CGRP-dependent sensitization of PKC-δ positive neurons in central amygdala mediates chronic migraine

Background: To investigate specific brain regions and neural circuits that are responsible for migraine chronification. Methods: We established a mouse model of chronic migraine with intermittent injections of clinically-relevant dose of nitroglycerin (0.1 mg/kg for 9 days) and validated the model with cephalic and extracephalic mechanical sensitivity, calcitonin gene-related peptide (CGRP) expression in trigeminal ganglion, and responsiveness to sumatriptan or central CGRP blockade. We explored the neurons that were sensitized along with migraine chronification and investigated their roles on migraine phenotypes with chemogenetics. Results: After repetitive nitroglycerin injections, mice displayed sustained supraorbital and hind paw mechanical hyperalgesia, which lasted beyond discontinuation of nitroglycerin infusion and could be transiently reversed by sumatriptan. The CGRP expression in trigeminal ganglion was also upregulated. We found the pERK positive cells were significantly increased in the central nucleus of the amygdala (CeA), and these sensitized cells in the CeA were predominantly protein kinase C-delta (PKC-δ) positive neurons co-expressing CGRP receptors. Remarkably, blockade of the parabrachial nucleus (PBN)-CeA CGRP neurotransmission by CGRP_8–37 microinjection to the CeA attenuated the sustained cephalic and extracephalic mechanical hyperalgesia. Furthermore, chemogenetic silencing of the sensitized CeA PKC-δ positive neurons reversed the mechanical hyperalgesia and CGRP expression in the trigeminal ganglion. In contrast, repetitive chemogenetic activation of the CeA PKC-δ positive neurons recapitulated chronic migraine-like phenotypes in naïve mice. Conclusions: Our data suggest that CeA PKC-δ positive neurons innervated by PBN CGRP positive neurons might contribute to the chronification of migraine, which may serve as future therapeutic targets for chronic migraine.