PROJECT SUMMARY/ABSTRACT: Chronic musculoskeletal (MSK) pain is leading contributor to disability. Many conditions that cause MSK pain have a higher prevalence in females. Females often report heightened sensitivity and lower pain tolerance in conditions where their male counterparts do not. Ischemia with reperfusion (I/R) injury is a common cause of MSK pain and exercise intolerance (through altered exercise pressor reflexes (EPRs)). Repeated I/R injury is often seen in complex regional pain syndrome and fibromyalgia, both of which have higher prevalence and prolonged pain in females compared to males. Clinical and preclinical research have linked sex hormones to pain. Prolactin (PRL) and the Prolactin receptor (Prlr) play a prominent role in inflammatory pain in females through dorsal root ganglia (DRGs) neuron sensitization. However, it is not yet known what roles PRL and Prlr play in ischemic myalgia. Pilot data demonstrated that prolonged behavioral hypersensitivity in females with repeated I/R correlates with upregulation in STAT3, TRPV1 and IL1R1 mRNA in the DRGs, while males show different transcriptional mechanisms. This may be due to sex-specific activation of AU-rich element RNA binding protein 1 (AUF1) in female DRGs. AUF1 regulates I/R-related behaviors and IL1r1 and TRPV1 expression and is known to be modulated by STAT3. Finally, Prlr antagonists not only inhibited female pain-like behaviors after I/R, but they also blocked the upregulation of STAT3, IL1r1, TRPV1 and AUF1. Therefore, we will test the central hypothesis that prolactin receptor modulates prolonged hypersensitivity following repeated ischemic injury in a sex- dependent manner. Transgenic and pharmacological approaches will be used to determine effects of Prlr on nociception and cardiovascular reflexes through specific neuronal subpopulations following repeated ischemic injury in males and females. Pain-related behavioral assays, EPR tests, ex vivo forepaw muscle/median and ulnar nerves/DRG/spinal cord single unit electrophysiological recording preparations, and molecular analyses (realtime PCR and RNA-Seq) of DRGs will be conducted. Aim 1 will determine the neuronal subtypes containing Prlr that modulate hypersensitivity after repeated I/R injury through knockout (KO) of Prlr in TRPV1+ sensory neurons. Aim 2 will determine the transcriptional mechanisms within sensory neurons that modulate prolonged hypersensitivity following repeated I/R injury and test the working hypothesis that neuronal (TRPV1+ vs all sensory neurons) STAT3, downstream of Prlr, regulates pain-like behaviors and EPRs after repeated I/R injury in females through activation of AUF1. This research will be the first to assess the sex-dependent role of Prlr in MSK pain and will provide some of the first evidence to support development of sex-based, treatment strategies for ischemic myalgia.