CORE--SCIENTIFIC CORE

可卡因和安非他明的许多急性和慢性作用是 被认为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脑桥核中。 在分子 水平，这些精神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途径， 其调节cAMP依赖性蛋白激酶（PKA）的活性。 一 基底神经节富集PKA底物家族的成员，称为 DARPP-32（多巴胺和环AMP调节的磷蛋白，M = 32，000） 在调节PKA的下游效应中起着核心作用- 依赖的途径，通过其能力，在磷酸化，以抑制 蛋白磷酸酶-1（PP-1）的活性。 确定和 这种D1/PKA/DARPP-32/PP-1的特异性靶标的表征 磷酸化级联，其磷酸化状态受 可卡因和安非他明的管理，将有助于更大的 了解这些滥用药物的作用方式，并可能导致 涉及用于治疗药物成瘾的新的治疗靶点。 一个多- 计划项目将采取纪律措施， 研究可卡因和苯丙胺对磷酸化的影响 这些多巴胺能神经元中关键分子状态和功能调节 信号通路 研究将在以下几个水平进行： 组织复杂性，包括体外生物化学研究， 纯化的分子，细胞系统的研究，以及 完整的动物，旨在识别和表征药物- 诱导的生化和行为表型之间的野生型和 基因敲除小鼠，携带关键效应分子的靶向缺失， D1/PKA磷酸化级联反应。 科学的总体目标 核心是为该计划的所有成员提供一系列支持 项目 核心的一个研究部分将产生小鼠品系， 将使得能够组织特异性诱导靶向缺失（“敲除”） 关键效应分子的基因，包括PP-1抑制剂、DARPP-32 以及DARPP家族成员DARPP-21和DARPP-16 （具体目标1）。 为了确保有足够的老鼠供应，核心将 为所有转基因的菌落提供育种、维护和基因分型 小鼠，以及常规和组织特异性诱导型敲除（“TIKO”） 生产和研究小鼠品系（具体目标2）。 芯会 维持关键试剂的库存，包括纯化的PKA和蛋白质 磷酸酶、DARPP-32和抗体，包括磷酸化状态- 特异性抗体，并将产生额外的抗体， 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).