Intracellular signaling mechanisms underlying opioid modulation of pain

项目总结 阿片类药物使用障碍是一种公共健康危机，源于高度成瘾的性质和强大的 阿片类药物的止痛特性。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，诱导多个 细胞内信号通路。其中，环磷酸腺苷(CAMP)和蛋白质 蛋白激酶A(PKA)通路是镇痛、疼痛相关厌恶和阿片类药物产生的关键机制。 诱发性痛觉过敏。大多数检测PKA信号对阿片类药物或疼痛反应的研究都受到以下限制 不能同时考虑细胞类型特异性PKA信号的体外或体外方法， 复杂的电路级调节，以及行为对PKA动力学的影响。因此，目前仍不清楚 对阿片类药物的反应，PKA在何时何地被调节；目前也不清楚PKA的功能效应是什么 在这些时空动态中，PKA是关于止痛的。了解的功能意义 阿片类药物诱导的细胞内信号转导及其在特定细胞类型和脑区的差异 将使我们更好地理解阿片类药物如何不同地影响疼痛和成瘾回路。目标 本研究的主要内容如下：首先，定义了Mu阿片类药物诱导的pKa的时间动力学。 丘脑内侧背侧(MD)向扣带回前部(ACC)回路发出信号。这条线路 高表达u阿片受体，整合感觉性和情感性疼痛。然后，我会决定 这些PKA动态与疼痛缓解之间是否存在因果关系。最后，我将检查 这些PKA动力学的细胞类型特异性。我的中心假设是，PKA的动态将取决于 关于阿片类药物暴露的持续时间，并将决定疼痛反应的程度，具有特定的细胞类型 作为PKA调控的关键部位。这一假设将使用一种新的基因编码进行验证 用于对行为正常的小鼠的PKA活性进行体内成像的传感器。考察中国的地区差异 时间动态对急性和慢性阿片类药物暴露的反应，PKA将在之前进行成像， MD和ACC在阿片类药物给药期间和之后。成像将与疼痛分析配对，以 评估镇痛和痛觉过敏。为了测试PKA动力学在止痛中的必要性和充分性， PKA活性将由基因编码的PKA抑制剂或可光激活的腺苷调节 循环，同时进行疼痛的行为分析。最后，检测PKA的细胞特异性。 动态，传感器表达将以依赖于Cre的方式被分离到每种感兴趣的细胞类型，并且 在PKA成像过程中，将局部输注Mu阿片受体的多肽激动剂和拮抗剂。这 研究将确定MD到ACC电路的特定组件中的PKA信令如何响应 阿片类药物，并调节疼痛缓解。实现这些目标将提供对细胞内信号如何 受阿片类药物的时空调节，有助于镇痛。

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.