br Introduction Migraine is a chronic neurological

Introduction Migraine is a chronic neurological disorder characterized by recurrent episodes of headache. It affects more than 10% of the population worldwide, resulting in a staggering amount of episodic disability, a substantial decrease in the quality of life and a concomitant loss in productivity (Akerman et al., 2011). Our understanding of migraine pathophysiology is still not sufficient to treat all of the pathological symptoms of migrainers. Based on the observation that vasodilation induced by serotonin (5-HT) modulation has an essential role in migraine, 5-HT receptor agonists (e.g. ‘triptans’ including sumatriptan) have been developed as first line therapeutics for severe migraine (Akerman et al., 2011). However, not all patients benefit from triptan, especially when taken at a late stage of the attack. Many patients also show recurrences, which require additional medication. Prolonged use of triptans can even induce severe migraine-like headache, known as medication overuse headache (MOH, (Russell, 2014)). Thus, there is a medical need for the development of novel pharmacotherapies that target different signaling mechanisms involved in migraine. One system with a potential clinical relevance for migraine is the endocannabinoid system (Greco et al., 2010). It consists of the G-protein coupled cannabinoid receptors CB1 and CB2, the lipid signaling molecules arachidonoylethanolamide (anandamide, AEA) and 2-arachidonoylglycerol (2-AG), and their biosynthetic and metabolic enzymes. Endocannabinoids are produced “on demand” through difference biosynthetic pathways from phospholipids. AEA is generated mainly from N-arachidonoylphosphatidylethanolamine (NAPE) through hydrolysis by different phospholipases (e.g. NAPE-PLD) and metabolized by enzyme fatty 8-Methoxypsoralen amide hydrolase (FAAH), as well as cyclooxygenase-2. The inhibition of FAAH activity through small molecules inhibitors or the genetic deletion of the FAAH gene, results in a profound increase in brain AEA levels and enhanced endocannabinoid signaling (Cravatt et al., 2001). A large body of evidence has firmly established that the endocannabinoid system is an important modulator of acute and chronic pain states. Activation of cannabinoid receptors expressed on nociceptive neurons and central areas involved in nociception can evoke analgesic effects in tonic or phasic pain including migraine (Pertwee, 2001). Thus, cannabis (or marijuana) has been used as effective remedy for headaches around the world at least since the 6th century (Russo, 1998). Interestingly, a number of reports showed that endocannabinoid deficiency may contribute to the pathophysiology of several chronic painful diseases including migraine (Russo, 2004, Smith and Wagner, 2014). Further clinical studies revealed a down-regulation of AEA and 2-AG, along with an up-regulation of the metabolic enzyme fatty acid amide hydrolase (FAAH) (Cupini et al., 2008, Rossi et al., 2008) in migraine patients. Animal studies have also shown that an acute injection of nitroglycerin (NTG), an animal model of migraine headache, increased the levels of FAAH and the 2-AG metabolic enzyme monoacylglycerol lipase (MAGL) in several brain regions (Greco et al., 2010). NTG injection evokes a peripheral hyperalgesia and neuronal hyperactivation in trigeminal nucleus, which was significantly inhibited by enxogenous AEA administration (Greco et al., 2010). The purpose of this study was to investigate the role of FAAH in the NTG animal model and to determine if this enzyme would be a valid target for migraine therapies. FAAH can be the better therapeutic target for migraine compare to cannabinoid receptors, as FAAH inhibitors are known to have analgesic effect in peripheral inflammatory and neuropathic pain models with no undesirable side effects (Ahn et al., 2009a). To this aim, we compared in vivo effect of NTG in mutant mice lacking FAAH of CB1 receptors, as well as after pharmacological blockade of FAAH or CB1 activity. We demonstrate that genetic or pharmacological inhibition of FAAH profoundly reduced NTG-induced migraine-like hyperalgesia and trigeminal neuronal activity. This effect was absent in CB1 deficient mice and after administration of the CB1 inverse agonist rimonabant, thus strongly suggesting that anti-NTG pain effect is due to CB1 receptor activation induced by increased AEA.