Chronic alcohol use is prevalent among the veteran population and can permanently alter brain function and exacerbate existing health conditions. There is a growing literature that alcohol use may exacerbate chronic pain and predispose to pain disorders, but the neural mechanisms are still unknown. Our preliminary data suggests that mice exposed to chronic intermittent ethanol (CIE) exhibit increased pain sensitivity, hyperexcitability of serotonin (5-HT) neurons, and elevated 5-HT immunoreactivity in the dorsal raphe nucleus (DRN). Ablation of 5-HT neurons in the DRN also reduced nociception in the Von Frey test, suggesting that hyperactivity in these neurons following CIE may facilitate pain. DRN neurons project principally to the forebrain and have excitatory, 5-HT-mediated input to the paraventricular nucleus of the thalamus (PVT), which can also facilitate pain via projections to the central amygdala (CeA). At the same time, CIE reduced tryptophan-hydroxylase 2 (Tph2) expression in the raphe magnus (RMg), which is another serotonergic nucleus that projects to the dorsal horn (DH) of the spinal cord and may promote anti-nociception. The overarching goal of this application is to investigate two discrete serotonergic mechanisms of alcohol- induced hyperalgesia. We propose that CIE increases activity in the 5-HT DRN→PVT pathway that facilities pain and decreases activity in the 5HT RMg→DH pathway that inhibits pain. In Aim 1, we will examine the effects of CIE on activity in 5-HT DRN→PVT projection neurons using in vivo fiber photometry and ex vivo slice electrophysiology as complementary approaches. We will then examine 5-HT release dynamics in the PVT with fiber photometry, followed by slice electrophysiology to dissect the effect of this enhanced 5-HT release on neural activity in PVT→CeA neurons. We will then determine whether CIE can alter 5-HT receptor expression in PVT→CeA projection neurons using translating ribosome affinity purification (TRAP) and RT-PCR. These experiments will reveal whether CIE can promote 5-HT signaling in the PVT and activate PVT projections to the CeA that facilitate pain. Aim 2 will investigate the effects of CIE on 5-HT RMg neurons using similar approaches as in Aim 1. We will also examine pain-evoked activity in DH neurons receiving input from 5-HT RMg neurons using fos immunohistochemistry. 5-HT receptor gene expression in DH neurons receiving input from the RMg will also be examined using a combination of TRAP and RT-PCR. Finally in Aim 3, we will evaluate the respective contributions of the 5-HT DRN→ PVT and 5-HT RMg→DH pathways to pain and alcohol-induced hyperalgesia using multiplexed DREADDS that enable simultaneous manipulation of both pathways in the same animals. Together, these experiments will generate mechanistic insight into the role of chronic alcohol abuse in predisposing to and precipitating pain disorders.