EXPRESSION PROFILING OF PSYCHOSTIMULANT-REGULATED GENES

在DNA微阵列上评估基因表达的不断发展的技术将研究范围从单基因转移到全基因组，并提供了在全球范围内检查不同药物治疗后基因表达变化的机会。这项探索性拨款申请旨在采用基因微阵列方法来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一直被认为是滥用精神兴奋剂、抗抑郁药和治疗注意缺陷多动障碍（ADHD）药物的主要靶点。然而，我们对具有不同选择性的药物如何抑制运输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其他单胺运输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经过自我使用可卡因训练的动物基因表达谱的差异。我们选择了从Affymetrix/TM获得的最先进的、可商用的DNA阵列，而不是为自己开发DNA阵列（一项我们宁愿留给工程师的任务），作为并行分析10/4-10/5个不同基因表达的最有效和可靠的方法。该系统使用在玻璃底物上合成的基因特异性寡核苷酸微阵列，与生物素化探针杂交，代表感兴趣组织中表达的序列。比较表达谱将完成使用一套程序可用于基因芯片数据的分析。在微阵列上观察到的表达变化将使用包括Northern印迹分析和原位杂交在内的常规方法进行确认。最终目的是比较使用不同类型的转运抑制剂获得的表达谱的全局变化。后一项研究将评估阵列技术在确定作用于相关靶标的不同类别药物的“特征”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表征和理解其深刻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.