Exploiting mouse models to understand female hypersensitivity to cocaine

描述（由申请人提供）：从最初的生理反应、获得药物寻求或自我给药行为的速度以及复发倾向的强弱来判断，女性和雌性实验动物对可卡因的敏感性远远高于男性和雄性实验动物。众所周知，女性在许多情感和认知反应上与男性不同，但我们对这些差异的分子基础的理解有限。我们将使用深度测序来深入了解在女性和男性中观察到的对可卡因戒断的不同反应的机制。我们之前将这种方法应用于从实验者给药的可卡因中退出的雄性小鼠伏隔核，并确定了Wnt/cadherin途径和miR-8家族成员是关键参与者。特异性通路的靶向分析揭示了可卡因介导的伏隔核中编码多巴胺、谷氨酸、GABA、乙酰胆碱、神经肽和内源性大麻素信号通路多个组分的mrna表达的变化。在第1项实验中，将对四组小鼠进行研究：雄性、雌性、去卵巢的雌性和雌二醇替代去卵巢的雌性。小鼠注射生理盐水或可卡因一周，停药四周后处死；伏隔核，前额叶皮层和腹侧被盖区将被收集用于制备RNA。从表现出运动敏化的小鼠伏隔核中制备的重复条形码文库将同时测序，并进行技术复制。生物信息学分析将用于确定所有群体共同的可卡因反应转录本和途径（核心可卡因反应），这是女性独有的，对雌激素敏感。在目标2中，我们将从核心可卡因反应组和女性敏感组中选择转录本和途径进行验证和进一步分析。该数据集将允许分析性别、雌激素和可卡因对选择性剪接和RNA编辑的影响。生物信息学分析雌激素反应元件和转录因子结合位点的启动子区域的可卡因反应基因将进行。有了这个高质量的、经过验证的数据集，集中的测序研究可以用来分析个体小鼠自我施用可卡因的反应。这种广泛的方法应该可以确定女性独有的或对雌激素敏感的治疗靶点。

DESCRIPTION (provided by applicant): Judging by initial physiological responses, rapidity of acquisition of drug-seeking or self-administration behavior, and strength of tendency to relapse, women and female laboratory animals are far more sensitive to cocaine than men and male laboratory animals. While it is well known that women differ from men in many emotional and cognitive responses, our understanding of the molecular underpinnings of these differences is limited. We will use deep sequencing to provide insight into the mechanisms that contribute to the different responses to withdrawal from cocaine observed in females and males. We previously applied this approach to the nucleus accumbens of male mice withdrawing from experimenter administered cocaine and identified the Wnt/cadherin pathway and miR-8 family members as key players. Targeted analysis of specific pathways revealed cocaine-mediated changes in the expression of mRNAs encoding multiple components of the dopamine, glutamate, GABA, acetylcholine, neuropeptide and endocannabinoid signaling pathways in the nucleus accumbens. In Aim 1, four groups of mice will be examined: males, cycling females, ovariectomized females and estradiol replaced ovariectomized females. Mice injected with saline or cocaine for a week will be sacrificed after four weeks of withdrawal; nucleus accumbens, prefrontal cortex and ventral tegmental area will be harvested for preparation of RNA. Duplicate bar-coded libraries prepared from the nucleus accumbens of mice exhibiting locomotor sensitization will be sequenced simultaneously, with technical replicates. Bioinformatic analysis will be used to identify cocaine-responsive transcripts and pathways common to all groups (core cocaine response), unique to females and sensitive to estrogen. In Aim 2 we will select transcripts and pathways from the core cocaine response group and female sensitivity group for validation and further analysis. The data set will allow analysis of the effets of sex, estrogen and cocaine on alternative splicing and RNA editing. Bioinformatic analysis of estrogen responsive elements and transcription factor binding sites in the promoter regions of cocaine-responsive genes will be undertaken. With this high quality, validated data set, focused sequencing studies can be used to analyze the response of individual mice self-administering cocaine. This broad approach should allow identification of therapeutic targets unique to females or sensitive to estrogen.