Role of endocannabinoid signaling in a preference/aversion circuitry

项目总结 寻求奖励和避免潜在威胁的能力是所有动物健康和生存的基础 从早期的生命阶段开始。我们的研究旨在通过细胞和分子的清晰度来解决电路范围的机制 使用幼虫斑马鱼。作为脊椎动物的遗传模式生物，斑马鱼有相当大的相似性 和哺乳动物在一起。在哺乳动物和斑马鱼幼体(见初步数据部分)中，脂质神经递质 内源性大麻素(ECB)和下丘脑促肾上腺皮质激素释放因子(Hy CRF)是已知的 规范有动机的行为。然而，在细胞分辨率上理解它们在电路范围内的作用 目前是缺乏的。斑马鱼幼体，其大脑相对简单和透明，由约100K的神经元组成(与 在老鼠体内达到~7500万，在人脑中达到~1000亿)非常适合解决这个问题。新的 在更简单的系统中发现的监管原则将为研究更复杂的系统奠定基础。 自由生活，需要捕食和躲避捕食者，斑马鱼的幼体很容易被观察到 对环境刺激、药物或社会暗示作出反应的接近和回避行为。在此，我建议 为了阐明ECB和Hy CRF在全脑细胞分辨率中的作用，在明/暗偏好的背景下， 这是一种基本的动机行为，在不同物种之间都是保守的。斑马鱼幼体避免黑暗，这可以 因应激源而增强，经抗焦虑药物缓解。我们的初步数据显示，Hy CRF神经元的消融 改善，而大麻素受体CB1的抑制则增强了黑暗回避。我们有遗传上的 破坏了欧洲央行信号通路中的主要基因，包括CB1(主要是神经)和CB2(主要是 免疫)受体、受体相互作用蛋白(CNRIP1a和CNRIP1b)、ECB合成酶(如： DAGLa、DAGLb、ABHD4)和ECB降解酶(MG11、FAAH)。这些被淘汰的动物很值钱 通过揭示哪些受体和配体以及相关的 调节蛋白参与特定的行为调节。此外，我们还建立了全脑范围的 检测分布式神经回路活性和可塑性的钙成像和计算平台 在细胞分辨率下。 在这项应用中，基于这些初步数据和我们在研究大脑发育和功能方面的专业知识 利用斑马鱼，我们将测试欧洲央行信号调节黑暗避免电路的假设 涉及Hy CRF神经元。我们将获得电路范围的了解，并发现新的细胞类型/分子 未来对高度可及的大脑中的电路组装和在压力或药物治疗下的可塑性的研究。 影响和结果：如果成功，据我们所知，这个项目将第一次实现 解决电路范围内对ECB信号与Hy CRF相关的基本动机行为的理解。 这种对整个电路水平的理解的提高将为未来的大麻政策奠定基础 并用于处理与CRF或ECB信号扰动相关的疾病状态。

PROJECT SUMMARY The ability to seek reward and avoid potential threats is fundamental to the fitness and survival of all animals from early life stages. Our research aims to address circuit-wide mechanisms with cellular and molecular clarity employing larval zebrafish. As a vertebrate genetic model organism, zebrafish shares considerable similarity with mammals. In both mammals and larval zebrafish (See Preliminary data section), the lipid neurotransmitters endocannabinoids (eCB) and the neuropeptide hypothalamic corticotropin releasing factor (Hy CRF) are known to regulate motivated behaviors. However, an understanding of their roles circuit-wide at cellular resolution is currently lacking. Larval zebrafish with a relatively simple and transparent brain of ~100K neurons (compared to ~75 million in the mouse, and ~100 billion in the human brain) is well suited to address this question. New regulatory principles uncovered in simpler systems will lay foundation for studying more complex systems. Free-living with the need to hunt for food and avoid predators, larval zebrafish display readily observable approach and avoidance behaviors in response to environmental stimuli, drugs, or social cues. Here I propose to elucidate the role of eCB and Hy CRF, brain-wide at cellular resolution, in the context of light/dark preference, a fundamental motivated behavior conserved across species. Larval zebrafish avoid dark, which can be enhanced by stressors and alleviated by anxiolytics. Our preliminary data show that ablation of Hy CRF neurons ameliorates, whereas inhibition of the cannabinoid receptor CB1 enhances, dark avoidance. We have genetically disrupted major genes in the eCB signaling pathway, including CB1 (primarily neural) and CB2 (primarily immune) receptors, receptor-interacting proteins (CNRIP1a and CNRIP1b), eCB synthesis enzymes (e.g. DAGLa, DAGLb, ABHD4), and eCB degradation enzymes (MGLL, FAAH). These knockout animals are valuable resources for understanding signaling specificity by uncovering which receptors and ligands and associated regulatory proteins are involved in specific behavioral regulation. Furthermore, we have established brain-wide calcium imaging and computational platforms for examining the activity and plasticity of distributed neural circuits at cellular resolution. In this application, built on these preliminary data and our expertise in studying brain development and function employing zebrafish, we will test the hypothesis that eCB signaling regulates dark avoidance circuitry that involves Hy CRF neurons. We will gain circuit-wide understanding and uncover new cell types/molecules for future studies of circuit assembly and plasticity under stress or drug treatment in a highly accessible brain. Impact and Outcomes: If successful, this project will achieve, for the first time to our knowledge, a cellular resolution circuit-wide understanding of eCB signaling in relation to Hy CRF in a fundamental motivated behavior. Such improved understanding at the whole circuitry level shall lay foundation for informing future marijuana policy and for tackling disease states associated with perturbed CRF or eCB signaling.