Alterations in the endocannabinoid levels have been found in animal models of pain, neurological and neurodegenerative states, disorders and inflammatory conditions [
1,
2]. There is strong evidence that cannabinoids (CB) can induce antinociceptive effects in models of phasic or tonic pain, through activation of CB receptors located on neurons both within and outside the brain and spinal cord [
3]. It has been shown that CB suppress spinal Fos expression, a neurochemical marker of neuronal activation [
4], in a variety of animal models of persistent pain [
5,
6]. The role of the endocannabinoid system in the pathogenesis of headaches has been recently put under scrutiny. Migraine may be caused by cerebral vasodilatation or by abnormal neurological firing or by neurogenic dural inflammation [
7]. Trigeminal sensory nerve fibers that innervate the cranial vasculature contain calcitonin gene-related peptide (CGRP), substance P and neurokinin [
8]. Endocannabinoid deficiency has been hypothesized to underlie the pathophysiology of migraine and several clinical studies [
9] support this idea although biochemical studies providing a scientific basis for the potential efficacy of (endo)cannabinoids in migraine are really limited. In a previous study, it was reported that anandamide (AEA), an endogenous ligand to the CB receptor, inhibits CGRP-induced and nitric oxide (NO)-induced neurogenic dural vasodilatation, suggesting that AEA may be tonically released to modulate the trigeminovascular system [
10]. Theoretically, the reduction of AEA levels, and thus the reduced inhibitory effect of endocannabinoid system (ECS), may contribute to facilitate/maintain central sensitization in chronic head pain, therefore providing an additional mechanism which contributes to CGRP release and NO production [
11,
12]. Systemic administration of nitroglycerin (NTG), a NO donor, provokes spontaneous-like migraine attacks in migraine sufferers. NTG also induces a condition of hyperalgesia in the rat, through the activation of spinal and brainstem structures involved in nociception [
13‐
15]; As such, NTG has been extensively used to investigate the neurobiological correlates of migraine pain, in rodents [
13‐
15]; Recently, we have shown that NTG-induced hyperalgesia is associated with an alteration of ECS in some areas of rat brain [
16]. In the mesencephalon, an increased activity of both the hydrolases that are involved in degradation of the 2-arachidonoylglycerol (2-AG) and AEA, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), has been observed, together with an increased density of CB binding sites in the mesencephalon. In the hypothalamus, NTG caused an increase in the activity of FAAH associated with an increase in density of CB binding sites, while, in the medulla only the activity of FAAH was increased [
16]. In the present study we have investigated the possible role for AEA in the mechanisms mediating NTG-induced hyperalgesia in the formalin test, a well-established model of persistent somatic pain widely used in rats [
17]. Additionally, we evaluated the effect of AEA on the cerebral expression of Fos protein elicited by NTG-induced hyperalgesia.