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Design of genetically encoded sensors for detecting endogenous opioid peptides

用于检测内源性阿片肽的基因编码传感器的设计

基本信息

批准号：
10682579
负责人：
Wenjing Wang
金额：
$ 33.69万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-01 至 2027-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10682579
关键词：
Address Adverse effects Agonist Alcohol abuse Analgesics Animal Model Autopsy Behavior Biological Brain Brain region Cells Central Nervous System Complement Complex Constipation Detection Directed Molecular Evolution Euphoria Fluorescence Genetic Transcription Goals Image Analysis Knowledge Label Light Maps Mental Depression Methods Microdialysis Monitor Mus Neurons Opiate Addiction Opioid Opioid Peptide Opioid Receptor Opioid agonist Pain Pathologic Processes Peptide Receptor Peptide Signal Sequences Peptides Peripheral Nervous System Pharmaceutical Preparations Physiological Processes Receptor Signaling Regulation Reporter Resolution Respiration Rewards Role Signal Transduction Sleep Specificity Stimulus System Time Virus Diseases addiction biological adaptation to stress brain tissue brain volume design detection method endogenous opioids improved in vivo irradiation meter mu opioid receptors neural circuit neuronal cell body neuronal circuitry neuroregulation novel opioid epidemic pain reduction respiratory response sensor side effect spatiotemporal temporal measurement tool

项目摘要

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.

用于检测内源性阿片肽的基因编码传感器的设计针对 mu-阿片受体 (MOR) 的阿片类药物仍然是最有效的止痛药，但具有严重的副作用，如成瘾、便秘和呼吸抑制。副作用这是因为目前可用的阿片类药物在疼痛调节回路中缺乏 MOR 特异性。由于内源性阿片肽不会导致此类不良反应，因此了解它们在不同方面的作用神经元回路可以增进我们对阿片肽如何不同作用的了解，并可能促进减少副作用的新型止痛药的设计。研究内源性阿片肽如何内源性阿片类药物作用于不同的神经回路，我们需要检测内源性阿片类药物何时何地发挥作用肽以高时空分辨率和电路水平在大脑中释放。微透析，检测小鼠大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 的神经调节肽通常在几秒到几分钟内释放并发挥作用。需要更高时空检测内源性阿片肽释放的方法解决。因此，我们建议设计两类阿片类传感器：1）转录报告基因这将能够在大范围内以细胞分辨率检测内源性阿片肽脑组织体积，用于研究内源性阿片肽如何在回路中发挥作用等级; 2) 实时荧光传感器将能够检测内源性阿片肽具有亚细胞空间分辨率和时间分辨率（秒级）。这两个传感器将相辅相成，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到底在哪里释放？对不同疼痛和奖赏刺激的反应？疼痛或奖赏刺激后多久产生内源性阿片类药物释放肽？完成该提案将有助于我们设计工具的长期目标增进我们对内源性阿片类药物信号传导的理解，以设计微型止痛药物妈妈的副作用。