RESUMO
Addiction develops from the gradual adaptation of the brain to chronic drug exposure, and involves genetic reprogramming of neuronal function. The central extended amygdala (EAc) is a network formed by the central amygdala and the bed nucleus of the stria terminalis. This key site controls drug craving and seeking behaviors, and has not been investigated at the gene regulation level. We used Affymetrix microarrays to analyze transcriptional activity in the murine EAc, with a focus on mu-opioid receptor-associated events because these receptors mediate drug reward and dependence. We identified 132 genes whose expression is regulated by a chronic escalating morphine regimen in the EAc from wild-type but not mu-opioid receptor knockout mice. These modifications are mostly EAc-specific. Gene ontology analysis reveals an overrepresentation of neurogenesis, cell growth and signaling protein categories. A separate quantitative PCR analysis of genes in the last of these groups confirms the dysregulation of both orphan (Gpr88) and known (DrD1A, Adora2A, Cnr1, Grm5, Gpr6) G protein-coupled receptors, scaffolding (PSD95, Homer1) and signaling (Sgk, Cap1) proteins, and neuropeptides (CCK, galanin). These transcriptional modifications do not occur following a single morphine injection, and hence result from long-term adaptation to excessive mu receptor activation. Proteins encoded by these genes are classically associated with spine modules function in other brain areas, and therefore our data suggest a remodeling of EAc circuits at sites where glutamatergic and monoaminergic afferences interact. Together, mu receptor-dependent genes identified in this study potentially contribute to drug-induced neural plasticity, and provide a unique molecular repertoire towards understanding drug craving and relapse.
Assuntos
Tonsila do Cerebelo/efeitos dos fármacos , Analgésicos Opioides/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Receptores Opioides mu/agonistas , Ativação Transcricional/efeitos dos fármacos , Tonsila do Cerebelo/metabolismo , Animais , Regulação da Expressão Gênica/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfina/farmacologia , Vias Neurais/efeitos dos fármacos , Vias Neurais/metabolismo , Plasticidade Neuronal/genética , Análise de Sequência com Séries de Oligonucleotídeos , Transtornos Relacionados ao Uso de Opioides/genética , Transtornos Relacionados ao Uso de Opioides/metabolismo , Transtornos Relacionados ao Uso de Opioides/fisiopatologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Opioides mu/metabolismo , Núcleos Septais/efeitos dos fármacos , Núcleos Septais/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Ativação Transcricional/genéticaRESUMO
The lateral hypothalamus (LH) is a brain structure that controls hedonic properties of both natural rewards and drugs of abuse. Mu opioid receptors are known to mediate drug reward, but whether overstimulation of these receptors impacts on LH function has not been studied. Here we have used a genome-wide microarray approach to identify LH responses to chronic mu opioid receptor activation at the transcriptional level. We have subjected wild-type and mu opioid receptor knockout mice to an escalating morphine regimen, which produces severe physical dependence in wild-type but not mutant animals. We have analyzed gene profiles in LH samples using the 430A.2 Affymetrix array and identified a set of 25 genes whose expression is altered by morphine in wild-type mice only. The regulation was confirmed for a subset of these genes using real-time quantitative PCR on samples from independent treatments. Altered expression of aquaporin 4, apolipoprotein D, and prostaglandin synthase is indicative of modified LH physiology. The regulation of two signaling genes (the serum glucocorticoid kinase and the regulator of G protein signaling 4) suggests that neurotransmission is altered in LH circuitry. Finally, the downregulation of apelin may indicate a potential role for this neuropeptide in opioid signaling and hedonic homeostasis. Altogether, our study shows that chronic mu opioid receptor stimulation induces gene expression plasticity in the LH and provides a unique collection of mu opioid receptor-dependent genes that potentially contribute to alter reward processes in addictive diseases.
Assuntos
Regulação da Expressão Gênica , Hipotálamo/metabolismo , Receptores Opioides mu/genética , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Hipotálamo/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfina/farmacologia , Reação em Cadeia da Polimerase , Receptores Opioides mu/metabolismo , Reprodutibilidade dos TestesRESUMO
Delta opioid (DOP) receptors participate to the control of chronic pain and emotional responses. Recent data also identified their implication in spatial memory and drug-context associations pointing to a critical role of hippocampal delta receptors. To better appreciate the impact of repeated drug exposure on their modulatory activity, we used fluorescent knock-in mice that express a functional delta receptor fused at its carboxy-terminus with the green fluorescent protein in place of the native receptor. We then tested the impact of chronic morphine treatment on the density and distribution of delta receptor-expressing cells in the hippocampus. A decrease in delta receptor-positive cell density was observed in the CA1, CA3 and dentate gyrus without alteration of the distribution across the different GABAergic populations that mainly express delta receptors. This effect partly persisted after four weeks of morphine abstinence. In addition, we observed increased DOP receptor expression at the cell surface compared to saline-treated animals. In the hippocampus, chronic morphine administration thus induces DOP receptor cellular redistribution and durably decreases delta receptor-expressing cell density. Such modifications are likely to alter hippocampal physiology, and to contribute to long-term cognitive deficits.
Assuntos
Hipocampo/efeitos dos fármacos , Morfina/farmacologia , Entorpecentes/farmacologia , Neurônios/efeitos dos fármacos , Receptores Opioides delta/metabolismo , Animais , Doença Crônica , Modelos Animais de Doenças , Feminino , Técnicas de Introdução de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/metabolismo , Hipocampo/patologia , Imuno-Histoquímica , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dependência de Morfina/metabolismo , Dependência de Morfina/patologia , Neurônios/metabolismo , Neurônios/patologia , Receptores Opioides delta/genéticaRESUMO
The habenular complex, encompassing medial (MHb) and lateral (LHb) divisions, is a highly conserved epithalamic structure involved in the dorsal diencephalic conduction system (DDC). These brain nuclei regulate information flow between the limbic forebrain and the mid- and hindbrain, integrating cognitive with emotional and sensory processes. The MHb is also one of the strongest expression sites for mu opioid receptors (MORs), which mediate analgesic and rewarding properties of opiates. At present however, anatomical distribution and function of these receptors have been poorly studied in MHb pathways. Here we took advantage of a newly generated MOR-mcherry knock-in mouse line to characterize MOR expression sites in the DDC. MOR-mcherry fluorescent signal is weak in the LHb, but strong expression is visible in the MHb, fasciculus retroflexus (fr) and interpeduncular nucleus (IPN), indicating that MOR is mainly present in the MHb-IPN pathway. MOR-mcherry cell bodies are detected both in basolateral and apical parts of MHb, where the receptor co-localizes with cholinergic and substance P (SP) neurons, respectively, representing two main MHb neuronal populations. MOR-mcherry is expressed in most MHb-SP neurons, and is present in only a subpopulation of MHb-cholinergic neurons. Intense diffuse fluorescence detected in lateral and rostral parts of the IPN further suggests that MOR-mcherry is transported to terminals of these SP and cholinergic neurons. Finally, MOR-mcherry is present in septal regions projecting to the MHb, and in neurons of the central and intermediate IPN. Together, this study describes MOR expression in several compartments of the MHb-IPN circuitry. The remarkably high MOR density in the MHb-IPN pathway suggests that these receptors are in a unique position to mediate analgesic, autonomic and reward responses.
Assuntos
Habenula/metabolismo , Núcleo Interpeduncular/metabolismo , Receptores Opioides mu/metabolismo , Acetilcolina/metabolismo , Animais , Encefalinas/metabolismo , Feminino , Técnicas de Introdução de Genes , Imuno-Histoquímica , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Vias Neurais/metabolismo , Neurônios/metabolismo , Precursores de Proteínas/metabolismo , Receptores Opioides mu/genética , Substância P/metabolismo , Proteína Vermelha FluorescenteRESUMO
BACKGROUND AND PURPOSE: Activation of δ opioid (DOP) receptors regulates pain and emotional responses, and also displays ligand-biased agonism. KNT-127 (1,2,3,4,4a,5,12,12a-octahydro-2-methyl-4aß,1ß-([1,2]benzenomethano)-2,6-diazanaphthacene-12aß,17-diol) is a novel DOP receptor agonist inducing analgesia and antidepressant effects in mice. Here, we have assessed KNT-127 for (i) analgesia against chronic inflammatory pain; (ii) effects on depression, locomotion and DOP receptor internalization; and (iii) for cross-tolerance to analgesic and antidepressant effects of acute treatment by other DOP receptor agonists. EXPERIMENTAL APPROACH: Inflammatory pain was induced by complete Freund's adjuvant injection into tail or hindpaw, and thermal and mechanical sensitivities were determined in mice. Locomotor and antidepressant-like effects were measured using actimetry and forced swim test respectively. In vivoâ KNT-127 selectivity and internalization were assessed using DOP receptor knockout mice and knock-in mice expressing fluorescent-tagged DOP receptors. KNT-127 was injected acutely at 0.1-10.0 mg·kg(-1) or administered chronically at 5 mg·kg(-1) daily over 5 days. KEY RESULTS: Acute treatment with KNT-127 reversed inflammatory hyperalgesia, produced an antidepressant-like effect but induced neither hyperlocomotion nor receptor sequestration. Chronic treatment with KNT-127 induced tolerance and cross-tolerance to SNC80-induced analgesia, but no tolerance to SNC80-evoked hyperlocomotor or antidepressant-like effects. CONCLUSIONS AND IMPLICATIONS: The DOP receptor agonist KNT-127 induced agonist-specific acute and chronic responses, at both behavioural and cellular levels. It displays activities similar to the other recently reported DOP agonists, AR-M1000390, ADL5747 and ADL5859, and differs from SNC80. SNC80 differs from the other DOP receptor agonists including KNT-127, by exhibiting ligand-biased tolerance at this receptor.
Assuntos
Analgésicos/uso terapêutico , Antidepressivos/uso terapêutico , Depressão/tratamento farmacológico , Morfinanos/uso terapêutico , Dor/tratamento farmacológico , Receptores Opioides delta/agonistas , Analgésicos/farmacologia , Animais , Antidepressivos/farmacologia , Comportamento Animal/efeitos dos fármacos , Benzamidas/farmacologia , Tolerância a Medicamentos , Adjuvante de Freund , Temperatura Alta , Hiperalgesia/tratamento farmacológico , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfinanos/farmacologia , Atividade Motora/efeitos dos fármacos , Dor/etiologia , Piperazinas/farmacologia , Receptores Opioides delta/genética , Receptores Opioides delta/metabolismoRESUMO
Delta opioid receptors participate to the control of chronic pain and emotional responses. Recent data also identified their implication in spatial memory and drug-context associations pointing to a critical role of hippocampal delta receptors. We examined the distribution of delta receptor-expressing cells in the hippocampus using fluorescent knock-in mice that express a functional delta receptor fused at its carboxyterminus with the green fluorescent protein in place of the native receptor. Colocalization with markers for different neuronal populations was performed by immunohistochemical detection. Fine mapping in the dorsal hippocampus confirmed that delta opioid receptors are mainly present in GABAergic neurons. Indeed, they are mostly expressed in parvalbumin-immunopositive neurons both in the Ammon's horn and dentate gyrus. These receptors, therefore, most likely participate in the dynamic regulation of hippocampal activity.