RESUMO
Glutamatergic systems, including AMPA receptors (AMPARs), are involved in opiate-induced neuronal and behavioral plasticity, although the mechanisms underlying these effects are not fully understood. In the present study, we investigated the effects of repeated morphine administration on AMPAR expression, synaptic plasticity, and context-dependent behavioral sensitization to morphine. We found that morphine treatment produced changes of synaptic AMPAR expression in the hippocampus, a brain area that is critically involved in learning and memory. These changes could be observed 1 week after the treatment, but only when mice developed context-dependent behavioral sensitization to morphine in which morphine treatment was associated with drug administration environment. Context-dependent behavioral sensitization to morphine was also associated with increased basal synaptic transmission and disrupted hippocampal long-term potentiation (LTP), whereas these effects were less robust when morphine administration was not paired with the drug administration environment. Interestingly, some effects may be related to the prior history of morphine exposure in the drug-associated environment, since alterations of AMPAR expression, basal synaptic transmission, and LTP were observed in mice that received a saline challenge 1 week after discontinuation of morphine treatment. Furthermore, we demonstrated that phosphorylation of GluA1 AMPAR subunit plays a critical role in the acquisition and expression of context-dependent behavioral sensitization, as this behavior is blocked by a viral vector that disrupts GluA1 phosphorylation. These data provide evidence that glutamatergic signaling in the hippocampus plays an important role in context-dependent sensitization to morphine and supports further investigation of glutamate-based strategies for treating opiate addiction.
Assuntos
Hipocampo/efeitos dos fármacos , Dependência de Morfina/metabolismo , Morfina/farmacologia , Atividade Motora/efeitos dos fármacos , Entorpecentes/farmacologia , Receptores de AMPA/metabolismo , Alanina/genética , Análise de Variância , Animais , Comportamento Animal/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Proteína 4 Homóloga a Disks-Large , Relação Dose-Resposta a Droga , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/genética , Guanilato Quinases/metabolismo , Marcação In Situ das Extremidades Cortadas/métodos , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Proteínas Nucleares/metabolismo , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Distribuição Aleatória , Receptores de AMPA/genética , Serina/genética , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismoRESUMO
Kainate receptors (KARs) mediate postsynaptic responses in CA3 pyramidal cells and CA1 interneurones in the hippocampus. In CA1 pyramidal cells knockout studies have inidcated the presence of functional GluR6-containing KARs, however in this region they made no ionotropic contribution to the synaptic responses. In the meantime, a metabotropic function was reported for presynaptic KARs modulating transmitter release in CA1. We examined the possibility that KARs in CA1 pyramidal cells have a metabotropic function. Kainate is known to inhibit a slow afterhyperpolarization current that regulates excitability in hippocampus and can be modulated by a number of G protein coupled receptors. We showed that KARs activation reduces slow afterhyperpolarization current in CA1 pyramidal cells via metabotropic action and elucidated the transduction mechanism(s) underlying this action.
Assuntos
Células Piramidais/metabolismo , Receptores de Ácido Caínico/metabolismo , Animais , Humanos , Transdução de Sinais , Sinapses/metabolismoRESUMO
In mammals, the melanopsin gene (Opn4) encodes a sensory photopigment that underpins newly discovered inner retinal photoreceptors. Since its first discovery in Xenopus laevis and subsequent description in humans and mice, melanopsin genes have been described in all vertebrate classes. Until now, all of these sequences have been considered representatives of a single orthologous gene (albeit with duplications in the teleost fish). Here, we describe the discovery and functional characterisation of a new melanopsin gene in fish, bird, and amphibian genomes, demonstrating that, in fact, the vertebrates have evolved two quite separate melanopsins. On the basis of sequence similarity, chromosomal localisation, and phylogeny, we identify our new melanopsins as the true orthologs of the melanopsin gene previously described in mammals and term this grouping Opn4m. By contrast, the previously published melanopsin genes in nonmammalian vertebrates represent a separate branch of the melanopsin family which we term Opn4x. RT-PCR analysis in chicken, zebrafish, and Xenopus identifies expression of both Opn4m and Opn4x genes in tissues known to be photosensitive (eye, brain, and skin). In the day-14 chicken eye, Opn4m mRNA is found in a subset of cells in the outer nuclear, inner nuclear, and ganglion cell layers, the vast majority of which also express Opn4x. Importantly, we show that a representative of the new melanopsins (chicken Opn4m) encodes a photosensory pigment capable of activating G protein signalling cascades in a light- and retinaldehyde-dependent manner under heterologous expression in Neuro-2a cells. A comprehensive in silico analysis of vertebrate genomes indicates that while most vertebrate species have both Opn4m and Opn4x genes, the latter is absent from eutherian and, possibly, marsupial mammals, lost in the course of their evolution as a result of chromosomal reorganisation. Thus, our findings show for the first time that nonmammalian vertebrates retain two quite separate melanopsin genes, while mammals have just one. These data raise important questions regarding the functional differences between Opn4x and Opn4m pigments, the associated adaptive advantages for most vertebrate species in retaining both melanopsins, and the implications for mammalian biology of lacking Opn4x.
Assuntos
Galinhas/genética , Evolução Molecular , Células Fotorreceptoras de Vertebrados , Opsinas de Bastonetes/genética , Xenopus laevis/genética , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Filogenia , Retina/química , Opsinas de Bastonetes/química , Opsinas de Bastonetes/fisiologia , Alinhamento de Sequência , Transfecção , VertebradosRESUMO
The use of opioids in the treatment of chronic pain has increased dramatically in the past few decades making them one of the most commonly prescribed medications in the US. However, long-term use of opioids is limited by development of tolerance (decreased antinociceptive efficacy) and opioid-induced hyperalgesia - paradoxical sensitization to noxious (hyperalgesia) and non-noxious (allodynia) stimuli. Novel adjunctive therapies are needed to increase the efficacy and prolong the duration of action of opioids in chronic pain treatment. Acupuncture is often used as an adjunct therapy for the treatment of symptoms induced by non-clinical use of opioids. The National Acupuncture Detoxification Association (NADA) auricular acupuncture protocol is the most common form of acupuncture treatment for substance abuse. The standardized, easy to use and virtually painless procedure make it an attractive complementary treatment option for patients suffering from opioid-induced adverse effects. Clinical trials designed to test the efficacy of the NADA protocol yielded contradictory results. The mechanism by which NADA acupuncture could serve as a successful treatment remains unknown. Therefore, establishing an animal model of NADA acupuncture can provide a tool for investigating the efficacy and cellular mechanisms of NADA treatment. Previous studies have shown that repeated morphine administration in rodents can produce locomotor sensitization and reduce analgesic potency of a challenge dose of morphine, indicating development of morphine tolerance. Here we show that NADA acupuncture treatment can both reduce morphine-induced locomotor sensitization and prevent the development of morphine tolerance in rats, thus validating a new model for NADA acupuncture studies. Our data provides support for evidence-based use of NADA acupuncture as a new adjunctive approach that can potentially improve the side-effect profile of morphine and other prescription opioids.
Assuntos
Analgesia por Acupuntura/métodos , Acupuntura Auricular/métodos , Analgésicos Opioides/administração & dosagem , Tolerância a Medicamentos , Modelos Animais , Morfina/administração & dosagem , Animais , Locomoção/efeitos dos fármacos , Masculino , Limiar da Dor/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
N-methyl-D-aspartate receptor (NMDAR) antagonists have been shown to reduce mechanical hypersensitivity in animal models of inflammatory pain. However, their clinical use is associated with significant dose-limiting side effects. Small-conductance Ca-activated K channels (SK) have been shown to modulate NMDAR activity in the brain. We demonstrate that in vivo activation of SK channels in the spinal cord can alleviate mechanical hypersensitivity in a rat model of inflammatory pain. Intrathecal (i.t.) administration of the SK channel activator, 6,7-dichloro-1H-indole-2,3-dione 3-oxime (NS309), attenuates complete Freund adjuvant (CFA)-induced mechanical hypersensitivity in a dose-dependent manner. Postsynaptic expression of the SK channel subunit, SK3, and apamin-sensitive SK channel-mediated currents recorded from superficial laminae are significantly reduced in the dorsal horn (DH) after CFA. Complete Freund adjuvant-induced decrease in SK-mediated currents can be reversed in vitro by bath application of NS309. In addition, immunostaining for the SK3 subunit indicates that SK3-containing channels within DH neurons can have both somatic and dendritic localization. Double immunostaining shows coexpression of SK3 and NMDAR subunit, NR1, compatible with functional interaction. Moreover, we demonstrate that i.t. coadministration of NS309 with an NMDAR antagonist reduces the dose of NMDAR antagonist, DL-2-amino-5-phosphonopentanoic acid (DL-AP5), required to produce antinociceptive effects in the CFA model. This reduction could attenuate the unwanted side effects associated with NMDAR antagonists, giving this combination potential clinical implications.
Assuntos
Indóis/farmacologia , Inflamação/induzido quimicamente , Oximas/farmacologia , Dor/tratamento farmacológico , Células do Corno Posterior/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Canais de Potássio Ativados por Cálcio de Condutância Baixa/antagonistas & inibidores , Animais , Modelos Animais de Doenças , Adjuvante de Freund/toxicidade , Indóis/administração & dosagem , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/fisiopatologia , Injeções Espinhais , Masculino , Oximas/administração & dosagem , Dor/induzido quimicamente , Dor/metabolismo , Dor/fisiopatologia , Limiar da Dor , Células do Corno Posterior/metabolismo , Ratos , Ratos Sprague-Dawley , Medula Espinal/citologia , Resultado do TratamentoRESUMO
Withdrawal from prescribed opioids results in increased pain sensitivity, which prolongs the treatment. This pain sensitivity is attributed to neuroplastic changes that converge at the spinal cord dorsal horn. We have recently reported that repeated morphine administration triggers an insertion of GluA2-lacking (Ca(2+)-permeable) α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPAR) in the hippocampus. This finding together with the reported involvement of AMPAR in the mechanisms underlying inflammatory pain led us to hypothesize a role for spinal AMPAR in opioid-induced pain behavior. Mice treated with escalating doses of morphine showed hypersensitivity to mechanical stimulation. Intrathecal administration of a Ca(2+)-permeable AMPAR selective blocker disrupted morphine-induced mechanical sensitivity. Analysis of the expression and phosphorylation levels of AMPAR subunits (GluA1/2/3/4) in homogenates and in postsynaptic density fractions from spinal cord dorsal horns showed an increase in GluA4 expression and phosphorylation in the postsynaptic density after morphine. Co-immunoprecipitation analyses suggested an increase in GluA4 homomers (Ca(2+)-permeable AMPAR) and immunohistochemical staining localized the increase in GluA4 levels in laminae III-V. The excitatory postsynaptic currents (EPSCs) recorded in laminae III-V showed enhanced sensitivity to Ca(2+)-permeable AMPAR blockers in morphine-treated mice. Furthermore, current-voltage relationships of AMPAR-mediated EPSCs showed that rectification index (an indicator of Ca(2+)-permeable AMPAR contribution) is increased in morphine-treated but not in saline-treated mice. These effects could be reversed by infusion of GluA4 antibody through patch pipette. This is the first direct evidence for a role of GluA4-containing AMPAR in morphine-induced pain and highlights spinal GluA4-containing AMPAR as targets to prevent the morphine-induced pain sensitivity.