SummaryMigraine is a prevalent neurological disorder affecting millions of people worldwide. The underlyingpathophysiology of migraine likely involves diverse mechanisms within the trigeminal pain pathways and pain-related structures in the brain. Recently introduced medications targeting CGRP mechanisms includingantibodies and small molecule CGRP receptor antagonists appear to act outside of the blood brain barrier. Thesemedications are effective as preventive treatment in some patients implicating the role of meningeal CGRP inmigraine pathology. However many patients do not respond to these therapies and even those that do oftenexperience breakthrough migraines suggesting the existence of non-CGRP peripheral migraine mechanisms.The brain circuits mediating migraine pain remain understudied. Cephalic nociceptive pathways involveactivation of trigeminal afferents and transmission of the nociceptive signal through the second order cells in thetrigeminocervical complex (TCC) to multiple brain regions. Human neuroimaging studies during a migraine attackhave demonstrated activations in the amygdala and dorsal pons encompassing the parabrachial nucleus (PBN).The PBN receives both exteroceptive and interoceptive sensory inputs and projects to multiple sites includingthe central nucleus of the amygdala (CeA) an area mediating emotional aspects of pain. Our preclinical studiesusing pharmacological and optogenetic activation of dural afferents neuronal tracing and behavior suggest afunctional pathway from the dura mater to TCC PBN and CeA that may promote migraine-like pain.In this proposal we will use opto/chemo-genetic methods microscopy with immunostaining and RNAscopeCRISPR-Cas9 (i.e. CRISPR) genetic manipulations electrophysiology calcium imaging and pain behavior toinvestigate if and how the PBNCeA pathway may promote migraine-like pain elicited by activation of duralafferents using several different approaches in male and female mice. Aim 1 will measure the effects on markersof neural activation in brain networks (microscopy) and consequences on pain behavior; Aim 2 will use brainslice electrophysiology and calcium imaging with pharmacological CRISPR or opto/chemo-geneticmanipulations of PBN outputs to evaluate plasticity of CeA cell types; Aim 3 will use chemogenetic inhibition ofPBN outputs or CRISPR deletions in the CeA to evaluate possible inhibition of migraine pain behaviors.Our studies aim to address significant gaps in our knowledge of central pathways of migraine pain. We willdetermine the potential relevance of the central PBNCeA circuit as a common pathway of migraine pain thatcan be engaged by CGRP-dependent and CGRP-independent peripheral mechanisms.