GENE EXPRESSION AND COCAINE IN PROLONGED ABSTINENCE

尽管许多可卡因成瘾者可以在短时间内戒毒，但在较长的戒毒期内，复吸率非常高，有时超过90%。我们发现，在长期戒断的模型中，大鼠也表现出更多的寻求可卡因的行为倾向。在这个模型中，训练大鼠自我给药2-3周，然后在不允许动物进入自我给药室时进行短期或长期的强制禁欲。在每个戒断期结束时，通过非增强的药物配对杠杆反应和线索诱导的恢复测试来衡量可卡因寻找行为的水平。使用这个模型，大鼠在强制戒毒的1天到6周期间，寻找可卡因的行为表现出时间依赖性的增加。这种“可卡因戒断效应”代表了诱因敏感化的现象，即随着戒除的进行，与药物相关的记忆获得了激励性的突显。拟议中的研究将利用新开发的寡核苷酸微阵列来识别边缘脑区基因表达的变化，这些变化与可卡因寻找行为的时序依赖增加相一致。重要的是，行为模型的设计针对的是基因表达的变化，这些变化与长期戒毒期间药物渴求的增加直接相关，而不是针对暴露于可卡因和短期戒毒所产生的大量变化。微阵列技术的出现将允许同时测量7000多种基因产品，这比以前的表达谱技术有了实质性的改进。基因表达的变化将通过实时荧光聚合酶链式反应和/或蛋白质水平的蛋白质印迹来验证。对最有希望的候选基因的二次分析将试图通过原位杂交和/或单细胞PCR/微阵列策略确定每个广泛区域内发生表达变化的神经元亚群。这一正向遗传策略旨在识别边缘脑区与渴求相关的新的和特定的细胞变化，这些变化随后将确定反向遗传或药理学策略，以研究它们在调控药物寻找行为中的功能作用。这些研究的另一个主要目的是测试早期和晚期“消亡训练”对与长期戒酒相关的分子变化的发展和表达的影响。这一目的是基于我们最近的研究，该研究表明，戒除期间的消退训练除了减弱与可卡因相关的刺激诱导药物寻找的能力外，还逆转或正常化了与可卡因戒断相关的许多神经适应。这些研究将进一步加深我们对药物渴求和环境背景之间复杂相互作用的理解，并可能提出新的治疗行为方法。

Although many cocaine addicts can abstain from drug use for short periods of time, relapse rates at longer periods of abstinence are remarkably high sometimes exceeding 90 percent. We have found that rats also exhibit an increased propensity for cocaine- seeking behavior in a model of prolonged abstinence. In this model, rats are trained to self-administer cocaine for 2-3 weeks, followed by short or long periods of forced abstinence when the animals are not allowed access to the self-administration chambers. At the end of each abstinence period, the level of cocaine-seeking behavior is measured by non-reinforced drug- paired lever responding during extinction and cue-induced reinstatement testing. Using this model, rats exhibit time- dependent increases in cocaine-seeking behavior from 1 day to 6 weeks of forced abstinence. This "Cocaine Abstinence Effect" represents the phenomenon of incentive sensitization, whereby drug-associated memories gain motivational salience as abstinence proceeds. The proposed studies will utilize newly developed oligonucleotide microarrays to identify changes in gene expression in limbic brain regions that coincide with tune-dependent increase in cocaine-seeking behavior. Importantly, the behavioral model is designed to target changes in gene expression that are directly related to increased drug craving during prolonged abstinence, and not the multitude of changes that are produced by cocaine exposure and short-term withdrawal. The advent of microarray technology will allow for more than 7000 gene products to be measured simultaneously, a substantial improvement over previous expression profiling techniques. Changes in gene expression will be verified by real-time PCR and/or at the protein level by western blot. Secondary analysis of the most promising gene candidates will attempt to identify subpopulations of neurons within each broad region where expression changes occur by in situ hybridization and/or single cell PCR/microarray strategies. This forward genetic strategy is aimed at identifying novel and specific craving-related cellular changes in limbic brain regions that subsequently will determine reverse genetic or pharmalogical strategies to study their functional role in regulating drug- seeking behavior. Another major aim of these studies is to test the effects of early and late "extinction training" on both the development and expression of molecular changes associated with prolonged abstinence. This aim is based on our recent studies showing that extinction training during abstinence reverses or normalizes many neuroadaptations associated with cocaine withdrawal, in addition to attenuating the ability of contextual cocaine-related stimuli to elicit drug-seeking. These studies will further our understanding of the complex interaction between drug craving and environmental context, and may suggest novel behavioral approaches to treatment.