Endocannabinoids Block Headache and Anxiety Comorbidity via Two-Pronged Anterior Insular Projections

The mechanism of and effective treatment for the headache, with the global prevalence of 52%, and its common anxiety comorbidity remain elusive. Here, we found that chronic isosorbide dinitrate (ISDN) injections induce c-fos expression in the anterior insula (AI), prelimbic cortex (PrL), and oval nucleus of the bed nucleus of the stria terminalis (ovBNST), suggesting their contributions to headache and anxiety comorbidity. This hypothesis is substantiated by our findings that chronic ISDN injection-induced headache and anxiety are blocked by inhibition of ventral AI (vAI)-PrL and dorsal AI (dAI)-ovBNST circuits, respectively. Headache and anxiety stimuli in chronic ISDN-injected mice markedly increase endocannabinoid (eCB) release at both glutamatergic vAI-PrL synapses and dAI-ovBNST synapses, indicating the role of eCB signaling in modulating headache and anxiety. Indeed, presynaptic knockdown of eCB hydrolase or presynaptic activation of cannabinoid type 1 receptors (CB1Rs) in vAI-PrL and dAI-ovBNST circuits separately alleviates headache and anxiety. A systemic application of eCB degradation enzyme inhibitors blocks chronic ISDN-induced headache and anxiety comorbidity, which are separately blocked by CB1R antagonist application in PrLs and ovBNSTs. Our findings reveal divergent counteracting effects of elevated eCB signaling in vAI-PrL and dAI-ovBNST circuits on comorbid headache and anxiety.