Intracellular signaling mechanisms underlying opioid modulation of pain

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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.