RESUMEN
Methamphetamine (METH) is a psychostimulant drug with significant abuse potential and neurotoxic effects. A high percentage of users relapse to use after detoxification and no effective medication has been developed for treatment of METH addiction. Developing evidences indicated the role of glial cells in drugs abused related phenomena. However, little is known about the role of these cells in the maintenance and reinstatement of METH-seeking behaviors. Therefore, the current study was conducted to clarify the role of glial cells in the maintenance and reinstatement of METH-induced conditioned place preference (CPP) in rats. Astrocyte condition medium (ACM) and neuroglia conditioned medium (NCM) are liquid mediums prepared from primary astrocyte and neuroglia cells. These mediums seem to contain many factors that release by glia cells. CPP was induced by systemic administration of METH (1mg/kg for 5days, s.c.). Following the establishment of CPP, the rats were given daily bilateral injections (0.5µl/side) of either vehicle, ACM or NCM into the nucleus accumbens (NAc) and then were tested for the maintenance and reinstatement. Intra-NAc administration of ACM treated with METH, could extend the extinction period and also, intensified the magnitude of METH reinstatement. Furthermore, intra-accumbal administration of NCM treated with METH notably delayed the extinction period by four days and significantly increased the magnitude of CPP score in the reinstatement phase compared to the post-test phase. Collectively, these findings suggested that activation of glial cells may be involved in the maintenance and reinstatement of METH-seeking behaviors. It provides new evidence that glia cells might be considered as a potential target for the treatment of METH addiction.
Asunto(s)
Condicionamiento Operante/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Extinción Psicológica/efectos de los fármacos , Núcleo Accumbens/efectos de los fármacos , Animales , Animales Recién Nacidos , Estimulantes del Sistema Nervioso Central/farmacología , Corteza Cerebral/citología , Medios de Cultivo Condicionados/química , Medios de Cultivo Condicionados/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Masculino , Metanfetamina/farmacología , Microinyecciones , Neuroglía/química , Neuroglía/metabolismo , Núcleo Accumbens/fisiología , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Methamphetamine (METH) is a major criminal justice and public health problem. Repeated use of METH causes dependence in humans and there are currently no particular pharmacological treatments for METH addiction. Glial cell activation is linked with METH abuse and METH administration causes activation of these cells in many areas of the brain. Many studies have demonstrated that glial cell modulators can modulate drug abuse effects. In this study, we examined the effect of the putative microglial inhibitor, minocycline on maintenance and prime-induced reinstatement of METH seeking behavior using the conditioned place preference (CPP) paradigm. CPP induced with METH (1 mg/kg, i.p. for 3 days) lasted for 11 days after cessation of METH treatment and priming dose of METH (0.5 mg/kg, i.p.) reinstated the extinguished METH-induced CPP. Daily treatment of minocycline (40 mg/kg, i.p.) followed by establishment of CPP blocked the maintenance of METH-induced CPP and also could attenuate priming-induced reinstatement. Furthermore, daily bilateral intra-accumbal injection of minocycline (10 and 20 µg/0.5 µl saline), during extinction period blocked the maintenance of METH CPP but just the highest dose of that could attenuate priming-induced reinstatement. We showed that minocycline administration during extinction period could facilitate extinction and maybe abolish the ability of drug-related cues evoke reinstatement, suggesting that minocycline might be considered as a promising therapeutic agent in preventing relapse in METH dependent individuals.
Asunto(s)
Antibacterianos/farmacología , Estimulantes del Sistema Nervioso Central/efectos adversos , Condicionamiento Operante/efectos de los fármacos , Comportamiento de Búsqueda de Drogas/efectos de los fármacos , Metanfetamina/efectos adversos , Minociclina/farmacología , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Extinción Psicológica/efectos de los fármacos , Masculino , Ratas , Ratas Wistar , Refuerzo en Psicología , Trastornos Relacionados con Sustancias/tratamiento farmacológicoRESUMEN
Addiction is a common psychiatric disease and stress has an important role in the drug seeking and relapse behaviors. The involvement of basolateral amygdala (BLA) in the effects of stress on reward pathway is discussed in several studies. In this study, we tried to find out the involvement of glucocorticoid receptors (GRs) in the BLA in stress-induced reinstatement of extinguished morphine-induced conditioned place preference (CPP) in rats. The CPP paradigm was done in adult male Wistar rats weighing 220-320 g, and conditioning score and locomotor activity were recorded by Ethovision software. Animals received effective dose of morphine (5mg/kg) daily, during the 3-day conditioning phase. In extinction phase, rats were put in the CPP box for 30 min a day for 8 days. After extinction, animals were injected by corticosterone (10 m/kg) or exposed to forced swim stress (FSS) 10 min before subcutaneous administration of ineffective dose of morphine (0.5mg/kg) in order to reinstate the extinguished morphine-CPP. To block the glucocorticoid receptors in the BLA, after stereotaxic surgery and placing two cannulae in this area bilaterally, animals received GR antagonist mifepristone (RU38486; 0.3, 3 and 30 ng/0.3 µl DMSO per side) prior to exposure to FSS then each animal received ineffective dose of morphine (0.5mg/kg) as drug-induced reinstatement. The results revealed that physical stress (FSS) but not exogenous corticosterone can significantly induce reinstatement of extinguished morphine-CPP, and intra-BLA mifepristone prevents the stress-induced reinstatement. It can be proposed that stress partially exerts its effect on the reward pathway via glucocorticoid receptors in the BLA.
Asunto(s)
Amígdala del Cerebelo/metabolismo , Condicionamiento Operante/fisiología , Extinción Psicológica/fisiología , Morfina/administración & dosificación , Narcóticos/administración & dosificación , Receptores de Glucocorticoides/metabolismo , Estrés Psicológico/psicología , Natación/psicología , Amígdala del Cerebelo/efectos de los fármacos , Análisis de Varianza , Animales , Condicionamiento Operante/efectos de los fármacos , Corticosterona/farmacología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Extinción Psicológica/efectos de los fármacos , Antagonistas de Hormonas/farmacología , Masculino , Mifepristona/farmacología , Ratas , Ratas Wistar , Estrés Psicológico/etiologíaRESUMEN
Addiction is a common chronic psychiatric disease which represents a global problem and stress has an important role to increase drug addiction and relapse. In the present study, we investigated the effects of physical stress and exogenous corticosterone on the acquisition and expression of morphine-induced conditioned place preference (CPP). Also, we tried to find out the role of glucocorticoid receptors (GRs) of basolateral amygdala (BLA) in this regard. In the CPP paradigm, conditioning score and locomotion activity were recorded by Ethovision software. Male adult rats received forced swim stress (FSS) as a physical stress or corticosterone (10 mg/kg; ip) as a dominant stress hormone in rodents, 10min before morphine injection (5 mg/kg; sc) during three conditioning days (acquisition) or just prior to CPP test in the post-conditioning day (expression). In FSS procedure, animals were forced to swim for 6 min in cylinder filled with water (24-27 °C). To evaluate the role of glucocorticoid receptors in the BLA, different doses of mifepristone (RU38486) as a GR antagonist were injected into the BLA (0.3, 3 and 30 ng/side) during 3-day conditioning phase before FSS or injection of corticosterone in morphine-CPP paradigm. The results showed that FSS and corticosterone reduce the acquisition but not expression of morphine-induced CPP. Moreover, blockade of GRs in the BLA could diminish the inhibitory effects of FSS or corticosterone on the acquisition of morphine-induced CPP. It seems that stress exerts its effect on reward pathway via glucocorticoid receptors in the BLA.
Asunto(s)
Amígdala del Cerebelo/metabolismo , Condicionamiento Operante/efectos de los fármacos , Corticosterona/metabolismo , Morfina/farmacología , Narcóticos/farmacología , Receptores de Glucocorticoides/metabolismo , Estrés Psicológico/fisiopatología , Amígdala del Cerebelo/efectos de los fármacos , Análisis de Varianza , Animales , Corticosterona/farmacología , Antagonistas de Hormonas/farmacología , Masculino , Mifepristona/farmacología , Actividad Motora/efectos de los fármacos , Ratas , Ratas Wistar , Natación/psicologíaRESUMEN
RNA interference (RNAi) is an important mosquito defense mechanism against arbovirus infection. In this paper we study the processes underlying antiviral RNAi in Aedes albopictus-derived U4.4 mosquito cells infected with Semliki Forest virus (SFV) (Togaviridae; Alphavirus). The production of virus-derived small interfering RNAs (viRNAs) from viral double-stranded RNA (dsRNA) is a key event in this host response. dsRNA could be formed by RNA replication intermediates, by secondary structures in RNA genomes or antigenomes, or by both. Which of these dsRNAs is the substrate for the generation of viRNAs is a fundamental question. Here we used deep sequencing of viRNAs and bioinformatic analysis of RNA secondary structures to gain insights into the characteristics and origins of viRNAs. An asymmetric distribution of SFV-derived viRNAs with notable areas of high-level viRNA production (hot spots) and no or a low frequency of viRNA production (cold spots) along the length of the viral genome with a slight bias toward the production of genome-derived viRNAs over antigenome-derived viRNAs was observed. Bioinformatic analysis suggests that hot spots of viRNA production are rarely but not generally associated with putative secondary structures in the SFV genome, suggesting that most viRNAs are derived from replicative dsRNA. A pattern of viRNAs almost identical to those of A. albopictus cells was observed for Aedes aegypti-derived Aag2 cells, suggesting common mechanisms that lead to viRNA production. Hot-spot viRNAs were found to be significantly less efficient at mediating antiviral RNAi than cold-spot viRNAs, pointing toward a nucleic acid-based viral decoy mechanism to evade the RNAi response.
Asunto(s)
Aedes/fisiología , Aedes/virología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Virus de los Bosques Semliki/crecimiento & desarrollo , Aedes/inmunología , Animales , Línea Celular , ARN Bicatenario/genética , ARN Bicatenario/metabolismo , ARN Interferente Pequeño/genética , ARN Viral/genética , ARN Viral/metabolismo , Virus de los Bosques Semliki/genéticaRESUMEN
Arthropod-borne viruses - arboviruses - are a significant threat to public health. Whilst there is considerable knowledge about arbovirus interactions with vertebrate immunity, relatively little is known about how vectors such as mosquitoes control arbovirus infections. In this review, we discuss novel findings in the field of mosquito antiviral responses to arboviruses, in particular RNA interference, the up-and-coming field of general immune-signalling pathways, and cell death/apoptosis.
Asunto(s)
Infecciones por Arbovirus/virología , Arbovirus/inmunología , Culicidae/inmunología , Culicidae/virología , Inmunidad Innata , Insectos Vectores/inmunología , Animales , Infecciones por Arbovirus/transmisión , Culicidae/genética , Humanos , Insectos Vectores/genética , Insectos Vectores/virologíaRESUMEN
In their vertebrate hosts, arboviruses such as Semliki Forest virus (SFV) (Togaviridae) generally counteract innate defenses and trigger cell death. In contrast, in mosquito cells, following an early phase of efficient virus production, a persistent infection with low levels of virus production is established. Whether arboviruses counteract RNA interference (RNAi), which provides an important antiviral defense system in mosquitoes, is an important question. Here we show that in Aedes albopictus-derived mosquito cells, SFV cannot prevent the establishment of an antiviral RNAi response or prevent the spread of protective antiviral double-stranded RNA/small interfering RNA (siRNA) from cell to cell, which can inhibit the replication of incoming virus. The expression of tombusvirus siRNA-binding protein p19 by SFV strongly enhanced virus spread between cultured cells rather than virus replication in initially infected cells. Our results indicate that the spread of the RNAi signal contributes to limiting virus dissemination.
Asunto(s)
Culicidae/virología , Interferencia de ARN , Virus de los Bosques Semliki/genética , Animales , Línea Celular , Cricetinae , Regulación Viral de la Expresión Génica , ARN Viral/genética , Replicación ViralRESUMEN
The double-stranded RNA-activated protein kinase (PKR) is a key regulator of protein translation, interferon (IFN) expression and cell survival. Upon infection of vertebrate cells in continuous culture, the alphavirus Semliki Forest virus (SFV) initiates apoptosis and IFN synthesis. To determine the effect of PKR on SFV infection, we studied the course of infection in wild-type (wt) mice, mice with a genetic deletion of PKR (PKR-/-) and mouse embryo fibroblasts (MEFs) derived from these mice. In MEFs, PKR delayed virus protein synthesis, production of infectious virus and caspase-3-activated cell death and reduced the yield of infectious virus by 90%. Small interfering RNA suppression of PKR levels in NIH-3T3 cells also reduced virus production and apoptosis. In MEFs, PKR was not required for initiation of IFN-beta gene transcription, but contributed strongly to the magnitude of this response. Levels of IFN-beta transcripts in PKR-/- MEFs at 8 h were 80% lower than those in wt MEFs and levels of functional IFN at 24 h were 95% lower. Following infection of wt and PKR-/- mice, SFV4 and SFV A7(74) were avirulent. PKR increased levels of serum IFN and the rate of clearance of infectious virus from the brain. In summary, in response to SFV, PKR exerts an early antiviral effect that delays virus protein production and release of infectious virus and, whilst PKR is not required for induction of apoptosis or activation of the type I IFN response, it strongly augments the type I IFN response and contributes to clearance of infectious virus from the mouse brain.
Asunto(s)
Infecciones por Alphavirus/virología , Interferón Tipo I/inmunología , Virus de los Bosques Semliki/inmunología , eIF-2 Quinasa/metabolismo , Animales , Encéfalo/virología , Células Cultivadas , Fibroblastos/virología , Interferón Tipo I/sangre , Ratones , Ratones Noqueados , eIF-2 Quinasa/deficienciaRESUMEN
The molecular and cellular basis of the psychotropic actions of adrenal corticosteroids is poorly understood. Previously, we reported that modulation of large conductance Ca2+-activated potassium channel (BK-channel) function by glucocorticoids can be recapitulated in human embryonic kidney293 (HEK293) cells (J Physiol 537:57, 2001). In the present paper, we examined the effect of dexamethasone on the expression of candidate mediator proteins of glucocorticoid action, dex-ras1 and serum and glucocorticoid inducible protein kinase 1 (SGK), in HEK293 cells. Dex-ras1 mRNA was readily detectable under basal conditions however, no changes of dex-ras1 mRNA expression occurred upon exposure to 100 nM of dexamethasone for 2 h. In contrast, a 2.5-fold increase of SGK mRNA was found under similar conditions. Total levels of cellular SGK protein were unaltered upon exposure to dexamethasone, but a marked increase of SGK in a Triton-X100 insoluble fraction was observed. BK-channel alpha-subunits could not be co-immunoprecipitated with SGK. In summary, SGK, but not dex-ras1, mRNA is rapidly induced by glucocorticoid stimulation in HEK293 cells. However, there appears to be no direct protein-protein interaction between SGK and BK-channel alpha-subunits.
Asunto(s)
Dexametasona/farmacología , Proteínas Inmediatas-Precoces/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas ras/genética , Northern Blotting , Línea Celular Tumoral , Genes Reporteros/efectos de los fármacos , Genes Reporteros/genética , Humanos , Inmunoprecipitación , Luciferasas/genética , Canales de Potasio Calcio-Activados/efectos de los fármacos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
La Crosse virus (LACV) is a mosquito-transmitted member of the Bunyaviridae family that causes severe encephalitis in children. For the LACV nonstructural protein NSs, previous overexpression studies with mammalian cells had suggested two different functions, namely induction of apoptosis and inhibition of RNA interference (RNAi). Here, we demonstrate that mosquito cells persistently infected with LACV do not undergo apoptosis and mount a specific RNAi response. Recombinant viruses that either express (rLACV) or lack (rLACVdelNSs) the NSs gene similarly persisted and were prone to the RNAi-mediated resistance to superinfection. Furthermore, in mosquito cells overexpressed LACV NSs was unable to inhibit RNAi against Semliki Forest virus. In mammalian cells, however, the rLACVdelNSs mutant virus strongly activated the antiviral type I interferon (IFN) system, whereas rLACV as well as overexpressed NSs suppressed IFN induction. Consequently, rLACVdelNSs was attenuated in IFN-competent mouse embryo fibroblasts and animals but not in systems lacking the type I IFN receptor. In situ analyses of mouse brains demonstrated that wild-type and mutant LACV mainly infect neuronal cells and that NSs is able to suppress IFN induction in the central nervous system. Thus, our data suggest little relevance of the NSs-induced apoptosis or RNAi inhibition for growth or pathogenesis of LACV in the mammalian host and indicate that NSs has no function in the insect vector. Since deletion of the viral NSs gene can be fully complemented by inactivation of the host's IFN system, we propose that the major biological function of NSs is suppression of the mammalian innate immune response.