Project Summary/Abstract Migraine is a highly prevalent, disabling, chronic episodic and progressive disorder affecting up to a fifth of the entire world population with tremendous socioeconomic impact. Despite recent advances in our understanding of migraine pathophysiology, treatment options are limited and have poor efficacy. Novel therapeutic modalities in migraine are an urgent unmet need. Cortical spreading depression (CSD) is an intense depolarization wave that is the electrophysiological substrate of migraine aura and a headache trigger. CSD is considered among the most reliable and robust experimental models of migraine, and CSD susceptibility is widely accepted as a validated platform to screen for migraine therapies. We recently discovered that vagus nerve stimulation (VNS), a novel neuromodulatory treatment already in clinical use for epilepsy and depression, acutely suppresses CSD susceptibility, suggesting potential therapeutic efficacy in migraine. More importantly, non-invasive cervical transcutaneous VNS (nVNS) was at least as effective as invasive VNS (iVNS) by an implanted electrode, increasing the translational potential. Pilot data show that nVNS inhibition of CSD is mediated by vagal afferent fibers projecting to the brainstem, and involves, at least in part, central serotonergic and norepinephrinergic systems. Building on these discoveries, we propose two aims to (1) establish the therapeutic profile and (2) gain insight into the mechanisms of action of VNS as a novel neuromodulatory intervention targeting CSD. Aim 1 will determine dose/frequency-response, side-specificity, duration of action, additive or synergistic interactions with migraine prophylactic drugs, and chronic daily prophylaxis. In addition, using optogenetics to induce CSD non- invasively, we will test VNS efficacy in clinically more relevant freely behaving female mice expressing human migraine mutations. This translational aim will inform future clinical trials. Aim 2 will interrogate the cerebral circuitry in a logical and linear fashion to understand how VNS inhibits CSD. We will determine the contributions of efferent vs. afferent vagal fibers, map VNS-induced brain activation/inhibition by fMRI, lesion the nucleus tractus solitarius to show its relay role, pharmacologically interrogate the central neurotransmitter systems that may contribute to VNS efficacy on CSD, and using in vivo microdialysis, we will link these to curbing cortical glutamate release as the final common step in CSD suppression by VNS. Altogether, we will fill significant gaps in our knowledge on the therapeutic potential of VNS in migraine and its mechanisms of action on CSD using validated models and innovative, proprietary nVNS technology. The knowledge we gain will also shed light on other diseases where CSD plays a significant role, including traumatic brain injury and ischemic or hemorrhagic stroke, as collateral benefits.