Project Summary/ Abstract Opioids like morphine are the most effective pain relieving drugs available but their rewarding properties lead to misuse. The rewarding and analgesic properties of opioids are mediated by activation of mu opioid receptors (MOR). Hyperalgesia is an exaggerated pain perception in response to a stimulus that is normally mildly uncomfortable. Hyperalgesia occurs when opioid-dependent people discontinue opioid use, known as opioid withdrawal-induced hyperalgesia (OWIH). Opioids also relieve this hyperalgesia providing motivation for continued opioid use and dose escalation. Midline thalamic (MThal) nuclei are key hubs responsible for activating brain regions involved in opioid modulation of pain and motivation. However, we lack critical details about what aspects of opioid-mediated behavior and physiology are due to MOR activation on thalamic neurons. We have found that glutamate transmission from MThal neurons to cortical and striatal brain regions is inhibited by MOR agonists like morphine. We hypothesize that MThal neurons are a heterogeneous mix of MOR-expressing and MOR-lacking neurons. We further hypothesize that prolonged inhibition of MThal neurons by opioids can induce adaptations leading to hyperalgesia upon opioid withdrawal (OWIH) while acute opioid-inhibition of MThal neurons will attenuate hyperalgesia. The long term goal of this project is to understand what neurons in the MThal express MOR, how these MOR-expressing MThal neurons respond to conditions that cause hyperalgesia, including OWIH, and whether inhibition of these neurons is sufficient to suppress hyperalgesia. We will use behavioral pharmacology, imaging, whole-cell electrophysiology, and optogenetic manipulations in mice to gain a foundational understanding of how opioid action in thalamo- cortico-striatal brain circuits ultimately affect hyperalgesia and antihyperalgesia. Aim 1 will determine the relative abundance of both MOR-expressing and MOR-lacking medial thalami