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

 描述：最近，大麻使用合法化一直是公众关注的一个重要话题，但我们对基本潜在机制的理解仍然处于初级阶段。Δ-9-四氢大麻酚是大麻中的主要精神活性成分，可与内源性大麻素系统相互作用，包括大麻素受体、内源性配体以及参与这些配体的合成和代谢的酶。这一发现表明，人类发育中的大麻暴露与晚年的精神脆弱和成瘾有关，这表明ECB可能在显著方面影响大脑发育和可塑性。然而，欧洲央行是如何发挥这种影响力的，人们仍然知之甚少。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，使得结构性基因敲除表型难以解释。因此，需要新的工具和技术来阐明ECB的特定细胞类型和时间依赖功能。斑马鱼Danio rerio是一种脊椎动物模式生物，非常适合将大脑发育与功能联系起来。透明和容易接触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，斑马鱼幼体就需要捕食和躲避捕食者，因此有了一个简单但实用的奖励系统。胚胎斑马鱼和幼体斑马鱼可以方便地暴露在化学品(例如滥用药物)中，以研究它们的生物效应。以时空精确度控制基因活性的能力将极大地促进生物学和医学的新发现。最近，一种名为CRISPR的RNA引导的基因组编辑技术的发展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方案中，我们将采用体外CRISPR基因调控技术，利用光在体内实现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检验的具体假设是：大麻素信号调节与奖赏回路相关的多巴胺(DA)神经元的发育。影响：如果该项目成功，将在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。我们将向研究界广泛提供这项技术。2)揭示ECB是否调节与奖赏回路有关的DA神经元的发育。这些新知识将有助于为政策决策提供信息，并促进治疗药物滥用和成瘾的创新。对CEBRA的适用性：(1)本项目将检验一个非常新颖和重要的假说，旨在评估ECB在与奖赏回路相关的DA神经元发育中的作用。目前，关于这一主题的先例或初步数据很少，然而，如果得到证实，这将对目前关于ECB信号在奖赏回路发展中的想法产生重大影响。(2)该项目将开发高时空精度的基因沉默和激活的创新技术。这将对药物滥用研究和其他领域具有很好的适用性。

 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.