Design of genetically encoded sensors for detecting endogenous opioid peptides

内源性阿片肽基因编码传感器的设计 靶向μ阿片受体（莫尔）的阿片类药物仍然是最有效的止痛药， 有严重的副作用，如成瘾，便秘和呼吸抑制。副作用 这是由于目前可用的阿片类药物对疼痛调节回路中的莫尔缺乏特异性。 由于内源性阿片肽不会导致这种不良反应，因此了解它们在不同的 神经元回路可以推进我们对阿片肽如何不同地起作用的知识，并可能促进 设计新的止痛药，减少副作用。研究内源性阿片肽 在不同的神经回路中发挥作用，我们需要检测内源性阿片类药物何时何地 肽在脑中以高时空分辨率和回路水平释放。微透析， 用于检测小鼠脑中阿片肽的最佳可用方法，可以检测阿片肽， 空间分辨率约为400 µm，时间分辨率约为20分钟。然而，神经元胞体是~ 20 µm和神经调节肽通常在几秒到几分钟的时间内释放和发挥作用。 因此，需要一种能够在更高的时空分辨率下检测内源性阿片肽释放的方法 分辨率因此，我们建议设计两类阿片受体：1）转录报告基因 这将使内源性阿片肽的检测在细胞分辨率跨越大的 用于研究内源性阿片肽如何在回路中发挥作用的脑组织体积 水平; 2）真实的时间荧光传感器，其将使得能够检测内源性阿片肽 具有亚细胞空间分辨率和秒级的时间分辨率。这两个传感器 将相互补充，以解决有关内源性阿片类药物的长期悬而未决的问题， 肽调节和信号传导。例如，什么样的疼痛和奖赏刺激会刺激阿片类药物 肽释放？阿片肽在细胞或亚细胞分辨率下释放的确切位置是什么？ 对不同的疼痛和奖励刺激的反应疼痛或奖赏刺激后多久内源性阿片样物质 释放的肽？完成这一提案将有助于我们设计工具的长期目标， 推进我们对内源性阿片信号传导的理解，以设计具有小剂量的止痛药。 副作用。

Design of genetically encoded sensors for detecting endogenous opioid peptides Opioids that target the mu-opioid receptors (MOR) remain the most effective pain medication but with severe side effects, such as addiction, constipation and respiratory suppression. The side effects result from a lack of specificity for the MOR in the pain modulation circuit by currently available opioids. Since endogenous opioid peptides do not lead to such adverse effects, understanding their role in different neuronal circuits could advance our knowledge of how opioid peptides act differently, and possibly facilitate the design of novel pain medications with reduced side effects. To study how endogenous opioid peptides exert their effects on different neural circuits, we need to detect when and where the endogenous opioid peptides are released in the brain at a high spatiotemporal resolution and at the circuit level. Microdialysis, the best available method for detecting opioid peptides in the mouse brain, can detect opioid peptides with a spatial resolution of ~ 400 µm and a temporal resolution of ~20 minutes. However, neuron somas are ~ 20 µm and neuromodulating peptides are usually released and function on the order of seconds to minutes. There is a need of methods to detect the endogenous opioid peptide release with higher spatiotemporal resolution. Therefore, we propose to design two classes of opioid sensors: 1) A transcriptional reporter that will enable the detection of the endogenous opioid peptides at a cellular resolution across a large volume of the brain tissue for studying how endogenous opioid peptides exert their effects at the circuit level; 2) Real time fluorescent sensors that will enable the detection of the endogenous opioid peptides with subcellular spatial resolution and a temporal resolution on the order of seconds. These two sensors will complement each other to address the long unanswered questions regarding the endogenous opioid peptide regulation and signaling. For example, what kind of pain and reward stimuli will stimulate the opioid peptide release? Where exactly are the opioid peptides released at a cellular or sub-cellular resolution in response to different pain and reward stimuli? How soon after pain or reward stimuli are endogenous opioid peptides released? Completion of this proposal will contribute to our long-term goal of designing tools to advance our understanding of the endogenous opioid signaling for designing pain medications with mini- mum side effects.