EXPRESSION PROFILING OF PSYCHOSTIMULANT-REGULATED GENES

不断发展的DNA微阵列基因表达评估技术将研究规模从单个基因转移到整个基因组，并提供了机会来全面检查不同药物治疗后发生的基因表达变化。这项探索性的拨款申请旨在调整基因微阵列方法，以评估精神兴奋剂药物给药后基因表达的适应性变化。生物胺载体长期以来被认为是滥用的精神兴奋剂药物以及抗抑郁药和用于治疗注意缺陷多动障碍（ADHD）的药物的主要靶标。然而，我们对不同选择性的药物如何抑制转运导致如此惊人而多样的行为后果知之甚少。拟议的研究使用大鼠基因芯片阵列来检查可卡因和最终其他单胺转运抑制剂对关键大脑区域基因表达谱的影响。该提案的第二个目的是研究训练自我施用可卡因的动物的基因表达谱的差异。我们没有为自己开发DNA阵列（我们更愿意将这项任务留给工程师），而是选择了最先进的、从Affytron/TM获得的商业可用阵列作为并行分析10/4-10/5不同基因表达的最有效和可靠的方法。该系统使用在玻璃基板上合成的基因特异性寡核苷酸的微阵列，其与代表感兴趣的组织中表达的序列的生物素化探针杂交。比较表达谱将使用一套可用于分析基因芯片数据的程序来完成。将使用常规方法包括北方印迹分析和原位杂交来确认在微阵列上观察到的表达变化。最后一个目标是比较使用不同类型的转运抑制剂获得的表达谱的全球变化。后一项研究将评估阵列技术在确定作用于相关靶点的不同类别药物的“签名”基因表达谱方面的效用。从全球基因组的角度理解药物的共同和独特作用，应该使我们能够描述和理解其深刻的行为后果背后的机制。

The evolving technologies for assessing gene expression on DNA microarrays shift the scale of study from single genes to whole genomes and provide opportunities to globally examine the alterations in gene expression that occur following different drug treatments. This exploratory grant application aims to adapt gene microarray approaches to assess the adaptive changes in gene expression that take place after psychostimulant drug administration. The biogenic amine carriers have long been recognized as the primary targets for psychostimulant drugs of abuse, as well as anti-depressants, and drugs used to treat attention deficit hyperactivity disorder (ADHD). However, we know relatively little about how inhibition of transport by drugs with varying selectivities leads to such striking yet diverse behavioral consequences. The proposed studies use rat gene chip arrays to examine the impact of cocaine and eventually other monoamine transport inhibitors on gene expression profiles in key brain regions. A second aim of the proposal will examine differences in the profiles of gene expression in animals trained to self-administer cocaine. Rather than developing the DNA arrays for ourselves (a task we prefer to leave to engineers), we have chosen state-of-the-art, commercially available arrays obtained from Affymetrix/TM as the most efficient and reliable method for analyzing the expression of 10/4-10/5 different genes in parallel. The system uses microarrays of gene-specific oligonucleotides synthesized on a glass substrate which are hybridized with biotinylated probes representing the sequences expressed in the tissues of interest. Comparisons expression profiles will be accomplishes using a suite of programs available for the analysis of gene chip data. Changes in expression observed on microarrays will be confirmed using conventional approaches including Northern blotting analyses and in situ hybridization. A final aim will compare global changes in the expression profiles obtained using different classes of transport inhibitors. This latter study will assess the utility of array technology in determining "signature" gene expression profiles for different classes of drugs that act on related targets. Understanding the common and unique actions of drugs from a global, genomic perspective should enable us to characterize and understand the mechanisms that underlie their profound behavioral consequences.