Functional Imaging of Mu and Delta Opioid Receptors In Vivo: Receptor Dynamics

首席研究员/项目主任：Evans，Christopher J.，Ph.D.，Component I 成分II：Muand Delta阿片受体的功能成像 在体内：受体动力学和对滥用阿片类药物的适应 摘要： 鸦片类药物是治疗疼痛的重要治疗化合物，但它们也有可能 导致上瘾。研究表明，不同的阿片受体在中枢神经系统中的特殊作用。 阿片类药物的治疗和成瘾作用我们最近在遗传缺乏阿片类药物的小鼠身上进行的研究 受体建立(I)u受体作为人工和自然奖励的关键分子触发器。 刺激，以及(Ii)Delta受体活性可能是药物滥用和环境诱导易感性的一个因素 渴望。这些受体如何在神经回路中运作并适应慢性阿片类药物的问题 在生物化学、电生理和行为学水平上得到了广泛的研究。不过，这个 人们对这一重要问题仍知之甚少。将这些技术应用于 阿片受体和成瘾研究的问题一直是缺乏可靠的方法直接 在生理相关模型中可视化阿片受体。基因编码的发展， 荧光标记的阿片受体代表了克服这一障碍的直接途径。我们有 成功开发出一种表达用荧光标记标记的增量阿片受体的小鼠， 绿色荧光蛋白，并获得了引人入胜的成像数据。我们已经证明了…… 在小鼠体内直接显示受体是一个可以实现的目标。我们现在计划开发 表达u阿片受体的小鼠被标记了不同的荧光标记物mCherry，以及 表达这两种标记受体的组合小鼠。我们将在体外和体内使用尖端技术 成像技术与先进的分子技术相结合，实现了以下具体目标 目的：1.建立Mor-mCherry敲入小鼠模型，并研究其受体表达及功能。 分发。2.研究Mor-mCherry和DOR-EGFP突变小鼠的神经解剖学 荧光显微镜。3.研究MOR-MCherry和DOR-EGFP在急性反应中的动态变化 鸦片类药物。4.研究慢性阿片类药物对MOR-mCherry和DOR-EGFP反应的动态变化。 我们的项目提供了一种全新的方法来实现u和Delta阿片受体的功能成像。 在活体内。明确识别表达每个受体亚型的神经元的能力将代表 我们在表征阿片受体神经元生理反应的能力方面取得了重大进展 基础条件，以及对急性和慢性阿片类药物和其他神经递质的反应。我们还将 能够直接可视化这些受体在大脑中的表达位置，它们在 单个细胞的特定隔间，以及受体的大脑和细胞分布如何变化 在不同的生理和药理条件下。结合了最新的成像技术 阿片受体可视化的技术和强大的新分子工具有可能 为阿片剂发挥其治疗和治疗作用的细胞机制提供了重要的新见解 令人上瘾的效果。 主要

Principal Investigator/Program Director: Evans, Christopher J., Ph.D., Component I Component II: Functional Imaging of Muand Delta Opioid Receptors In Vivo: Receptor Dynamics and Adaptation to Abused Opiates ABSTRACT: Opiates are important therapeutic compounds for treatment of pain, but they also have the potential to cause addiction. Studies have demonstrated specific roles for the different opioid receptors in the therapeutic and addictive effects of opiates Our recent studies using mice genetically lacking opioid receptors establish (i) the mu receptor as a key molecular trigger for both artificial and natural rewarding stimuli, and (ii)delta receptor activity as a possible factor in vulnerability to drug abuse and context-induced craving. The question of how these receptors operate within neural circuits and adapt to chronic opiates in vivo, is being widely studied at biochemical, electrophysiological and behavioral levels. However, this important issue remains poorly understood. A significant obstacle to the application of these techniques to the question of opiate receptor and addiction research has been the lack of reliable methods for directly visualizing opiate receptors in physiologically relevant models. The development of genetically encoded, fluorescent- labeled opioid receptors represents a straightforward path to overcome this obstacle. We have successfully developed a mouse that expresses the delta opioid receptor labeled with a fluorescent marker, Green Fluorescent Protein, and obtained fascinating imaging data. We have established the proof of principle that direct receptor visualization in mice in vivo is an achievable goal. We now plan to develop mice that express mu opioid receptors labeled with a different fluorescent marker, mCherry, as well as combinatorial mice expressing both types of labeled receptors. We will use cutting-edge in vitro and in vivo imaging techniques, in combination with advanced molecular techniques, to achieve the following Specific Aims: 1. To generate the MOR-mcherry knock-in mouse model and characterize receptor expression and distribution. 2. To study MOR-mcherry and DOR-eGFP neuroanatomy in mutant mouse lines by fluorescence microscopy. 3. To study MOR-mcherry and DOR-eGFP dynamics in response to acute opiates. 4. To study MOR-mcherry and DOR-eGFP dynamics in response to chronic opiates. Our project offers an entirely novel approach to achieve functional imaging of mu and delta opioid receptors in vivo. The ability to definitively identify neurons expressing each individual receptor subtype will represent a major advance in our ability to characterize the physiological responses of opioid receptor neurons under basal conditions, as well in response to acute and chronic opioids and other neurotransmitters. We will also be able to directly visualize the location of the expression of these receptors in the brain, their location in specific compartments of individual cells, and how the brain and cellular distribution of receptors changes under different physiological and pharmacological conditions. The combination of the latest imaging technologies and powerful new molecular tools for visualization of opioid receptors has the potential to provide important new insight into the cellular mechanisms by which opiates exert their therapeutic and addictive effects. Primary