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

主要研究者/项目负责人：Evans，Christopher J.，哲学博士、组分I 组件II：μ和δ阿片受体的功能成像 在体内：受体动力学和适应滥用阿片类药物 摘要： 阿片类药物是治疗疼痛的重要治疗化合物，但它们也有可能 导致成瘾。研究已经证明了不同阿片受体在脑内的特定作用。 阿片类药物的治疗和成瘾作用我们最近的研究使用遗传缺乏阿片类药物的小鼠 受体确立了（i）μ受体作为人工和天然奖赏的关键分子触发器 刺激，和（ii）δ受体活性作为一个可能的因素，在脆弱性药物滥用和环境引起的 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， 体内，正在生物化学、电生理和行为水平上进行广泛研究。但这 对重要问题仍然知之甚少。应用这些技术的一个重大障碍是 阿片受体和成瘾研究的问题一直是缺乏可靠的方法， 在生理学相关模型中可视化阿片受体。基因编码， 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 成功开发了表达用荧光标记物标记的δ阿片受体的小鼠， 绿色荧光蛋白，并获得了迷人的成像数据。我们已经证明了 原理，直接受体可视化在小鼠体内是一个可实现的目标。我们现在计划开发 表达用不同荧光标记物mCherry标记的μ阿片受体的小鼠，以及 表达两种类型标记受体的组合小鼠。我们将使用尖端的体外和体内 成像技术，结合先进的分子技术，以实现以下具体 目的：1.为了产生MOR-mcherry敲入小鼠模型并表征受体表达， 分布2.为了研究突变小鼠系中的MOR-mcherry和DOR-eGFP神经解剖学， 荧光显微镜3.为了研究MOR-mcherry和DOR-eGFP在急性炎症反应中的动力学， 鸦片类药物4.研究MOR-mcherry和DOR-eGFP对慢性阿片类药物的响应动力学。 我们的项目提供了一种全新的方法来实现μ和δ阿片受体的功能成像 in vivo.明确识别表达每种受体亚型的神经元的能力将代表 这是我们在描述阿片受体神经元的生理反应方面的一个重大进展， 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表达的位置，它们在大脑中的位置， 单个细胞的特定区室，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。结合最新的成像技术 技术和强大的新的分子工具，阿片受体的可视化有可能， 为阿片类药物发挥其治疗作用的细胞机制提供了重要的新见解， 上瘾的效果。 初级

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