MOLECULAR BASIS OF OPIOID PHARMACODYNAMICS

这是一份申请ADAMHA研究科学家奖的申请。应用 要求资金，让我继续收购的技术， 分子生物学和神经科学及其在2个项目中的应用。 第一个项目将通过溶液杂交、北方印迹 原位杂交和免疫细胞化学技术 和前脑啡肽（Penk）前强啡肽NMDA受体的肽水平， 一氧化氮合酶、c-fos和酪氨酸羟化酶在15个选定地区 与阿片类镇痛、耐受和依赖有关。这些 将在耐受和/或依赖以下药物的动物中进行研究： μ、κ和δ阿片类药物以及在存在和不存在药物的情况下 （受体拮抗剂和一氧化氮合酶抑制剂）， 减弱或防止阿片耐受性和依赖性的发展。 此外，将使用消减杂交方法克隆 并识别因阿片耐受性和依赖性而改变的蛋白质。 第二个项目将定义包括膜在内的因素 去极化，第二信使系统和糖皮质激素，调节 仓鼠、大鼠和人类的Penk基因。糖皮质激素反应元件 仓鼠、大鼠和人Penk基因将通过克隆选择 DNA片段 这些项目需要获得和应用一些 分子和神经科学技术包括原位杂交， 消减杂交，组织培养，融合基因构建，基因 转移、凝胶迁移率测定和转基因分析。的 这些技术的获得将来自与 在这些领域都有专业知识的科学家， 支持这些技术的获取和转移到我的实验室。 这个RSA奖将使我有机会减少我的教学， 行政责任，并增加我可以投入的时间 提高我的科学技能

This is a request for an ADAMHA Research Scientist Award. The application requests funds to allow me to pursue the acquisition of techniques of molecular biology and neuroscience and their application to 2 projects. The first project will examine by solution hybridization, Northern blot analysis, in situ hybridization and immunocytochemical techniques the mRNA and peptide levels for proenkephalin (Penk) prodynorphin NMDA receptor, nitric oxide synthase, c-fos and tyrosine hydroxylase in 15 select areas that are associated with opioid analgesia, tolerance and dependence. These studies will be conducted in animals made tolerant and/or dependent with mu, kappa and delta opioids and in the presence and absence of drugs (receptor antagonists and nitric oxide synthase inhibitors) which attenuate or prevent the development of opioid tolerance and dependence. In addition, a subtractive hybridization approach will be used to clone and identify proteins altered by opioid tolerance and dependence. The second project will define the factors including membrane depolarizing,second messenger systems and glucocorticoid that regulate the hamster, rat and human Penk genes. Glucocorticoid response elements in the hamster, rat and human Penk genes will be identified by cloning selected DNA fragments. These projects require the acquisition and application of a number of molecular and neuroscience techniques including in situ hybridization, subtractive hybridization, tissue culture, fusion gene construction, gene transfer, gel-shift mobility assays and transgenic analysis. The acquisition of these techniques will result from collaborations with scientists with expertise in each of these areas who have agreed to support the acquisition and transfer of these techniques to my laboratory. This RSA award will allow me the opportunity to reduce my teaching and administrative responsibilities and increase the time I can devote to enhancing my scientific skills.