RESUMEN
BACKGROUND: Drugs activating the mu opioid receptor are routinely used to treat severe acute and chronic pain. Unfortunately, side effects including nausea, constipation, respiratory depression, addiction and tolerance can limit clinical utility. In contrast, kappa opioid receptor (KOPr) agonists, such as Salvinorin A (SalA), have analgesic properties with little potential for abuse. METHODS: We evaluated SalA and the novel analogue ß-tetrahydropyran Salvinorin B (ß-THP SalB) for the ability to modulate pain and inflammation in vivo. The hot water tail-withdrawal assay, intradermal formalin-induced inflammatory pain and paclitaxel-induced neuropathic pain models were used to evaluate analgesic properties in mice. Tissue infiltration of inflammatory cells was measured by histology and flow cytometry. RESULTS: ß-tetrahydropyran Salvinorin B produced a longer duration of action in the tail-withdrawal assay compared to the parent compound SalA, and, like SalA and U50,488, ß-THP SalB is a full agonist at the KOPr. In the formalin-induced inflammatory pain model, ß-THP SalB and SalA significantly reduced pain score, paw oedema and limited the infiltration of neutrophils into the inflamed tissue. ß-THP SalB and SalA supressed both mechanical and cold allodynia in the paclitaxel-induced neuropathic pain model, in a dose-dependent manner. CONCLUSIONS: Structural modification of SalA at the C-2 position alters its analgesic potency and efficacy in vivo. Substitution with a tetrahydropyran group at C-2 produced potent analgesic and anti-inflammatory effects, including a reduction in paclitaxel-induced neuropathic pain. This study highlights the potential for KOPr agonists as analgesics with anti-inflammatory action and little risk of abuse. SIGNIFICANCE: Salvinorin A and the novel analogue ß-THP Salvinorin B show analgesic effects in the tail-withdrawal and formalin assays. They reduce oedema and decrease neutrophil infiltration into inflamed tissue, and suppress mechanical and cold allodynia in paclitaxel-induced neuropathic pain.
Asunto(s)
Analgésicos/uso terapéutico , Antiinflamatorios/uso terapéutico , Diterpenos/uso terapéutico , Hiperalgesia/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Neuralgia/tratamiento farmacológico , Receptores Opioides kappa/agonistas , Animales , Diterpenos de Tipo Clerodano , Masculino , RatonesRESUMEN
BACKGROUND: Methamphetamine is a highly addictive central nervous system stimulant with increasing levels of abuse worldwide. Alterations to mRNA and miRNA expression within the mesolimbic system can affect addiction-like behaviors and thus play a role in the development of drug addiction. While many studies have investigated the effects of high-dose methamphetamine, and identified neurotoxic effects, few have looked at the role that persistent changes in gene regulation play following methamphetamine self-administration. Therefore, the aim of this study was to identify RNA changes in the ventral tegmental area following methamphetamine self-administration. We performed microarray analyses on RNA extracted from the ventral tegmental area of Sprague-Dawley rats following methamphetamine self-administration training (2 h/day) and 14 days of abstinence. RESULTS: We identified 78 miRNA and 150 mRNA transcripts that were differentially expressed (fdr adjusted p < 0.05, absolute log2 fold change >0.5); these included genes not previously associated with addiction (miR-125a-5p, miR-145 and Foxa1), loci encoding receptors related to drug addiction behaviors and genes with previously recognized roles in addiction such as miR-124, miR-181a, DAT and Ret. CONCLUSION: This study provides insight into the effects of methamphetamine on RNA expression in a key brain region associated with addiction, highlighting the possibility that persistent changes in the expression of genes with both known and previously unknown roles in addiction occur.
Asunto(s)
Estimulantes del Sistema Nervioso Central/administración & dosificación , Metanfetamina/administración & dosificación , MicroARNs/metabolismo , ARN Mensajero/metabolismo , Área Tegmental Ventral/efectos de los fármacos , Área Tegmental Ventral/metabolismo , Trastornos Relacionados con Anfetaminas/metabolismo , Animales , Catéteres de Permanencia , Comportamiento de Búsqueda de Drogas/fisiología , Masculino , Análisis por Micromatrices , Distribución Aleatoria , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , AutoadministraciónRESUMEN
BACKGROUND AND PURPOSE: Acute activation of κ opioid (KOP) receptors results in anticocaine-like effects, but adverse effects, such as dysphoria, aversion, sedation and depression, limit their clinical development. Salvinorin A, isolated from the plant Salvia divinorum, and its semi-synthetic analogues have been shown to have potent KOP receptor agonist activity and may induce a unique response with similar anticocaine addiction effects as the classic KOP receptor agonists, but with a different side effect profile. EXPERIMENTAL APPROACH: We evaluated the duration of effects of Mesyl Sal Bâ in vivo utilizing antinociception assays and screened for cocaine-prime induced cocaine-seeking behaviour in self-administering rats to predict anti-addiction effects. Cellular transporter uptake assays and in vitro voltammetry were used to assess modulation of dopamine transporter (DAT) function and to investigate transporter trafficking and kinase signalling pathways modulated by KOP receptor agonists. KEY RESULTS: Mesyl Sal B had a longer duration of action than SalA, had anti-addiction properties and increased DAT function in vitro in a KOP receptor-dependent and Pertussis toxin-sensitive manner. These effects on DAT function required ERK1/2 activation. We identified differences between Mesyl Sal B and SalA, with Mesyl Sal B increasing the Vmax of dopamine uptake without altering cell-surface expression of DAT. CONCLUSIONS AND IMPLICATIONS: SalA analogues, such as Mesyl Sal B, have potential for development as anticocaine agents. Further tests are warranted to elucidate the mechanisms by which the novel salvinorin-based neoclerodane diterpene KOP receptor ligands produce both anti-addiction and adverse side effects. LINKED ARTICLES: This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.
Asunto(s)
Trastornos Relacionados con Cocaína/tratamiento farmacológico , Diterpenos/farmacología , Diterpenos/uso terapéutico , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Mesilatos/farmacología , Mesilatos/uso terapéutico , Receptores Opioides kappa/agonistas , Animales , Conducta Animal/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Cocaína/administración & dosificación , Trastornos Relacionados con Cocaína/metabolismo , Diterpenos de Tipo Clerodano , Comportamiento de Búsqueda de Drogas/efectos de los fármacos , Células HEK293 , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratones , Actividad Motora/efectos de los fármacos , Dolor/tratamiento farmacológico , Dolor/metabolismo , Ratas Sprague-Dawley , Receptores Opioides kappa/metabolismo , AutoadministraciónRESUMEN
Primary culture of postnatal brainstem neurons in defined medium has not been described in the literature. Successful primary culture of brainstem neurons is typically restricted to embryonic ages E14-E18. This study describes a method for culture of late fetal and early postnatal brainstem neurons using a serum-free culture medium. The culture system is based on Neurobasal medium supplemented with antioxidant-rich B27 (Life Technologies). Neuron survival was optimized by replacing glutamine with GlutaMaxI, by matching osmolality with neuronal age, and by using Hibernate medium to increase neuron survival during tissue dissociation. This paper describes the first reliable method for culturing brainstem neurons from late fetal and early postnatal stages of the rat for up to 6 days postpartum.
Asunto(s)
Tronco Encefálico/citología , Técnicas de Cultivo de Célula/métodos , Medio de Cultivo Libre de Suero/farmacología , Neuronas/citología , Animales , Animales Recién Nacidos , Supervivencia Celular/efectos de los fármacos , Femenino , Feto/citología , Embarazo , Ratas , Ratas WistarRESUMEN
Serum-free medium is essential for cell culture studies in which complete control of the environment is required. Primary culture of post-natal brainstem neurons in defined medium has not been described in the literature, and successful culture of primary brainstem neurons is typically restricted to embryonic ages E14-E18. This study describes a method for culture of fetal and post-natal brainstem neurons using a serum-free culture medium. The culture system is based on Neurobasal medium supplemented with antioxidant-rich B27. Media and supplements are commercially available products from Life Technologies. Neuron survival was optimized by replacing glutamine with GlutaMaxI, by matching osmolality with neuronal age, and by using Hibernate medium to increase neuron survival during tissue dissociation. Fetal E14, E16, E20, and post-natal P3 and P6 cultures were examined after 4, 7, and 9 days in culture. Neuron and glial cells present in the cultures were identified using immunocytochemistry with antibodies raised against microtubule-associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP), respectively. Fetal E14 cultures had more bipolar neurons than multipolar neurons compared with developmentally older P6 cultures. Early fetal cultures had a higher percentage of neurons than late fetal and early post-natal cultures. Neuron survival was similar between 4 and 9 days in culture for all age groups tested. This is the first reliable, defined culture medium that supports brainstem neurons from late fetal and early post-natal stages of the rat for up to 6 days post-partum.