain region selective activation of the AKT/mTORC1 pathway, which is likely to contribute to NAc shell and OFC-dependent mechanisms that underlie the development and maintenance of alcohol drinking behavior.
Assuntos
Consumo Excessivo de Bebidas Alcoólicas/metabolismo , Encéfalo/efeitos dos fármacos , Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Animais , Comportamento Animal/efeitos dos fármacos , Western Blotting , Encéfalo/metabolismo , Modelos Animais de Doenças , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Long-Evans , Transdução de Sinais/efeitos dos fármacosRESUMO
RACK1 is a scaffolding protein that contributes to the specificity and propagation of several signaling cascades including the cAMP pathway. As such, RACK1 participates in numerous cellular functions ranging from cell migration and morphology to gene transcription. To obtain further insights on the mechanisms whereby RACK1 regulates cAMP-dependent processes, we set out to identify new binding partners of RACK1 during activation of the cAMP signaling using a proteomics strategy. We identified ß-actin as a direct RACK1 binding partner and found that the association between ß-actin and RACK1 is increased in response to the activation of the cAMP pathway. Furthermore, we show that cAMP-dependent increase in BDNF expression requires filamentous actin. We further report that ß-actin associates with the BDNF promoter IV upon the activation of the cAMP pathway and present data to suggest that the association of ß-actin with BDNF promoter IV is RACK1-dependent. Taken together, our data suggest that ß-actin is a new RACK1 binding partner and that the RACK1 and ß-actin association participate in the cAMP-dependent regulation of BDNF transcription.
Assuntos
Actinas/metabolismo , Fator Neurotrófico Derivado do Encéfalo/genética , AMP Cíclico/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Neoplasias/metabolismo , Regiões Promotoras Genéticas/genética , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Citoesqueleto de Actina/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Humanos , Ligação Proteica , Transporte Proteico , Receptores de Quinase C Ativada , Transcrição GênicaRESUMO
Glial cell line-derived neurotrophic factor (GDNF) is a potent inhibitor of ethanol consumption and relapse, and GDNF heterozygous knockout mice display increased reward sensitivity to ethanol and consume more ethanol after a period of abstinence than their wild-type littermates. Here, we tested whether ethanol alters GDNF expression in the ventral tegmental area (VTA; GDNF's site of action) and/or the nucleus accumbens (NAc; the main source of GDNF), and if so, determine the role of the endogenous growth factor in the regulation of ethanol consumption. Systemic administration of ethanol increased GDNF expression and protein levels in the VTA, but not the NAc. Additionally, GDNF levels were elevated after an ethanol-drinking session in rats that consumed ethanol in the intermittent-access two-bottle choice procedure for 1 week, but not 7 weeks. Deprivation following 7 weeks of excessive ethanol intake reduced GDNF levels, while a short ethanol binge drinking period following deprivation upregulated GDNF expression. Importantly, knockdown of GDNF within the VTA using adenovirus expressing short hairpin RNA facilitated the escalation of ethanol drinking by ethanol-naïve rats, but not by rats with a history of excessive ethanol consumption. These results suggest that during initial ethanol-drinking experiences, GDNF in the VTA is increased and protects against the development of excessive ethanol intake. However, the growth factor's protective response to ethanol breaks down after protracted excessive ethanol intake and withdrawal, resulting in persistent, excessive ethanol consumption.
Assuntos
Transtornos Relacionados ao Uso de Álcool/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Área Tegmentar Ventral/metabolismo , Consumo de Bebidas Alcoólicas/genética , Transtornos Relacionados ao Uso de Álcool/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Western Blotting/métodos , Depressores do Sistema Nervoso Central/administração & dosagem , Depressores do Sistema Nervoso Central/farmacologia , Condicionamento Operante , Modelos Animais de Doenças , Etanol/administração & dosagem , Etanol/farmacologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Masculino , Dados de Sequência Molecular , Núcleo Accumbens/metabolismo , Ratos , Ratos Long-Evans , Reação em Cadeia da Polimerase em Tempo Real/métodos , Recompensa , AutoadministraçãoRESUMO
We previously found that excessive ethanol drinking activates Fyn in the dorsomedial striatum (DMS) (Wang et al., 2010; Gibb et al., 2011). Ethanol-mediated Fyn activation in the DMS leads to the phosphorylation of the GluN2B subunit of the NMDA receptor, to the enhancement of the channel's activity, and to the development and/or maintenance of ethanol drinking behaviors (Wang et al., 2007, 2010). Protein tyrosine phosphatase α (PTPα) is essential for Fyn kinase activation (Bhandari et al., 1998), and we showed that ethanol-mediated Fyn activation is facilitated by the recruitment of PTPα to synaptic membranes, the compartment where Fyn resides (Gibb et al., 2011). Here we tested the hypothesis that PTPα in the DMS is part of the Fyn/GluN2B pathway and is thus a major contributor to the neuroadaptations underlying excessive ethanol intake behaviors. We found that RNA interference (RNAi)-mediated PTPα knockdown in the DMS reduces excessive ethanol intake and preference in rodents. Importantly, no alterations in water, saccharine/sucrose, or quinine intake were observed. Furthermore, downregulation of PTPα in the DMS of mice significantly reduces ethanol-mediated Fyn activation, GluN2B phosphorylation, and ethanol withdrawal-induced long-term facilitation of NMDAR activity without altering the intrinsic features of DMS neurons. Together, these results position PTPα upstream of Fyn within the DMS and demonstrate the important contribution of the phosphatase to the maladaptive synaptic changes that lead to excessive ethanol intake.
Assuntos
Consumo de Bebidas Alcoólicas/metabolismo , Corpo Estriado/fisiologia , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/metabolismo , Adaptação Fisiológica , Animais , Corpo Estriado/citologia , Corpo Estriado/metabolismo , Regulação para Baixo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/enzimologia , Neurônios/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Ratos , Ratos Long-Evans , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Transcrição GênicaRESUMO
This perspective summarises the first and long overdue RACK1 meeting held at the University of Limerick, Ireland, May 2013, in which RACK1's role in the immune system, the heart and the brain were discussed and its contribution to disease states such as cancer, cardiac hypertrophy and addiction were described. RACK1 is a scaffolding protein and a member of the WD repeat family of proteins. These proteins have a unique architectural assembly that facilitates protein anchoring and the stabilisation of protein activity. A large body of evidence is accumulating which is helping to define the versatile role of RACK1 in assembling and dismantling complex signaling pathways from the cell membrane to the nucleus in health and disease. In this commentary, we first provide a historical perspective on RACK1. We also address many of the pertinent and topical questions about this protein such as its role in transcription, epigenetics and translation, its cytoskeletal contribution and the merits of targeting RACK1 in disease.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Proteínas do Citoesqueleto/metabolismo , Epigenômica , Humanos , Biossíntese de Proteínas , Receptores de Quinase C Ativada , Transcrição GênicaRESUMO
Glial cell line-derived neurotrophic factor (GDNF) is a potent growth factor essential to the development, survival, and function of dopaminergic neurons (Airaksinen and Saarma 2002). The molecular mechanisms underlying GDNF expression remain elusive; thus, we set out to identify a signaling pathway that governs GDNF levels. We found that treatment of both differentiated dopaminergic-like SH-SY5Y cells and rat midbrain slices with the dopamine D2 receptor (D2R) agonist, quinpirole, triggered an increase in the expression of GDNF that was temporally preceded by an increase in the levels of zinc-finger protein 268 (Zif268), a DNA-binding transcription factor encoded by an immediate-early gene. Moreover, the D2R inhibitor raclopride blocked the increase of both GDNF and Zif268 expression following potassium-evoked dopamine release in SH-SY5Y cells. We used adenoviral delivery of small hairpin RNA (shRNA) targeting Zif268 to down-regulate its expression and found that Zif268 is specifically required for the D2R-mediated up-regulation of GDNF. Furthermore, the D2R-mediated induction of GDNF and Zif268 expression was dependent on Gßγ-mediated signaling and activation of extracellular signal-regulated kinase 1/2. Importantly, using chromatin immunoprecipitation assay, we identified a direct association of Zif268 with the GDNF promoter. These results suggest that D2R activation induces a Gßγ- and extracellular signal-regulated kinase 1/2-dependent increase in the level of Zif268, which functions to directly up-regulate the expression of GDNF.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/biossíntese , Receptores de Dopamina D2/metabolismo , Transdução de Sinais/fisiologia , Animais , Western Blotting , Linhagem Celular , Imunoprecipitação da Cromatina , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
Uncontrolled consumption of alcohol is a hallmark of alcohol abuse disorders; however, the central molecular mechanisms underlying excessive alcohol consumption are still unclear. Here, we report that the GTP binding protein, H-Ras in the nucleus accumbens (NAc) plays a key role in neuroadaptations that underlie excessive alcohol-drinking behaviors. Specifically, acute (15 min) systemic administration of alcohol (2.5 g/kg) leads to the activation of H-Ras in the NAc of mice, which is observed even 24 h later. Similarly, rat operant self-administration of alcohol (20%) also results in the activation of H-Ras in the NAc. Using the same procedures, we provide evidence suggesting that the exchange factor GRF1 is upstream of H-Ras activation by alcohol. Importantly, we show that infection of mice NAc with lentivirus expressing a short hairpin RNA that targets the H-Ras gene produces a significant reduction of voluntary consumption of 20% alcohol. In contrast, knockdown of H-Ras in the NAc of mice did not alter water, quinine, and saccharin intake. Furthermore, using two-bottle choice and operant self-administration procedures, we show that inhibiting H-Ras activity by intra-NAc infusion of the farnesyltransferase inhibitor, FTI-276, produced a robust decrease of rats' alcohol drinking; however, sucrose consumption was unaltered. Finally, intra-NAc infusion of FTI-276 also resulted in an attenuation of seeking for alcohol. Together, these results position H-Ras as a central molecular mediator of alcohol's actions within the mesolimbic system and put forward the potential value of the enzyme as a novel target to treat alcohol use disorders.
Assuntos
Consumo de Bebidas Alcoólicas/metabolismo , Consumo Excessivo de Bebidas Alcoólicas/metabolismo , Etanol/farmacologia , Núcleo Accumbens/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Consumo de Bebidas Alcoólicas/genética , Animais , Consumo Excessivo de Bebidas Alcoólicas/genética , Comportamento de Escolha/efeitos dos fármacos , Comportamento de Escolha/fisiologia , Farnesiltranstransferase/antagonistas & inibidores , Masculino , Metionina/análogos & derivados , Metionina/farmacologia , Camundongos , Camundongos Transgênicos , Núcleo Accumbens/efeitos dos fármacos , Fosforilação , Proteínas Proto-Oncogênicas p21(ras)/genética , Ratos , Ratos Long-Evans , Sacarose/farmacologia , ras-GRF1/genética , ras-GRF1/metabolismoRESUMO
The α4ßδ gamma-aminobutyric acid A receptor (GABA(A) R) has been proposed to mediate the rewarding effects of low-to-moderate concentrations of alcohol (ethanol) that approximate those achieved by social drinking. If this is true, then this receptor should be necessary for the reinforcing effects of ethanol as assessed in an instrumental self-administration procedure in which rats are trained to lever press for oral ethanol. We used viral-mediated RNA interference to transiently reduce expression of the α4 GABA(A) R subunit in the shell region of the nucleus accumbens (NAc). We found that responding for ethanol was significantly reduced after α4 reductions in the NAc shell, but not NAc core. This reduction was specific to ethanol, as responding for sucrose was not altered. The presence of ethanol was also required as unreinforced responding for ethanol in subjects previously trained to respond for ethanol (i.e. responding during an extinction test) was not altered. In addition, responding during reinforced sessions was not altered during the initial 5 minutes of the session, but decreased after 5 minutes, following multiple reinforced responses. Together, these findings indicate that the α4 GABA(A) R subunit in the NAc shell is necessary for the instrumental reinforcing effects of oral ethanol, further supporting a role for α4-containing GABA(A) Rs in the rewarding/reinforcing effects of ethanol. Possible pharmacological and non-pharmacological explanations for these effects are considered.
Assuntos
Etanol/farmacologia , Receptores de GABA-A/fisiologia , Reforço Psicológico , Adenoviridae , Consumo de Bebidas Alcoólicas/psicologia , Animais , Condicionamento Operante/efeitos dos fármacos , Etanol/administração & dosagem , Técnicas de Silenciamento de Genes , Vetores Genéticos , Masculino , Atividade Motora/efeitos dos fármacos , Desempenho Psicomotor/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Long-Evans , Sacarose/administração & dosagem , Edulcorantes/administração & dosagemRESUMO
RACK1 is a scaffolding protein that spatially and temporally regulates numerous signaling cascades. We previously found that activation of the cAMP signaling pathway induces the translocation of RACK1 to the nucleus. We further showed that nuclear RACK1 is required to promote the transcription of the brain-derived neurotrophic factor (BDNF). Here, we set out to elucidate the mechanism underlying cAMP-dependent RACK1 nuclear translocation and BDNF transcription. We identified the scaffolding protein 14-3-3ζ as a direct binding partner of RACK1. Moreover, we found that 14-3-3ζ was necessary for the cAMP-dependent translocation of RACK1 to the nucleus. We further observed that the disruption of RACK1/14-3-3ζ interaction with a peptide derived from the RACK1/14-3-3ζ binding site or shRNA-mediated 14-3-3ζ knockdown inhibited cAMP induction of BDNF transcription. Together, these data reveal that the function of nuclear RACK1 is mediated through its interaction with 14-3-3ζ. As RACK1 and 14-3-3ζ are two multifunctional scaffolding proteins that coordinate a wide variety of signaling events, their interaction is likely to regulate other essential cellular functions.
Assuntos
Proteínas 14-3-3/metabolismo , Fator Neurotrófico Derivado do Encéfalo/genética , Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica , Proteínas de Neoplasias/metabolismo , Receptores de Superfície Celular/metabolismo , Transcrição Gênica , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Ligação ao GTP/química , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Neoplasias/química , Neurônios/citologia , Neurônios/metabolismo , Fosforilação , Ligação Proteica , Conformação Proteica , Ratos , Receptores de Quinase C Ativada , Receptores de Superfície Celular/química , Reprodutibilidade dos Testes , Transdução de SinaisRESUMO
BACKGROUND: Neuroadaptations within the nucleus accumbens (NAc) have been implicated in molecular mechanisms underlying the development and/or maintenance of alcohol abuse disorders. We recently reported that the activation of mammalian target of rapamycin complex 1 (mTORC1) signaling pathway in the NAc of rodents, after exposure to alcohol, contributes to alcohol drinking behaviors. The kinase AKT is a main upstream activator of the mTORC1 pathway. We therefore hypothesized that the activation of AKT in the NAc in response to alcohol exposure plays an important role in mechanisms that underlie excessive alcohol consumption. METHODS: Western blot analysis was used to assess the phosphorylation levels of enzymes. Acute exposure of mice to alcohol was achieved by the administration of 2 g/kg alcohol intraperitoneally (i.p.). Two-bottle choice and operant self-administration procedures were used to assess drinking behaviors in rats. RESULTS: We found that acute systemic administration of alcohol and recurring cycles of excessive voluntary consumption of alcohol and withdrawal result in the activation of AKT signaling in the NAc of rodents. We show that inhibition of AKT or its upstream activator, phosphatidylinositol-3-kinase (PI3K), within the NAc of rats attenuates binge drinking as well as alcohol but not sucrose operant self-administration. CONCLUSIONS: Our results suggest that the activation of the AKT pathway in the NAc in response to alcohol exposure is an important contributor to the molecular mechanisms underlying alcohol-drinking behaviors. AKT signaling pathway inhibitors are therefore potential candidates for drug development for the treatment of alcohol use and abuse disorders.
Assuntos
Consumo de Bebidas Alcoólicas/metabolismo , Comportamento Aditivo/metabolismo , Comportamento Aditivo/fisiopatologia , Condicionamento Operante/efeitos dos fármacos , Núcleo Accumbens/fisiopatologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Transdução de Sinais/fisiologia , Consumo de Bebidas Alcoólicas/fisiopatologia , Androstadienos/administração & dosagem , Androstadienos/farmacologia , Animais , Comportamento de Escolha/efeitos dos fármacos , Comportamento de Escolha/fisiologia , Condicionamento Operante/fisiologia , Etanol/administração & dosagem , Etanol/antagonistas & inibidores , Etanol/intoxicação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microinjeções , Núcleo Accumbens/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Long-Evans , Ribonucleosídeos/administração & dosagem , Ribonucleosídeos/farmacologia , Autoadministração/métodos , Autoadministração/psicologia , Transdução de Sinais/efeitos dos fármacos , Sacarose/administração & dosagem , WortmaninaRESUMO
Recent findings suggest that extrasynaptic δ-subunit-containing GABA(A) receptors are sensitive to low-to-moderate concentrations of alcohol, raising the possibility that these receptors mediate the reinforcing effects of alcohol after consumption of one or a few drinks. We used the technique of viral-mediated RNAi to reduce expression of the GABA(A) receptor δ-subunit in adult rats in localized regions of the nucleus accumbens (NAc) to test the hypothesis that δ-subunit-containing GABA(A) receptors in the NAc are necessary for oral alcohol consumption. We found that knockdown of the δ-subunit in the medial shell region of the NAc, but not in the ventral or lateral shell or in the core, reduced alcohol intake. In contrast, δ-subunit knockdown in the medial shell did not affect intake of a 2% sucrose solution, suggesting that the effects of GABA(A) receptor δ-subunit reduction are specific to alcohol. These results provide strong evidence that extrasynaptic δ-subunit-containing GABA(A) receptors in the medial shell of the NAc are critical for the reinforcing effects of oral ethanol.
Assuntos
Consumo de Bebidas Alcoólicas/metabolismo , Núcleo Mediodorsal do Tálamo/metabolismo , Núcleo Accumbens/metabolismo , Receptores de GABA-A/metabolismo , Sinapses/metabolismo , Adenoviridae/genética , Administração Oral , Animais , Técnicas de Silenciamento de Genes , Masculino , Núcleo Mediodorsal do Tálamo/patologia , Núcleo Accumbens/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Long-Evans , Receptores de GABA-A/genética , Reprodutibilidade dos Testes , Sacarose/metabolismoRESUMO
We recently found that ethanol-induced long-term facilitation (LTF) of NMDAR activity is mediated by NR2B-NMDARs and is observed in the dorsomedial striatum (DMS) but not in the dorsolateral striatum (DLS). We also showed that repeated administration of ethanol causes a long-lasting increase in NMDAR activity in the DMS, resulting from ethanol-mediated Fyn phosphorylation of NR2B subunits. In this addendum, we report that the different sensitivity of NMDARs to ethanol between the DMS and DLS is not attributed to the abundance of synaptic NR2B-NMDARs or differences in Fyn levels. We further show that LTF is specific for NR2B-, but not NR2A-NMDARs, and that the duration of the in vivo ethanol-mediated increase in NMDAR activity is associated with the period of ethanol exposure, but not with alteration in NR1 or NR2A protein levels. Together, these results suggest that upregulation of NR2B-NMDAR activity by ethanol is selective and that ethanol's effect on NMDAR activity is gradual and cumulative.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Corpo Estriado/metabolismo , Etanol/farmacologia , Receptores de N-Metil-D-Aspartato/metabolismo , Regulação para Cima/efeitos dos fármacos , Animais , Camundongos , Camundongos Knockout , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Proteínas Proto-Oncogênicas c-fyn/genética , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Regulação para Cima/genéticaRESUMO
Ibogaine is a naturally occurring alkaloid that has been reported to decrease various adverse phenotypes associated with exposure to drugs of abuse and alcohol in human and rodent models. Unfortunately, ibogaine cannot be used as a medication to treat addiction because of severe side effects. Previously, we reported that the desirable actions of ibogaine to reduce self-administration of, and relapse to, alcohol consumption are mediated via the upregulation of the expression of the glial cell line-derived neurotrophic factor (GDNF) in the midbrain ventral tegmental area (VTA), and the consequent activation of the GDNF pathway. The ibogaine metabolite, noribogaine, and a synthetic derivative of ibogaine, 18-Methoxycoronaridine (18-MC), possess a similar anti-addictive profile as ibogaine in rodent models, but without some of its adverse side effects. Here, we determined whether noribogaine and/or 18-MC, like ibogaine, increase GDNF expression, and whether their site of action to reduce alcohol consumption is the VTA. We used SH-SY5Y cells as a cell culture model and found that noribogaine, like ibogaine, but not 18-MC, induces a robust increase in GDNF mRNA levels. Next, we tested the effect of intra-VTA infusion of noribogaine and 18-MC on rat operant alcohol self-administration and found that noribogaine, but not 18-MC, in the VTA decreases responding for alcohol. Together, our results suggest that noribogaine and 18-MC have different mechanisms and sites of action.
Assuntos
Dissuasores de Álcool/farmacologia , Consumo de Bebidas Alcoólicas/genética , Alcoolismo/genética , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Ibogaína/análogos & derivados , Ibogaína/farmacologia , Área Tegmentar Ventral/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Condicionamento Operante/efeitos dos fármacos , Humanos , Masculino , Motivação/efeitos dos fármacos , RNA Mensageiro/genética , Ratos , Ratos Long-Evans , AutoadministraçãoRESUMO
A growing number of studies suggest that the development of compulsive drug seeking and taking depends on dorsostriatal mechanisms. We previously observed that ex vivo acute exposure of the dorsal striatum to, and withdrawal from, alcohol induces long-term facilitation (LTF) of the activity of NR2B-containing NMDA receptors (NR2B-NMDARs) in a mechanism that requires the Src family protein tyrosine kinase (PTK), Fyn (Wang et al., 2007). In the present study, we first compared alcohol's actions in rat dorsomedial (DMS) and the dorsolateral (DLS) subregions of the striatum, which differ in their anatomical connectivity and function. We found that alcohol-mediated induction of LTF of NR2B-NMDAR activity is centered in the DMS. Next, we tested whether in vivo exposure of rats to alcohol leads to long-term adaptations of the NMDAR system in the DMS. We observed that repeated daily administration of alcohol results in a long-lasting increase in the activity of the NR2B-NMDARs in the DMS. The same procedure leads to a prolonged activation of Fyn, increased NR2B phosphorylation, and membrane localization of the subunit. Importantly, similar electrophysiological and biochemical modifications were observed in the DMS of rats that consumed large quantities of alcohol. Finally, we show that inhibition of NR2B-NMDARs or Src family PTKs in the DMS, but not in the DLS, significantly decreases operant self-administration of alcohol and reduces alcohol-priming-induced reinstatement of alcohol seeking. Our results suggest that the upregulation of NR2B-NMDAR activity within the DMS by alcohol contributes to the maladaptive synaptic changes that lead to excessive alcohol intake and relapse.
Assuntos
Adaptação Fisiológica , Consumo de Bebidas Alcoólicas/patologia , Corpo Estriado/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Consumo de Bebidas Alcoólicas/sangue , Consumo de Bebidas Alcoólicas/psicologia , Análise de Variância , Animais , Comportamento Animal , Depressores do Sistema Nervoso Central/administração & dosagem , Depressores do Sistema Nervoso Central/efeitos adversos , Depressores do Sistema Nervoso Central/sangue , Comportamento de Escolha , Condicionamento Operante/efeitos dos fármacos , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/patologia , Etanol/administração & dosagem , Etanol/efeitos adversos , Etanol/sangue , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Imunoprecipitação/métodos , Técnicas In Vitro , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp , Fenóis/farmacologia , Fosforilação/efeitos dos fármacos , Piperidinas/farmacologia , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Ratos , Ratos Long-Evans , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Recidiva , Autoadministração/métodos , Sacarose/administração & dosagem , Edulcorantes/administração & dosagem , Sinaptossomos/metabolismoRESUMO
Scaffolding proteins are major contributors to the spatial and temporal orchestration of signaling cascades and hence cellular functions. RACK1 is a scaffolding protein that plays an important role in the regulation of, and cross-talk between, various signaling pathways. Here we report that RACK1 is a mediator of chromatin remodeling, resulting in an exon-specific expression of the brain-derived neurotrophic factor (BDNF) gene. Specifically, we found that following the activation of the cAMP pathway, nuclear RACK1 localizes at the promoter IV region of the BDNF gene by its association with histones H3 and H4, leading to the dissociation of the transcription repressor methyl-CpG-binding protein 2 (MeCP2) from the promoter, resulting in the acetylation of histone H4. These chromatin modifications lead to the activation of the promoter and to the subsequent promoter-controlled transcription of BDNF exon IV. Our findings expand our knowledge regarding the function of scaffolding proteins such as RACK1. Furthermore, this novel mechanism for the regulation of exon-specific expression of the BDNF gene by RACK1 could have implications on the neuronal functions of the growth factor including synaptic plasticity, learning, and memory.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/biossíntese , Fator Neurotrófico Derivado do Encéfalo/genética , Epigênese Genética , Proteínas de Ligação ao GTP/biossíntese , Proteínas de Ligação ao GTP/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/genética , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Ilhas de CpG , Histonas/química , Humanos , Aprendizagem , Memória , Proteína 2 de Ligação a Metil-CpG/genética , Ratos , Ratos Sprague-Dawley , Receptores de Quinase C Ativada , Transdução de SinaisRESUMO
We previously found that brain-derived neurotrophic factor (BDNF)-haplodeficient mice exhibit greater ethanol-induced place preference and psychomotor sensitization, and greater ethanol consumption after deprivation, than control mice. We further observed that, in mice, voluntary ethanol intake increases BDNF expression in the dorsal striatum (DS). Here, we determined whether BDNF within the DS regulates ethanol self-administration in Long-Evans rats trained to self-administer a 10% ethanol solution. We observed a greater increase in BDNF expression after ethanol self-administration in the dorsolateral striatum (DLS) than in the dorsomedial striatum (DMS). We further found that downregulation of endogenous BDNF using viral-mediated siRNA in the DLS, but not in the DMS, significantly increased ethanol self-administration. Infusion of exogenous BDNF (0.25 microg/microl/side into the DMS; 0.25 and 0.75 microg/microl/side into the DLS) attenuated responding for ethanol when infused 3 h before the beginning of the self-administration session. Although the decrease in ethanol intake was similar in the DLS and DMS, BDNF infused in the DLS, but not in the DMS, induced an early termination of the drinking episode. Furthermore, the action of BDNF in the DLS was specific for ethanol, as infusion of the neurotrophic factor in the DMS, but not DLS, resulted in a reduction of sucrose intake. Together, these findings demonstrate that the BDNF pathway within the DLS controls the level of ethanol self-administration. Importantly, our results suggest that an endogenous signaling pathway within the same brain region that mediates drug-taking behavior also plays a critical role in gating the level of ethanol intake.