CORE--SCIENTIFIC CORE

可卡因和安非他明的许多急性和慢性作用是 据信是由增强的多巴胺能神经传递介导的， 特别是在新纹状体和伏隔核。在分子层面 水平，这些精神运动兴奋剂的影响似乎涉及 激活D1和/或D2受体介导的信号转导通路， 它们调节cAMP依赖的蛋白激酶(PKA)的活性。一 富含基底节的PKA底物家族的成员，称为 DARPP-32(多巴胺和环磷酸腺苷调节的磷蛋白，M=32,000) 在调节这种PKA的下游效应中发挥核心作用- 依赖途径通过它的能力，在磷酸化，以抑制 蛋白磷酸酶-1(PP-1)活性。身份识别和 D1/PKA/DARPP-32/PP-1特异性靶标的表征 磷化物级联，其磷化态受 管理可卡因和安非他明，将有助于更大 了解这些药物的滥用行为模式，并可能导致 为治疗药物成瘾提供新的治疗靶点。一个多- 计划项目将采取纪律方法来 研究可卡因和安非他明对磷酸化的影响 这些多巴胺能系统中关键分子的状态和功能调节 信号通路。研究将在几个层面上进行 组织复杂性，包括体外生化研究 纯化的分子、细胞系统的研究和比较研究 完整的动物被设计来识别和表征药物的差异- 野生型与野生型的诱导生化和行为表型 基因敲除小鼠，携带靶向缺失的关键效应分子 D1/PKA磷酸化级联反应。科学发展观的总体目标 核心是向该方案的所有成员提供一系列支持 项目。Core的一个研究组件将生产鼠标线， 将实现组织特异性可诱导的靶向缺失(敲除) 关键效应分子的基因，包括PP-1抑制剂DARPP-32 和抑制物-1，以及DARPP家族成员DARPP-21和DARPP-16 (具体目标1)。为确保有足够的会展中心供应，中心将 为所有转基因群体提供育种、维护和基因分型 小鼠，以及常规和组织特异性的可诱导基因敲除(“TIKO”) 要培育和研究的小鼠品系(特定目标2)。核心意志 保持关键试剂的库存，包括纯化的PKA和蛋白质 磷酸酶，DARPP-32和抗体，包括磷酸盐状态- 特异性抗体，并将根据需要产生额外的抗体 支持其他项目(具体目标3)。

Many of the acute and chronic actions of cocaine and amphetamines are believed to be mediated by enhanced dopaminergic neurotransmission, particularly in the neostriatum and nucleus accumbens. At the molecular level, the effects of these psychomotor stimulants appear to involve activation of D1 and/or D2 receptor-mediated signal transduction pathways, which regulate the activity of cAMP-dependent protein kinase (PKA). One member of a family of basal ganglia-enriched PKA substrates, known as DARPP-32 (dopamine- and cyclic AMP-regulated phosphoproteins, M,=32,000) plays a central role in the regulation of downstream effects of this PKA- dependent pathway through its ability, upon phosphorylation, to inhibit the activity of protein phosphatase-1 (PP-1). The identification and characterization of specific targets of this D1/PKA/DARPP-32/PP-1 phosphorylaiton cascade, whose phosphorylaiton state is affected by administration of cocaine and amphetamines, will contribute to a greater understanding of the mode of action of these drugs of abuse, and may lead to novel therapeutic targets for the treatment of drug addiction. A multi- disciplinary approach will be undertaken by the Program Project to investigate the effects of cocaine and amphetamine on the phosphorylation state and functional regulation of key molecules in these dopaminergic signaling pathways. Studies will be performed at several levels of organizational complexity, encompassing in vitro biochemical studies with purified molecules, studies in cellular systems, and comparative studies in intact animals designed to identify and characterize differences in drug- induced biochemical and behavioral phenotypes between wild-type and knockout mice, harboring targeted deletions of key effector molecules in the D1/PKA phosphorylation cascade. The overall goal of the Scientific Core is to provide a range of support to all members of the Program Project. A research component of the Core will produce mouse lines which will enable tissue-specific inducible targeted deletion ("knockout") of the genes for key effector molecules, including the PP-1 inhibitors, DARPP-32 and inhibitor-1, and the DARPP family members, DARPP-21 and DARPP-16 (Specific Aim 1). To ensure adequate supplies of mice, the Core will provide breeding, maintenance and genotyping for all colonies of transgenic mice, and the conventional and tissue-specific inducible knockout ("TIKO") mouse lines to be produced and studied (Specific Aim 2). The Core will maintain stocks of key reagents, including purified PKA and protein phosphatases, DARPP-32 and antibodies, including phosphorylaiton state- specific antibodies, and will produce additional antibodies as required to support the other Projects (Specific Aim 3).