Linking cocaine induced synaptic changes to the single neuron transcriptome

描述(申请人提供)：突触是细胞间的连接，调节神经元的交流，许多突触分子调节它们的功能。最近的证据表明，可卡因暴露会导致少数神经元的突触传递异常，这些神经元在可卡因诱导的精神运动敏化中起着因果作用。更好地了解可卡因对少数神经元及其突触功能的影响，可能会为成瘾提供更好的治疗策略，从而可能减轻疾病的负担。为了达到这些目的，识别与行为相关的回路，然后分析与这些回路相关的神经元的单个转录，可以提供关于成瘾潜在机制的丰富信息。为了实现这一目标，我建议使用FosCreer转基因小鼠系来从基因上追踪因接触可卡因或上下文而激活的神经元电路，这使得当他莫昔芬存在时，活跃的神经元可以被基因标记。利用FosCreer小鼠，我还将试图通过阻断依赖Cre重组酶病毒传递的破伤风毒素轻链表达的可卡因激活回路的活性，来确定可卡因激活回路与可卡因相关行为的因果关系。同时，我将通过RNAseq和Q-PCR分析同一回路中激活的单个神经元的转录组，寻找其表达变化与突触特性改变共同变化的候选基因。在初步实验中，我开发了一种新的单个神经元分离方法，将FACS分选与集成微流控系统(C1Fluidigm)相结合。与以前的方法相比，这种新的方法使我能够提高分析单个神经元转录组的纯度和效率。对于独立阶段，我将首先探讨多巴胺在可卡因诱导的基因表达变化中的作用。有证据表明，可卡因的强化作用取决于它快速阻断多巴胺转运体的能力。为了进一步测试观察到的基因表达变化是否与这些增强效应有关，我将测试具有功能性但对可卡因不敏感的多巴胺转运体的转基因小鼠的关键基因表达变化。同时，我将使用在培训阶段发展起来的方法来研究可卡因自我给药模型。最后，将通过体外和体内操作来探索个体转录变化的作用。该提案中的一系列广泛的实验结合了新的工具来提取关于可卡因成瘾过程的生物学见解，这将指导未来基于机制的有针对性的药理学干预措施的设计。

DESCRIPTION (provided by applicant): Synapses are intercellular junctions that mediate neuronal communication, and many synaptic molecules regulate their functions. Recent evidence suggests that cocaine exposure causes abnormalities in synaptic transmission in a minority of neurons that play a causal role in cocaine-induced psychomotor sensitization. A better understanding of cocaine effects on that minority of neurons and their synapse function may provide better treatment strategies for addiction, and thereby might reduce the burden of disease. Towards these ends, identifying behaviorally relevant circuits and then analyzing individual transcript me of neurons associated with those circuits can provide a wealth of information about potential mechanisms of addiction. To achieve such goal, I propose to genetically trace neuronal circuits that are activated by cocaine exposure or context using a FosCreER transgenic mouse line, which allows active neurons to be genetically labeled when the tamoxifen is present. Using FosCreER mouse I will also try to establish the causality of cocaine activated circuits for cocaine related behaviors by blocking activity of those circuits wit virally delivered Cre recombinase dependent expression of the light chain of tetanus toxin. In parallel, I will analyze the transcriptome of activated single neurons in the same circuit by RNAseq and Q-PCR and searching for candidate genes whose expression changes co-vary with altered synaptic properties. In preliminary experiments, I have developed a novel single neuron isolation method using FACS sorting combined with an integrated microfluidic system (C1 Fluidigm). This novel approach enabled me to achieve enhanced purity and efficiency of analyzing single neurons' transcriptome when compared to previous methods. For the independent phase, I will first explore the role of dopamine in cocaine induced gene expression changes. Evidence suggests that cocaine's reinforcing effects depend on its ability to rapidly block the dopamine transporter. To further test if the observed gene expression changes are related to those reinforcing effects, I will test the key gene expression changes in the knock-in mice that have a functional but "cocaine-insensitive" dopamine transporter. In parallel, I will employ the methods developed in training phase to study cocaine self-administration model. Finally, the role of individual transcriptional changes will be explored by in vitro and in vivo manipulations. The extensive set of experiments in this proposal combines new tools to extract biological insights about the process of cocaine addiction, which will guide the future design of mechanism based targeted pharmacological interventions.