While animal models of headache have been able to recreate migraine symptoms, including tactile sensitivity, modeling headache induction is still elusive. This novel demonstration of inducible headache like pain using a dietary headache trigger represents an important step for characterizing mechanisms and developing therapeutics. We show the effects of ethanol and its two major metabolites, acetaldehyde and acetate, in an animal model of trigeminal pain. Ethanol initially decreased nociceptive behaviors and sensitivity. This was followed by increased sensitivity to trigeminal mechanical stimuli. The initial decrease in nociceptive behaviors is due to ethanol induced analgesia. In the 19th
century, ethanol was used during human surgery as an anesthetic (summarized in 
). Ethanol induces analgesia in many rat nociceptive behaviors, including tail flick and cold water test at doses ranging from 0.5 to 1.5 g/kg 
. In addition, Friedman and colleagues found analgesia in other behaviors such as shock induced startle, vocalizations and overt movements in rats 
. Similar behavioral phenotypes were found in mice given ethanol 
. Ethanol induced analgesia is mediated through the GABA neurotransmitter system 
. Molecular studies have suggested a role for the G-protein coupled inwardly rectifying potassium channels in analgesia 
. While the ethanol induced analgesia is well documented in the literature, little is known regarding its pronociceptive properties.
Ethanol induced delayed trigeminal hypersensitivity 4 to 6 hours after administration, when blood ethanol levels are expected to be close to zero 
. The decrease in periorbital pressure threshold is analogous to the mechanical allodynia seen in patients during a headache 
. Ketorolac reversed this decrease in threshold, suggesting that the rats have inflammatory pain. Interestingly, the decrease in threshold was only found in sensitized rats with a prior history of repetitive trigeminal nociceptor stimulation. This is in consensus with clinical studies that show an increase in hangover vulnerability in patients with a history of recurrent headaches 
. Low dose ethanol, equivalent to approximately one standard drink in humans, was chosen to differentiate between sensitized rats and those naive to trigeminal pain. In humans, a delayed alcohol induced headache (DAIH) is characterized by pain occurring from 4 to 24 hours following ingestion and is correlated with blood ethanol levels at or returning to zero 
. In addition, migraineurs can experience DAIH with a modest intake of alcohol, whereas non migraineurs require larger amounts to produce the phenomenon 
. Therefore, our findings model the sensitivities seen in migraineurs.
Most authors claim that acetaldehyde is responsible for hangover symptoms, but they neglected to consider the rapid formation of acetate from acetaldehyde 
. Our results provide direct evidence that acetate, not acetaldehyde, contributes to the delayed increase in trigeminal sensitivity. Ethanol plus disulfiram, which is known to increase acetaldehyde levels, did not produce delayed hypersensitivity for nociceptive behaviors. This suggests that acetaldehyde may not directly contribute to the pain seen in our model. Alternatively, the low dose of ethanol may not have been sufficient to produce a toxic build up of acetaldehyde when given with disulfiram. Disulfiram at 100 mg/kg provides approximately 80% inhibition of aldehyde dehydrogenase 1 and 2 and 61% inhibition of aldehyde dehydrogenase 3 in the rat 
. Since a majority of aldehyde dehydrogenase was inhibited by disulfiram and headache pain is reported with the combination of disulfiram and ethanol in alcoholics, acetaldehyde accumulation did not cause the pain seen in our model.
Acetate administration induced an immediate decrease in trigeminal pressure thresholds in sensitized rats. The dose of acetate administered (60 mg/kg) produces serum acetate levels of 1.09 mM in rats, which is equivalent to acetate levels attained with our dose of ethanol (0.95 mM) and consistent with previously published data in rats 
and humans 
. Thus, we used physiologically relevant doses of acetate to induce increased sensitivity for nociceptive behaviors, indicating that acetate may contribute to DAIH. The similar effects of ethanol and acetate have been shown in behaviors including motor coordination and sensitivity to anesthesia 
. Patients experience headaches when acetate is used as a buffer for kidney dialysis at serum concentrations ranging from 2 to 4 mM, but headache history and their susceptibility to headache was not measured 
These data do not completely exclude the role of acetaldehyde in hangover. Although treatment with ethanol in the presence of disulfiram did not induce a headache in our sensitized rats, these data provide indirect evidence that acetaldehyde may not be involved in ethanol induced trigeminal pain. Acetaldehyde may still cause hangover symptoms in humans including headache. The data in this study demonstrate that acetate also contributes to the headache component of the hangover.
Signaling cascades downstream of acetate can promote pain. Acetate increases adenosine in many tissues, including the brain 
. The adenosine receptor antagonist, caffeine, administered after ethanol, blocked the nociceptive behaviors associated with ethanol. This suggests that adenosine contributes to ethanol induced hypersensitivity. Caffeine and ketorolac are used to treat hangover symptoms in humans. Non-steroidal anti-inflammatory drugs (ketorolac) alleviate the headache pain associated with alcohol hangovers and caffeine is indicated to ease other hangover symptoms, including fatigue and malaise 
Our findings challenge the concept that dehydration and congeners alone are the cause of hangover headache. Our rats were normally hydrated and administered pure ethanol. While dehydration and congeners may contribute to the hangover in humans, this is the first demonstration that acetate accumulation may play a role in hangover headache induction.
We have shown that ethanol, a migraine trigger in humans, lowers nociceptive threshold in sensitized rats with a history of trigeminal nociceptor stimulation. This is similar to the effect of nitroglycerin, a nitric oxide donor, both clinically and in our model 
. Nitroglycerin induces a short dull headache in non migraineurs but in migraine patients can trigger a long lasting migraine attack 
. In naive rats, nitroglycerin causes a decrease in threshold for nociceptive behaviors for about 30 minutes. However, in rats with repeated dura stimulation, nitroglycerin administration produces significantly decreased thresholds for more than two hours 
. Our model is thus sensitive to two common triggers of headache, nitric oxide donors and ethanol. Interestingly, the timing of delayed nociception in our model allows for future studies of the molecular mechanism of headache induction with ethanol or other triggers.
Many studies have used inflammatory dura stimulation to model different types of headaches. Migraine, a primary headache disorder, is characterized by sensitivity to light, sound and touch on the head and face. Facial allodynia is sensitivity to innocuous stimuli that is mediated by the trigeminal neurovascular system 
. Reports from various laboratories indicate that inflammatory stimulation of the dura causes allodynia on the face in animals, through convergence of dura and periorbital afferents at the level of the trigeminal system 
. This phenomenon is consistent with human data 
. Our laboratory has also shown increased sensitivity to sound or phonophobia following repeated dura stimulation, which has been demonstrated in humans both during and between migraine attacks 
. As described in the current study, our model is sensitive to common headache triggers including ethanol and nitroglycerin.
Ethanol in sensitized rats induces analgesia, followed by increased sensitivity for nociceptive behaviors, modeling delayed alcohol induced headache. Acetate contributes to the pain portion of the ethanol hangover, in part through adenosine receptors. This is the first demonstration of a potential mechanism for ethanol hangover headache.