Intracellular signaling mechanisms underlying opioid modulation of pain

项目摘要 阿片类药物使用障碍是一种公共卫生危机，源于其高度成瘾性和强效 阿片类药物的镇痛特性。阿片类药物调节参与镇痛的回路，疼痛诱导的负性 情感、动机、奖励和成瘾。它们作用于G蛋白偶联的阿片受体，诱导多种阿片受体， 细胞内信号通路。其中，环磷酸腺苷（cAMP）和蛋白质 激酶A（PKA）通路是镇痛、疼痛相关厌恶和阿片类药物作用的关键机制。 诱发痛觉过敏。大多数研究PKA信号对阿片类药物或疼痛的反应受到限制， 不能同时考虑细胞类型特异性PKA信号传导的体外或离体方法， 复杂的电路水平的调节，以及行为对PKA动力学的影响。因此，目前尚不清楚 确切地说，PKA在阿片类药物的作用下何时何地被调节;也不清楚PKA的功能作用是什么。 这些时空PKA动力学是镇痛。理解的功能意义 阿片类药物诱导的细胞内信号传导以及这种信号传导在独特的细胞类型和大脑区域中的差异 将使我们更好地理解阿片类药物如何区别影响疼痛和成瘾回路。的目标 首先，我将定义μ阿片诱导的PKA的时间动力学 中背丘脑（MD）至前扣带皮层（ACC）回路内的信号传导。该电路 高度表达μ阿片受体并整合感觉和情感疼痛。然后我会决定 这些PKA动力学和疼痛缓解之间是否存在因果关系。最后，我将研究 这些PKA动力学的细胞类型特异性。我的中心假设是PKA动力学将取决于 阿片类药物暴露的持续时间，并将决定疼痛反应的程度，与特定的细胞类型 作为PKA调节的关键位点。这一假设将被测试使用一种新的基因编码， 设计用于在行为小鼠中PKA活性的体内成像的传感器。研究区域差异， 响应于急性和慢性阿片样物质暴露的时间PKA动力学，PKA将在 在MD和ACC中给予阿片类药物期间和之后。成像将与疼痛测定配对， 评估镇痛和痛觉过敏。为了验证PKA动力学在疼痛缓解中的必要性和充分性， PKA活性将由基因编码的PKA抑制剂或光活化腺苷酸调节。 环化酶，同时进行疼痛的行为测定。最后，为了检测PKA的细胞特异性， 动力学，传感器表达将以Cre依赖性方式被隔离到每种感兴趣的细胞类型，并且 μ阿片样物质受体的肽激动剂和拮抗剂将在PKA成像期间局部输注。这 这项研究将定义MD到ACC电路的特定组件中的PKA信号如何响应 阿片类药物和介导的疼痛缓解。实现这些目标将有助于深入了解细胞内信号是如何传递的 受阿片类药物的时空调节并促进镇痛。

PROJECT SUMMARY Opioid use disorder is a public health crisis that stems from the highly addictive nature and potent analgesic properties of opioids. Opioids modulate circuitry involved in analgesia, pain-induced negative affect, motivation, reward, and addiction. They act on G-protein coupled opioid receptors, inducing multiple intracellular signaling pathways. Of these, the cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) pathway is known to be a key mechanism in analgesia, pain-related aversion, and opioid- induced hyperalgesia. Most studies examining PKA signaling in response to opioids or pain are limited by in vitro or ex vivo approaches that cannot simultaneously consider cell-type specific PKA signaling, complex circuit-level regulation, and effects of behavior on PKA dynamics. As a result, it remains unclear exactly where and when PKA is modulated in response to opioids; nor is it clear what the functional effects of these spatiotemporal PKA dynamics are on analgesia. Understanding the functional significance of opioid-induced intracellular signaling and how this signaling differs in unique cell types and brain regions will allow us to better comprehend how opioids differentially effect pain and addiction circuitry. The goals of this proposal are as follows: First, I will define the temporal dynamics of mu opioid-induced PKA signaling within the mediodorsal thalamus (MD) to anterior cingulate cortex (ACC) circuitry. This circuitry highly expresses mu opioid receptors and integrates sensory and affective pain. Then, I will determine whether there is a causal relationship between these PKA dynamics and pain relief. Finally, I will examine the cell-type specificity of these PKA dynamics. My central hypothesis is that PKA dynamics will depend on the duration of opioid exposure and will determine the extent of pain response, with specific cell types acting as key sites of PKA modulation. This hypothesis will be tested using a novel genetically encoded sensor designed for in vivo imaging of PKA activity in behaving mice. To examine regional differences in temporal PKA dynamics in response to acute and chronic opioid exposure, PKA will be imaged before, during, and after opioid administration in the MD and ACC. Imaging will be paired with pain assays to assess analgesia and hyperalgesia. To test the necessity and sufficiency of PKA dynamics in pain relief, PKA activity will be modulated by either a genetically encoded PKA inhibitor or photoactivatable adenylyl cyclase while conducting behavioral assays of pain. Finally, to examine the cell-specificity of PKA dynamics, sensor expression will be isolated to each cell type of interest in a Cre-dependent manner, and peptide agonists and antagonists of mu opioid receptors will be locally infused during PKA imaging. This study will define how PKA signaling in specific components of the MD to ACC circuitry both responds to opioids and mediates pain relief. Achieving these goals will provide insight into how intracellular signaling is spatiotemporally regulated by opioids and facilitates analgesia.