GENE EXPRESSION AND COCAINE IN PROLONGED ABSTINENCE

虽然许多可卡因成瘾者可以在短时间内戒断毒品，但在较长时间的戒断后，复发率非常高，有时超过90%。 我们发现，在长期禁欲的模型中，大鼠也表现出增加的可卡因寻求行为倾向。 在该模型中，训练大鼠自我施用可卡因2-3周，然后当动物不被允许进入自我施用室时，进行短时间或长时间的强迫禁欲。 在每个戒断期结束时，通过在消退和线索诱导的恢复测试期间的非增强药物配对杠杆反应来测量可卡因寻求行为的水平。 使用该模型，大鼠在强制戒断1天至6周期间表现出可卡因寻求行为的时间依赖性增加。 这种“药物戒断效应”代表了激励敏感化现象，即随着戒断的进行，与药物相关的记忆获得了激励性的显著性。拟议的研究将利用新开发的寡核苷酸微阵列来确定边缘脑区域基因表达的变化，这些变化与可卡因寻求行为的调谐依赖性增加相一致。 重要的是，行为模型的设计目标是在长期禁欲期间与增加药物渴望直接相关的基因表达变化，而不是可卡因暴露和短期戒断产生的众多变化。 微阵列技术的出现将允许同时测量超过7000个基因产物，这是对先前表达谱分析技术的实质性改进。 将通过实时PCR和/或蛋白质印迹法在蛋白质水平验证基因表达的变化。 最有希望的候选基因的二次分析将试图确定在每个广泛的区域内的神经元亚群的表达变化发生的原位杂交和/或单细胞PCR/微阵列策略。这种正向遗传策略旨在鉴定边缘脑区域中新的和特异性的与渴望相关的细胞变化，其随后将确定反向遗传或药理学策略以研究它们在调节药物寻求行为中的功能作用。这些研究的另一个主要目的是测试早期和晚期“灭绝训练”对与长期禁欲相关的分子变化的发展和表达的影响。 这一目标是基于我们最近的研究表明，戒断期间的消退训练逆转或正常化了许多与可卡因戒断相关的神经适应，除了减弱背景可卡因相关刺激引起药物寻求的能力。 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，并可能提出新的行为治疗方法。

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.