A novel RNA-guided platform for dissecting cannabinoid signaling in reward circuit development

 产品说明：最近，大麻使用合法化一直是公众关注的话题，但我们对基本机制的理解仍然是初步的。Δ9-四氢大麻酚（THC）是大麻中的主要精神活性成分，可以与内源性大麻素系统（eCB）相互作用，该系统包括大麻素受体，内源性配体以及参与这些配体合成和代谢的酶。人类在发育过程中接触大麻与晚年的精神脆弱性和成瘾有关，这一发现表明eCB可能会以显着的方式影响大脑发育和可塑性。然而，eCB如何发挥这种影响仍然知之甚少。这在很大程度上是由于eCB在空间和时间上的广泛表达，由于发育补偿和缺乏特异性，使得组成型敲除表型难以解释。因此，需要新的工具和技术来阐明eCB的细胞类型特异性和时间依赖性功能。 斑马鱼Danio rerio是一种非常适合将大脑发育与功能联系起来的脊椎动物模式生物。透明且易于接近的胚胎和幼虫提供了在单细胞水平上观察大脑发育的机会。早在受精后5天（dpf），斑马鱼幼体就需要寻找食物和逃避捕食者，因此一个简单而功能性的奖励系统就已经存在。胚胎和幼体斑马鱼可以方便地暴露于化学品（如滥用药物），以研究其生物效应。 时空精确控制基因活动的能力将极大地促进生物学和医学的新发现。最近开发的一种名为CRISPR的新型RNA引导基因组编辑技术为研究体内基因功能提供了前所未有的便利。在这个探索性的R21提案中，我们将采用体外CRISPR基因调控技术，利用光在体内神经元中进行时空可控的基因沉默和激活，我们称之为Opto-CRISPR。我们将用这项新技术测试的具体假设是：大麻素信号调节与奖赏回路相关的多巴胺能（DA）神经元的发育。影响：如果成功，该项目将对以下研究领域产生重大影响：1）它将推进体内神经元时空靶向基因沉默和激活的技术。我们将把这项技术广泛地提供给研究界。2)它将揭示eCB是否调节与奖赏回路相关的DA神经元的发育。这些新知识将有助于为政策决定提供信息，并促进治疗药物滥用和成瘾的创新。CEBRA的适用性：（1）本项目将测试一个非常新颖和重要的假设，旨在评估eCB在与奖励回路相关的DA神经元发育中的作用。目前，关于这个主题的先例或初步数据很少，但是，如果得到证实，它将对目前关于eCB信号在奖励电路发展中的思想产生重大影响。(2)该项目将开发具有高时空精度的基因沉默和激活创新技术。这将有希望适用于药物滥用研究及其他领域。

 DESCRIPTION: Recently, legalization of marijuana use has been a topic of significant public attention, yet our understanding of the fundamental underlying mechanisms remains rudimentary. Δ9-tetrahydrocannabinol (THC), the main psychoactive ingredient in marijuana, can interact with the endogenous cannabinoid system (eCBs), which includes cannabinoid receptors, endogenous ligands, and enzymes involved in the synthesis and metabolism of these ligands. The finding that developmental cannabis exposure in humans is associated with psychiatric vulnerability and addiction later in life suggests that eCBs may influence brain development and plasticity in significant ways. However, how eCBs exert such influences remains poorly understood. This is in large part due to the broad expression of eCBs both spatially and temporally, making constitutive knockout phenotypes difficult to interpret, due to developmental compensation and lack of specificity. Thus, it requires new tools and technologies to elucidate cell type-specific and time-dependent functions of eCBs. The zebrafish Danio rerio is a vertebrate model organism well suited to connect brain development with function. The transparent and easily accessible embryos and larvae offer the opportunity to observe brain development at single-cell levels. As early as 5 days post fertilization (dpf), larva zebrafish need to hunt for food and escape from predators, thus a simple yet functional reward system is in place. Embryonic and larval zebrafish can be conveniently exposed to chemicals (e.g. drugs of abuse) for studying their biological effects. The ability to control gene activity ith spatiotemporal precision shall greatly facilitate new discoveries in biology and medicine. The recent development of a novel RNA-guided genome-editing technology named CRISPR offers unprecedented ease for studying gene function in vivo. In this exploratory R21 proposal, we will adapt the in vitro CRISPR gene regulation technologies for spatiotemporally controllable gene silencing and activation in neurons in vivo using light, which we term Opto-CRISPR. The specific hypothesis that we will test with this new technology is: the cannabinoid signaling regulates the development of dopaminergic (DA) neurons related to reward circuitry. Impact: If successful, this project will exert a major impact on the following research fields: 1) It will advance technologies for spatiotemporally targetable gene silencing and activation in neurons in vivo. We will make this technology widely available to the research community. 2) It will reveal whether eCBs regulate the development of DA neurons related to reward circuitry. Such new knowledge will help inform policy decisions and foster innovations for treating drug abuse and addiction. Suitability for CEBRA: (1) This project will test a highly novel and significant hypothesis, aimed at evaluating the role of eCBs in the development of DA neurons related to reward circuitry. At the moment, there are scant precedents or preliminary data regarding this topic, however, if confirmed, it would have a substantial impact on current thinking of eCBs signaling in the reward circuit development. (2) This project will develop innovative techniques for gene silencing and activation with high spatiotemporal precision. This will have promising applicability to drug abuse research and beyond.