Insular cortex CaMKIIα neurons drive migraine-related behaviours in chronic migraine mice

The insular cortex (IC) is recognized as a critical neural structure involved in the regulation of pain perception and emotional responses, with its dysfunction being closely associated with the development of chronic migraine (CM). The specific cellular substrates or mechanisms underlying its contributions to CM pathogenesis are poorly understood. The IC is predominantly composed of CaMKIIα-expressing neurons, which are key molecules involved in synaptic plasticity. It remains unknown whether these IC CaMKIIα-expressing neurons modulate CM development, pain-associated behaviours, and synaptic plasticity within the IC. Adult mice received repeated intraperitoneal nitroglycerin (NTG) injections to establish the CM model, and pain sensitivity and photophobia-like behaviour were evaluated by von-Frey filaments and the light/dark box test. Western blotting and immunofluorescence staining were performed to evaluate IC involvement and quantify activated CaMKIIα-expressing neurons. In vivo calcium imaging was used to monitor the activity dynamics of the right IC CaMKIIα-expressing neurons following the final NTG challenge in mice with established CM, and the MED64 multichannel recording system was applied to demonstrate the extracellular field-potential responses between CM and control groups. The chemogenetic approaches were employed to selectively modulate the IC CaMKIIα neuronal activity and examine pain-related behavioural outcomes. Repeated NTG administration induced robust pain sensitization and light-aversion in adult mice. Enhanced expression of c-Fos, calcitonin gene-related peptide (CGRP), and CGRP receptor component protein (CRCP) was found in the IC of CM mice. Co-localization analysis and in vivo calcium imaging demonstrated significant increases in both the number and activity of activated IC CaMKIIα-expressing neurons in the NTG-induced CM model. Electrophysiological recordings from the IC revealed enhanced synaptic potentiation, characterized by increased long-term potentiation (LTP) after theta-burst stimulation, with a greater proportion of LTP-responsive channels in the IC area compared to controls. Chemogenetic inhibition of IC CaMKIIα-expressing neurons attenuated pain sensitization and light-aversion, whereas activation in naïve mice did not induce these phenotypes. Our findings reveal that IC CaMKIIα-expressing neurons contribute significantly to the regulation of pain sensitization and light-aversion in CM pathophysiology. Enhanced insula synaptic plasticity may represent a potential mechanism underlying CM, providing a promising therapeutic target for migraine.